Information

Removing a restriction site and introducing other at its place


What would you do if you want to remove an EcoRI restriction site and introduce BamHI restriction site at apprx. the same location ?

One of the answers in my textbook was : To construct a DNA fragment with the structure:

5'AATTGGATCC3' 3'CCTAGGTTAA5'

I understand that this would ligate efficiently to the sticky ends generated by EcoRI cleavage and would introduce a BamHI site but I don't understand how this will remove/not regenerate the EcoRI site.


The recognition site for EcoRI isGAATTC, and the enzyme cuts after the first base. See this picture from NEB:

The overhang is: AATT, which is supplied by your oligo and fits into the overhang. If the next nucleotide whould be aCthan the site would be recreated, since its aGits not. The new sequence of the old EcoRI site isGGATTGwhich is not recognized by EcoRI.

One technical note: To avoid religation of the digested DNA which carries this site, I would treat it with alkaline phosphatase to remove the phosphate end at the 5' end of the DNA.


Just in case you are having difficulty visualising what happens in the answer from @Chris, here are the steps with the linker shown in lower case

… NNNNNGAATTCNNNNN… ds DNA… NNNNNCTTAAGNNNNN… | V… NNNNG AATTCNNNNN… after EcoRI digestion… NNNNCTTAA GNNNNN… | V… NNNNG aattggatcc AATTCNNNNN… incoming linker… NNNNCTTAA cctaggttaa GNNNNN… | V… NNNNGaattggatccAATTCNNNNN… after ligation… NNNNCTTAAcctaggttaaGNNNNN…

The left hand "EcoRI" is now GAATTG and the right hand "EcoRI" is CAATTC.


Plasmids 101: Restriction Cloning

When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of how to do perform restriction digests.

Before beginning the restriction digest and ligation process, you should carefully choose your backbone and insert - these both must have compatible cut sites for restriction enzymes that allow your insert to be placed into the backbone in the proper orientation. For instance, if you were cloning a gene into an expression vector, you would want the start of the gene to end up just downstream of the promoter found in the backbone. Ideally, the backbone will contain a variety of restriction enzyme cut sites (restriction sites) downstream of the promoter as part of a multiple cloning site (MCS). Having multiple sites allows you to easily orient your gene insert with respect to the promoter.

For example, let’s say your plasmid backbone looks like the one found on the left side of the image below. It has a promoter (blue arrow) followed by the restriction sites EcoRI, XhoI, and HindIII. To place your gene in the proper orientation downstream of the promoter, you can add an EcoRI site just 5’ of the start of the gene and a HindIII site just 3’ of the end of the gene. This way you can then cut the plasmid backbone as well as the insert with EcoRI and HindIII and, when you mix the cut products together, the two EcoRI digested ends will anneal and the two HindIII digested ends will anneal leaving the 5’ end of your gene just downstream of the promoter and placing the gene in the proper orientation. You then add ligase to the mixture to covalently link the backbone and insert and, PRESTO, you have a full plasmid ready to be used in your experiments.

Alternatively, this whole process can be completed using a single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert can then anneal to the backbone in either a forward or reverse orientation so you’ll need some way to verify that the insert ended up in the direction you want - usually by S anger sequencing or further restriction digests.

Of course there’s much more detail and verification required for the process to work well, so let ’ s go over the details step-by-step.


Health Care Policy Analyst - John Locke Foundation

While the search for a global vaccination to cure the disease is in process, the stress on medical providers and hospitals prompted a historic move toward the authorization and adoption of telehealth services. Embroiled in decades-old debates over its effectiveness in providing patient care, telehealth has also faced other obstacles to its adoption and use, including licensure, reimbursement, and eligible services. Yet, in response to the coronavirus outbreak, the Trump administration and the U.S. Department of Health and Human Services (HHS) sweepingly approved the use of telehealth services as part of the Coronavirus Preparedness and Response Supplemental Appropriations Act. 7 As part of this newly granted permission, most Medicare payment requirements were waived and recipients were able to access remote care, regardless of where they live. During the pandemic, telehealth services were also charged at the same rate of in-person medical services, or at parity. The move to accelerate the use of telehealth services also included other exemptions, including some HIPAA exceptions for providers when Facetime or Skype was used by doctors to communicate with patients.

Before COVID-19, telehealth initiatives provided a platform to combat the shortcomings of cost, quality, and access ingrained in American health care. The breadth of telehealth services includes remote clinical health care, patient and professional health-related education, public health, and health administration via electronic information and telecommunication technologies. 8 Health-care delivery services are also integrating artificial intelligence (AI) systems into the suite of telehealth services, as both doctors and patients move from solely remote patient monitoring for continuous recording of vital signs to real-time alerts from a patient sensor when there is a deteriorating change in condition. Further, AI is assisting in the management of chronic conditions, including diabetes and heart disease, and when patients require care from multiple specialists working at different times and locations. In these instances, existing applications, AI, and other emerging technologies are coordinated under the guise of telemedicine for complex treatments, like virtual assistants to help patients carry out treatment plans by sending reminders to take medications and providing relevant health information. 9

“State and federal barriers in the use of telehealth and AI have served as hindrances to the launch of its full capabilities.”

Prior to the coronavirus outbreak, telehealth and integrated AI were somewhat familiar though not common in practice. But the increasing use of technology has not necessarily been embraced by the long-standing rules and regulations governing the full body of the health-care system. Until recently, telehealth use has largely been limited, stifled by the ambiguous and often changing regulations on the reimbursement of doctors and licensure, especially across state lines. State and federal barriers in the use of telehealth and AI have served as hindrances to the launch of its full capabilities, particularly those laws that present a patchwork of accepted and non-eligible costs and services. Given that telehealth now has a critical role in the mitigation of COVID-19, how well will the U.S. take guidance from its rapid adoption and use? More specifically, how can telehealth be more visibly positioned as an important aspect of health-care delivery in a post COVID-19 health-care ecosystem? And, will telehealth practices be continued without the previously applied restrictions of state and federal laws, especially those around service reimbursement or parity agreements?

This paper explores these questions to extrapolate what state and federal policies will need to be adopted to potentially prepare for more ubiquitous adoption and use of telehealth services in an expanded set of use cases than those recorded by law. The authors also explore the application of existing and emerging state parity laws, which could serve as an obstacle to telehealth delivery in the future. Despite their application as a framework for reimbursement of COVID-19 expenses during the current application, the paper will provide guidance on these and other state and federal laws that will run counter to the long-term promotion and patient access of digital technologies, particularly those that aid in the management of primary care, chronic health conditions, and prevention. The paper concludes with a set of policy and pragmatic proposals that combine the recent lessons learned by the health-care community and patients, along with larger issues, including broadband access, that set the stage for future use. These recommendations were compiled after a structured focus group with medical practitioners, associations, and health policymakers working on the matters described in this paper.


How is DNA Manipulated?

1. Explain the following terms and their role in recombinant DNA technology

a) Restriction enzyme ___ cuts DNA into fragments _____
b) Recognition site ___ specific site or spot where the enzyme cuts __
c) Sticky end ______ area of DNA where bases are ready to be paired, they will "stick" to matching DNA __
d) Ligation _____________ the joining of two different DNA ________________
e) rDNA ________ the new DNA is called DNA, or recombinant DNA _____________

Here are some restriction enzymes and their sites.

G T C / G A C
C A G / C T G

2. On each of the sequences below, determine which restriction enzyme could be used to splice the DNA and indicate where the cut will be made and the enzyme used. This is actually tedious to accomplish, I've found it easier if you actually cut the enzymes from the above chart and use them to find the corresponding locations on the sequences below, you may also wish to only assign the first couple because it takes so long to find the matches.

5' T T T G / A A T T C A G A T 3'
3' A A A C T T A A / G T C T A 5'

5' G T G G / G A T C C C T T A 3'
3' C A C C C T A G / G G A A T 5'

Enzyme: ___ BAMHI ________

5' A C G C C T C / C G G A G A 3'
3' T G C G G A G G C / C T C T 5'

5' T T A / A G C T T A A G A A G C T T 3'
3' A A T T C G A / A T T C T T C G A A 5'

Enzyme: __ HindIII ________

5' A A G C G / C G T C G A C T T A T A 3'
3' T T C / G C G C A G C T G A A T A T 5'

Enzyme: ____ HhaI ________

3. Create a restriction enzyme that will remove the gene of interst. Give it a name too!

4. The following DNA sequence is from a virus that is dangerous, scientists want to use a restriction enzyme to cut the virus into bits. They do not need sticky ends because the do not plan to combine it with other DNA. Use HindII to show how this DNA would be cut. How many pieces would you have? __ 4 __

G A A A A G T C / G A C A A G G C A G T C / G A C T T T T A A A A G T C / G A C A T G C
C T T T T C A G / C T G T T C C G T C A G / C T G A A A A T T T T C A G / C T G T A C G


3. Restrictions

3.1 The nature and effect of restrictions

3.1.1 The general effect of restrictions

Restrictions prohibit the making of an entry in respect of a disposition or a disposition of a specified kind. The prohibition may be indefinite or for a specified period and it may be absolute or conditional on something happening (for example, on getting the consent of a third-party).

The term ‘disposition’ is not defined in the Land Registration Act 2002. Most restrictions refer to dispositions by the proprietor of the registered estate or of a registered charge, implying some action by that proprietor to make the disposition. A disposition may also occur by operation of law, and a restriction that refers merely to “no disposition” would also catch such a disposition. Please note, however, that a discharge of a registered charge is not a disposition and cannot be prevented by a restriction.

A restriction makes it apparent from the register that either the powers of the relevant proprietor are limited, or that a prior condition must be met before a disposition can be registered. The purpose of restrictions is to regulate registration, not regulate dispositions. So, where a restriction entered in the register requires consent or has an option requiring consent, the consent given in relation to the restriction should expressly consent to the registration of the disposition, not consent to the disposition. This requirement does not apply to a restriction that requires a certificate. In that case, all that is needed is that the certificate complies with the requirements of the restriction.

Once entered, a restriction will remain in the register until it is cancelled or withdrawn. Restrictions are not automatically cancelled following a disposition, although we may cancel any restriction that has clearly become superfluous.

3.1.2 Restrictions that affect a registered estate

A restriction that is entered to regulate dispositions of a registered estate will be entered in the proprietorship register.

Such restrictions do not have any effect on existing registered charges or the powers of the registered chargee. Since 10 November 2008, the wording of standard restrictions makes this clear, but the principle also applies to restrictions entered before that date. However, a restriction entered in the proprietorship register may affect a charge that is registered afterwards. The date entered in brackets at the beginning of an entry shows the date of its registration. You can tell by comparing the date of a restriction and the date of registration of a registered charge whether the chargee’s powers may be affected.

When you are applying to register more than one transaction, we need to be clear as to the order of the applications. For instance, where a transfer and a charge are lodged together, and the transfer includes a request to enter a restriction, we will not automatically assume that the charge application has priority over a restriction requested in a transfer. The order of applications must be clearly stated, and this is the customer’s responsibility. Where required, the restrictioner’s consent to the registration of the charge must be lodged.

3.1.3 Restrictions that affect an existing registered charge

A restriction that affects an existing registered charge will be entered in the charges register and will refer specifically to the entries relating to the affected charge.

Even though a restriction entered in the proprietorship register may appear to restrict dispositions by ‘the proprietor of any registered charge’ (see, for example, the Form O restriction), it will not have any effect on a charge that was registered prior to the entry of the restriction.

If you intend to restrict all dispositions whether by the proprietor of the registered estate or the proprietor of an existing registered charge, you must apply for separate restrictions in the 2 parts of the register.

3.1.4 Entries that may be prevented by a restriction

The standard form restrictions prescribed in Schedule 4 to the Land Registration Rules 2003 each regulate or prohibit the registration of a disposition. Registration in this context is defined as meaning the completion by registration of a registrable disposition . None of the standard form restrictions prevent the mere entry of a notice.

Note, however, that some restrictions entered under the Land Registration Act 1925 expressly prevented the entry of a notice. We will not accept an application for a restriction not in a standard form under the Land Registration Act 2002 that expressly prevents the entry of a notice, as this would have the effect of preventing the protection of a third-party interest by an agreed or unilateral notice.

3.1.5 Compliance with a restriction

3.1.5.1 General

Any certificate or consent required by a restriction must be signed in ‘wet ink’ subject to Certificate or consents by conveyancers and Emailed certificates or consents.

Where a restriction catches 2 or more dispositions being lodged at the same time, the consent or certificate of compliance must be to all of the dispositions being registered. For example, if both a transfer and charge are being registered and both are caught by a restriction that requires consent, the consent given must be to the registration of both the transfer and the charge.

A consent required by a restriction must state that it is to the registration of the disposition, and not just to the disposition itself.

If an application is being made by post and the certificate or consent is being lodged with that application, the original certificate or consent, or a certified copy of it, must be lodged by post.

If an application is being made via the portal or Business Gateway and a copy of the certificate or consent is being lodged with that application, or the restriction relates to a pending application, a scanned copy of the certificate or consent should be lodged by uploading it via our portal or Business Gateway. The scanned copy of the certificate or consent should be certified to be a true copy of the original using the certification statements available through our portal or Business Gateway. We will not accept a certificate or consent that is given only in the notes function within ‘reply to requisition’ in our portal or Business Gateway.

3.1.5.2 Certificate or consent by corporation aggregate

Where the terms of a restriction require a certificate or consent signed by a corporation aggregate, (which includes overseas companies), unless a contrary intention appears in the restriction or the certificate or consent is given in a deed executed by the corporation in question, the certificate or consent must be signed by either:

  • its clerk, secretary or other permanent officer
  • a member of its board of directors, council or other governing body
  • its conveyancer
  • its duly authorised employee or agent (rule 91B of the Land Registration Rules 2003)

If a restriction requires a certificate or consent to be signed on behalf of a corporation by its secretary but that corporation has no secretary, the certificate or consent should be signed by one of the other people listed above (rule 91B(4) of the Land Registration Rules 2003).

The certificate or consent must state the full name of the signatory and the capacity in which the signatory signs (rule 91B(5) of the Land Registration Rules 2003). The certificate or consent must be signed by the individual in their own name.

3.1.5.3 Certificate or consent by a conveyancer

The certificate or consent (again with the exception of the certificate required by a restriction in standard Form LL) can also be signed by the firm’s name being typed or stamped at the end, but the individual completing the certificate or consent must also provide their full name and status in the firm.

3.1.5.4 Emailed certificates or consents

A certificates or consent by an individual (on their own behalf or on behalf of a corporation aggregate) can be given in an email to their conveyancer .

The email must not be forwarded or sent to us by a further email. Instead, the normal lodging process set out in General, must be followed so that a copy of the email containing the consent or certificate is lodged either:

3.1.5.5 Restriction in Form LL

3.2 Entry of restrictions

3.2.1 Restrictions entered at the registrar’s discretion

Section 42(1) of the Land Registration Act 2002 sets out the registrar’s general power to enter a restriction as follows.

“The registrar may enter a restriction in the register if it appears to him that it is necessary or desirable to do so for the purpose of –

(a) preventing invalidity or unlawfulness in relation to dispositions of a registered estate or charge,

(b) securing that interests which are capable of being overreached on a disposition of a registered estate or charge are overreached, or

(c) protecting a right or claim in relation to a registered estate or charge.”

The registrar may enter a restriction to fulfil one of these purposes whether or not an application is made to do so. However, the registrar will always notify the relevant proprietor when a restriction is entered without an application having been made to do so. See Notifiable applications for information about notices served when an application has been made to enter a restriction.

It will usually be clear whether a restriction is necessary or desirable for one of the three permitted purposes, but this will not always be the case. Note that the purpose set out in section 42(1)(c) of the Land Registration Act 2002 does not permit the registrar to enter a restriction in respect of any right or claim but is limited as follows.

  • Firstly, the registrar may not enter a restriction to protect the priority of an interest that is or could be protected by notice (section 42(2) of the Land Registration Act 2002). However, a restriction might still be necessary or desirable for one of the other permitted purposes set out in section 42(1)(a) or (b) of the Land Registration Act 2002, for example to prevent the unlawful breach of a provision in a contract that has been protected by notice
  • Secondly, a right or claim may only be protected under the third permitted purpose if it relates to a registered estate or charge. As only the legal estate will be registered, this does not include rights or claims that relate only to a beneficial interest in property

A charging order affecting a beneficial interest under a trust may still be protected by a restriction entered under section 42(1)(c) of the Land Registration Act 2002, such as Form K, as this is expressly confirmed in the Land Registration Act 2002 (section 42(4) of the Land Registration Act 2002). However, generally a restriction can only be entered in respect of an interest under a trust for one of the other two permitted purposes. In most cases, a restriction in Form A will be appropriate for the second purpose – to ensure that overreaching takes place on a disposition that gives rise to capital monies. See practice guide 24: private trusts of land for more information.

3.2.2 Where we are obliged to enter a restriction

We are obliged to enter a restriction in certain circumstances. These are where:

  • we enter 2 or more people as joint proprietors of a registered estate in land and the survivor will not be able to give a valid receipt for capital money (section 44(1) of the Land Registration Act 2002) – see practice guide 24: private trusts of land for more information
  • some other statute requires the registrar to enter a restriction (section 44(2) of the Land Registration Act 2002)
  • a bankruptcy order is registered under the Land Charges Act 1972 and it appears that a registered estate or charge is affected (section 86(4) of the Land Registration Act 2002). Practice guide 34: personal insolvency provides further detail about the entry of a bankruptcy restriction
  • the court makes an order that requires the registrar to enter a restriction (section 46 of the Land Registration Act 2002) - see Court orders requiring the entry of a restriction for more detail about restrictions required by the court

Rule 95 of the Land Registration Rules 2003 specifies the forms of restriction that we are obliged to enter under various statutory provisions.

3.3 The form of a restriction

3.3.1 Standard form restrictions

The effect of a restriction must be clear from its wording and its administration must not place us under an unreasonable burden. Schedule 4 to the Land Registration Rules 2003 prescribes a number of standard form restrictions that are intended to cover the vast majority of applications made.

The standard form restrictions are worded in a clear manner so that we, and someone inspecting the register, will be able to determine whether a given application will be caught by its terms and, if so, what action needs to be taken to allow the application to proceed.

When applying for a standard application, remember that:

  • words in [square brackets] in ordinary type are optional parts of the form the brackets are not to be included in the restriction
  • words in re instructions for completion of the form, and are not to be included in the restriction
  • where (round brackets) enclose one or more words, the brackets and all words in ordinary type enclosed in them are part of the form and, unless also enclosed in [square brackets], must be included in the restriction
  • where a form contains a group of clauses introduced by bullets, only one of the clauses may be used the bullets are not to be included in the restriction
  • where a restriction in Form J, K, Q, S, T, BB, DD, FF, HH, JJ, LL or OO relates to a registered charge, which is one of two or more registered charges bearing the same date and affecting the same registered estate, the words ‘in favour of’ followed by the name of the registered proprietor of the charge must be inserted in the restriction after the date of the charge
  • while some standard restrictions allow reference to compliance with a specific clause in a deed, they do not allow wording which refers to the deed as a whole, “all clauses” or similar: such references would make the restriction non-standard
  • where the wording of a restriction in Schedule 4 to the Land Registration Rules 2003 provides for a certificate or consent to be given by the restrictioner ‘[or [their conveyancer or specify appropriate details]’, the words ‘or specify appropriate details’ should refer to a class (or classes) of person who may be expected to act on behalf of the restrictioner rather than to a particular named individual
  • a standard restriction cannot be amended to commence with the words ‘no dealing’ or ‘no disposition or dealing’, nor can wording be used to prevent noting (as opposed to registration) of a disposition

Note that restrictions in standard Form N and T were amended on 10 November 2008. Prior to that date these restrictions provided for the option of either a consent or a certificate to be lodged. They now provide only for a consent to be given. Forms NN and OO have been introduced and these allow for either a consent or a certificate.

