Information

Self pairing in DNA


I know that ssRNA molecule can fold over itself (e.g. in t-RNA). Can DNA do the same? Is there any example of this in nature?

Why is this phenomenon more common in RNA than in DNA?


DNA can adopt secondary structures like RNA, the main difference is that DNA is usually present as double-stranded DNA while RNA is in most cases present as single-stranded RNA. Double-stranded DNA won't easily adopt any other conformation than the well-known double helix as this one is more stable than possible structures each single strand could adopt on its own.

One example that occurs in nature are G-Quadruplexes which for example occur in telomeres.

There are also artificially created DNA enzymes (also called DNAzymes or deoxyribozymes) that adopt tertiary structures like ribozymes do. But there are no known DNA enzymes that occur in nature as far as I know.


Self pairing in DNA - Biology

Distinguish between unique or single-copy genes and highly repetitive sequences in nuclear DNA.

Highly repetitive sequences (satellite DNA) constitutes 5–45% of the genome. The sequences are typically between 5 and 300 base pairs per repeat, and may be duplicated as many as 10 5 times per genome.

TOK: Highly repetitive sequences were once classified as “junk DNA”, showing a degree of confidence that it had no role. This addresses the question: To what extent do the labels and categories used in the pursuit of knowledge affect the knowledge we obtain?

State that eukaryotic genes can contain exons and introns.

Eukaryotic genes contain coding fragments known as exons and non-coding fragments known as introns. Exons are sequences of bases that are transcribed and translated, and introns are sequences that are transcribed but not translated.

Cite all sources using the CSE method (or ISO 690 Numerical in Word). Highlight all objective 1 command terms in yellow and complete these before class. Highlight all objective 2 and 3 command terms in green – these will be part of the discussions in class. After class, go back and review them.

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Abstract

While the solution assembly of amphiphilic copolymers has been studied extensively, the assembly of DNA-containing copolymers is only recently emerging as a promising new area. DNA, a natural hydrophilic biopolymer that is highly predictable in its hybridization characteristics, brings to the field several useful and unique properties including monodispersity, the ability to functionalize in a site-specific manner, and programmability with Watson–Crick base pairing. The inclusion of DNA as a segment in the copolymer not only adds to the current knowledge base in the block copolymer assembly but also creates new modalities of assembly that have already led to novel and technologically useful structures. In this Review, we discuss recent progress in the self-assembly of DNA-containing copolymers, including assemblies driven by hydrophobicity via amphiphilic constructs, programmed assemblies mediated by DNA hybridization, and assemblies involving both of these interactions.


Abstract

Some proteins have the property of self-assembly, known to be an important mechanism in constructing supramolecular architectures for cellular functions. However, as yet, the ability of double-stranded (ds) DNA molecules to self-assemble has not been established. Here we report that dsDNA molecules also have a property of self-assembly in aqueous solutions containing physiological concentrations of Mg 2+ . We show that DNA molecules preferentially interact with molecules with an identical sequence and length even in a solution composed of heterogeneous DNA species. Curved DNA and DNA with an unusual conformation and property also exhibit this phenomenon, indicating that it is not specific to usual B-form DNA. Atomic force microscopy (AFM) directly reveals the assembled DNA molecules formed at concentrations of 10 nM but rarely at 1 nM. The self-assembly is concentration-dependent. We suggest that the attractive force causing DNA self-assembly may function in biological processes such as folding of repetitive DNA, recombination between homologous sequences, and synapsis in meiosis.

This study was supported in part by JSPS and MEXT research grants to T.O.

National Food Research Institute.

MRC Laboratory of Molecular Biology.

To whom correspondence should be addressed. E-mail: [email protected] waseda.jp. Telephone: +81 3 5286 1520. Fax: +81 3 3207 9694.

Faculty of Education and Integrated Arts and Sciences and Graduate School of Science and Engineering, Waseda University.


DNA: Types, Structure and Function of DNA

Nucleic acids were first isolated by Friedrich Miescher (1869) from pus cells.

They were named nuclein. Hertwig (1884) proposed nuclein to be the carrier of hereditary traits. Because of their acidic nature they were named nucleinic acids and then nucleic acids (Altmann, 1899).

Fisher (1880s) discovered the presence of purine and pyri­midine bases in nucleic acids. Levene (1910) found deoxyribose nucleic acid to contain phosphoric acid as well as deoxyribose sugar.

He characterised four types of nucleotides present in DNA. In 1950, Chargaff found that purine and pyrimidine content of DNA was equal. By this time W.T. Astbury had found through X-ray diffraction that DNA is a polynucleotide with nucleotides arranged perpendicular to the long axis of the molecule and separated from one another by a distance of 0.34 nm.

In 1953, Wilkins and Franklin got very fine X-ray photographs of DNA. The photographs showed that DNA was a helix with a width of 2.0 nm. One turn of the helix was 3.4 nm with 10 layers of bases stacked in it. Watson and Crick (1953) worked out the first correct double helix model from the X-ray photo­graphs of Wilkins and Franklin. Wilkins, Watson and Crick were awarded Nobel Prize for the same in 1962.

Watson and Crick (1953) built a 3D, molecular model of DNA that satisfied all the details obtained from X-ray photographs. They proposed that DNA consisted of a double helix with two chains having sugar phosphate on the outside and nitrogen bases on the inner side.

The nitrogen bases of the two chains formed complementary pairs with purine of one and pyrimidine of the other held together by hydrogen bonds (A-T, C-G). Comple­mentary base pairing between the two polynucleotide chains is considered to be hall mark of their proposition. It is of course based on early finding of Chargaff that A = T and С = G Their second big proposal was that the two chains are antiparallel with 5’→ 3′ orien­tation of one and У → 5’orientation of the other.

The two chains are twisted helically just as a rope ladder with rigid steps twisted into a spiral. Each turn of the spiral contains 10 nucleotides. This double helix or duplex model of DNA with antiparallel polynucleotide chains having complementary bases has an implicit mechanism of its replication and copying.

Here both the polynucleotide chains function as templates forming two double helices, each with one parent chain and one new but complementary strand. The phenomenon is called semi conservative replication. In vitro synthesis of DNA has been carried out by Kornberg in 1959.

Types of DNA:

DNA duplex model proposed by Watson and Crick is right handed spiral and is called B-DNA (Balanced DNA). In the model the base pairs lie at nearly right angles to the axis of helix (Fig. 6.5 D). Another right handed duplex model is A-DNA (Alternate DNA). Here, a single turn of helix has 11 base pairs.

The base pairs lie 20° away from perpendicular to the axis. C-DNA has 9 base pairs per turn of spiral while in D-DNA the number is only 8 base pairs. Both are right handed. Z-DNA (Zigzag DNA) is left-handed double helix with zigzag back-bone, alternate purine and pyrimidine bases, single turn of 45 A length with 12 base pairs and a single groove.

B-DNA is more hydrated and most frequently found DNA in living cells. It is physiologically and biologically active form. However, it can get changed into other forms. Right handed DNA is known to change temporarily into the left handed form at least for a short distance. Such changes may cause changes in gene expression.

Circular and Linear DNA:

In many prokaryotes the two ends of a DNA duplex are covalently linked to form circular DNA. Circular DNA is naked, that is, without association with histone proteins, though polyamines do occur. In linear DNA the two ends are free. It is found in eukaryotic nuclei where it is associated with histone proteins.

Linear DNA, without association with histone proteins, also occurs in some prokaryotes, e.g., Myco­plasma. In semi-autonomous cell organelles (mitochondria, plastids) DNA is circular, less commonly linear. It is always naked.

Chargaff’s Rules:

Chargaff (1950) made observations on the bases and other compo­nents of DNA. These observations or generalizations are called Chargaff’s base equivalence rule.

(i) Purine and pyrimidine base pairs are in equal amount, that is, adenine + guanine = thymine + cytosine. [A + G] = [T + C], i.e., [A+G] / [T+C] = 1

(ii) Molar amount of adenine is always equal to the molar amount of thymine. Similarly, molar concentration of guanine is equalled by molar concentration of cytosine.

[A] = [T], i.e., [A] / [T] = 1 [G] = [C], i.e., [G] / [C] = 1

(iii) Sugar deoxyribose and phosphate occur in equimolar proportions.

(iv) A-T base pairs are rarely equal to С—G base pairs.

(v) The ratio of [A+T] / [G+C] is variable but constant for a species (Table 6.2). It can be used to identify the source of DNA. The ratio is low in primitive organisms and higher in advanced ones.

Table 6.2. Base Composition of DNA from Various Sources:

Species A G С T A+T/C+G
1. Man 30.4 19.0 19.9 30.1 1.55
2. Calf 29.0 21.2 21.2 28.5 1.35
3. Wheat germ 28.1 21.8 22.7 27.4 1.25
4. Pea 30.8 19.2 18.5 30.5 1.62
5. Euglena 22.6 27.7 25.8 24.4 0.88
6 Escherichia coli 24.7 26.0 25.7 23.6 0.93

Structure of DNA:

DNA or deoxyribonucleic acid is a helically twisted double chain polydeoxyribonucleotide macromolecule which constitutes the genetic material of all organisms with the exception of rhinoviruses. In prokaryotes it occurs in nucleoid and plasmids. This DNA is usually circular. In eukaryotes, most of the DNA is found in chromatin of nucleus.