3.3.1.1 Adapt the restriction to suit your circumstances

Rule 91A of the Land Registration Rules 2003 allows the following amendments to the standard restrictions. They are:

where a standard form restriction is intended to affect part of a registered estate the words ‘No [disposition ] of the registered estate’ [should be replaced by ‘No disposition of the part of the registered estate]’ followed by a sufficient description, by reference to a plan or otherwise, to clearly identify the part affected

As well as the options allowed by rule 91A referred to above, which include provisions for restrictions that will cease to have effect on the death of a named person or people, the wording of restrictions in standard Form L, N. S, T, NN and OO allow for the inclusion of wording to show who should give the certificate or consent if the restriction is to continue to have effect after the death of the person named in the restriction. Thought should always be given to making use of these options when the restrictioner is a person as they can make later applications for cancellation, withdrawal or modification considerably more straightforward for both the applicant and HM Land Registry. The options include:

  • adding ‘or their personal representatives’ after the name and address of the person required to give a certificate or consent by the restriction
  • adding ‘or after that person’s death by of
    ’ after the name of the restrictioner if the restriction is to continue to have effect after the death of the restrictioner but it is not appropriate that the restrictioner’s personal representatives give the consent or certificate
  • using ‘ of
    and of
    or the survivor of them’ where more than one person is named in the restriction and there is a right of survivorship to the interest protected by the restriction
  • using ‘ of
    and of
    or the survivor of them or by the personal representatives of the survivor’ if the restriction will continue to have effect after the death of the survivor of the people named
  • ‘or by of
    ’ may be added after the name of the restrictioner if they wished to allow an alternative second person to give the consent or certificate

The additional wording should only be used where it is shown as an option in the wording of the standard restrictions.

Other practice guides in this series provide information about standard form restrictions that should or may be applied for or entered in particular situations.

Any amendment not provided by rule 91A or which goes beyond those explained in Standard form restrictions will make the restriction non-standard. For example, the field in the standard restrictions does not allow for additional descriptive text such as details of the particular office or function of the restrictioner. If a restriction is required in favour of, for example, ‘X, the supervisor of …’ application should be made for a non-standard restriction.

Restrictions in Form L, M, O, P, S, NN, OO and PP that require compliance with all of the provisions in a deed or other document (rather than certain specified clauses) will also make the restriction non-standard.

3.3.2 Restrictions not in a standard form

You should only apply for a restriction that is not in a standard form if none of the standard form restrictions is appropriate.

Where there is no appropriate standard form available we will only approve the form that you have applied for if:

  • it is reasonable
  • its application would be straightforward
  • its application would not place us under an unreasonable burden (section 43(3) of the Land Registration Act 2002)

Please remember that when applying for a restriction not in standard form:

  • it must always contain the words ‘is to be completed by registration’ rather than ‘is to be registered’. This will serve to make the effect of the restriction clear. The term ‘registered’, where used in any of the standard form restrictions, means the completion of a registrable disposition by complying with the relevant registration requirements prescribed in Schedule 2 to the Land Registration Act 2002 (rule 91(3) of the Land Registration Rules 2003), but this statutory definition only applies to standard form restrictions. Please note that we will not accept restrictions not in standard form for registration that contain the words ‘is to be registered’
  • if the restriction affects part only of a registered extent it must contain a sufficient description, by reference to a plan or otherwise, the part affected must be clearly identified
  • do not commence the wording of the restriction with ‘No dealing’ or ‘No disposition or dealing’ or use wording to prevent noting (as opposed to registration) of a disposition
  • before you finalise an agreement in which the parties agree to apply for a non-standard restriction in a specified form, check with us the proposed form is acceptable it can prove difficult to renegotiate the terms of an unacceptable restriction after an agreement has been made

As we must consider the appropriateness of any restriction applied for that is not in a standard form, the application fee prescribed in the current Land Registration Fee Order , see HM Land Registry: Registration Services fees

3.4 Apply for a restriction

3.4.1 Those who may apply for a restriction

You can only apply for the entry of a restriction if you:

  • are the relevant proprietor
  • are entitled to be registered as the relevant proprietor (see Applications made with the cooperation of the relevant proprietor)
  • have got the consent of the relevant proprietor or someone entitled to be registered as such
  • otherwise have a sufficient interest in the making of the entry

3.4.2 Compulsory applications

Rule 94 of the Land Registration Rules 2003 prescribes certain situations where a person must apply for a restriction. In particular, where a new trust is set up or there is a change in a trust of land and as a result a sole proprietor will not be able to give a valid receipt for capital money, a proprietor must apply for a restriction in Form A (Schedule 4 to the Land Registration Rules 2003). This is the standard joint proprietorship restriction – see practice guide 24: private trusts of land for more information.

Note that where 2 or more people are under an obligation under rule 94 of the Land Registration Rules 2003 to apply for entry of a restriction in Form A, that obligation will be satisfied by an application by one of those people. In this case however the application should be made as if it was made by a person with sufficient interest (see Applications made without the cooperation of the relevant proprietor: the need to show a sufficient interest) and HM Land Registry will serve a notice for information only on the other proprietor(s).

3.4.3 Applications made without the cooperation of the relevant proprietor: the need to show a sufficient interest

Where the applicant does not have the cooperation of the relevant proprietor, they may only apply for a restriction if they can satisfy us that they have a sufficient interest in the making of the entry.

Rule 93 of the Land Registration Rules 2003 contains a list of standard situations where a class of person will be regarded as having a sufficient interest in the making of an entry. In most cases, the rule identifies which of the standard form restrictions will be appropriate to each situation covered.

The applicant must give details of the nature of their interest and how that interest arose. We will require evidence to show sufficient interest in support of an application.

If there is more than one applicant and they chose to give a statement, that statement must be given by all the applicants. Where the applicant is a corporation the person giving the statement should confirm their position and that they are authorised to give the statement on behalf of the corporation.

3.4.4 Interests under trusts

An interest under a trust of land cannot be protected by an agreed or unilateral notice (section 33(a)(i) of the Land Registration Act 2002) but may be protected by a restriction. Generally, a beneficiary under a trust of land may apply for a Form A restriction if one has not already been entered in the register. A Form A restriction ensures that any capital money must be paid to 2 trustees or a trust corporation. A second Form A restriction cannot be entered because the purpose of a Form A is to ensure that interests behind the trust are overreached it does not give notice of an individual’s interest under a trust.

An interest under a trust of land means the interest of a person under such a trust who stands only one step away from the registered estate. Examples include:

  • where A and B are the proprietors of the registered estate and hold on trust for themselves (both have interests under a trust of land)
  • where C and D are the proprietors of the registered estate and hold on trust for E for life and for F thereafter (E and F have interests under a trust of land)
  • where G is the proprietor of the registered estate and holds on a bare trust for H (H has an interest under a trust of land)

If another form of restriction is required either in place of or in addition to a Form A restriction, evidence will have to be lodged showing that the applicant has a sufficient interest in the making of the entry. Whether the application can be accepted will depend on the restriction applied for, the nature of the applicant’s interest and the circumstances of the case.

If the application was for a consent restriction such as Form N, the registrar would have to be satisfied that it was necessary or desirable (for one of the purposes in section 42(1) of the Land Registration Act 2002) for such a restriction to be entered. To allow a consent restriction to be entered (except, for example, when it was required under the trust) would be to give the beneficiary a right they are not entitled to and could result, in practice, in thwarting the clear intention of sections 42(1)(b) and 44(1) of the Land Registration Act 2002 and sections 2 and 27 of the Law of Property Act 1925 that overreaching should take place.

Where a Form A restriction is considered insufficient to protect a beneficiary’s interest under a trust of land, they may also apply for a restriction in Form II. A restriction in this form should ensure that the person named in the restriction receives notice of the disposition, thereby giving them the opportunity of pursuing the proceeds of sale.

If the beneficiary’s consent is required under the terms of the trust, an application may be made for a Form B restriction.

Further information about protecting an interest under a trust of land can be found in Appendix A: some possible means of protection for common third-party interests.

The interest of a person who is two or more steps away from a registered estate which is subject to a trust of land is termed a ‘derivative interest’ in this guide. Examples include:

  • proprietors J and K hold on trust for L and M and M holds on trust for herself, N and O (N and O will have derivative interests)
  • proprietors P and Q hold on trust for R and S, and S holds on trust for T and U (T and U have derivative interests)
  • proprietors V and W hold on trust for X and Y and X charges their interest to Z (Z has a derivative interest)

A person with a derivative interest may apply for a Form A restriction provided such a restriction has not already been entered in the register.

It is difficult to see how an applicant with a derivative interest would be able to satisfy the registrar that they have sufficient interest under section 42(1) of the Land Registration Act 2002 (see Restrictions entered at the registrar’s discretion) for any other form of restriction, for example a consent restriction, to be entered. Generally, a person with a derivative interest will not be able to apply for a different form of restriction because:

  • the holder of a derivative interest cannot apply under section 42(1)(a) of the Land Registration Act 2002 on the basis that the restriction might prevent the trustees from misapplying the proceeds of sale following a disposition which overreaches the beneficial interests, as subsection (1)(a) is concerned only with preventing unlawfulness or invalid dispositions of registered estates and not with subsequent dealings with the proceeds of sale
  • a derivative interest is not a right or claim in relation to a registered estate or charge within section 42(1)(c) of the Land Registration Act 2002 as it is a right or claim in relation to the beneficial interest under the trust of land (not in relation to the registered estate or charge)

A person with the benefit of a charging order over a beneficial interest under a trust of land may apply for a Form K restriction, even though their interest is a derivative interest, because of the provisions of section 42(4) of the Land Registration Act 2002 and rule 93(k) of the Land Registration Rules 2003.

The Legal Aid Agency, where it has a statutory charge over a beneficial interest under a trust of land, may apply for a restriction in Form JJ.

3.4.5 Notifiable applications

We will notify the relevant proprietor before we complete an application for a restriction unless it is either:

  • made by or with the consent of the relevant proprietor or someone entitled to be registered as such
  • one of the compulsory applications listed in rule 94 of the Land Registration Rules 2003
  • applied for to reflect a limitation under a court order or an order of the registrar (or an undertaking given in place of such an order) (section 45 of the Land Registration Act 2002)

The notice will give the relevant proprietor 15 working days to object to the application. If a dispute arises from an objection to an application made within that period and it cannot be resolved by agreement, it will be referred to the tribunal . See practice guide 37: objections and disputes: HM Land Registry practice and procedures for more information about the resolution of disputes by the tribunal .

3.5 How to apply for a restriction

3.5.1 Application form and fee

Most applications for restrictions must be made in form RX1. Before applying, please think carefully about the disposition that you wish to restrict. Is it against a disposition of the registered estate or against a disposition of a registered charge? Select the standard form restriction as appropriate to your circumstances. Remember that a standard form restriction against a disposition of a registered charge will appear in the charges register of the title concerned.

However, you may apply for any standard form restriction by making the application in:

  • the additional provisions panel of any of the following forms: form TP1, form TP2, form TR1, form TR2, form TR4, form TR5, form AS1, form AS2, form AS3
  • the following forms of charge:
    • panel 9 of form CH1
    • an electronic legal charge
    • a charge where we have approved the form of the charge in advance (including the application for the restriction)

    Note: You must include words of application such as “the transferor applies to register the following restriction…”. It is not sufficient to enter just the wording of the restriction without saying who is applying for it.

    Note any application to register a restriction not in one of the forms of charge above, or to register a non-standard restriction,must be made using form RX1.

    • a lease containing clauses LR1 to LR14 of Schedule 1A to the Land Registration Rules 2003 (see Applications for a restriction contained in a lease)

    An application for a Form A restriction can also be made using form SEV.

    The application must be accompanied by the fixed fee prescribed under the current Land Registration Fee Order , see HM Land Registry: Registration Services fees.

    Form RX1 is intended to be used for applying for one restriction only but we will accept an application if a single form RX1 is used to apply for different restrictions provided (a) the applicant, and (b) the reason given as to the entitlement to apply for the restrictions, are the same. If the applicant or the entitlement to apply are different, separate forms must always be used.

    3.5.2 Applications for a restriction contained in a lease

    In general, no effect will be given to an application to register a restriction contained in the body of a lease.

    However, any lease containing clauses LR1 to LR14 of Schedule 1A to the Land Registration Rules 2003 may be used, at clause LR13, to apply for entry of a standard form restriction. This includes prescribed clauses leases granted on or after 19 June 2006. If clause LR13 is not completed in such a lease, any application to register a restriction contained within the body of the lease will be ignored. For information on drafting the restriction where it relates to covenants contained in a lease, please see practice guide 19A: restrictions and leasehold properties.

    Where a lease containing clauses LR1 to LR14 of Schedule 1A to the Land Registration Rules 2003 is lodged for registration, and the standard form restriction is to be entered against titles other than the landlord’s or that created by the registration of the lease, it will only be registered if clause LR2.2 is also completed (rule 72A(4) of the Land Registration Rules 2003).

    Where application is made against a title other than the landlord’s or that created by the registration of the lease, evidence may be required of the consent of the registered proprietor, or of the person entitled to be registered as proprietor, or that the person applying has sufficient interest in the making of the entry. Where clause LR13 is used to apply for a standard form restriction and such evidence is required it should be lodged under a covering letter with the application to register the lease.

    Clause LR13 may not be used to apply for a non-standard form restriction and a form RX1 should continue to be used.

    Practice guide 64: prescribed clauses leases provides further information about prescribed clauses leases.

    3.5.3 Information that must accompany the application

    Your application must be accompanied by (see Retention of documents lodged with applications regarding retention of documents sent to us):

    • full details of the restriction you are applying for
    • an address for service (to be included within the text of the restriction applied for at any appropriate point) for:
      • anyone named in a standard form restriction whose address is required by that restriction
      • anyone named in any other restriction whose consent or certificate is required or to whom notice must be given by the registrar or another person
      • the consent
      • a certificate given by a conveyancer confirming that he holds the relevant consent: this should be entered in panel 13 of form RX1 or the additional provisions panel of the forms mentioned in Application form and fee
      • evidence of their entitlement
      • a certificate given by a conveyancer confirming that they are satisfied that person is entitled to be registered and that either the conveyancer holds original documentary evidence of the entitlement or that there is a pending application to register that person as proprietor at HM Land Registry: this should be entered in panel 13 of form RX1 or the additional provisions panel of the forms mentioned in Application form and fee
      • evidence of your interest in the making of the application (details of the nature of the interest and of how that interest arose must be given either as a statement by the applicant in panel 12 or as a conveyancer ’s certificate in panel 13 of form RX1. When referring to the registered owner of the property, you should refer to them by name and not just as “the registered proprietor”. Where it is available, documentary evidence of the interest should be lodged with the application. Although there may be no documentary evidence where the interest is a resulting or constructive trust we would expect to see documentary evidence for all other restrictions (apart from restrictions in Forms D, E, or F))

      3.6 Court orders requiring the entry of a restriction

      3.6.1 The court’s power to order the entry of a restriction

      The court may make an order requiring the registrar to enter a restriction (section 46 of the Land Registration Act 2002). Forms AA to HH are the standard form restrictions that the court is most likely to order the registrar to enter, but it may also order the entry of a restriction in a different form.

      If you intend to apply to court for an order that will require the registrar to enter a restriction that is not in one of the standard forms, please contact us first to ensure that the proposed form will be straightforward and will not place us under an unreasonable burden.

      3.6.2 Application form and fee

      Although the order may be addressed directly to the Chief Land Registrar, you should make a formal application for the restriction to be entered. This will ensure that the restriction is entered against the correct titles.

      Your application should be made in form AP1 (not RX1) (rule 92(8) of the Land Registration Rules 2003) and should be accompanied by the fixed fee prescribed under the current Land Registration Fee Order , see HM Land Registry: Registration Services fees.

      3.6.3 Overriding priority

      The court may direct that the terms of the restriction must take priority over that afforded by any official search with priority that is pending when we process the application for the restriction (section 46(3) of the Land Registration Act 2002).

      This direction may be appropriate if there is a risk that somebody may apply for an official search ‘with priority’ before the restriction can be entered so that they can register a disposition of the property without being caught by the terms of the restriction.

      3.7 Removal of a restriction entry

      3.7.1 Removal of restrictions

      Restrictions may be removed from the register by:

      • being withdrawn voluntarily by the appropriate people interested in the restriction in form RX4 (section 47 of the Land Registration Act 2002 and rule 98 of the Land Registration Rules 2003)
      • an application by anyone to cancel a restriction that is no longer required in form RX3 (rule 97 of the Land Registration Rules 2003)
      • being cancelled by ourselves if it is clear that it is superfluous (paragraph 5 of Schedule 4 to the Land Registration Act 2002)
      • being cancelled by ourselves if it is a restriction entered in respect of a trust of land and we are satisfied the affected estate is no longer subject to the trust (rule 99 of the Land Registration Rules 2003) see practice guide 24: private trusts of land - Cancellation and withdrawal of restrictions for more information about cancellation of trust restrictions

      Depending on its terms, a restriction may continue to have effect despite numerous changes of proprietorship, other dispositions and the lapse of time.

      Someone intending to take a disposition of an estate or charge against which a restriction has been registered should therefore consider whether:

      • the disposition will be affected by the restriction and, if so, whether they can comply with its terms
      • the restriction may affect any later disposition they may wish to make

      In appropriate circumstances they should take steps to ensure the restriction will be cancelled or withdrawn before committing themselves to complete the disposition.

      3.7.2 Removal of Form LL restriction

      We may cancel a Form LL restriction automatically where it appears superfluous due to a transfer of the whole of a registered title to a new proprietor, and the certificate required by the restriction has been lodged – see Restrictions cancelled without application.

      Where an application to cancel or withdraw the restriction accompanies an application to register a disposition other than a transfer of whole, you should lodge evidence of compliance with the restriction in order for the restriction to be cancelled or withdrawn – see Restriction in Form LL.

      3.7.2.1 Application to cancel or withdraw a Form LL restriction that does not accompany a disposition for registration

      All the registered proprietors may agree to cancel or withdraw a Form LL restriction. We will require a certificate from an individual conveyancer that they are satisfied that the people applying for or consenting to the application are the same people as the registered proprietors.

      If we receive an application to cancel a Form LL restriction without the involvement of all registered proprietors, we will need to consider carefully whether we can be satisfied that it is no longer required.

      If we are not satisfied on the evidence lodged that the restriction should be cancelled or withdrawn, we will cancel the application and the restriction will remain in the register.

      When giving a certificate, you must comply with the requirements detailed in Restriction in Form LL.

      3.7.3 Applications to cancel a restriction

      Cancellation is the term used in rule 97 of the Land Registration Rules 2003 to refer to an application to cancel a restriction that is no longer required.

      Any person may apply to cancel a restriction. The application must be made in form RX3 and no fee is payable.

      If the restriction being cancelled is a Form A restriction, you can use form ST5 to supply the necessary evidence in support of your application.

      Where the application to remove the restriction is made by or with the consent of the people having the benefit of the restriction, application should be made to withdraw the restriction using form RX4 unless it is one of those restrictions referred to in Applications to withdraw a restriction, which cannot be withdrawn.

      Cancelling a landlord/management company restriction where a right to manage company has been appointed cannot be done without consent of the landlord, the right to manage company and the tenant.

      We will cancel the restriction if we are satisfied that the restriction is no longer required. The application must be accompanied by evidence to show that this is the case. If anyone is referred to in the restriction and if an address for service is listed for that person, we will usually notify them of the application and give them an opportunity to object to the application before cancelling the restriction.

      3.7.4 Applications to withdraw a restriction

      Withdrawal of a restriction is the term used in section 47 of the Land Registration Act 2002 and rule 98 of the Land Registration Rules 2003. Rule 98(1) and (2) of the Land Registration Rules 2003 requires an application for the withdrawal of a restriction to be accompanied by ‘the required consent’. If we are satisfied the required consent has been given, we will remove the entry without investigating whether the restriction continues to serve any purpose.

      The application must be made in form RX4 no fee is payable.

      Restrictions cannot be withdrawn (rule 98(3) of the Land Registration Rules 2003) when they are:

      • those entered to prevent an unlawful or invalid disposition by a proprietor whose powers are limited by statute or under the general law
      • those entered as a result of an application by someone who was obliged to apply under rule 94 of the Land Registration Rules 2003
      • any the registrar is obliged to enter
      • those entered to reflect a limitation in an order of the court or the registrar or a limitation in an undertaking given in place of an order
      • any the court has ordered the registrar to enter

      If one of these restrictions has ceased to apply, application to cancel the restriction should be made as described in Applications to cancel a restriction. It should be noted that a restriction in standard Forms U, V, W, X, Y, JJ and QQ will always fall within the above list and Forms A, B and C will very often come within rule 98(3) of the Land Registration Rules 2003.