It is linear. Smaller quantities of circular, double stranded DNA are found in mitochondria and plastids (organelle DNA). Small sized DNAs occur in viruses, ф x 174 bacteriophage has 5386 nucleotides. Bacteriophage lambda (Phage X) possesses 48502 base pairs (bp) while number of base pairs in Escherichia coli is 4.6 x 10 6 . A single genome (haploid set of 23 chromosomes) has about 3.3 x 10 9 bp in human beings. Single-stranded DNA occurs as a genetic material in some viruses (e.g., phage ф x 174, coliphage fd, M13). DNA is the largest macromolecule with a diameter of 2 nm (20Å or 2 x 10 -9 m) and often having 3 length in millimetres.

It 15 negatively charged due to phosphate groups. It is a long chain polymer of generally several hundred thousands of deoxyribonucleotides. A DNA molecule has two un-branched comple­mentary strands. They are spirally coiled. The two spiral strands of DNA are collectively called DNA duplex (Fig. 6.5).

The two strands are not coiled upon each other but the whole double strand (DNA duplex) is coiled upon itself around a common axis like a rope stair case with solid steps twisted into a spiral. Due to spiral twisting, the DNA duplex comes to have two types of alternate grooves, major (22 Å) and minor (12 Å).

In B-DNA, one turn of the spiral has about 10 nucleotides on each strand of DNA. It occupies a distance of about 3.4 nm (34 Å or 3.4吆 -9 m) so that adjacent nucleotides or their bases are separated by a space of about 0.34 nm (0.34吆 -9 m or 3.4 Å).

A deoxyribonucleotide of DNA is formed by cross-linking of three chemicals ortho- phosphoric acid (H3PO4), deoxyribose sugar (C5H10O4) and a nitrogen base. Four types of nitrogen bases occur in DNA. They belong to two groups, purines (9-membered double rings with nitrogen at 1,3,7 and 9 positions) and pyrimidines (six membered rings with nitrogen at 1 and 3 positions). DNA has two types of purines (adenine or A and guanine or G) and two types of pyrimidines (cytosine or С and thymine or T).

Depending upon the type of nitrogen base, DNA has four kinds of deoxyribonucleotides —deoxy adenosine 5- monophosphate (d AMP), deoxy guaninosine 5-monophosphate (d GMP), deoxy thymidine 5-monophosphate (d TMP) and deoxy cytidine 5- monophosphate (d CMP).

The back bone of a DNA chain or strand is built up of alternate deoxyribose sugar and phosphoric acid groups. The phosphate group is connected to carbon 5′ of the sugar residue of its own nucleotide and carbon У of the sugar residue of the next nucleotide by (3 ‘—5’) phosphodiester bonds. -H of phosphate and -OH of sugar are eliminated as H20 during each ester formation.

Phosphate group provides acidity to the nucleic acids because at least one of its side group is free to dissociate. Nitrogen bases lie at right angles to the longitudinal axis of DNA chains. They are attached to carbon atom 1 of the sugars by N-glycosidic bonds. Pyrimidine (C or T) is attached to deoxyribose by its N-atom at 1 position while a purine (A or G) does so by N-atom at 9 position.

The two DNA chains are antiparallel that is, they run parallel but in opposite directions. In one chain the direction is 5’→ У while in the opposite one it is 3′ →5′ (Fig. 6.5). The two chains are held together by hydrogen bonds between their bases. Adenine (A), a purine of one chain lies exactly opposite thymine (T), a pyramidine of the other chain. Similarly, cytosine (C, a pyrimidine) lies opposite guanine (G a purine). This allows a sort of lock and key arrangement between large sized purine and small sized pyrimidine.

It is strengthened by the appearance of hydrogen bonds between the two. Three hydrogen bonds occur between cytosine and guanine (C = G) at positions 1’-1’, 2′- 6′ and 6′-2′. There are two such hydrogen bonds between adenine and thymine (A=T) which are formed at positions 1’-3′ and 6′-4′. Hydrogen bonds occur between hydrogen of one base and oxygen or nitrogen of the other base. Since specific and different nitrogen bases occur on the two DNA chains, the latter are complementary.

Thus the sequence of say AAGCTCAG of one chain would have a complementary sequence of TTCGAGTC on the other chain. In other words, the two DNA chains are not identical but complementary to each other. It is because of specific base pairing with a purine lying opposite a pyrimidine. This makes the two chains 2 nm thick.

A purine- purine base pair will make it thicker while a pyrimidine- pyrimidine base pair will make it narrower than 2 nm. Further, A and С or G and T do not pair because they fail to form hydrogen bonds between them. 5′ end of each chain bears phosphate radical while the 3′ end possesses a sugar residue (З’-ОН).

Salient Features of В model of DNA of Watson and Crick:

1. DNA is the largest biomolecule in the cell.

2. DNA is negatively charged and dextrorotatory.

3. Molecular configuration of DNA is 3D.

4. DNA has two polynucleotide chains.

5. The two chains of DNA have antiparallel polarity, 5′ —> 3′ in one and 3′ —> 5′ in other.

6. Backbone of each polynucleotide chain is made of alternate sugar-phosphate groups. The nitrogen bases project inwardly.

7. Nitrogen bases of two polynucleotide chains form complementary pairs, A opposite T and С opposite G.

8. A large sized purine always comes opposite a small sized pyrimidine. This generates uniform distance between two strands of helix.

9. Adenine (A) of one polynucleotide chain is held to thymine (T) of opposite chain by two hydrogen bonds. Cytosine (C) of one chain is similarly held to guanine of the other chain by three hydrogen bonds.

10. The double chain is coiled in a helical fashion. The coiling is right handed. This coiling produces minor and major grooves alternately.

11. The pitch of helix is 3.4 nm (34 A) with roughly 10 base pairs in each turn. The average distance between two adjacent base pairs comes to about 0.34 nm (0.34 x 10 -9 m or 3.4 A).

12. Planes of adjacent base pairs are stacked over one another. Alongwith hydrogen bonding, the stacking confers stability to the helical structure.

13. DNA is acidic. For its compaction, it requires basic (histone) proteins. The histone proteins are +vely charged and occupy the major grooves of DNA at an angle of 30° to helix axis.

Sense and Antisense Strands:

Both the strands of DNA do not take part in controlling heredity and metabolism. Only one of them does so. The DNA strand which functions as template for RNA synthesis is known as template strand, minus (-) strand or antisense strand.

Its complementary strand is named nontemplate strand, plus (+) strand, sense and coding strand. The latter name is given because by convention DNA genetic code is written according to its sequence.

DNA Nontemplate, Sense (+) or Coding Strand

DNA Template, Antisense, or Noncoding or (-) Strand

RNA is transcribed on 3’→5′ (-) strand (template/antistrand) of DNA in 5 → 3 direction.

The term antisense is also used in wider prospective for any sequence or strand of DNA (or RNA) which is complementary to mRNA.

Denaturation and Renaturation:

The H-bonds between nitrogen bases of two strands of DNA can break due to high temperature (82-90°C) or low or high pH, so that the two strands separate from each other. It is called denaturation or melting. Since A-T base pair has only 2H bonds, the area rich in A-T base pairs can undergo easy denaturation (melting). These areas are called low melting areas because they denature at comparatively low temperature. The area rich in G- C base pairs (called high melting area) is comparatively more stable and dense because three hydrogen bonds connect the G-C bases.

These areas have high temperature of melting (Tm). On melting the viscosity of DNA decreases. The denatured DNA has the tendency to reassociate, i.e., the DNA strands separated by melting at 82-90°C can reassociate and form duplex on cooling to temperature at 65°C. It is called renaturation or annealing.

Denatured or separated DNA strands absorb more light energy than the intact DNA double strand. The increased absorption of light energy by separated or denatured DNA strands is called hyperchromatic effect. The effect is used in knowing whether DNA is single or double stranded.

DNA duplex possesses areas where sequence of nucleotides is the same but opposite in the two strands. These sequences are recognised by restriction endonucleases and are used in genetic engineering. Given hereunder sequence of bases in one strand (3′ → 5′) is GAATTC. It is same in other strand when read in 5′ → 3′ direction. It is similar to palindrome words having same words in both forward and backward direction, e.g., NITIN, MALAYALAM.

It is the DNA having multiple copies of identical sequences of nitrogen bases. The number of copies of the same base sequence varies from a few to millions. DNA having single copy of base sequences is called unique DNA. It is made of functional genes. rRNA genes are, however, repeated several times. Repetitive DNA may occur in tandem or inter­spersed with unique sequences.