      • where the restriction requires the consent of a specified person, the consent of that person
      • where the restriction requires a certificate to be given by a specified person, the consent of that person
      • where the restriction requires notice to be given to a specified person, the consent of that person
      • where the restriction requires the consent of a specified person, or alternatively a certificate to be given by a specified person, the consent of all such people
      • in any other case, the consent of all people who appear to the registrar to have an interest in the restriction

      Where the person consenting is not the same as the person referred to in the restriction, you must supply appropriate evidence of devolution of the right to consent.

      However, where a restrictioner has died and the terms of the restriction do not indicate that it will end upon death, or who is to have the benefit after death, then in practice it will usually be impossible to withdraw the restriction. Application must be made for its cancellation.

      Where the application is to withdraw part of the land within an affected estate or charge from the effect of the restriction (for example in readiness for a transfer of that part), the part in question must be clearly identifiable from the application.

      3.7.5 Restrictions cancelled without application

      We may cancel a restriction without any application being made if it is clear that the restriction has become superfluous (paragraph 5(d) of Schedule 4 to the Land Registration Act 2002). We might cancel a restriction automatically where:

      • the restriction is limited in time and the relevant period has expired
      • the restriction was entered in connection with the registration of a charge which has now been discharged
      • the restriction was entered to protect an interest that has since been overreached by the payment of capital money arising on a registrable disposition to the proprietors who have given a valid receipt (for example Form A)
      • the restriction was entered in relation to a limitation on the powers of a previous proprietor
      • we register a transfer under a power of sale by the proprietor of a registered charge whose powers were not affected by the restriction
      • a Form LL restriction was entered at the request of a registered proprietor, an application is subsequently received to register a transfer of the whole of the registered title to different proprietors, and the necessary evidence of compliance with the restriction has been lodged

      3.8 Applications to update a restriction

      Sometimes it is necessary to alter the wording of a restriction to allow it to continue to work as intended when circumstances have changed.

      Common examples include when the address of the person with the benefit of the restriction has changed or where the title number referred to in a restriction has changed. However, there may be other situations where this is applicable.

      In these situations, an application to alter the restriction to bring it up to date may be appropriate.

      An application to update a restriction must be made in form AP1 and be accompanied by evidence to justify the alteration requested.

      3.9 Applications to disapply or modify a restriction

      3.9.1 Disapplying a restriction

      Anyone who has a sufficient interest in a restriction may apply for an order that it is disapplied to enable a disposition or dispositions of a specified kind to be registered.

      For example, a registered estate might be subject to a restriction prohibiting the registration of any transfer without the consent of a management company. If the company has been dissolved (see practice guide 35: corporate insolvency - restrictions in favour of dissolved companies) but the applicant can show that an application has been made to restore the company to the companies’ register, and there is no reason why the transfer should not proceed, the registrar may make an order permitting the transfer to be registered. In those circumstances it might not be appropriate to cancel the restriction completely, but see practice guide 19A: restrictions and leasehold property for when it might be appropriate to apply to cancel a restriction.

      In considering an application to disapply the registrar will require evidence that the applicant has used their best endeavours to comply with the terms of the restriction.

      If the restriction is disapplied, the transfer can be registered but the restriction would remain in the register.

      3.9.2 Modifying a restriction

      Anyone who has a sufficient interest in a restriction may apply for an order that its terms be modified. The modification might relate to a specific disposition or to dispositions of a specified class.

      For example, an applicant may wish to modify the terms of the restriction so that it no longer ‘catches’ a charge. Please note, however, that we cannot accept an application to modify a restriction that extends its effect. To achieve this, the restrictioner must first apply in form RX4 to withdraw it and then in form RX1 for a new restriction.

      The registrar’s power to make an order is discretionary and, in general terms, will only be used when it is not practicable for the restriction to be withdrawn and a new restriction entered or for the register to be altered under Schedule 4 to the Land Registration Act 2002.

      Where a restriction is withdrawn and replaced by a new restriction, remember that a restriction that is worded to catch dispositions by the proprietor of any registered charge will not apply to a charge that was registered before that entry of that restriction. Where the existing restriction to be withdrawn applies to dispositions by the proprietor of any registered charge and a charge has been registered since the entry of the restriction, you should consider whether you also need to apply for an additional restriction against the charge concerned. For instance, if a Form L restriction is being withdrawn and re-entered, entry of a Form S restriction may also be required.

      Where a restriction is to be updated, to reflect for example a change of name or of address, application for alteration of the register should be made on form AP1.

      3.9.3 The application

      An application to disapply or modify a restriction must be made in form RX2. The application must be accompanied by the fixed fee prescribed under the current Land Registration Fee Order , see HM Land Registry: Registration Services fees.

      • state whether the application is to disapply or to modify the restriction
      • explain their interest and why it is sufficient to make the application
      • state why the applicant considers that the registrar should make the order
      • give details of the disposition or kind of dispositions that will be affected by the order. If the application is to modify the restriction, give details of the modification requested

      The application may be made before, or at the same time as, an application to register the disposition that is caught by the restriction.

      When considering whether to make the order, the registrar will additionally consider any available evidence to clarify what purpose the restriction still serves. The registrar may ask for further evidence from the applicant and may serve appropriate notices.


      Removing a restriction site and introducing other at its place - Biology

      Project 1: Screening for P-Transposable Elements in a Wild-Type
      Strain of Drosophila melanogaster

      Project 2: Isolation and Characterization of Mutations
      In Drosophila melanogaster

      Project 1: Screening for P-Transposable Elements in a Wild-Type Strain of Drosophila melanogaster

      Project Summary
      The genomes of many wild-type strains of the fruit fly, Drosophila melanogaste r, contain P-elements. P-elements, like other transposable elements, are a major cause of mutation and play an important role in evolution. P-elements are short stretches of DNA that can move around in the genome. Just how prevalent are P-elements in wild fruit fly populations? Will every strain of D. melanogaster isolated in the wild have the transposons? In an attempt to address this question, the goal of this project is to determine whether or not a wild-type strain of D. melanogaster , isolated on this campus, contains P-elements in its genome. You will perform this project over the course of 7 lab sessions. You will isolate genomic DNA from wild-type fruit flies and cut the Drosophila genome into many small fragments using restriction enzymes. You will do Southern blot hybridization analysis to probe for our sequence of interest, the Drosophila P-element. To prepare for hybridizing your Southern blot, you will prepare large amounts of the probe DNA by transforming bacterial cells with a recombinant plasmid containing P-element sequences, isolating the plasmid from the cells, and purifying the P-element-containing fragments by restricition digestion and gel electrophoresis. You will then use your pure probe DNA in an attempt to identify P-elements on your Southern blot.
      You will also use an alternative approach, the Polymerase Chain Reaction (PCR), to try to detect P-elements in the fruit flies from campus.

      Drosophila P-elements
      The fruit fly, Drosophila melanogaster , is an ideal model organism for use in genetic and molecular analyses (for more information on Drosophila melanogaster , see Project 2). The genomes of many wild-type strains of Drosophila melanogaste r contain P-elements. P-elements are short stretches of DNA (<=2.9kb) that are transposable elements, or transposons (or "jumping genes") that is, they can physically excise from one chromosome and move to another, or they can move from point to point within a chromosome. P-elements vary in length but are all derived from the 2.9kb complete P-element sequence, which encodes a transposase (the enzyme that cuts out and moves its own stretch of DNA). The complete P-element is actually more than just the transposase gene, and its derivatives are usually incomplete but for simplicity's sake, we will henceforth refer to the P-element as our "gene of interest." Transposons are common in nature and relatively easy to detect in a known DNA sequence, since the transposase works by recognition of a characteristic transposon feature: flanking inverted repeats. When a transposon moves, oftentimes only a portion of the DNA sequence will "jump" to a new location. Some of the gene sequence is usually left behind in the original position. Therefore, while P-elements can vary in length from 0.5-2.9kb, all are recognizable by the 31bp flanking inverted repeats (the "transposon footprint"). Incomplete excision and movement of P-elements also leads to high copy number, that is, to the presence of many copies of some or all of the P-element scattered throughout the Drosophila genome. Since the goal of our experiment is to detect P-elements in a wild-type population of Drosophila melanogaster , their high copy number suits our purpose well.

      Recombinant DNA Technology
      The relatively recent development of recombinant DNA technology has enabled biological researchers to make great strides in our understanding of the structures and functions of genes. Before the development of recombinant DNA technology, the complex genomes of eukaryotes were extremely difficult to analyze. Recombinant DNA technology enables researchers to break large genomes into specific fragments, which can then be inserted into the small genome or into a DNA molecule from a different species, such as a bacterium, and analyzed with relative ease. The small genome or DNA molecule into which the fragments are inserted is called a vector, and recombinant DNA molecules can be made by inserting DNA fragments from almost any species into a vector. Recombinant DNA molecules are commonly introduced into bacterial "host" cells by the process of transformation. The vectors used to construct recombinant DNA molecules are usually capable of replication, so once inside a bacterial cell, the recombinant DNA molecule will be replicated, resulting in the amplification (replication of many copies) of a specific DNA fragment. The insertion of a fragment of DNA into a vector, and the subsequent replication of the recombinant DNA molecule is often called "cloning". The ability to produce many copies of a given DNA sequence has been extremely helpful in the analysis of gene structure and function. The Human Genome Project and other genome projects would be impossible without recombinant DNA technology. In addition, genes encoding medically- and industrially-important polypeptides can be inserted into vectors, and maintained and amplified in host cells. Host cells capable of synthesizing the polypeptide products of these recombinant genes provide a means of producing large quantities of important molecules. For example, insulin, which is necessary for the treatment of some types of diabetes, is produced inexpensively and in large quantities by bacterial cells that express the human insulin gene from a recombinant vector. Recombinant DNA technology has been made possible by the discovery and development of a number of important "tools" - some of which are discussed below.

      Restriction Enzymes
      Restriction endonucleases (or restriction enzymes), discovered in the 1970s, are valuable tools for characterizing and manipulating DNA molecules. Restriction endonucleases are enzymes that recognize specific nucleotide sequences, often 4, 6, or 8 base-pairs long, and cut DNA at these sequences only. Each restriction enzyme recognizes its own, specific sequence of nucleotides, called a "restriction site". There are many different restriction enzymes that recognize and cut at many different nucleotide sequences. Restriction enzymes are produced by bacteria as a defense against invasion from foreign DNA - especially from bacteriophages. A bacterium modifies its own DNA to protect the DNA from restriction enzymes. Foreign DNA that finds its way into the bacterial cell will be recognized and digested, or "restricted" by the cell's restriction enzymes. Restriction enzymes are purified from bacterial cells for use in molecular biology experiments.
      By cutting a given piece of DNA with specific restriction enzymes, we can determine the locations of the restriction sites for those enzymes on that DNA, and generate a "restriction map" of the given DNA. We can identify the different-sized fragments resulting from the digestion of a given piece of DNA with a certain restriction enzyme by "electrophoresing" the restriction-digested DNA on an agarose gel, as shown in figure 1 . Briefly, the digested DNA (consisting of a mixture of many copies of each these three fragments) is loaded into one of the sample wells at one end of an agarose electrophoretic gel. A voltage is set up across the gel such that the sample wells are closest to the negative electrode, and the far end of the gel is closest to the positive electrode. DNA has a net negative charge, and thus migrates (moves) through the gel toward the positive electrode (this process is called electrophoresis). The gel is composed of a porous matrix that acts like a molecular sieve. Smaller DNA fragments move through the gel faster than do larger DNA fragments. After the electrophoresis of the digested DNA has been carried out for a certain period of time, it is stopped, and the DNA in the gel is stained to make it visible. The DNA is often visible as discrete bands. Each band represents a collection of many DNA fragments that are all of the same size and have thus migrated the same distance in the gel. Restriction enzymes also enable us to break large genomes into specific, small fragments that can be inserted into vectors to make recombinant DNA molecules.

      Generation of Recombinant DNA Molecules
      The combining of two different DNA molecules is facilitated by the fact that many restriction enzymes leave short regions of single-stranded DNA at their sites of cutting. This results in the generation of cohesive, or "sticky" ends at the restriction sites. If two DNA molecules are cut with the same restriction enzyme, they will have complementary sticky ends that can be joined together (ligated) by base-pairing with the help of enzymes called DNA ligases.
      Figure 2 shows an example of a case in which a circular vector DNA, and human genomic DNA are both cut with the restriction enzyme, Eco R I, which recognizes the restriction site

      and cuts between G and A on both strands, leaving short overhangs of single-stranded DNA on either side of the restriction site. If this particular vector has only one EcoR I site, the digest results in a linear vector with two sticky ends, which looks like (N stands for a nucleotide of any type):

      A sticky end from one DNA molecule can link with, or anneal with a sticky end from another DNA molecule by virtue of complementary pairing between nucleotide bases in the single-stranded overhangs that are generated at the restriction sites:

      These bases can pair with these bases

      5'-NNNNNNNNNNNNNNG-3' 5'-AATTCNNNNNNNNNNNN-3'
      3'-NNNNNNNNNNNNNNCTTAA-5' 3'-GNNNNNNNNNNNN-5'

      The digestion of human genomic DNA with EcoR I results in a very large number of linear DNA molecules with sticky ends that are complementary to the sticky ends of the digested vector DNA. Any one of these human DNA fragments can be combined with the vector DNA by base-pairing between complementary sticky ends. The digested DNA molecules are put into solution together, and the sticky ends meet as the DNA molecules move randomly about in the solution and bump into one another. Treatment with a DNA ligase enzyme will covalently join the DNA molecules that have base-paired, to form one circular, recombinant DNA molecule. The fragment of DNA that is ligated to the vector DNA is called an "insert". Recombinant molecules can also be made by digesting vector DNA with a mixture of two different restriction enzymes, and digesting the DNA to be cloned with the same two restriction enzymes. This process is called "directional" cloning, since the insert DNA will be spliced into the vector DNA in a specific orientation, as shown in figure 3 .

      Vectors
      Several different types of vector can be used in the generation of recombinant DNA molecules. These vectors originate from bacteriophages (viruses that infect bacterial cells), bacteria, and also from eukaryotes, such as yeasts. In this lab, we will be using a bacterial vector, and this discussion will be restricted to phage and bacterial vectors. A good discussion of eukaryotic vectors can be found in Chapter 10 of the textbook, Griffiths, A.J.F., W.M. Gelbart, J.H. Miller, and R.C. Lewontin (1999). Modern Genetic Analysis. New York. W.H. Freeman and Co. Some non-eukaryotic vectors that are used commonly include:

      Plasmids
      Plasmids are small, circular DNA molecules. Plasmids can be introduced into competent bacterial cells by transformation. Inside the bacterial cell, a plasmid exists and replicates independently from the much larger bacterial genome, as depicted in figure 4 . Plasmids can (and have been) engineered to carry genes that confer on the cells containing them resistance to specific antibiotics. Plasmids can also carry genes encoding certain enzymes that can be used to "mark" bacterial cells by assaying the cells for the presence of those enzymes. Since plasmids replicate in bacterial cells, they allow amplification of the inserted DNA molecule into many copies. One disadvantage of plasmid vectors is they that cannot contain large inserts. Most plasmid vectors can hold only inserts smaller than 10 kilobases (kb) (1 kb = 1,000 base-pairs).

      Bacteriophage l Vectors
      Derivatives of the bacteriophage l can be used to clone larger fragments of DNA - fragments on the order of 15 to 20 kb. The linear Phage l genome can be made into a cloning vector by removing much of its central portion, which can then be replaced with foreign DNA fragments, resulting in recombinant molecules. The recombinant phage are then replicated in host E. coli bacterial cells.

      Cosmid Vectors
      Cosmid vectors are hybrids between plasmid and phage vectors. Cosmids can be used to clone insert fragments of up to 45 kb in length. Cosmids can be maintained in bacterial cells in the circular plasmid form, and they can be purified from the cells by packaging into phage particles.

      Southern Blot Hybridization Analysis
      One way to check for the presence of a specific sequence (a partial or complete gene, for example) in a genomic DNA sample is to perform Southern blot hybridization analysis (When using genomic DNA, the technique is called genomic Southern blot hybridization analysis). Like finding a needle in a haystack, this powerful technique detects specific

      DNA sequences in the total DNA of an organism. Southern blots can also be used for restriction mapping of DNA sequences. The first step in genomic Southern blot hybridization analysis is to digest genomic DNA from the organism of choice with restriction enzymes and run it out on a gel. The next step is blot the gel onto a nitrocellulose membrane, as shown in figure 5 . When you then "hybridize" this membrane to a radiolabeled probe specific for the sequence of interest, the probe should stick ­ not just anywhere on your membrane, but specifically where there is DNA of the proper sequence. The resulting point sources of radioactivity will expose corresponding spots on a piece of X-ray film (this is called autoradiography). When the X-ray film is developed, dark bands will appear corresponding to the locations of DNA sequences of interest on your membrane. A DNA size marker run on the same gel, hybridized to its own radiolabeled probe, will allow you to measure the sizes (in base pairs) of your element restriction fragments.


      Polymerase Chain Reaction (PCR)
      Polymerase Chain Reaction (PCR) is a method for making many copies of, or amplifying, a specific DNA sequences (such as a particular gene or region of the genome). PCR is performed using a pair of specific primers, which are themselves single-stranded sequences of DNA, usually about 20 bases long. A primer is designed to be complementary in sequence to a specific region of the genome, and a pair of primers that flanks a specific region of the genome is used to amplify (make many copies of) that genomic region, which is called the template. The primers prime replication of the genomic DNA by an enzyme called Taq DNA polymerase, or Taq . Taq DNA polymerase is isolated from a bacterium called Thermus aquaticus , which lives in the very hot water of geothermal vents, and all of whose enzymes are active at high temperatures. Taq DNA polymerase is stable at 94oC and optimally active at 72oC. A PCR reaction takes place in a microfuge tube (also called an "Eppendorf" tube). The reaction mixture usually contains genomic DNA (template), primer pairs, Taq , free deoxynucleotide triphosphates (dNTPs ­ A, C, T, and G) and the appropriate buffers and salts, including magnesium ­ a necessary cofactor for the Taq enzyme. The microfuge tube containing the reaction mixture is placed in a thermocycler (a machine for varying temperature through a preset number of cycles. In PCR, genomic DNA is heated to denature the double-stranded DNA molecules, making them single-stranded (This is the Denaturation step). The reaction mix is then cooled, allowing primers to anneal to complementary sequences on opposite strands of the template genomic DNA (by hydrogen bonding between complementary bases: A-T, G-C) flanking the DNA segment to be amplified (This is the Annealing step). The reaction is then brought to an intermediate temperature, and, using free deoxyribonucleotides added to the reaction mixture, Taq DNA polymerase extends these primers from their 3' ends toward each other, as shown in figure 6 (This is called the Extension, or Elongation step) . This replicates the region between the two primers, and generates two double stranded DNA molecules from the original one. After this round of replication is completed, the reaction mixture is heated to denature the double stranded DNA molecules, and then the temperature is lowered to allow the primers to anneal again - this time with double the number of templates. The reaction is then brought to an intermediate temperature again, allowing extension. This process is repeated for a number of cycles (usually 20-30 cycles), resulting in the production of many copies of the template DNA sequence. These copies are called the PCR product(s), or "amplicons". If all goes as planned, the number of amplicons should double every cycle. So, given one molecule of template DNA, how many copies of a given amplicon should we have at the end of 40 cycles?
      Depending on the number of nucleotide base-pairs present between the two primers used in a PCR reaction, different-sized fragments of DNA (products) will be generated in the reaction. We can identify the different-sized fragments of DNA resulting from PCR reactions by electrophoresing the product(s) of that reaction on an agarose gel.

      Transformation of bacterial cells with recombinant molecules
      We have a very small amount of a recombinant plasmid called p25.1 (kindly provided by B. Engels). p25.1 contains P-element probe sequences. We will be using E. coli bacterial cells to produce the mass quantities of this plasmid that we need. To do this, the plasmid will be introduced into E. coli cells by the process of transformation. The bacterial cells to be transformed must first be treated in a special way to make them "competent" to take up foreign DNA. In the process of transformation, under special conditions, competent bacterial cells take up foreign DNA molecules from the surrounding medium.