It is of two types, highly repetitive and moderately repetitive. Highly repetitive DNA consists of short sequences of less than 10 base pairs which are repeated millions of times. They occur in preeentromeric regions, heterochromatic re­gions of Y-chromosomes and satellite regions. Moderately repetitive dna consists of a few hundred base pairs repeated at least 1000 times. It occurs in telomeres, centromeres and transposons.

Tandemly repeated sequences are especially liable to undergo misalignments during chromosome pairing, and thus the size of tandem clusters tends to be highly poly­morphic, with wide variations between individuals. Smaller clusters of such sequences can be used to characterize individual genomes in the technique of “DNA-finger-printing”.

It is that part of repetitive DNA which has long repetitive nucleotide sequences in tandem that forms a separate fraction on density ultracentrifugation. Depending upon the number of base pairs involved in repeat regions, satellite DNA is of two types, microsatellite sequences (1-6 bp repeat units flanked by conserved sequences) and minisatellite sequences (11-60 bp flanked by conserved restriction sites). The latter are hyper variable and are specific for each individual. They are being used for DNA matching or finger printing as first found out by Jeffreys et al (1985).

The arrangement of nitrogen bases of DNA (and its product mRNA) determines the sequence of amino acid groups in polypeptides or proteins formed over ribosomes. One amino acid is specified by the sequence of three adjacent nitrogen bases. The latter is called codon. The segment of DNA which determines the synthesis of complete polypeptide is known as cistron.

In procaryotes, a cistron has a continuous coding sequence from beginning to end. In eucaryotes a cistron contains noncoding regions which do not produce part of gene product. They are called introns. Introns are often variable. The coding parts are known as exons. Cistrons having introns are called split genes.

Coding and Noncoding DNA:

Depending on the ability to form functional or nonfunc­tional products, DNA has two types of segments, coding and noncoding. In eukaryotes a greater part of DNA is noncoding since it does not form any functional product. They often possess repeated sequences or repetitive DNA. Most of them have fixed positions.

Some can move from one place to another. The mobile sequences are called jumping genes or transposons. In prokaryotes the amount of noncoding or nonfunctional DNA is small. Coding DNA consists of coding DNA sequences. These are of 2 types — protein coding sequences coding for all proteins except histone and nonprotein coding sequences for tRNA, rRNA and histones.

Functions of DNA:

1. Genetic Information (Genetic Blue Print):

DNA is the genetic material which car­ries all the hereditary information. The genetic information is coded in the arrangement of its nitrogen bases.

DNA has unique property of replication or production of carbon copies (Autocatalytic function). This is essential for transfer of genetic information from one cell to its daughters and from one generation to the next.

DNA occurs inside chromosomes. This is essential for equitable distribution of DNA during cell division.

During meiosis, crossing over gives rise to new combination of genes called recombinations.

Changes in sequence of nitrogen bases due to addition, deletion or wrong replication give rise to mutations. Mutations are the fountain head of all variations and evolution.

DNA gives rise to RNAs through the process of transcription. It is heterocatalytic activity of DNA.

7. Cellular Metabolism:

It controls the metabolic reactions of the cells through the help of specific RNAs, synthesis of specific proteins, enzymes and hormones.

Due to differential functioning of some specific regions of DNA or genes, different parts of the organisms get differentiated in shape, size and functions.

DNA controls development of an organism through working of an internal genetic clock with or without the help of extrinsic information.

10. DNA Finger Printing:

Hypervariable microsatellite DNA sequences of each indi­vidual are distinct. They are used in identification of individuals and deciphering their relationships. The mechanism is called DNA finger printing.

Defective heredity can be rectified by incorporating correct genes in place of defective ones.

Excess availability of anti-mRNA or antisense RNAs will not allow the pathogenic genes to express themselves. By this technique failure of angioplasty has been checked. A modification of this technique is RNA interference (RNAi).


Materials and methods

Strains and cell culture

Inbred strains B2086 (mating type II), CU438 (Pmr/Pmr [mating type IV, pm-s]), CU427 (Chx/Chx [mating type VI, cy-s]), and CU428 (Mpr/Mpr [mating type VII, mp-s]) were obtained originally from Peter Bruns (Cornell University, Ithaca, NY). The mature tester strains of mating type III and mating type V were F1 progeny of CU427 and CU428. TKU80 homozygous germline knockout strains were generated as described before [23]. ΔTKU80 strains that lacked TKU80 in both the germline and macronucleus were generated by mating 2 TKU80 homozygous germline knockout strains at 30 °C. Mating pairs were isolated and incubated in SPP medium at 5–6 hours post-mixing. TKU70-1, TKU70-2, TKU80, and GFP hairpin RNA-generating strains were generated by using CU427 and CU428 as parental strains through the method described before [40]. Cells were cultured in axenic media as previously described [41].