      Selection of cells that contain plasmids
      The plasmid vector that is part of the recombinant plasmid containing the P-element probe is called pBR322. A simple map of pBR322 will be provided. pBR322 contains a gene that confers resistance to the antibiotic ampicillin upon the cells that carry the plasmid. Cells that carry pBR322 can therefore be distinguished and purified from cells without pBR322 by growing the mixture of cells with and without the plasmid on medium that includes ampicillin. Cells with pBR322 will thrive on the ampicillin-containing medium, while the antibiotic will prevent cells lacking the plasmid from growing on the plates. PBR322 also contains another gene that provides resisitance to the antibiotic tetracycline. In addition, pBR322 has single restriction sites for many commonly-used restriction enzymes (each of these enzymes will thus cut pBR322 in only one distinct site, linearizing the plasmid). Many of these restriction sites are within the tetracycline-resistance gene. Insertion of foreign DNA fragments (such as Drosophila P-element sequences) into these restriction sites will thus disrupt the the tetracycycline-ressistance gene, rendering it incapable of providing resistance to tetracycline. Cells that carry recombinant plasmids with P-element sequence inserts in the the tetracycycline-resistance gene will not grow on plates that contain tetracycline.
      As another interesting experiment, you will transform E. coli cells with a plasmid called pUC19. PUC19 contains an ampicillin-resistance gene, and it also contains the bacterial lacZ gene, which encodes a b-galactosidase enzyme. The enzyme encoded by the lacZ gene can cleave a substrate molecule called X-gal, yielding a dark blue precipitate as a product. E. coli that do not produce ß-galactosidase form white colonies, but cells that carry pUC19 form dark blue colonies when grown on medium that includes X-gal.
      In this project, you will plate cells that you have attempted to transform with p25.1, and with the control plasmid pBR322 on two different types of nutrient agar plates. The first type of plates will contain ampicillin. The second type of plates will contain tetracycline. You will also plate cells that you have attempted to transform with pUC19 on plates that contain ampicillin and X-gal (the plates also contain IPTG, a molecule necessary for the activation of the lacZ gene).

      What do you expect to see on each of the different plates you'll be setting up?

      Analysis of recombinant plasmids by restriction digestion and gel electrophoresis
      After you have purified your p25.1 plasmid DNA, you will digest the plasmid with a restriciton enzyme or enzymes that will separate pBR322 plasmid DNA from P-element sequences. You will run the sample of digested p25.1 on an agarose electrophoretic gel. You will determine which band(s) on the gel contains P-element sequences, and actually cut that band out of the gel with a razor blade, thus preparing a pure sample of P-element DNA.

      Southern blot for the Drosophila P-element
      To check for the presence of P-elements in your genomic DNA sample, you will perform a Southern blot. You will first isolate genomic DNA from the local strain of Drosophila melanogaster . You will then digest your genomic fly DNA with restriction enzymes and run it out on a gel. Under the appropriate conditions, you will then blot the gel onto a nitrocellulose membrane. Your instructor will hybridize this membrane to a radiolabeled P-element probe and do the autoradiography, and you will analyze the resulting data.

      Using Polymerase Chain Reaction (PCR) in an attempt to detect P-elements
      In addition to using Southern blot hybridization analysis to screen for P-elements, you will also be using a PCR-based strategy toward achieving the same goal. After examining the nucleotide base-pair sequence of the P-element (available on the World Wide Web), you will design a pair of PCR primers that you will then use in an attempt to amplify and detect P-elements in the genome of the local strain of Drosophila .

      Outline of the Lab Project

      Lab Session 1
      1) Transform E. coli cells with plasmid containing probe DNA and plate the cells.

      Lab Session 2
      1) Begin isolating genomic DNA from the fruit fly, Drosophila melanogaster .
      2) Identify transformant colonies on your plates from Lab Session 1 and calculate transformation efficiencies.

      Lab Session 3
      1) Finish isolating genomic DNA from the fruit fly, Drosophila melanogaster.
      2) Digest the genomic DNA with restriction enzymes.

      Lab Session 4
      1) Your instructor will grow 2 ml cultures of selected colonies from your plates that you set up during Session 1.
      2) You will isolate recombinant plasmid DNA containing the probe from these cultured cells.
      3) Set-up restriction digests of the purified plasmids. Save these digests for electrophoretic analysis during Session 5.

      Lab Session 5
      1) Run a gel on the digested Drosophila genomic DNA and set up a Southern blot.

      Lab Session 6
      1) Run a gel on the digested plasmid DNA and isolate the probe DNA fragment.
      2) Set up and run your PCR reactions.

      Lab Session 7
      1) Run your PCR products on a gel and anayze the data.
      2) Analyze your Southern blot hybridization analysis data.

      Lab Session 1
      1. Transform E. coli cells with plasmid containing probe DNA and plate the cells.

      During this lab session, you will begin the process of preparing large amounts of the P-element probe DNA. You will introduce a recombinant plasmid called p25.1, which contains P-element probe sequences, into competent E. coli cells, "transforming" the bacterial cells. You will then plate the cells on nutrient agar plates that contain the antibiotic, ampicillin, which will kill untransformed cells. You will also be doing some control experiments. You will transform E. coli cells with a pure plasmid called pBR322, which is the vector plasmid of p25.1 (pBR322 = p25.1 ­ P-element sequences). You will plate your two sets of transformed cells on two different types of plates. One type of plate will contain ampicillin, while the other will contain a different antibiotic called tetracycline. PBR322 contains genes for both ampicillin resisitance and tetracycline resistance, so cells transformed with pBR322 should grow on both types of plates. After observing restriction maps of p25.1 and pBR322, make a prediction about which plates cells transformed with this recombinant plasmid will grow on.
      In addition to the above experiments, you'll do yet another test. The plates you'll be using in this test will contain X-gal, which serves as substrate for the ß-galactosidase enzyme that is encoded by the lacZ gene. The lacZ gene is part of some plasmid vectors, such as pUC19. You will transform a third set of E. coli cells with pUC19. When you plate cells transformed with pUC19, the ß-galactosidase enzyme will cleave the X-gal substrate in the presence of IPTG to yield a blue precipitate, and thus, cells transformed with pUC19 will appear blue.

      Procedure
      1) Obtain three microfuge tubes of plasmid DNA, labelled pBR322, p25.1, and pUC19, and three microfuge tubes containing 90µl dilution fluid (sterile saline). Pipet 10 µl of one plasmid DNA sample into one of the tubes with 90µl dilution fluid to make a 10-1 dilution. Repeat for the other two plasmid DNA samples, then set these three diluted DNA samples aside for a while.

      2) Obtain a microfuge tube containing competent E. coli cells, and place it on ice.

      3) Gently tap the tube of competent cells with the tip of your finger to ensure that the cells are well suspended.

      4) Using a sterile pipet, transfer 0.2 ml (200 µl) of competent cells into the tube containing what is left of the undiluted pBR322 plasmid DNA. Cap the tube, and tap it with the tip of your finger to mix the cells with the DNA. Put the tube on ice for 20 minutes. The competent cells, which are suspended in CaCl2, will begin to take-up the recombinant plasmid DNA.

      5) Similarly, using sterile pipets, transfer 0.2 ml (200 µl) of competent cells into each of the remaining two tubes of undiluted plasmid DNAs. Cap and tap as before, and put the tubes on ice for 20 minutes.

      6) After the 20 minute ice treatment, place each of the three tubes in a 42oC water bath for 1.5 minutes. This heat shock facilitates the uptake of DNA by the bacterial cells.

      7) Transfer the tubes to ice for 1.5 minutes.

      8) Using a sterile pipet, add 0.8 ml (800 µl) of LB (L Broth, a nutrient-rich bacterial growth medium) to each tube.

      9) Incubate the tubes at 37oC for 20 minutes, then tap each tube to mix its contents well. Place them back at 37oC for an additional 20 minutes. This incubation period allows the cells to recover from the CaCl2 treatment, and to begin expressing the ampicillin resistance and/or the tetracycline resistance genes on the plasmid. Mix the contents of each of the tubes by tapping .

      10) You will now dilute your transformation mixtures prior to plating them on selective agar medium. Transfer 0.1 ml (100 µl) of the pBR322 transformation mixture into a tube containing 9.9 ml of dilution fluid. This is a 1:100 (or 10-2) dilution. Also make a 10-3 dilution by transferring 1.0ml of the 10-2 dilution into a tube containing 9.0 ml of dilution fluid

      11) Repeat step 10 for each of the other two transformation mixtures.

      12) Using the "spread plating technique" (which your lab instructor will explain and demonstrate for you), plate 0.1 ml (100 µl) of each of the three dilutions of the pBR322 transformation mixture (undiluted, 10-2, 10-3) onto LBAmp plates and also onto LBTet plates.

      13) Repeat step 12 for the three dilutions of the p25.1 transformation mixture, using LBAmp and LBTet plates.

      14) Repeat step 12 for the three dilutions of the pUC19 transformation mixture, using LBAmp/Xgal/IPTG plates.

      15) Plate 0.1 ml (100 µl) of the competent cells alone on both LBAmp and LBTet plates. Also, plate the entire contents of the three 10-1 dilutions of plasmid DNA samples you made earlier onto three LB plates.

      16) There should be a total of 20 plates altogether. Incubate these plates upside-down at 37oC. Tomorrow morning, your instructor will move the plates to the refrigerator for storage until next week.

      Lab Session 2
      1) Begin isolating genomic DNA from the fruit fly, Drosophila melanogaster .
      2) Identify transformant colonies on your plates from Lab Session 1 and calculate transformation efficiencies.

      During this lab session, you will also begin the process of isolating genomic DNA from fruit flies. You will anesthetize about 50 adult flies, then grind them up (homogenize them) in a special buffer that keeps DNA stable. You will then treat the resulting homogenate with a detergent to disrupt the cell membranes, and a protease enzyme to destroy proteins. Finally, you will mix the homogenate with a combination of phenol, chloroform, and isoamyl alcohol, and store it in the freezer until next lab session.
      You will also examine the plates that you prepared last session, looking for bacterial colonies. You will count the numbers of colonies on each of your plates and determine transformation efficiencies. You should pay attention to the colors of the colonies.

      Part 1. Isolation of Genomic DNA from Drosophila melanogaster

      Procedure
      1) You will first be provided with a vial containing wild-type fruit flies. Anesthetize all of the flies in the vial as demonstrated by your lab instructor. Place the anesthetized flies on a white paper card, and view them using a dissecting microscope. For more information about fruit flies, see Project 2.

      2) Practice moving the flies around on the white paper card with a fine paint brush. Transfer approximately 50 flies into a 1.7 ml microfuge tube. Keep the tube on ice.

      3) Add 1 ml of HOM* buffer to the flies in the microfuge tube. Using a blue plastic homogenizer, homogenize the flies. Use several strokes, and do not be too rough.

      (*HOM: 80mM EDTA, 100mM Tris-Cl, 160 mM sucrose, pH 8.0)

      4) Transfer the homogenate to a 15 ml centrifuge tube. Add another 500 µl of HOM to the original microfuge tube, then transfer this to the 15 ml centrifuge tube.

      5) Add 75 µl of 10% SDS and 25 µl of 10 mg/ml Proteinase K.

      6) Incubate this for at least 1 hour at 68oC.

      During this incubation, do Part 2 of today's lab session.

      7) After the 68oC incubation, cool it to room temperature.

      2 ml) of phenol:chloroform:isoamyl alcohol (25:24:1) and mix. Store this in the freezer until next lab period.

      Part 2. Examination of Transformation Plates

      1) Obtain the plates that you prepared last session.

      2) Your instructor will show you how to effectively count the number of colonies on each plate, and determine the transformation efficiencies.

      What color are the colonies on each plate? Explain why the colonies have their particular colors.

      Finish Part 1 of today's lab session.

      Lab Session 3
      1. Finish isolating genomic DNA from the fruit fly, Drosophila melanogaster.
      2. Digest the genomic DNA with restriction enzymes.

      During this lab session, you will complete the process of isolating genomic DNA from fruit flies. You will perform a series of phenol:chloroform:isoamyl alcohol extractions to remove impurities from the DNA, and you will precipitate the DNA away from other impurities with ethanol. You will dissolve your fly genomic DNA in a buffer called TE. Finally, you will digest your Drosophila genomic DNA with restriction endonucleases.

      Procedure
      1) Remove your sample from lab session 1 from the freezer, and allow it to thaw at room temperature.

      2) Centrifuge your sample at 5,000 rpm for 5 minutes.

      3) Transfer the aqueous (top) layer to a clean 15 ml centrifuge tube, and add 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) to it. Mix, and centrifuge at 5,000 rpm for 5 minutes.

      4) Transfer the aqueous (top) layer to a clean centrifuge tube, and add 1 volume of chloroform to it. Mix, and centrifuge at 5,000 rpm for 5 minutes.

      5) Transfer the aqueous (top) layer to a clean centrifuge tube, and add 1 volume of chloroform to it. Mix, and centrifuge at 5,000 rpm for 5 minutes.

      6) Transfer the aqueous (top) layer to a clean centrifuge tube, and add 225 µl of 8M potassium acetate (KOAc) to it. Mix, and place this on ice for at least 30 minutes.

      7) Centrifuge this at 13,000 rpm for 20 minutes.

      8) Transfer the supernatant to a clean tube, and add 1 volume of 95% ethanol to it. Incubate this for 10 minutes at room temperature to precipitate the Drosophila genomic DNA.

      9) Centrifuge this at 10,000 rpm for 10 minutes. Remove the ethanol, and allow the pellet to air-dry slightly.

      10) Resuspend the pellet in 50 µl of TE buffer.

      11) Transfer all of the resuspended DNA into a clean microfuge tube and label the tube appropriately.

      12) Pipet 14 ml of the DNA into a new microfuge tube. Label the tube "Digested DNA".

      Save the rest of your Drosophila genomic DNA in the freezer. It will be used for template in PCR reactions that you'll set up during Lab Session 6.

      13) Pipet 6 ml of restriction digestion mix in to the tube labeled "Digested DNA"..

      14) Tap the tubes gently to mix the contents, and place them at 37oC until tomorrow morning.

      Lab Session 4
      1. Your instructor will have grown 2 ml cultures of selected colonies from your plates that you set up during Session 1.
      2. You will isolate recombinant plasmid DNA containing the probe from these cultured cells.
      3. Set-up restriction digests of the purified plasmids. Save these digests for electrophoretic analysis during Session 5.

      On the afternoon prior to this lab session, your instructor selected bacterial colonies from your plates that you set up during Session 1. These bacterial colonies consist of cells that were transformed with the recombinant plasmid p25.1 (contains P-element probe sequences). Your instructor inoculated 2 ml of LB (nutrient broth) + ampicillin with each colony. The inoculated cultures were grown overnight at 37oC. During this lab session , you will isolate plasmid DNA from these overnight liquid cultures. You will then digest the plasmids with restriction enzymes.

      Part 1. Isolation of recombinant plasmid DNA

      Procedure
      1) Each pair of students should obtain two test tubes containing 2 ml overnight cultures, each started from a single colony of cells transformed with the recombinant plasmid p25.1 (contains P-element probe sequences).

      2) Pour 1.5 ml of one overnight into an approporiately-labeled microfuge tube. Pour 1.5 ml of the other overnight culture into a second appropriately-labeled microfuge tube. The following instructions apply to each of the two microfuge tubes.

      3) Centrifuge the tube at full speed for 20 seconds to pellet the cells.

      4) Pour the supernatant out of the tube. Discard the supernatant.

      5) Resuspend the bacteria in 100 µl (0.1 ml) of GTE buffer by vortexing well.

      6) Add 200 µl (0.2 ml) of NS solution to each tube to lyse the bacteria. Mix by inverting the tube several times. Leave the tube at room temperature for 5 minutes.

      7) Add 100 µl (0.1 ml) of potassium acetate (KOAc) to the tube. Mix by shaking the tube vigorously. You should see a heavy, clotted precipitate of bacterial genomic DNA and protein.

      8) Centrifuge the tube at full speed for 2 minutes.

      9) Remove the supernatant with a plastic transfer pipet. Try to avoid the floating white debris and the pellet. Transfer the supernatant to a new, appropriately-labeled microfuge tube.

      10) Add 500 µl (0.5 ml) of isopropanol to the tube. Mix by inverting the tube several times.

      11) Centrifuge the tube at full speed for 2 minutes.

      12) Remove the supernatant with a plastic transfer pipet, and discard the supernatant. The pellet contains plasmid DNA. Resuspend the pellet in 100 µl (0.1 ml) of 50 mM Tris, pH 8.3.

      13) Add 50 µl of 8 M ammonium acetate (NH4Ac). Mix the contents of the tube, and place it in a dry ice/ethanol bath until the contents are frozen solid (

      14) Let the contents of the tube thaw at room temperature, then centrifuge it at full speed for 2 minutes.

      15) The supernatant contains the plasmid DNA. Transfer this supernatant to a new, appropriately-labeled microfuge tube.

      16) Add 500 µl of ethanol. Mix the contents by inverting the tube several times. Centrifuge the tube at full speed for 2 minutes.

      17) Pour the ethanol supernatant out of the tube. Discard the ethanol. Give the tube a quick spin, then remove the rest of the ethanol from the tube, leaving the plasmid DNA pellet undisturbed.

      18) Allow the pellet to air-dry for about 5 minutes, then resuspend the pellet in 25 µl of TE buffer.

      Part 2. Restriction digestion of recombinant plasmid DNA

      Procedure
      1) Label two microfuge tubes appropriately.

      2) Pipet 5 µl of each preparation of recombinant plasmid DNA from Part 1 into an appropriately-labeled new microfuge tube.

      3) Pipet 10 µl of restriction digestion mix into each tube.

      4) Tap the tubes gently to mix the contents, and place them at 37oC. Your instructor will move them to the freezer tomorrow morning.

      Lab Session 5
      1. Run a gel on the digested Drosophila genomic DNA and set up a Southern blot.

      During this lab session , you will run your restriction-digested Drosophila genomic DNA on an electrophoretic gel. You will then set up a Southern blot to transfer the DNA on the gel to a nitrocellulose (or nylon) hybridization membrane.

      Procedure
      1) Remove the tube containing your restriction-digested Drosophila genomic DNA (from Lab Session 3) from the freezer, and place it in a 70°C water bath for 5 minutes.

      2) You will be provided with another microfuge tube, labelled "l- Hin d III". This tube contains phage l DNA completely digested with Hin d III. This digested DNA will consist of DNA fragments of known length, which will serve as DNA size-standards on your electrophoretic gel. Keep this tube on ice until you are ready to use it.

      3) Add 5 µl of electrophoresis sample buffer to the tube containing your restriction-digested Drosophila genomic DNA. Tap the tube to mix the contents.

      Caution: The gel and reservoir buffer used in steps 4-8 contain ethidium bromide (etBr), a carcinogen. Wear gloves when handling anything containing etBr!

      4) Using your pipetting device, load the entire contents of the "l- Hin d III", and the tube conaining your restriction-digested Drosophila genomic DNA into the sample wells of an agarose gel as shown (2 pairs of students will share each gel. One pair of students will use wells 1, 2, and 3. The other pair of students will use lanes 4, 5 and 6:

      Sample Well
      Number (Starting
      at left) Sample
      1 "l- Hind III"
      2 Leave Empty
      One Pair of Students 3 Digested Fly DNA
      ________________________________________________________________________
      4 "l- Hind III"
      5 Leave Empty
      Second Pair of Students 6 Digested Fly DNA

      5) Cover the electrophoresis unit and plug the leads into the power supply. The leads are color coded so that the red lead plugs into the positive terminal, and the black lead plugs into the negative terminal. The sample wells in the gel should be closest to the negative (black) electrode.

      6) Turn the power source on and set the voltage at about 120 volts. Electrophorese until the bromphenol blue (dark blue dye) in the samples has migrated to within 5 mm of the positive (red) electrode end of the gel. At 120 volts, this should take about 1 hour.

      7) After your gel run is complete, turn the power supply off and unplug the leads. Remove the gel from the unit and place it on a ultraviolet (UV) transilluminator.

      8) Turn the UV transilluminator on, and photograph your gel using a gel imager as demonstrated by your instructor.

      1) Rinse the gel in distilled water.

      2) Soak the gel in Southern Denaturing Solution (0.5N NaOH, 1.5M NaCl) for 20 minutes on a gentle shaker. Repeat using fresh Southern Denaturing Solution for another 20 minutes.

      3) Soak the gel in Southern Neutralizing Solution (1M Tris-HCl, 1.5M NaCl, pH=8.0) for 20 minutes on a gentle shaker. Repeat using fresh Southern Neutralizing Solution for another 20 minutes.

      4) During soaks, prepare a Southern blotting stack as follows:
      a. Place a sponge in the center of a Tupperware dish.
      b. Cut a piece of Whatman 3MM (or equivalent) filter paper to the exact size of the sponge, and place this piece of filter paper on top of the sponge.
      c. Fill the dish with 20X SSC so as to completely soak the Whatman paper. This should lie flat against the sponge.
      d. Using a brand-new razor blade, cut a piece of nitrocellulose or nylon transfer membrane to the exact size of the gel. IMPORTANT: Never handle membrane without gloves. Keep membrane in its protective backing throughout the cutting process. "Measure twice, cut once!"
      e. Cut two pieces of Whatman paper to the exact size of the gel. Place them and the membrane in 2X SSC.

      5) When soaks are finished, place the gel upside-down onto the filter paper that covers the sponge. Remove any air bubbles from beneath the gel, using a glass roller.

      6) Surround the gel with Parafilm.

      7) Lay the wetted transfer membrane onto the gel, being careful to roll out any air bubbles. With a razor blade, cut off one corner ­ approximately 0.5cm2 ­ of both gel and membrane.

      8) Lay the two pieces of wetted Whatman paper ontothe transfer membrane. Remove bubbles.

      9) Place a stack of paper towels approximately 3" thick on top of the filter paper.

      10) Place a glass plate and appropriate weight (ask instructor) on top of the paper towels. Allow transfer overnight.

      Lab Session 6
      1. Run a gel on the digested plasmid DNA and isolate the probe DNA fragment.
      2. Set up and run your PCR reactions.

      In this lab session, you will analyze your restriction-digested p25.1 recombinant plasmids by electrophoresing them on a special low-melting temperature agarose gel. From the results of this experiment, you will be able to determine the size of the fly DNA fragment that resulted from your restriction digest of this plasmid. You will determine which fragments contain pure P-element DNA, and isolate them by cutting them out of the gel with a razor blade. In doing this, you will prepare pure P-element probe DNA.
      You will also set up PCR reactions to try to detect P-elements in your Drosophila genomic DNA. You will use the Drosophila genomic DNA you finished isolating in Lab Session 3 as template, and the primers that you designed.

      Part 1. Electrophoretic analysis of recombinant plasmids

      Procedure
      1) Remove the tubes containing your restriction-digetsed p25.1 plasmid DNA from the freezer, and heat them in a 70oC water bath for 5 minutes, then place them on ice.

      2) You will be provided with another microfuge tube, labelled "l- Hind III". This tube contains phage l DNA completely digested with Hind III. This digested DNA will consist of DNA fragments of known length, which will serve as DNA size-standards on your electrophoretic gel. Keep this tube on ice until you are ready to use it.

      3) Add 5 µl of electrophoresis sample buffer to tubes containing your restriction-digetsed p25.1 plasmid DNA. Tap the tubes to mix the contents.

      Caution: The gel and reservoir buffer used in steps 4-8 contain ethidium bromide (etBr), a carcinogen. Wear gloves when handling anything containing etBr!

      4) Load the entire contents of the tubes into the sample wells of an agarose gel as shown:

      Sample Well
      Number (Starting
      at left) Sample
      1 1 "l- Hind III "
      2 Leave Empty
      3 2 Digested p25.1 plasmid DNA
      4 3 Digested p25.1 plasmid DNA
      5 4 Digested p25.1 plasmid DNA
      6 5 Digested p25.1 plasmid DNA
      5) Cover the electrophoresis unit and plug the leads into the power supply. The leads are color coded so that the red lead plugs into the positive terminal, and the black lead plugs into the negative terminal. The sample wells in the gel should be closest to the negative (black) electrode.

      6) Turn the power source on and set the voltage at about 120 volts. Electrophorese until the bromphenol blue (dye) in the samples has migrated to within 5 mm of the positive (red) electrode end of the gel. At 120 volts, this should take about 1 hour.

      While your gel is running, do Part 2 of today's lab session.

      7) After your gel run is complete, turn the power supply off and unplug the leads. Remove the gel from the unit and place it on a ultraviolet (UV) transilluminator.

      8) Turn the UV transilluminator on, and photograph your gel using a gel imager or Polaroid camera as demonstrated by your instructor.

      9) Using information in the "Interpretation of Electrophoresis Data" part of this manual (pp. 22 -23), and the guidance of your lab instructor, detemine the sizes of the DNA fragments in any bands in the "Digested p25.1 plasmid DNA" lanes of your gel.

      10) Consult a restriction map od the plasmid, p25.1 and decide which bands on the gel contain fragments of pure P-element DNA. Your instructor will demonstrate how to cut these bands out of the gel using a clean razor blade, thus isolating pure P-element DNA for use as a hybridization probe for the Southern blot. After cutting out the appropriate probe DNA bands, place them in a microfuge tube and store the tube in the refrigerator.

      Procedure
      1) Remove the tube containing your leftover undigested Drosophila genomic DNA (from Lab Session 3) from the refrigerator and place it in a 70°C water bath for 5 minutes.

      2) Label a microfuge tube with your initials and the letter Q (for "quantification sample"). Dispense 250µl of distilled water into the tube.

      3) Using the 2µl micropipetter, add 0.5µl of DNA from your sample to the Q tube. Mix well by vortexing.

      4) Determine the concentration of your DNA samples using the UV spectrophotometer. Your instructor will demonstrate.

      5) Label a fresh microfuge tube with your initials, sample abbreviations and P (for PCR). From your quantification data, calculate the necessary sample volume to add 10-20ng of Drosophila genomic DNA to the tube (this will probably differ between samples, and you may need to dilute DNA aliquots with TE buffer). Add the appropriate amount of DNA, primer and PCR reaction mix to the tube, and place it in the thermocycler. When everyone's samples are in the thermocycler, it will be turned on and run for the proper number of cycles.

      After the PCR reactions are completed, your instructor will shut down the thermocycler and store your samples for you until next week.

      Lab Session 7
      1. Run your PCR products on a gel and anayze the data.
      2. Analyze your Southern blot hybridization analysis data.

      During this lab session, you will run the products of your PCR reactions on an electrophoretic gel and interpet the resulting data.
      In addition, during the past week, your instructor labeled your P-element probe DNA radioactively, hybridized this probe to your Southern blot, and placed the membrane in a cassette with X-ray film to detect spots of radioactivity (i.e., autoradiographed the hybridized southern blot, producing an "autoradiogram"). You will analyze the resulting data.

      Procedure
      1) Remove the tube containing your PCR reaction products (from Lab Session 6) from the freezer, and place it in a 70oC water bath for 5 minutes.

      2) You will be provided with another microfuge tube, labelled "l- Hin d III". This tube contains phage l DNA completely digested with Hin d III. This digested DNA will consist of DNA fragments of known length, which will serve as DNA size-standards on your electrophoretic gel. Keep this tube on ice until you are ready to use it.

      3) Add 5 µl of electrophoresis sample buffer to the tube containing your restriction-digested Drosophila genomic DNA. Tap the tube to mix the contents.

      Caution: The gel and reservoir buffer used in steps 4-8 contain ethidium bromide (etBr), a carcinogen. Wear gloves when handling anything containing etBr!

      4) Using your pipetting device, load the entire contents of the "l- Hin d III", and the tube conaining your restriction-digested Drosophila genomic DNA into the sample wells of an agarose gel as shown (2 pairs of students will share each gel. One pair of students will use wells 1, 2, and 3. The other pair of students will use lanes 4, 5 and 6:

      Sample Well
      Number (Starting
      at left) Sample
      1 "l- Hind III"
      2 Leave Empty
      One Pair of Students 3 PCR Reaction Products
      ________________________________________________________________________
      4 "l- Hind III"
      5 Leave Empty
      Second Pair of Students 6 PCR Reaction Products

      5) Cover the electrophoresis unit and plug the leads into the power supply. The leads are color coded so that the red lead plugs into the positive terminal, and the black lead plugs into the negative terminal. The sample wells in the gel should be closest to the negative (black) electrode.

      6) Turn the power source on and set the voltage at about 120 volts. Electrophorese until the bromphenol blue (dark blue dye) in the samples has migrated to within 5 mm of the positive (red) electrode end of the gel. At 120 volts, this should take about 1 hour.

      While you gel is running, do Part 2 of Today's lab session.

      7) After your gel run is complete, turn the power supply off and unplug the leads. Remove the gel from the unit and place it on a ultraviolet (UV) transilluminator.

      8) Turn the UV transilluminator on, and photograph your gel using a gel imager or Polaroid camera as demonstrated by your instructor.

      9) Using information in the "Interpretation of Electrophoresis Data" part of this manual (pp. 22-23), and the guidance of your lab instructor, detemine the sizes of the DNA fragments in any bands in the "PCR Reaction Products" lane of your gel. Did you detect P-elements in the genome of this strain of D. melanogaster ? If so, how many?

      Part 2. Interpretation of Southern Blot Hybridization Analysis Data

      1) Obtain the autoradiogram of your hybridized Southern blot. Using information in the "Interpretation of Electrophoresis Data" part of this manual (pp. 22-23), and the guidance of your lab instructor, detemine the sizes of the DNA fragments to which the P-element probe hybridized on your Southern blot. Did you detect P-elements in the genome of this strain of D. melanogaster ? If so, how many?

      Finish Part 1 of today's lab session.

      Interpretation of Electrophoresis Data

      1) Figure 7 shows the lengths (in kb) of the DNA fragments in each band resulting from the electrophoresis of l DNA cut with Hin d III alone (the DNA size-standards in this experiment). Examine the bands in the l- Hin d III lane of your gel, and determine the lengths of the fragments that make-up each band. For example, the band that is closest to the sample well represents the largest fragment, which is 23.1 kb long. Determine the distance (in mm) that each of these l- Hin d III bands has migrated from the sample wells. Using semi-log graph paper, plot migration distance (in mm) of each l- Hin d III band on the X-axis, and DNA fragment length (in kb) of that band on the Y-axis. Draw a line though the points on your graph. From this graph, determine the length of the fragments that make up each band in the experimental lanes of your gel or autoradiogram based on their migration distances. Do this as follows for each band:

      a) Find the migration distance of the band on the X-axis of your graph.
      b) Find the point on the line that is directly above this X-coordinate.
      c) Find the point on the Y-axis that is directly to the left of this point on the line. This Y-coordinate is the size of the fragment (in kb).

      Project 2: Isolation and Characterization of Mutations in Drosophila melanogaster

      One of the most widely used organisms in genetic studies is the fruit fly, Drosophila melanogaster . Thomas Hunt Morgan pioneered genetic studies with Drosophila at Columbia University in 1911. Today, there is a large body of knowledge regarding the genetics of the fruit fly. Drosophila has four pairs of chromosomes (a relatively small number) that have been characterized extensively. In less than two weeks, the fruit fly undergoes a specific developmental sequence from fertilized egg to larva, prepupa, pupa, then adult. The geneticist can follow the inheritance of morphological, physiological, and developmental patterns. In this project, you (the geneticist) will be performing a screen to identify and characterize Drosophila mutants that exhibit morphological abnormalities. You will then characterize the mutations that you identify, in the hope of learning something about the genes involved.

      The chromosomes of Drosophila melanogaster

      The individual Drosophila has four pairs of chromosomes. A female has two each of chromosomes 1 (more commonly called the X chromosome), 2, 3, and 4. A male has one X chromosome, one Y chromosome, and two each of chromosomes 2, 3, and 4. The Y chromosome and chromosome 4 are both very small, and carry few genes. The majority of the fly's genes are carried on chromosomes X, 2, and 3. The X and Y chromosomes are involved in sex determination, and are thus called the sex chromosomes. Chromosomes 2, 3, and 4 are called the autosomes. In the fruit fly, sex is determined by the relative number of X chromosomes and autosomes. If a fly has two X chromosomes, and two of each autosome (an X:autosome ratio of 1:1), it will develop as a female. If a fly has only one X chromosome, and two of each autosome (an X:autosome ratio of 1:2), it will develop as a male. In Drosophila , the Y chromosome does not determine maleness! (This is in contrast to the case in mammals, in which the presence of the Y chromosome determines maleness.). In fact, a fly that has two X chromosomes and a Y chromosome will develop as a female.

      Development of Drosophila melanogaster

      Mating in the fruit fly occurs 6-8 hours after the adult female emerges from her pupal case. Eggs may be laid at this time, or retained and laid later. A female receives about 4000 sperm from a male, and stores them in special sacs. The sperm are released gradually as the eggs are produced. Each female can lay several hundred fertilized eggs on the surface of a food source. Each fertilized egg develops over a period of 24 hours into a larva. The larva burrows into the food source, and eats yeast cells. Four to five days and two molts (shedding of the larva's exterior cuticle) later, the larva climbs onto a solid surface and pupariates to form a prepupa, which covers itself in a hard pupal case. The prepupa develops into a pupa in 12 hours. Over the next 4-5 days, the pupa develops into an adult, which emerges from the pupal case in the process of eclosion. Initially the fly is long and thin, with folded-up wings, and is light in color. Gradually, the wings expand and the fly takes on a more rounded form and darker color. The entire life cycle, which takes 10-14 days at 25 C, is illustrated in figure 1.

      Figure 1. The Drosophila life cycle.

      The study of mutations in Drosophila melanogaster

      Mutations are powerful tools in genetic analysis. The logic behind mutational analysis is that we can learn about the function of a gene by examining what goes wrong when that gene doesn't function properly. Genes can be altered from their wild-type forms by mutations, which often disrupt or completely eliminate gene function. Mutational analysis has been called "genetic dissection". The first stage in a genetic dissection is the hunt for mutants (individual organisms that carry mutant genes). Mutants occur spontaneously in any population at low frequency. Through the use of mutagens, however, we can dramatically increase the likelihood of finding useful mutants. The use of a mutagen to induce mutations is called mutagenesis . A mutagen that has proven extremely effective in inducing mutations in Drosophila is the chemical, ethyl methane sulfonate (EMS). EMS can add an ethyl group (-CH2CH3) to many positions on all four bases found in DNA, altering their pairing properties. The most common change induced by EMS is the addition of an ethyl group to guanine (G), enabling it to pair with thymine (T). This illegitimate pairing leads to GC --> AT transitions at the next round of replication (see Griffiths textbook, p. 596).
      EMS induces a high proportion of point mutations. This mutagen is easily administered to adult flies by placing them on filter paper saturated with a mixed aqueous solution of EMS and sugar. Males fed 0.025 M EMS produce sperm carrying lethal mutations on 70% of all X chromosomes, and on almost every chromosome 2 and 3. At these levels of mutation induction, it is feasible to look for (screen) for mutations at specific loci, or for mutants that display unusual phenotypes (see Griffiths textbook, p. 202). In this project, you will be screening flies that have been mutagenized with EMS, to find mutants with abnormal phenotypes.

      The use of the attached-X chromosome in Drosophila mutagenesis

      The majority of interesting mutations that will be detected in a screen following EMS mutagenesis will be recessive, thus yielding no mutant phenotype unless homozygous. An exception to this requirement of homozygosity for the expression of recessive mutant phenotypes is the case of X chromosome-linked mutations in male flies. Since a male fly has only one X chromosome (and is thus called hemizygous for all X-linked genes), he will express the abnormal phenotype associated with any recessive mutation on the X. By making use of a special chromosome called an attached-X chromosome (represented as X^X), it is possible to screen F1 generation males for interesting mutations on the X chromosome. The attached-X chromosome is a compound chromosome formed by the fusion of two X chromosomes. An X^X chromosome is inherited as a single unit, and flies carrying an X^X chromosome will be female. A female that has an X^X chromosome, and also has a Y chromosome can be mated with a normal male to produce X^X/Y female, and X/Y male F1 offspring, as shown in figure 2. Attached-X chromosomes make it possible to perform a rapid EMS mutagenesis and mutant screen by feeding EMS to Parental generation males, mating them to X^X females, and screening the resulting F1 progeny males for abnormal phenotypes. A scheme for performing this type of mutagenesis and screen is outlined in figure 3. In the F1 generation, only the males will carry mutagenized X chromosomes. Since males are hemizygous for all X-linked genes, recessive X-linked mutations will be expressed in these F1 males.

      In this laboratory project, you will be receiving a large group of F1 offspring resulting from the mating of EMS-treated wild-type males with attached-X females. This group of F1 flies should contain a number of X-linked mutants. You are on a search for interesting mutants. You will be working in pairs, but the entire class will cooperate in this search for mutants, and you can share mutants with each other. You will discover these mutants by looking for flies displaying phenotypes that vary from wild-type. An example of such a phenotype would be eyes that are a different color from the wild-type, dark red eye color. You may also find flies that have abnormal-looking wings, bristles, or other body parts. Once you have identified as many interesting mutants as you can, you will want to characterize them. For a given mutation, you will want to determine if that mutation can be transmitted to the next generation. You also want to determine if each mutation is, in fact, on the X chromosome. You should design experiments to answer these questions. Once you have determined which of your mutations are transmissible, it is time to choose one to examine in more detail. You will want to map the mutation to a specific region of the X chromosome. This laboratory manual is designed to provide you with guidance. It is not meant to lead you step-by-step through each experiment. You should perform this analysis as independently as possible! With the help of your instructors, you should design and carry out experiments to address the questions listed above. A potential schedule for this project is outlined below.

      Experimental Procedures (work in groups of 4)

      During this lab session, you will begin screening for mutant flies. You will first anesthetize and examine a group of wild-type Drosophila . You should familiarize yourself with the appearance of a wild-type fly, and learn to distinguish males from females. Once you feel comfortable working with flies, you will be given a culture bottle containing F1 progeny of EMS-treated wild-type males mated with attached-X females. The bottle will contain attached-X F1 females, and X*/Y F1 males. (See Figure 3) You will be concentrating on the males, looking for flies with abnormal phenotypes.

      You will be provided with:

      a vial containing wild-type fruit flies
      a fly anesthetizer
      fly anesthetic chemical
      a white paper card
      a paint brush
      a dissecting microscope
      a large group of F1 offspring resulting from the mating of EMS-treated wild-type males with
      attached-X females.
      a vial of virgin attached-X females [ C(1)A, y ]
      empty fly culture vials

      Examination of wild-type fruit flies

      You will first be provided with a vial containing wild-type fruit flies. Anesthetize all of the flies in the vial as described below, and as demonstrated by your lab instructor.

      1) Remove the bottom cap of your fly anesthetizer, and take out the foam rubber pad found inside the apparatus.

      2) "Charge" your fly anesthetizer by putting about 10 drops of Fly Nap anesthetic on the foam rubber pad, and placing the pad back inside the apparatus. Put the bottom cap back on.

      3) Remove the top cap from your fly anesthetizer. Tap the bottom of the vial of flies lightly and rapidly on a pad on your bench, then remove the plug from the vial. Quickly invert the fly vial over the top of the open anesthetizer, and tap the whole thing lightly and rapidly on a pad, so that the flies fall into the anesthetizer.

      4) Quickly cap the anesthetizer, and keep the flies in the anesthesia chamber until they all stop moving (this should take a couple of minutes).

      5) Dump the anesthetized flies out of the anesthetizer onto a white paper card, and view them using a dissecting microscope.

      6) Practice moving the flies around on the white paper card with a fine paint brush. Notice the wild-type, dark red color of the eyes.

      7) Using the diagrams in Figure 4 and provided in the lab room, separate the males from the females. Males have narrower abdomens than females, and the posterior end of the male abdomen is more darkly pigmented than that of the female. Males have dark genitalia on the extreme posterior ends of their abdomens that females lack. Males also have specialized bristles called "sex combs" on their most anterior pair of legs. If you are having trouble telling males from females by looking at the end of the abdomen, the sex combs will positively-identify a male.

      8) Once you feel comfortable working with flies and telling males apart from females, it is time to begin screening for mutant flies!