Selfing examination

Subcloned cells (approximately 2 × 10 6 cells) were washed and starved in 10 mL of 10 mM Tris-HCl buffer (pH 7.4) at 30 °C. Tetrahymena mating pairs were fixed in phosphate-buffered saline (PBS) containing 2% paraformaldehyde at 6 hours after cells were starved. Mating pair ratio was examined under a microscope (Zeiss Axio Imager Z1 Carl Zeiss, Jena, Germany). For mass screening of selfers, subclones that had been grown to saturation in Neff medium were diluted 50-fold in 10 mM Tris-HCl buffer (pH 7.4) and incubated at 30 °C. Mating pairs were examined 12–24 hours after dilution. Mating pairs were viewed under a dissecting microscope (Leica MZ 125 Leica Microsystems GmbH, Wetzlar, Germany).

MTA/MTB analysis

Whole cell DNA (approximately 95% was macronuclear DNA) was isolated for PCR analysis and Southern blot hybridization as previously described [42]. For PCR amplification of the MTA and MTB C-terminal junctions, we used 1 primer located on the MTA (or MTB) specific region and the other primer located on the constant region as described before [10]. For analysis by Southern hybridization, genomic DNA was digested by restriction enzymes and subjected to electrophoresis in a 0.8% agarose gel. DNA was transferred to a nylon membrane (IMMOBILON-NY+ Millipore, Bedford, MA) and hybridized with probes labeled with digoxigenin by a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). All primers used for the PCR reactions are listed in S5 Table. The membrane was washed first in 2× saline–sodium citrate (SSC) with 0.1% SDS and then in 0.5× SSC with 0.1% SDS at 65 °C several times. Luminescence signals was detected following manufacturer’s instructions.

Hairpin RNAi gene silencing (knockdown)

To generate TKU70-1, TKU70-2, TKU80, and GFP knockdown constructs, the regions within the respective ORFs were amplified by PCR and cloned into the PCRII-I vector with 2 sets of restriction enzyme sites, producing 2 copies in inverted orientation, and then were moved into the pIBF rDNA vectors. The expression of the inverted dimer was driven by the cadmium-inducible MTT1 promoter [40]. Each construct was transformed into mating Tetrahymena by electroporation using 10 μg of hairpin vector DNA [43]. Cells were transferred into SPP medium after electroporation (approximately 10 hours post-mixing) at 30 °C, followed by plating into 96-well plates (approximately 16 hours post-mixing). Transformants were selected in 120 μg/ml paromomycin (approximately 28 hours post-mixing). Silencing was induced using cadmium (1 μg/ml) at approximately 1 fission (approximately 25 hours post-mixing), approximately 16 fissions, and approximately 22 fissions. For continuously silencing of TKU70-1hp, TKU70-2hp, and TKU80hp, silencing were terminated at 69 fissions when cells were subcloned for selfing analysis after sexual maturation continuous silencing of GFP, the proper control for normal cells, was terminated at 82 fissions when cells were subcloned for selfing analysis. The silencing effect was analyzed by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from cells by using an RNA isolation kit (Roche). First-strand cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche) and oligo (dT)18 as a primer. The qRT-PCR analysis was carried out by using the LightCycler Carousel-Based PCR system and LightCycler FastStart DNA Master PLUS SYBR Green I (Roche). The relative expression levels were normalized by using α-tubulin mRNA as an internal control. Primer sequences are listed in S5 Table.

Assortment assay

Single cells were subcloned into individual drops of medium on Petri dishes and kept in a humid box at 30 °C. One cell propagating to saturation in a drop requires approximately 13 fissions. At this time, a single cell was subcloned again to a fresh drop, and the remaining cells were replicated to medium with cycloheximide (25 μg/mL) or 6-methylpurine (15 μg/mL) for the analysis of drug resistance [44]. The drug-sensitive proportions were examined in serial vegetative fissions and analyzed by linear regression.

Mating compatibility analysis

Tester cells of different mating types were starved in Dryl’s medium (Na citrate-2H2O (2 mM), NaH2PO4·H2O (1 mM), Na2HPO4 (1 mM), and CaCl2 (1.5 mM)) [45] at 30 °C for 5 hours and labeled with 40 μg/mL NHS-Rhodamine (Thermo Fisher Scientific) in Dryl’s medium for 2 hours. To avoid selfing, selfer strains were starved in 50 mM Tris-HCl (pH 7.4) at 30 °C for 7 hours. Before mixing, both selfer and tester cells were washed with 10 mM Tris-HCl (pH 7.4). Cells were collected at 4 hours post-mixing and fixed in PBS containing 2% paraformaldehyde, followed by DAPI staining (100 ng/mL). Images were captured using a fluorescent microscope (Zeiss Axio Imager Z1 Carl Zeiss, Jena, Germany). Unpaired single cells that showed meiotic nuclei were taken as indications of loose pairs that had initiated the mating reaction (including meiosis) but had separated prematurely. They were counted to determine the fraction of loose pairs.