      Figure 4. Distinguishing Male and Female Drosophila

      You will be given a large group of F1 offspring resulting from the mating of EMS-treated wild-type males with attached-X females.

      1) Anesthetize and examine the flies carefully, concentrating on the males. The males and females should be easy to tell apart, because the attached-X females have yellow bodies, while the males, unless altered by a newly-induced mutation affecting body color, will have dark tan bodies. The females will have yellow bodies, because the attached-X chromosomes carry the mutation, yellow (abbreviated, y ). The name of the attached-X chromosome that these females carry is C(1)A, y . This stands for "Compound Chromosome 1 (the X) of Armentrout (the scientist who made the chromosome), carrying the mutation, yellow ". Both of the X chromosomes that make up C(1)A, y carry the yellow mutation, so the females are homozygous for this recessive mutation, and display the mutant yellow body color. Keep in mind that, if you see a yellow-colored male, it is potentially due to a newly induced X-linked mutation, and you should examine it further.

      2) Look for males that show any differences from wild-type. Save each mutant-looking male that you find in his own culture vial. If you absolutely cannot find a mutant, DON'T WORRY! This will be a team effort, and you can get a mutant from another group of students if you need to. Your instructor will also provide additional mutants.

      3) With the help of your lab instructor (if you want it) design and begin a experiments to determine if the mutations you have identified are transmissible to the next generation. You'll be provided with everything (including flies) that you need to set up these experiments. You should discuss your approach with your instructor. You should also be thinking about how you will map your mutations to a region of the X chromosome.

      During this lab period, you will interpret the results of your experiment designed to determine if each of your newly identified mutations is transmissible.

      1) Anesthetize the flies in the vials that you set up crosses in during Lab Session 1. Examine all of the flies. These flies, the progeny of the cross(es) you set up during Lab Session 1, are the F2 generation. Look for F2 flies displaying the mutant phenotypes you identified during Lab Session 1. Pay attention to differences between males and females.

      -Which of your mutations are transmissible?

      -Of the mutations that are transmissible, can you tell if any are dominant or recessive? Can you tell if any are X-linked or autosomal? Explain your reasoning.

      -If a particular mutation did not transmit, think about why that may be.

      2) If a particular mutation is transmissible, begin your experiment to map it to a region of the X- chromosome. You will be provided with virgin female fruit flies homozygous for a multiply-marked X-chromosome. This multiply-marked X-chromosome carries several different recessive mutations that are easy to identify. An example of a multiply-marked X-chromosome is the
      y cv v f chromosome. This chromosome carries recessive mutant alleles of four genes that are spaced along the length of the chromosome. These genes are:

      y = yellow (maps to the telomere, or extreme left end of the X-chromosome, Map Position = 0) Female flies homozygous (or male flies hemizygous) for mutations in yellow have very light yellow body color, as opposed to the tan body color of wild-type flies.

      cv = crossveinless (Map Position = 13.7, which means it is 13.7 Map Units to the right of the telomere) Female flies homozygous (or male flies hemizygous) for mutations in crossveinless lack a certain set of veins that are supposed to be in their wings.

      v = vermillion (Map Position = 33.0, which means it is 33 Map Units to the right of the telomere) Female flies homozygous (or male flies hemizygous) for mutations in vermillion have an abnormal pinkish eye color, as opposed to the dark red eye color of wild-type flies.

      f = forked (Map Position = 56.7, which means it is 56.7 Map Units to the right of the telomere) Female flies homozygous (or male flies hemizygous) for mutations in forked have an abnormal sharp bend in the end of their bristles, which makes forked bristles appear quite different from the straight, pointed bristles of wild-type fruit flies.

      A schematic map of the y cv v f chromosome would look like this:

      If the mutation that you wish to map is X-linked, set up a cross of males hemizygous for your newly-identified mutation with virgin females homozygous for the multiply-marked X-chromosome. You will interpret the results of this experiment during Lab Session 3.

      During this lab period, you will continue your mapping experiments. You will interpret the results of the cross(es) that you set up during Lab Session 2, and set up another cross (or crosses).

      1) Anesthetize the flies in the vials that you set up crosses in during Lab Session 2. These flies, the progeny of the crosses you set up during Lab Session 2, are the F3 generation. Examine all of the flies. Look for flies displaying the mutant phenotypes you identified during Lab Session 1. Pay attention to differences between males and females.

      -What do the males flies look like?

      -What do the female flies look like?

      -Do any of the flies display the mutant phenotype that you identified in Lab Session 1?

      -From your results, can you determine whether your newly-identified mutation is dominant or recessive?

      -From your results, can you determine whether your newly-identified mutation is allelic to any known mutation?

      2) The F3 females are heterozygous for the X-chromosome carrying your newly-identified mutation, and the multiply-marked X-chromosome. During meiosis in these females, crossing-over can occur between these two X-chromosomes, resulting in eggs carrying recombinant X-chromosomes. By examining the F4 progeny resulting from a cross between these females and appropriate male flies, you can determine the recombination frequencies between your newly-identified mutation and the known mutations on the multiply-marked X-chromosomes. This will enable you to calculate a map position for your new mutation.
      Since you are dealing with X-linked mutations, you can plan to restrict your analysis of the F4 progeny to the F4 males. Each F4 male will inherit a single X-chromosome from his heterozygous F3 mother, and will display the phenotypes associated with any mutations on that X-chromosome. Each F4 male will either be parental-type or recombinant with respect to the several mutations you are working with. Determining the percentage of F4 male progeny that are recombinant will enable you to calculate a map position for your newly-identified mutation.
      Since you will be looking at only the males in the F4 generation, the genotype of the males that you cross with your F3 heterozygous females does not matter. You can consider these males to be merely sperm donors. Set up a cross(es) of your F3 heterozygous females with available males.

      During this lab period, you should be able to finish your proposed mapping experiment, and determine what region of the X chromosome each of your mutations maps to.

      1) Anesthetize the flies in the vials that you set up crosses in during Lab Session 3. These flies, the progeny of the crosses you set up during Lab Session 3, are the F4 generation. Examine all of the flies. Separate the males from the females, and discard the females. You will focus this analysis on the F4 males only.

      2) Pick three mutations to focus your attention on . These three mutaions must include your newly-isolated mutation and two of the mutations on the multiply-marked X-chromosome. You can now consider your study a three-point cross, as described on pages 142-144 of the textbook Griffiths et al. (1999) Modern Genetic Analysis . New York, W.H. Freeman and Company. Your first job will be to determine the phenotype (mutant or wild-type) of each of the F4 males with respect to the three mutations you are considering. You can then figure out the numbers of F4 males that are parental-type and recombinant-type with respect to each of the three mutations.

      3) After scoring all of the F4 males for phenotype, determine which class of progeny represent the double cross-overs. Determine the number of single cross-overs that occurred between each of the three mutations. With these numbers, you should be able to map the three mutations relative to one another. Since you know the map positions of two of the mutations, you should be able to determine a map position for your newly-isolated mutation.

      4) Draw a map of the X-chromosome showing the map positions of your newly-isolated mutation and the two other mutations you used.


      COMMON THEMES IN THE BIOLOGY OF TA AND RM SYSTEMS

      Classifying TA systems

      The genetic modules of TA systems are units that produce a functional, stable, toxic protein and its inhibitor, a more labile antitoxin. TA systems are divided into the following types based on the nature of the antitoxin’s inhibitory effect ( Figure 2):

      Type I has an antisense RNA as the antitoxin, which pairs with the toxin mRNA ( 19, 20)

      Type II has a protein antitoxin, which neutralizes the toxin’s effect by binding directly to it ( 21, 22)

      Type III has an antitoxin RNA (that is not an antisense RNA), which interacts directly with the toxin protein ( 23, 24)

      Type IV has a protein toxin and a protein antitoxin, which interferes with the binding of the toxin to its target (rather than inhibiting the toxin via direct binding) ( 25) and

      Type V has a protein antitoxin, which cleaves the toxin mRNA ( 26).

      TA systems and Type II RM systems. The toxin is a protein in all cases. The rightmost parts indicate the toxin action after the loss of the genes or an imbalance between the toxin and the antitoxin. Type II RMs have antitoxin modification enzymes that methylate the genomic DNA, protecting it from cleavage by a restriction enzyme (toxin) type I TA has an antisense RNA (the antitoxin) that pairs with the toxin mRNA ( 19, 20) type II TA has a protein antitoxin that binds to the toxin ( 21, 22) type III TA has an antitoxin RNA (not an antisense RNA) that binds to the toxin ( 23, 24) type IV TA has a protein antitoxin that binds to the toxin target ( 25) and type V TA has a protein antitoxin that cleaves the toxin mRNA ( 26). A, antitoxin T, toxin P, promoter Me, methyl group on a DNA base SD, Shine–Dalgarno sequence. Toxins and their genes are shown in dark red antitoxins and their genes are shown in light blue.

      TA systems and Type II RM systems. The toxin is a protein in all cases. The rightmost parts indicate the toxin action after the loss of the genes or an imbalance between the toxin and the antitoxin. Type II RMs have antitoxin modification enzymes that methylate the genomic DNA, protecting it from cleavage by a restriction enzyme (toxin) type I TA has an antisense RNA (the antitoxin) that pairs with the toxin mRNA ( 19, 20) type II TA has a protein antitoxin that binds to the toxin ( 21, 22) type III TA has an antitoxin RNA (not an antisense RNA) that binds to the toxin ( 23, 24) type IV TA has a protein antitoxin that binds to the toxin target ( 25) and type V TA has a protein antitoxin that cleaves the toxin mRNA ( 26). A, antitoxin T, toxin P, promoter Me, methyl group on a DNA base SD, Shine–Dalgarno sequence. Toxins and their genes are shown in dark red antitoxins and their genes are shown in light blue.

      Our conception of TA systems, presented in this review, has been formed primarily through research into type I and type II systems. Types IV and V have been identified recently, and more members must yet be evaluated. The recent classification of type II TA systems has defined 12 toxin superfamilies and 20 antitoxin superfamilies based on their specific functional capacities to associate with the TA partner and cross-neutralize it ( Tables 1 and 2) ( 22, 61). For type I TA systems, small hydrophobic candidate proteins are identified by similarities in their features beyond any sequence similarities ( 20).

      Type . TA system . Toxin . Activity . Reference .
      II mazE–mazMazF cleaving mRNA ( 27, 28)
      phd–docDoc binding to ribosomal 30S subunit (blocking translation elongation) ( 29)
      vapB–vapCVapC cleaving initiator tRNA ( 30, 31, 32)
      ccdA–ccdBCcdB inhibiting DNA gyrase ( 33–36)
      epsilon–zetaZeta ATP-dependent kinase ( 37–40)
      hipA–hipBHipA serine kinase ( 41–43)
      III toxN–toxIToxN cleaving mRNA ( 23, 44)
      IV cbtA–cbeACbtA inhibiting cytoskeleton polymerization ( 25)
      V ghoT–ghoSGhoT lyzing cell membrane ( 26)
      Type . TA system . Toxin . Activity . Reference .
      II mazE–mazMazF cleaving mRNA ( 27, 28)
      phd–docDoc binding to ribosomal 30S subunit (blocking translation elongation) ( 29)
      vapB–vapCVapC cleaving initiator tRNA ( 30, 31, 32)
      ccdA–ccdBCcdB inhibiting DNA gyrase ( 33–36)
      epsilon–zetaZeta ATP-dependent kinase ( 37–40)
      hipA–hipBHipA serine kinase ( 41–43)
      III toxN–toxIToxN cleaving mRNA ( 23, 44)
      IV cbtA–cbeACbtA inhibiting cytoskeleton polymerization ( 25)
      V ghoT–ghoSGhoT lyzing cell membrane ( 26)

      For type I TA systems, see Table 2.

      Type . TA system . Toxin . Activity . Reference .
      II mazE–mazMazF cleaving mRNA ( 27, 28)
      phd–docDoc binding to ribosomal 30S subunit (blocking translation elongation) ( 29)
      vapB–vapCVapC cleaving initiator tRNA ( 30, 31, 32)
      ccdA–ccdBCcdB inhibiting DNA gyrase ( 33–36)
      epsilon–zetaZeta ATP-dependent kinase ( 37–40)
      hipA–hipBHipA serine kinase ( 41–43)
      III toxN–toxIToxN cleaving mRNA ( 23, 44)
      IV cbtA–cbeACbtA inhibiting cytoskeleton polymerization ( 25)
      V ghoT–ghoSGhoT lyzing cell membrane ( 26)
      Type . TA system . Toxin . Activity . Reference .
      II mazE–mazMazF cleaving mRNA ( 27, 28)
      phd–docDoc binding to ribosomal 30S subunit (blocking translation elongation) ( 29)
      vapB–vapCVapC cleaving initiator tRNA ( 30, 31, 32)
      ccdA–ccdBCcdB inhibiting DNA gyrase ( 33–36)
      epsilon–zetaZeta ATP-dependent kinase ( 37–40)
      hipA–hipBHipA serine kinase ( 41–43)
      III toxN–toxIToxN cleaving mRNA ( 23, 44)
      IV cbtA–cbeACbtA inhibiting cytoskeleton polymerization ( 25)
      V ghoT–ghoSGhoT lyzing cell membrane ( 26)

      For type I TA systems, see Table 2.

      TA system . Host . RNA antitoxin (overlaps mRNA) . Protein toxin . Toxin target . Location . Reference .
      hok–sokE. colisok (5′ end)Hok Cell membrane Plasmid ( 4, 45)
      ldrD–rdlDE. colirdlD (5′ end)LdrD Cell membrane Chromosome ( 46)
      istR–tisBE. coliistR-1 (5′ end)TisB Cell membrane Chromosome ( 47, 48)
      symE–symRE. colisymR (5′ end)SymE mRNA Chromosome ( 49)
      ibsC–sibCE. colisibC (5′ end)IbsC Cell membrane Chromosome ( 50, 51)
      txpA–ratAB. SubtillisratA (3′ end)TxpA Cell membrane Chromosome (prophage) ( 52, 53)
      fst–RNAI–RNAII E. faecalisRNAII (3′ end)Fst Cell membrane Plasmid ( 54–58)
      srnB–srnCE. colisrnC (5′ end)SrnB Cell membrane?Plasmid ( 59)
      pndA–pndBE. colipndB (5′ end)PndA Cell membrane Plasmid ( 59)
      shoB–ohsCE. coliohsC (5′ end)ShoB Cell membrane Chromosome ( 50)
      bsrG–SR4 B. subtilisSR4 (3′ end)BsrG Cell membrane Chromosome (prophage) ( 60)
      TA system . Host . RNA antitoxin (overlaps mRNA) . Protein toxin . Toxin target . Location . Reference .
      hok–sokE. colisok (5′ end)Hok Cell membrane Plasmid ( 4, 45)
      ldrD–rdlDE. colirdlD (5′ end)LdrD Cell membrane Chromosome ( 46)
      istR–tisBE. coliistR-1 (5′ end)TisB Cell membrane Chromosome ( 47, 48)
      symE–symRE. colisymR (5′ end)SymE mRNA Chromosome ( 49)
      ibsC–sibCE. colisibC (5′ end)IbsC Cell membrane Chromosome ( 50, 51)
      txpA–ratAB. SubtillisratA (3′ end)TxpA Cell membrane Chromosome (prophage) ( 52, 53)
      fst–RNAI–RNAII E. faecalisRNAII (3′ end)Fst Cell membrane Plasmid ( 54–58)
      srnB–srnCE. colisrnC (5′ end)SrnB Cell membrane?Plasmid ( 59)
      pndA–pndBE. colipndB (5′ end)PndA Cell membrane Plasmid ( 59)
      shoB–ohsCE. coliohsC (5′ end)ShoB Cell membrane Chromosome ( 50)
      bsrG–SR4 B. subtilisSR4 (3′ end)BsrG Cell membrane Chromosome (prophage) ( 60)
      TA system . Host . RNA antitoxin (overlaps mRNA) . Protein toxin . Toxin target . Location . Reference .
      hok–sokE. colisok (5′ end)Hok Cell membrane Plasmid ( 4, 45)
      ldrD–rdlDE. colirdlD (5′ end)LdrD Cell membrane Chromosome ( 46)
      istR–tisBE. coliistR-1 (5′ end)TisB Cell membrane Chromosome ( 47, 48)
      symE–symRE. colisymR (5′ end)SymE mRNA Chromosome ( 49)
      ibsC–sibCE. colisibC (5′ end)IbsC Cell membrane Chromosome ( 50, 51)
      txpA–ratAB. SubtillisratA (3′ end)TxpA Cell membrane Chromosome (prophage) ( 52, 53)
      fst–RNAI–RNAII E. faecalisRNAII (3′ end)Fst Cell membrane Plasmid ( 54–58)
      srnB–srnCE. colisrnC (5′ end)SrnB Cell membrane?Plasmid ( 59)
      pndA–pndBE. colipndB (5′ end)PndA Cell membrane Plasmid ( 59)
      shoB–ohsCE. coliohsC (5′ end)ShoB Cell membrane Chromosome ( 50)
      bsrG–SR4 B. subtilisSR4 (3′ end)BsrG Cell membrane Chromosome (prophage) ( 60)
      TA system . Host . RNA antitoxin (overlaps mRNA) . Protein toxin . Toxin target . Location . Reference .
      hok–sokE. colisok (5′ end)Hok Cell membrane Plasmid ( 4, 45)
      ldrD–rdlDE. colirdlD (5′ end)LdrD Cell membrane Chromosome ( 46)
      istR–tisBE. coliistR-1 (5′ end)TisB Cell membrane Chromosome ( 47, 48)
      symE–symRE. colisymR (5′ end)SymE mRNA Chromosome ( 49)
      ibsC–sibCE. colisibC (5′ end)IbsC Cell membrane Chromosome ( 50, 51)
      txpA–ratAB. SubtillisratA (3′ end)TxpA Cell membrane Chromosome (prophage) ( 52, 53)
      fst–RNAI–RNAII E. faecalisRNAII (3′ end)Fst Cell membrane Plasmid ( 54–58)
      srnB–srnCE. colisrnC (5′ end)SrnB Cell membrane?Plasmid ( 59)
      pndA–pndBE. colipndB (5′ end)PndA Cell membrane Plasmid ( 59)
      shoB–ohsCE. coliohsC (5′ end)ShoB Cell membrane Chromosome ( 50)
      bsrG–SR4 B. subtilisSR4 (3′ end)BsrG Cell membrane Chromosome (prophage) ( 60)

      Sometimes, the homologue of a known toxin seems to occur in the absence of a linked gene for a cognate antitoxin homologue in the genome. Likewise, an antitoxin gene homologue can be found without a linked toxin gene. Their products are called ‘solitary toxins’ and ‘solitary antitoxins’, respectively, in this review.

      Classifying RM systems

      RM systems are, confusingly enough, also classified into Type I, Type II and Type III. The use of the upper case letter T in the word ‘Type’ is recommended in a new nomenclature system for RM systems ( 62).

      Among the RM systems, a typical Type II system ( 62), such as EcoRI, carries the restriction enzyme activity and the modification enzyme activity on two separate proteins, as in some TA systems. The Type II RM systems examined show postsegregational killing (see later in the text). Therefore, in this review, we primarily limit our discussion to typical Type II RM systems, calling them simply ‘RM systems’, unless otherwise stated. These RM systems act like type IV TA systems, where the modification enzyme (antitoxin) activity prevents the cleavage of its target by the restriction enzyme (toxin).

      The Type I RM systems examined do not display postsegregational killing ( 63), but they may attack the host chromosome under specific physiological/genetic conditions ( 9, 10, 13, 64, 65), putatively at an arrested replication fork ( 13). They are composed of three subunits: S (specificity), M (modification) and R (restriction). S and M form a complex with modification activity, whereas S, M and R form a complex with restriction activity. Type III RM systems are composed of a Mod protein and a Res protein. Mod has modification activity, whereas Mod bound to Res has restriction activity ( 66).

      Type IV ‘restriction systems’ (as opposed to ‘RM systems’) consist of a methyl-specific restriction enzyme (Type IV restriction enzyme), all of which cut DNA with moderate methylated-sequence specificity. Their role in programming host death in conflicts between epigenetic DNA methylation systems ( 10) is discussed later in the text.

      A modification enzyme activity is often accompanied by its cognate restriction enzyme activity in a genome. However, a modification activity may be present in the absence of its cognate restriction activity. Such an activity is called a ‘solitary DNA methyltransferase’ in this review. These activities are biologically important, as we see later in the text. Less frequently, a restriction enzyme (a ‘solitary restriction enzyme’) may be present in a genome in the absence of its cognate modification enzyme. The distinction between a complete RM system and a solitary enzyme is not trivial because genes of a single RM system may not be linked to each other ( 67).

      Abundance and mobility

      TA and RM systems are highly abundant in the prokaryotic world ( 11, 20, 22, 68–73).

      Specifically, type II TA systems are found in many bacterial species, with no clear correlation with their lifestyles or genome sizes ( 22, 72). However, many sequenced genomes that contain from several to >60 predicted chromosomally encoded type II TA systems are slow-growing organisms that thrive under nutrient-limiting conditions, such as Mycobacterium tuberculosis ( 30, 72). The type III TA systems seem to be more common in Fusobacteria and Firmicutes, and they are slightly less common in Proteobacteria ( 24). Many RM systems are found in naturally competent bacteria, including Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningititis and Haemophilus influenzae (http://tools.neb.com/∼vincze/genomes/). In contrast, small genomes usually lack TA systems, but this could be linked to the scale of the genome and may be biologically irrelevant to the function of the TA systems ( 74). Moreover, genomes with no or very few TA or RM systems are often those of intracellular host-associated organisms, such as Mycoplasma, Buchnera, Treponema and Chlamydia. However, there are exceptions to this: Mycobacteria have many TAs, although they are intracellular organisms, whereas Rickettsia have many TA systems but no RM systems ( 18, 22, 72, 75). This trend may suggest that TA or RM systems provide little, if any, selective advantage for those bacteria limited to intracellular growth ( 72). These bacteria already appear separate from the genetic flux that is the target of RMs and TAs and that allows their acquisition.

      Type II TA systems, like Type II RM systems, seem to be widely spread by horizontal gene transfer ( 22, 72, 74, 46–79), whereas type I TA systems have evolved by lineage-specific duplication ( 20). The biological significance of this contrast is not completely understood, but there are some clues to it, as we discuss later in the text. In the following paragraphs, we discuss cases in which the domains within a gene represent units of mobility.

      RM and TA systems are often found on potentially mobile genetic elements, including genomic islands. RM systems are found on plasmids, prophages, integrons and transposons ( 68–70). The type I and II TA systems are often located on plasmids, prophages and integrons ( 70, 71, 80). Their presence on these elements can be explained by the stabilization of their maintenance by postsegregational killing ( 8). RM and TA systems are also found in apparently regular chromosomal positions.

      Although the mobility of RM and TA systems is often ascribed to their carriage by a known type of mobile genetic element, some RM systems can move unlinked to any mobile element. They themselves appear to be mobile elements. Some RM units seem to insert into the genome with a short targeted duplication, as do some of the classical DNA transposons ( 81). Like many DNA transposons, they have imperfect inverted repeats at their ends. Some other RM systems can insert into a genome with long and variable (in the order of 100 bp) target duplications ( 81, 82). Movement of RM systems within a genome is often associated with extensive genomic rearrangements ( 69, 81, 83). In some RM systems, the individual component genes appear mobile ( 81).

      Type I and Type III RM systems appear less mobile than Type II RM systems in genome comparison ( 81). However, close examination has revealed that the target-recognition domain within their genes can be a unit of mobility in the following sense ( 64, 84). In Type III RM systems, a specific amino-acid sequence in the target-recognition domain of the modification protein can move between nonorthologous proteins within a species and also beyond species barriers to spread in the bacterial world ( 84). In the specificity subunit of Type I systems, an amino-acid sequence that recognizes a specific target DNA sequence may move between nonorthologous proteins. An amino acid sequence may even move between two target-recognition domain sites within one protein ( 64). The likely underlying mechanism of this movement (‘domain movement’) is recombination at shared DNA sequences flanking the two target-recognition domains.

      Postsegregational killing: toxin versus antitoxin

      The postsegregational killing by TA systems (in the narrow sense of the word, excluding RM systems) so far examined relies on the difference in stability of the toxin and the antitoxin. (Parenthetically, some antitoxins have a significant half-life that does not place them in the category of labile proteins.) Cells that do not receive a TA plasmid during cell division are killed by the amount of toxin remaining because they lack the protection of the antitoxin ( 21, 85).

      The difference in stability between the toxin and the antitoxin in type II TA systems has been shown to result from the susceptibility of the antitoxin to proteolytic degradation. A number of antitoxins are sensitive to degradation by Lon, ClpPX or ClpPA protease. For example, HipB antitoxin is stabilized in the absence of Lon in vivo and degraded by Lon in vitro. Under normal growth conditions, HipB neutralizes HipA toxin and represses the transcription of the hipBA operon ( 41, 86, 87). However, when no new HipB is produced or Lon activity is elevated, HipB turnover results in free HipA ( 41). A chaperon may interact with the antitoxin to prevent its aggregation and protect it from degradation ( 88). What starts the whole degradation cascade of the antitoxin protein is not at all clear in any of the TA systems.

      In contrast, postsegregational killing by Type II RM systems is expected to operate with no difference in stability between the modification enzyme and the restriction enzyme. After loss of the RM genes, the modification enzyme is diluted by cell division, leading to the exposure of unmethylated recognition sites on newly replicated chromosomes. This will result in DNA cleavage by the restriction enzyme activity remaining and cell death ( 89). The R and M proteins of the EcoRI system are similar in their metabolic stability ( 90). However, there may be selection for the instability of the modification enzyme to ensure stronger postsegregational killing. (Here, the unit of selection is the RM system, as opposed to the entire genome.) A mutation in the M protein of EcoRII makes this protein unstable and enhances the postsegregational killing by this RM system ( 91).

      Actions of toxins

      Examples of toxins are listed in Tables 1 and 2. Their classification into superfamilies has been proposed ( 22). It must be remembered that the overproduction of a toxin will not always result in cell death, but may lead instead to cell stasis. For example, cells with elevated RelE or MazF toxin remain viable but in stasis, and can be rescued by the subsequent induction of the cognate antitoxin ( 92).

      In most type II TA systems, the toxin inhibits translation ( 61) ( 31) ( 93). Some of these toxins are highly potent endoribonucleases that cleave cellular mRNA at specific sequences. Therefore, they are called mRNA interferases ( 94, 95). Some toxins of type II TA systems affect DNA replication by blocking DNA gyrase ( 33–36, 96).

      In the type II TA system epsilon–zeta, the zeta toxin is an ATP-dependent kinase that inhibits peptidoglycan synthesis. It phosphorylates uridine diphosphate-N-acetylglucosamine, a peptidoglycan precursor, so that phosphoenolpyruvate cannot be added subsequently. The resulting phosphorylated form also inhibits this addition ( 37–39). HipA toxin is a phosphatidylinositol/protein kinase and shows serine kinase activity that autophosphorylates it in vitro and in vivo ( 42, 43). When the expression of the toxin is elevated, a phosphorylation signal is transduced and cell-wall synthesis is impaired, which leads to cell lysis. Its pleiotropic effects include the inhibition of DNA replication, transcription and translation ( 40, 97). The LetA–LetS system and related TA systems carry serine-protease-like toxins and AAA-ATPase-like antitoxins ( 98).

      In contrast, most of the toxins of the type I and type V TA systems, such as TisB and GhoT, target the inner membrane ( 26, 47, 99–101) ( Table 2). They inhibit ATP synthesis by depleting the proton motive force, leading to dramatic RNA decay, thus halting protein synthesis ( 47). However, SymE toxin is thought to be a ribonuclease ( 49). The Ldr toxin (type I) contributes to nucleoid condensation ( 46) the Fst toxin (type I) targets the cell membrane, but at lower levels, it also affects chromosomal segregation and cell division ( 102). A type IV toxin, CbtA, binds to cytoskeletal proteins, MreB and FtsZ, and inhibits their polymerization, resulting in the loss of cell shape and polarity, incorrect cell division and finally death ( 25).

      Some TA systems induce the SOS response, a stress response triggered by DNA damage with RecA and LexA regulators. The actions of the type II TA systems DinJ–YafQ, YafN–YafO and ParE homologues ( 103–105) and of the type I TA systems (symE–symR, tisB–istR1) are, in turn, affected by the SOS response ( 47, 49, 48, 106).

      All toxins of the Type II RM systems (Type II restriction enzymes) so far examined are highly sequence-specific DNA endonucleases ( 78). Recent studies have shown that in some cases, cleavage of DNA–RNA hybrids can be achieved in vitro ( 107), although its significance in vivo remains unclear.

      The induction of the SOS response during postsegregational killing by Type II RM systems ( 103) and during unbalanced RM activities ( 104) is consistent with the fact that bacterial cells die when their genomes are cleaved. Cells form filaments and division is prevented. The RecBCD/RecA machinery can repair DNA damage to some extent to allow survival ( 108).

      Action of protein antitoxins

      More labile antitoxins must hold more stable toxins in check. In type II TA systems, such as Kid–Kis and MazF–MazE, the direct binding of the antitoxin to specific domains of the protein toxin, forming an oligomeric complex, inhibits the toxin activity ( 109). A toxin may have multiple antitoxin-binding domains with different binding affinities. Therefore, multiple protein complexes with different T and A stoichiometries are possible, such as T2:A2 and T2:A2:T2 ( 109, 110).

      In contrast, the type IV antitoxin protein YeeU (CbeA) does not form a complex with the toxin CbtA. Instead, YeeU binds directly to the targets of the cognate toxin, the cell filament-producing proteins MreB and FtsZ. YeeU binding stabilizes protein bundling and helps their polymerization into filaments. This process is inhibited by the CbtA toxin in the absence of antitoxin ( 25). This antitoxin action is somewhat similar to the action of the antitoxin modification enzyme in RM systems (of Type II), although the latter protects the target (DNA) by chemical modification (methylation).

      The antitoxin of one type V TA system, GhoS, is a sequence-specific endoribonuclease that cleaves the mRNA of its cognate toxin, GhoT, preventing its translation ( 26). In this activity, the GhoS antitoxin resembles the toxins of many type II TA systems, which are the mRNA interferases ( 94).

      Effects of antitoxins on global gene expression

      The effects of antitoxins on their own TA or RM systems will be discussed in later sections. Here, we discuss their effects on global gene expression.

      The antitoxin of the Escherichia coli MqsR–MqsA TA system directly represses the transcription of the gene encoding RpoS, the stationary-phase sigma factor and the master stress regulator ( 111). Furthermore, the degradation of the antitoxin during stress leads to a switch from the high-motility state to the low-motility state (leading to biofilm formation) ( 111). Similarly, the antitoxin DinJ of the YafQ–DinJ TA complex in E. coli reduces RpoS levels by an indirect mechanism ( 112). DinJ represses the cold-shock protein CspE, which boosts the translation of rpoS mRNA ( 112, 113).

      These TA systems are similar to the prototype type V TA system ghoS–ghoT and may be regarded as type V. Their action is similar to the action of RNA antitoxins of the type I TA systems, as described later in the text.

      RM systems affect the global gene expression of a genome ( 12). Each of the multiple DNA methyltransferases methylates many copies of a specific recognition sequence in the genome and they together define a specific methylome (or a series of related methylomes). Each of these methylation events may affect nearby gene expression. Overall, they may define a specific transcriptome/proteome.

      Methylation by Type III RM systems controls the expression of a group of genes (‘phasevarion’). Differential methylation places certain genes in an ON or OFF state, effectively generating two distinct cell types with two distinct phenotypes ( 114). Phasevarions are associated with lateral gene transfer, heat shock protein production, virulence factors, motility and colonization in Neisseria, Helicobacter and other pathogenic bacteria ( 114, 115).

      Solitary DNA methyltransferases have been studied extensively with respect to global gene expression ( 116–118). DNA adenine methylation by Dam (5′(-GmATC) affects the expression of several genes (99116–101118), and is required for the virulence of Salmonella, Haemophilus, Yersinia, Vibrio and pathogenic E. coli ( 118). It also acts to coordinate DNA replication and the cell cycle and for template-strand choice during DNA mismatch repair. The M.CcrII DNA methyltransferase [5′-GmANTC (N = A, C, G, or T)] regulates the cell cycle of Caulobacter crescentus ( 119). Methylation by Dcm (5′-Cm5CWGG [W = A or T]) affects the expression of genes in the stationary phase of E. coli. Its Dcm-defective mutants show increased expression of the stress response sigma factor, RpoS and many of its targets in the stationary phase ( 120).

      The transcriptome changes that occur during postsegregational killing by Type II RM systems were analyzed in E. coli ( 12). The induction of SOS genes and the RpoE regulon was followed by the induction of stress-response genes (including the RpoS regulon, and osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes and many hitherto uncharacterized genes. Death was accompanied by cell lysis and the release of cellular proteins. Some signal seemed to be transduced from the damaged genome to the cell surface, leading to its disintegration. These transcriptomal changes partly parallel the changes that occur in cells treated with bacteriocidal antibiotics. The RM systems and the bacteriocidal antibiotics may activate a single death program ( 12). We are not aware of any comparable transcriptome analysis of TA-mediated cell death.

      Defense against bacteriophages

      RM systems may block the entry of DNA from a lineage with a different improper epigenetic DNA methylation status. They inhibit bacteriophage infection if the bacteriophage is from such a lineage, whereas they do not, if it is from a lineage of the same epigenetic status. However, if the phage DNA of a different epigenetic status survives the attack by the restriction enzyme through recombination repair ( 121) or some other process (a phenomenon called ‘escape’), the phage DNA will be modified in the same way as the host DNA. The resulting phage and its progeny carrying the modified DNA can easily infect cells with the RM system. Once an epidemic starts within a bacterial population, the RM system is no longer effective against infection.

      Type IV restriction enzymes show coevolution, of the arms-race type, with bacteriophage DNA modification systems ( 122). However, cells are also equipped with other phage defense mechanisms that can be more effective, such as the acquired RNA-based immune system (‘clustered regularly interspaced short palindromic repeat’) ( 123) and phage exclusion (abortive infection) ( 124, 125). Phage exclusion is a system of altruistic death that protects the cell against infection: an infected cell promotes its own death to abort phage reproduction, thus preventing its spread within the population. Type IV restriction enzymes (methyl-specific restriction enzymes) may abort phage infection when the phage brings in a new DNA methylation system and starts methylating the host chromosome ( 125).

      The link between TA systems and phage resistance has not yet been explored thoroughly. Phage infection, in most cases, shuts off host gene expression, including that of the TA systems, which favors the activity of more stable toxins, as in postsegregational killing. In this context, TA systems might be considered antiphage (abortive) factors ( 126). [Although the modification enzyme is prone to proteolysis in some Type II RM systems ( 91), whether this leads to the restriction of the host chromosome after phage infection is unknown.]

      Few direct examples of this activity have so far been reported. One elegant study of a type III TA system suggests a mechanism of wide multiphage resistance that functions as an abortive infection system ( 24). The toxin protein ToxN, a member of the CcdB/MazF superfamily, induces reversible growth inhibition ( 23). Interestingly, its cognate antitoxin, ToxI, is the neutralizing RNA for the ToxN toxin, but it does not act as an antisense RNA interacting with the toxin mRNA. Rather, ToxI directly inhibits ToxN or outcompetes ToxN for certain cellular targets ( 23, 44).

      Other TA systems examined reduce infection by a single group of phages, such as the activity of the type II TA system MazE–MazF against P1 phage ( 2) and the type I TA system hok–sok against T4 phage ( 127). Overall, the importance of TA systems in the defense against bacteriophages still requires investigation.

      Selective advantage

      The biological roles or selective advantage of programmed death systems, such as phage exclusion, become clear only when we focus on the population level and on individual genes and genetic elements, rather than on the level of individual lineages or the entire genome. A property beneficial to a gene or a set of genes may not be as profitable to the entire genome. It is especially clear that genes are the units of selection when they are mobile with respect to the genome.

      An experimental/theoretical work on phage exclusion demonstrated that within the context of a spatial structure (as in a solid medium), cells that practice a suicide strategy win in competition with cells without such a strategy. However, the suicide strategy fails in the absence of a spatial structure (as in a well-mixed liquid culture) ( 125).

      In postsegregational killing, TA systems (and Type II RM systems) program the death of cells that have lost their genes. Postsegregational killing systems on plasmids may have been selected because they benefit the plasmids in environments in which multiple plasmids must compete during horizontal transfer and reproduction ( 3). Mochizuki et al. ( 128) analyzed the population dynamics of plasmids with analytical methods and computer simulations based on the methods of theoretical ecology. A genetic element (such as a plasmid) with a TA module has an advantage over a competitor genetic element (such as an incompatible plasmid) without a TA module. However, the advantage is limited in a population without a spatial structure. In contrast, in a structured habitat, the TA gene complex can increase in frequency, irrespective of its initial density. Several experiments have addressed the competitive advantage of TA systems for plasmids, but they have been undertaken in the absence of a spatial structure ( 129, 130).

      The postsegregational killing process probably occurs in chromosomal genes because chromosomal RM systems are resistant to replacement by an allelic DNA (lacking the restriction site) ( 131, 132) through homologous recombination. This process should be important because a chromosomal allele is frequently replaced by an allele transferred from another lineage in a bacterial species ( 133). The arguments presented earlier in the text on the advantages of postsegregational killing also apply to the competition between the alleles at any chromosomal locus, which occurs through homologous recombination.

      The chromosomally encoded TA units include those on superintegrons, which are involved in the stable maintenance of the superintegrons by minimizing the formation of superintegron-free cells ( 6, 7). In a structured habitat, postsegregational killing may provide an advantage in the competition between two integrons or between an integron and an integron-free allele.

      It has been suggested that many chromosomal type II TA systems function in the adjustment of gene expression in response to stress, to maintain overall bacterial fitness ( 72). This concept is not exclusive to the idea discussed earlier in the text that genes that program death confer a competitive advantage. The stress response and death process may form a continuous spectrum, with death as the final resolution. In support of the stress response hypothesis, some type II TA systems in M. tuberculosis are induced by hypoxia or macrophage infection, indicating their ability to adapt to stressful conditions ( 30).

      Researchers have analyzed the distinct phenotypes of E. coli strains from which all TA systems have been removed. In one study, the deletion of five TA systems in the E. coli K-12 strain generated no difference from the wild-type strain when exposed to stress in competitive experiments, and no correlation between the TA systems and greater bacterial fitness was observed ( 5). In other studies, removing the type II TA systems reduced the number of bacterial persisters when bacteria entered a state characterized by a high tolerance of antibiotics ( 48) and/or significant growth defects ( 63). The persister phenotype is not associated with a single mutation, although in selections from knockout libraries, a reduced persister frequency was associated with defects in a number of global regulators ( 47).

      In contrast, the mild overproduction of certain TA system toxins can make cells more tolerant of multiple antibiotics ( 43, 1, 135). Interestingly, in the case of the HipB–HipA system, the combination of two specific mutations within the hipA gene (hipA7 allele) produces a protein that is inactive as a toxin but confers a high-persistence phenotype on E. coli. The dormant state of such persistent cells depends on the increased synthesis of (p)ppGpp ( 136, 137).

      The abundance of TA systems has also been linked to a high level of virulence in bacteria with small genomes, although no direct evidence has been obtained supporting the idea that TA systems are responsible for the expression of any specific virulence factors ( 138, 139). A type II TA system carried by a uropathogenic E. coli strain affects its colonization of the bladder and its survival within the kidney, where it resists nutrient limitation and oxidative and nitrosative stress ( 140). A possible role of the type II solitary toxin MazF–Mx in the multicellular development of Myxococcus has also been examined ( 141, 142).

      As discussed earlier in the text, some RM systems, as well as solitary DNA methyltransferases, have specific effects on the transcriptome ( 12, 143). Changes in the sequence specificity of RM systems ( 64, 84) and the resulting changes in the methylome may alter global gene expression. Natural selection of the diverse resulting epigenomes may underlie adaptive evolution ( 17).