MTA/MTB expression analysis by RT-PCR

Total RNA was extracted from 3 hours’ starved cells by using an RNA isolation kit (Roche) and treated with DNAse followed by reverse transcription into cDNA using Transcriptor reverse transcriptase (Roche) with oligo (dT) primers. For examining the MTAs and MTBs expression, we used primers located on exons. The sequences of primers are used in RT-PCR are listed in S5 Table.

IES elimination and chromosome breakage analysis

Genomic DNA was purified from cells in conjugation, feeding after conjugation, and vegetative growth. Deletion of IESs and chromosome breakage were examined by PCR analysis [46–48]. The primers used in these experiments are listed (S5 Table).

Telomere-anchored PCR

Genomic DNA was extracted from vegetative cells collected at different fissions. Ten eliminated minichromosomes were examined by PCR analysis using a specific primer at 1 minichromosome end with a telomeric sequence primer [22]. The primers used in the experiment are listed in S5 Table.


Nanotechnology Tools for the Study of RNA

2.1.3 Scaffold Design (DNA Origami)

DNA origami takes a much different approach from that of multistranded and SST design. 42 Usually, 7249-nucleotides long single-strand circular genomic DNA of M13mp18 phage is used as scaffold and hundreds of short helper strands called staples are used to fold longer scaffold into specific structure. In the folding process, scaffold DNA acts as a guide or a seed, 51 which increases the efficiency of folding and robustness to the stoichiometry of strands.

In 2006, “Smiley face” shook the DNA nanotechnology field ( Fig. 3 A). 42 Rothemund changed the rule of the game from assembling short strands motif into a large structure to fold long scaffold into specific structure. 52 The long scaffold of DNA origami is fold and hold by crossover made by staple strands. Typically, the staple strands bind to three adjacent helices, and the length is commonly 32 nucleotides, in which central 16 nucleotides bind to one helix and the remaining two parts of 8-nucleotide ends bind to the adjacent helices ( Fig. 3 B). The helical turn of DNA is usually approximated to be 3–3.5 nm in length and 3.5 nm in width. One helical turn (10.67 nucleotides) is different from that of canonical DNA (10.4 nucleotides/turn), resulting in the slightly twisted structure. Therefore, to relax the strain, usually one nucleotide is omitted every 48 nucleotides. 53 The length (about 3.5 nm) is also slightly different from that of canonical DNA (3.4 nm), which might be due to the interhelix gap presumably induced by electrostatic repulsion. Folded structures with straight edges sometimes stick together due to ππ stacking. To prevent this aggregation, single-strand 4T hairpin loops (four thymidines) are introduced to the staple strands located at the edge and corner part. If the stacking of folded DNA origami cause severe problem, one can design the edge with concavity and convexity.

Figure 3 . Scaffold design (DNA origami). (A) Smiley face structure with DNA origami method. (B) In DNA origami method, long circular single-stranded DNA (black) is folded into the desired shape by many short single-stranded DNAs (termed “Staple”), the latter typically bind to three adjacent helices, and the length is commonly 32 nucleotides, in which the central 16 nucleotides bind to one helix and the remaining two parts of 8-nucleotide ends bind to the adjacent helices. Unit pixel size is with dimensions of 3.6 × 3.5 nm.

Part A, B: adapted from Rothemund (2006), images reproduced with permission from Nature Publishing Group (NPG). 42

Folding of DNA is performed by adding a 5- to 10-fold excess of each staple strand, and by annealing the sample using PCR machine with ramp method (decrease the temperature of the sample with time) or at constant temperature. 54 Folded DNA origami can be purified by column (ultrafiltration, gel filtration), by gel electrophoresis, 55,56 or by PEG precipitation. 57,58 The yield of folding is quite high (90–95%) and the homogeneity is also high. 59