      Interactions between multiple TA (or RM) systems

      In general, interference between multiple TA systems in postsegregational killing and in other contexts may occur when (i) the system components, toxins, antitoxins or regulatory elements are similar and/or (ii) the system components have the same target ( 10, 68). The interaction between two or more TA systems has not been explored thoroughly until recently, even though many organisms encode several TA systems in their genomes, and some cross-talk between TA system components is expected ( 35, 109). The recent classification of type II TA systems (see ‘Classifying TA systems’ section earlier in the text) is based on their specific functional capacity to associate with their TA partner and cross-neutralize it ( 22, 61).

      Such interference between RM systems is easily recognized because RM systems target specific DNA sequences and can affect cell survival and death in a dramatic way. Indeed, there is clear evidence for this interference, as detailed later in the text.

      Two RM systems with the same target sequence cannot ensure their maintenance by postsegregational killing because the loss of one RM system does not lead to the exposure of the target DNA sites, which are protected by methylation by the other RM system. Therefore, in the presence of another RM system, an RM system can be lost or inactivated without cell killing ( 144). This means that recognition sequence of an RM system defines an incompatibility group.

      An interaction can be asymmetric when the relationship between the recognition sequences is inclusive. For example, if an RM system recognizes 5′-CCWGG (W = A or T) and another recognizes 5′-CCNGG (N = A, C, G, or T), the loss of the former RM system does not lead to chromosomal cleavage because the latter system protects the sites of the former system. However, the loss of the latter RM system does lead to chromosomal cleavage because the former RM system cannot protect 5′-CCSGG (S = G or C) ( 145). In a similar way, a solitary antitoxin (modification enzyme) can attenuate host killing after the loss of an RM system with the same recognition sequence, as demonstrated for the Dcm methyltransferase and the EcoRII systems, which both recognize 5′-CCWGG (W = A or T) ( 146).

      In general, chromosomally encoded TA units and RM systems probably provide such ‘immunity’ to host killing following the loss of TA and RM plasmids ( 131). Chromosomally encoded TA systems may have evolved because of such antiaddiction or vaccination effects against addictive genetic elements. These TA systems on bacterial chromosomes could, in turn, have driven the evolution of plasmid-encoded TA systems, selecting for toxins that are no longer recognized by the antiaddiction module ( 147).

      The physical interaction between the toxin of one type II TA system and the antitoxin of another has been shown to occur between Ccd and ParD ( 148). Interestingly, these toxins act on different targets (one inhibits DNA gyrase and the other inhibits translation, as an endoribonuclease) and have distinct protein structures. However, the proteins share sequence similarities in certain functional modules. The TA cross-interaction leads to toxin cross-neutralization.

      Unexpectedly, the nature of the interaction does not follow the pattern seen in the toxin and antitoxin within the same TA system ( 148). This suggests that the toxin neutralization mechanism is relatively broad and might even occur between homologous TA systems in a single genome (paralogous TA systems) that have limited similarity.

      Several studies have demonstrated cross-activity between TA systems in postsegregational killing ( 35, 147). When the network of interactions overlaps, some TA units may control the activity of other units, as in the type II TA MqsR–MqsA system, which affects the type V TA system, GhoT–GhoS ( 149). The MqsR toxin, an endoribonuclease, degrades the ghoS mRNA by cleavage at 5′-GCU sites, and the reduced GhoS antitoxin cannot stop the expression of the GhoT toxin ( 149). In a sense, one TA system may interfere with the action of another TA unit, as long as it finds its specific target on the subject TA.

      Recent work has identified a solitary antitoxin, Dmd, encoded by bacteriophage T4. This unusual phage protein acts against antiviral TA systems of the host bacterium. As aforementioned, during T4 infection, the host genomic transcription is shut off, and the host antitoxins are quickly degraded, which leaves the toxins to kill the host and abort phage propagation. Dmd neutralizes the toxin activity of two types of TA systems (RnlA–RnlB and LsoA–LsoB) ( 126, 150). Dmd binds to the ribonuclease toxins, RlnA and LsoA, in place of their cognate antitoxins, RlnB and LsoB, even though it has no sequence similarity to these antitoxins. This association allows the phage to continue its replicative cycle.

      The excess toxin of one TA system may trigger a positive feedback loop of transcriptional activation of another TA unit. Such a cascade of interactions may cause bistability in growth and hence population heterogeneity ( 151). When two RM systems are similar in the mechanisms by which they regulate gene expression, their interaction can activate the toxin to kill cells, as detailed later in the text ( 152).


      EXPERIMENTAL PROCEDURES

      Expected Background Skills

      Students will need to be highly proficient in micropipettor use accurate measurement of small volumes will increase the likelihood of generating quality results. Alternatively, students may isolate DNA and add it to reactions that are prepared by the instructor. A background in restriction digests, agarose gel electrophoresis, and gel analysis would be useful, but it is not absolutely required.

      Web Sites

      An overview of the principles of the ABO blood group is present in the National Center for Biotechnology Information (NCBI) web site: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=rbcantigen.chapter.ch05ABO. The genetic, biochemical, and immunological aspects of the ABO phenotypes are outlined. There is also a comprehensive entry in the Online Mendelian Inheritance in Men website: http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=110300. NCBI also gives information about the value and utility of SNPs at http://www.ncbi.nlm.nih.gov/About/primer/snps.html. In addition, a database of human SNPs can be accessed through this web site. A PCR animation that requires Adobe Shockwave is maintained at the Dolan DNA Learning Center: http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html. A discussion of personalized medicine and pharmacogenomics is available from the Mayo Clinic: http://www.mayoclinic.com/health/personalized-medicine/CA00078.

      Materials

      Laboratory chemicals were purchased from Research Organics (Cleveland, OH), Fisher (Pittsburgh, PA), and VWR (West Chester, PA). Oragene DNA isolation kits were kindly provided by Genotek (Ottawa, Ontario, Canada). Taq DNA polymerase was purchased from USB (Cleveland, OH), whereas dNTPs, λ BstE II marker, and restriction enzymes were purchased from Promega Corporation (Madison, WI). Primers used in PCR were purchased from Operon Biotechnologies (Huntsville, AL). Agarose and pUC18 Hae III marker were acquired from Amresco (Solon, OH).

      Equipment and Software

      This exercise used the following equipments and software: microcentrifuge models 5415C and 5415D (Eppendorf, Hamburg, Germany), Bio-Rad DNA Engine PTC200 (Hercules, CA), BioMax QS710 and MP1015 gel electrophoresis systems, DC290 camera, and 1D version 3.6 gel analysis software (Kodak, Rochester, NY), Model 300 power supplies (VWR), and EB-15 UV transilluminator (Ultra-Lum, Carlsbad, CA).

      Safety Issues

      Permission to conduct this research was approved by the John Carroll University Institutional Review Board. Instructors should contact their university committee that deals with human studies for any special regulations. Collection of DNA from cells present in saliva is noninvasive. However, students should wear gloves whenever intact fluids are present. They should wash their hands and arms up to the elbow prior to leaving lab. Beakers containing 10% bleach should be used to decontaminate tips and tubes prior to disposal as biohazardous waste.

      Ethidium bromide is a known carcinogen, and it should be handled with care. Your institution may have special guidelines to dispose of ethidium bromide waste. Ethidium bromide decontamination protocols can be found in Sambrook et al. [ 7 ]. We disposed of the gels in a solid waste container, and the contaminated buffer is stirred with a destaining bag from Amresco. The destaining bag binds ethidium bromide from the solution subsequently, decontaminated buffer can be washed down the sink. The destaining bag is discarded in the solid waste container with gels. Finally, UV-light exposure should be minimized when observing the gels that are stained with ethidium bromide. UV-safe goggles or a UV shield should always be used.

      Scheduling

      Isolation of Genomic DNA

      For isolating genomic DNA, 2–2.5 hours were needed, with 1 hour open during the initial incubation at 50°C.

      Gel Electrophoresis of Genomic DNA

      Two hours were needed, for gel preparation, electrophoresis, and photography, with 20–30 minutes open during electrophoresis. Alternatively, gels may be prepared by the instructor prior to class. Students can prepare samples and reduce the necessary time to 1–1.5 hours.

      For PCR 1 hour was needed, followed by a 2.5–3-hour reaction that can be held overnight at 4°C in most PCR machines. The PCR reactions can be done on the same day as gel electrophoresis of the genomic DNA to save time.

      Gel Electrophoresis of PCR Products

      For gel electrophoresis of PCR products, 2 hours were needed, for gel preparation, electrophoresis, and photography, with 20–30 minutes open during electrophoresis.

      Restriction Digests

      Thirty minutes were needed to prepare the digests and 2 hours for incubation.

      Gel Electrophoresis of Restriction Digests

      Two hours were needed for gel preparation, electrophoresis, and photography, with 20–30 minutes open during electrophoresis. The 4% agarose (3:1 High Resolution Blend from Amresco)-1× TBE gels are prepared prior to the lab, and students load these gels immediately, as these have a longer running time than the 1% agarose-1× TBE gels that possess the exon 6 digests. Multiple groups can run their samples on the same gel to conserve materials.

      DNA Isolation

      Open an Oragene vial and spit into it until the level of saliva reaches the 4-mL line on the side of the vial.

      Incubate the sample in the Oragene vial at 50°C for 1 hour.

      Transfer 500 μL of sample into a 1.5-mL microcentrifuge tube. Label this sample with the sample number that is provided by the instructor. Store the remaining sample at room temperature.

      Add 20 μL of Oragene Purifier and mix by inverting four times. The sample will become turbid.

      Incubate on ice for 10 minutes.

      Centrifuge at top speed (16,000 × g) for 3 minutes at room temperature.

      Remove supernatant using a pipettor (without disturbing the pellet) and place in a new microcentrifuge tube.

      Add 500 μL of room temperature 95% ethanol to the supernatant and mix by inverting at least five times.

      Incubate for 10 minutes at room temperature.

      Centrifuge for 1 minute at top speed at room temperature place the hinge of the microcentrifuge tube facing up. The pellet is not always visible at this point, but the DNA will be pelleted at the bottom under the hinge. Remove the supernatant by pipetting while being careful not to disturb the pellet. If residual ethanol remains, centrifuge again for 10 seconds and remove the remaining ethanol.

      Place tube upside down on a Kimwipe to dry for 5 minutes.

      Add 100 μL of 1× TE (10 mM Tris-HCl, 1 mM EDTA (pH 8)) to dissolve the pellet. This volume will typically yield genomic DNA that is 50–100 ng/μL. EDTA will chelate divalent cations such as Mg 2+ that are required by DNA-degrading enzymes.

      Vortex the sample for 30 seconds and incubate overnight at room temperature. Alternatively, samples may be incubated at 50°C for 1 hour.

      Store the genomic DNA at 4°C. Freezing and thawing the genomic DNA samples can result in DNA shearing.

      Determination of Genomic DNA Concentration

      Prepare a 1% agarose/1× TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA (pH 8)) gel that contains 0.5 μg/mL ethidium bromide.

      Add 2 μL of genomic DNA, 8 μL of sterile water, and 2 μL of 6× loading dye to a microcentrifuge tube.

      Thaw a tube of “uncut λ DNA.” This tube contains 50 ng/μL uncut λ DNA.

      Make up the following three tubes:

      Load the gel with the three lambda dilutions (50, 150, and 300 ng) followed by the student DNA samples.

      Run the gel at a constant voltage of 10 V/cm for 45 minutes. Photograph as usual.

      Estimate the concentration of the genomic DNA by comparison to the known amounts in the lambda dilution lanes. Alternatively, comparison of genomic DNA intensity to a molecular weight standard can be performed to estimate the concentration (see later).

      Each two-person group will perform four PCR reactions for both exon 6 and 7 (eight total reactions). The four reactions for each exon contain genomic DNA from each student in the group, an unknown DNA that is provided by the instructor, or water to act as a negative control.

      Amplification of exon 6 is performed using the following conditions: 35 cycles at 94°C for 30 seconds, 53°C for 30 seconds, and 72°C for 60 seconds, followed by a 5-minute extension at 72°C and a post-dwell at 4°C. Exon 7 is amplified using the same conditions as exon 6, except the annealing temperature is 60°C instead of 53°C. Each PCR utilizes a 2-minute incubation at 94°C to ensure that DNA denatures prior to amplification.

      Exon 6-1: 5′-GGGCTGGGAATGATTTG-3′

      Exon 6-2: 5′-GGTGTCCCCCTCCTGCTATC-3′

      Exon 7-3: 5′-CCCCGTCCGCCTGCCTTGCAG-3′

      Exon 7-4: 5′-GGGCCTAGGCTTCAGTTACTC-3′

      Add 5 μL of template DNA or water (no DNA control) to labeled PCR tubes.

      Prepare the exon 6 premix as follows: 153.75 μL of sterile water, 25 μL of 10× Taq DNA polymerase buffer (without MgCl2), 15 μL of 25 mM MgCl2, 12.5 μL of 10 pmol/μL primer exon 6-1, 12.5 μL of 10 pmol/μL primer exon 6-2, 5 μL of 10 mM dNTPs, and 1.25 μL of Taq DNA polymerase (USB, 5 U/μL). Prepare the exon 7 premix this premix is identical to the exon 6 premix, except primers exon 7-3 and exon 7-4 are used in place of exon 6-1 and exon 6-2. Gently mix both premixes.

      Add 45 μL of exon 6 premix to the proper tubes and place the reactions in the thermocycler.

      Add 45 μL of exon 7 premix to the proper tubes and place the reactions in the thermocycler.

      Gel Electrophoresis of PCR Products

      Prepare a 1% agarose-1× TBE gel that contains 0.5 μg/mL ethidium bromide.

      While the gel is solidifying, students can prepare their PCR products for loading. Remove 10 μL of each PCR reaction and place in labeled microcentrifuge tubes.

      Add 2 μL of 6× loading dye (0.4% bromophenol blue, 30% glycerol) to each microcentrifuge tube.

      Prepare lambda BstE II and pUC18 Hae III markers (1 μL of marker, 9 μL of 1× TE, and 2 μL of 6× loading dye).

      Load the entire 12 μL for each sample and record the order.

      Run the gel at 10 V/cm for ∼40 minutes. Gels are visualized by exposure to UV light and photographed.

      Estimate the concentration of DNA produced by each of the PCR reactions of exons 6 and 7.

      This estimation is done by comparison of the PCR product to the pUC18 Hae III or lambda BstE II molecular weight marker (500 ng each). The 587-bp band of pUC18 Hae III represents ∼100 ng of DNA, whereas the 2,323-bp fragment in the lambda BstE II digest is ∼25 ng. If the PCR product and the 587-bp fragment are of approximately equal intensities, then we have 100 ng of PCR product. To determine the concentration of DNA in the PCR reactions, divide the mass (in ng) by the volume of DNA that was loaded (10 μL).

      Restriction Digests

      A premix for each restriction digest is prepped (one set of three for each lab group). For the Kpn I digest of exon 6, add 6 μL of 10× Promega buffer J, 6 μL of 10× BSA, and 2 μL Kpn I (10 U/μL) to a microcentrifuge tube. For the BstE II digest of exon 6, add 6 μL of 10× Promega buffer D, 6 μL of 10× BSA, and 2 μL BstE II (10 U/μL) to a microcentrifuge tube. For the Hpa II digest of exon 7, add 6 μL of 10× Promega buffer A, 6 μL of 10× BSA, and 2 μL Hpa II (10 U/μL) to a microcentrifuge tube. Mix all the premixes by tapping the tubes briefly centrifuge if necessary.

      Aliquot 3.5 μL of premix into three labeled microcentrifuge tubes.

      Add 11.5 μL of the appropriate PCR product. Exon 6 products are added into the tubes containing Kpn I and BstE II premixes, whereas exon 7 products are added to the tube containing Hpa II premix.

      Briefly centrifuge the reaction mixtures.

      Place reactions exon 6-Kpn I and exon 7-Hpa II at 37°C for 2 hours.

      Place reaction exon 6-BstE II at 60°C for 2 hours. This reaction should be briefly centrifuged every 30–45 minutes, as evaporation and condensation will occur under the cap.

      Once completed, place all reactions at −20°C.

      Gel Electrophoresis of Digests

      A 4% agarose-1× TBE gel has been poured prior to class. Two groups will share each gel one group will take the four lanes on the left and the other group will take the four lanes on the right.

      4% Agarose Gel for Exon 7 Hpa II Digests

      Add 3 μL of 6× loading dye to each digest from last lab meeting. Briefly centrifuge.

      Prepare a sample of pUC18 Hae III as usual (1 μL of DNA, 9 μL of 1× TE, and 2 μL of loading dye).

      Once both groups are ready to load the gel, begin loading. Load the entire 18 μL into a single well.

      Run the gel at a constant voltage of 9.5 V/cm and photograph as usual. The bromophenol blue tracking dye should travel 7 cm into the gel in order to resolve the 223- and 204-bp bands and also the 145- and 137-bp bands. There is no xylene cyanol in the loading dye, as it can obscure low-intensity bands.

      1% Agarose-1× TBE Gel for Exon 6 Digests

      Prepare a 1% agarose-1× TBE gel that contains 0.5 μg/mL ethidium bromide.

      Prepare an uncut sample of exon 6 PCR product. Add 5 μL of DNA from the exon 6 PCR reactions, 10 μL of sterile water, and 3 μL of 6× loading dye to a microcentrifuge tube. Briefly centrifuge.

      Add 3 μL of 6× loading dye to each of the exon 6 digests and briefly centrifuge.

      Prepare a sample of pUC18 Hae III and lambda BstE II as usual (1 μL of DNA, 9 μL of 1× TE, and 2 μL of loading dye).

      Load the gel making sure to position the uncut and digested PCR products for a given sample in three consecutive lanes for ease of analysis.

      Run the gel at 10 V/cm for 40 minutes and photograph the gel.


      Use in Biotechnology

      Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals. This is referred to as restriction fragment length polymorphism (RFLP). They're also used for gene cloning.

      RFLP techniques have been used to determine that individuals or groups of individuals have distinctive differences in gene sequences and restriction cleavage patterns in certain areas of the genome. Knowledge of these unique areas is the basis for DNA fingerprinting. Each of these methods depends on the use of agarose gel electrophoresis for the separation of the DNA fragments. TBE buffer, which is made up of Tris base, boric acid, and EDTA, is commonly used for agarose gel electrophoresis to examine DNA products.


      Assignments

      1. Read the following background information on restriction enzymes found at: SNP Restriction Enzymes and on gel electrophoresis at: Gel Electrophoresis.
      2. Read background information on SNPs in your textbook, pp. 678-684.
      3. Read the Abstract and Introduction of the following article, Wooding et al., Natural Selection and Molecular Evolution in PTC, a Bitter-Taste Receptor Gene. Am. J. Hum. Genet. 74:637–646, 2004. You should bring a hard copy of this article to lab next time.
      4. Read the following background information on ethical issues in Direct To Consumer genetic testing:

      PUBLIC HEALTH: A Case Study of Personalized Medicine by S. H. Katsanis, G. Javitt, and K. Hudson,published in Science, 4 April 2008 320: 53-54 [DOI: 10.1126/science.1156604] (in Policy Forum) found at: (http://0-www.sciencemag.org.luna.wellesley.edu/cgi/reprint/320/5872/53.pdf)

      The Answers to the following questions will be turned in at the beginning of Lab 7. You may download a .doc file of these questions here Media:Taster_DTC_questions110.doc‎ to use as a template for your answers.

      1. Think about the results you expect from next week's electrophoretic separation. After considering all you now know about PTC gene polymorphisms, draw the expected migration locations of the Fnu4H1 digested PCR product obtained from a homozygous taster PAV/PAV(left lane), a homozygous nontaster AVI/AVI (center lane) and a heterozygote PAV/AVI(right lane) on a diagram of an agarose gel (template shown below).


      2. Answer the following questions:

      a. Define the following terms:
      DTC genetic testing
      analytical validity
      clinical validity
      clinical utility
      pharmacogenetics

      b. What is the American Medical Association's position on DTC?

      c. Summarize the American College of Medical Genetics recommendations on DTC genetic testing.

      d. List 2 potential benefits and 2 potential risks/concerns of DTC genetic testing.

      e. Analytically valid tests are available for variants in the CYP450 genes. Are these tests clinically valid? Do they have clinical utility?

      3. Pick a disease for which one of the DTC companies on this list offers assessment of genetic risk. See if you can find outside verification that there is analytical validity, clinical validity or neither for this test from this service. Summarize your findings.