DNA origami also can be folded into 3D objects, 60,61 where the architecture is developed from a six-helix bundle (6HB) DNA nanotube. 62 In this architecture the unit length is 7 nucleotides and not 8 nucleotides. Seven nucleotides correspond exactly to 2/3 of a turn and 14 nucleotides correspond exactly to 4/3 of a turn, therefore, crossover between adjacent helix is allowed in honeycomb 6HB bundle structure, where six helices rotated 120 degrees to each other ( Fig. 4 A). These features allow connecting multiple honeycomb layers, enabling the formation of 3D objects ( Fig. 4 A). Further introduction of twist and curve, more complicated structure of gear, 63 box, 64–66 pot, 67 and sphere 68 were made ( Fig. 4 B). Recently, with the aid of graph theory and relaxation simulation, a general method of folding arbitrary polygonal digital meshes into the structure was reported, in which the design process is highly automated. 69 Thus, various types of structures could be made by scaffold design (DNA origami) methods.

Figure 4 . 3D DNA structure of Scaffold design. (A) Basic unit of scaffold designed 3D DNA structure. Cross-section (left) and side view (right) of honeycomb structure composed of six helices with sample staples using caDNAno ( http://cadnano.org/ , bottom). 60,70 See also Section 5.3 for detailed design processes. (B) Representative 3D structures such as gear and pod.

Part B: adapted from Deitz et al. (2009) (left) 63 and Han et al. (2011) (right). 67

Highly assembled structure of DNA origami is also possible. Using pole and joint approach Yin and coworkers made hexagonal prism (60 MDa, Fig. 5 A). 71 With 100-nm edges, the sizes of these structures become comparable to those of bacterial microcompartments such as carboxysomes. The joint pole termed DNA “tripod” is a 5-MDa 3-arm-junction origami tile, in which interarm angles and pole (arm) length can be controlled, so that, with the connector sequence design, many types of structures such as a tetrahedron (–20 MDa), a triangular prism (–30 MDa), a cube (–40 MDa), a pentagonal prism (–50 MDa), and a hexagonal prism (–60 MDa) can be self-assembled. In a tripod, each arm has an equal length (–50 nm) and contains 16 parallel double-helices packed on a honeycomb lattice with twofold rotational symmetry, and “struts” consisting of two double-helices support and control the angle between the two arms. The yields of tripod-assembled structures are highly dependent on the number of vertexes: 45, 24, 20, 4.2, and 0.11% for the tetrahedron, the triangular prism, the cube, the pentagonal prism, and the hexagonal prism, respectively. Dietz and coworkers took another approach to make polymerized structures with dynamic structure change. 72 Inspired by the interaction between an RNA-based enzyme ribonuclease P (RNase P) which cleaves the 5’ leader sequence for tRNA maturation and its substrate pretransfer RNA (tRNA). They used shape complementarity to assemble multiple DNA origami. In RNase P recognition, the acceptor stem and the TΨC loop of tRNA fit to the binding pocket of RNase P by a few nucleobase stacking interactions with the S domain of RNase P ( Fig. 5 B). 73 Similarly, in RNase P-inspired shape recognition method, blunt-ended double-helical DNA protrusions on one motif assume the role of the tRNA acceptor stem and corresponding concave on another motif mimic the RNase P binding pocket, and the nucleobase stacking bonds connect two motifs. 53,74–76 Upon two motifs engage, nucleobase stacking interactions occur at the double helical interface of the shape complementary protrusions and concave, but only when the helices fit correctly. Nucleobase stacking interaction method is sensitive to the concentration of counter ions, such as monovalent and divalent cations in the solution because repulsion between the negatively charged surfaces of DNA affects the equilibrium of the interaction, which allows on-off switching of the interface. All in all, without base pairing, RNase P-inspired method with nucleobase stacking bonds can build up micrometer-scale one- and two-stranded filaments and lattice, and transformable nanorobot. The merit of DNA origami is the design ability and robustness. As mentioned earlier, many types of structures have been made with high yield. In addition, long single-strand DNA scaffold can be a backbone, such that the structural rigidity to be ensured. 77 The limitation is based on the length of long scaffold. However, long scaffolds such as lambda DNA/M13 hybrid DNA scaffold (51,466 nucleotides), 78 PCR amplification-based scaffold [26 kb nucleotide fragment of lambda DNA (48,502 kb)], 79 and double strand form of lambda DNA itself 80 was used instead of M13mp18 scaffold (7,249 nucleotides). Combining these methods with higher order assemble method described previously, the limitation of scaffold may not be a problem from a practical point of view.

Figure 5 . Higher assembled DNA structure. (A) Pole and joint approach to construct higher assembled DNA structure. A tripod is composed of one set of DNA origami structure and used as a basic unit that has three arms and binds with each other at the apical point. The structure of the end product is defined by the angle between the arms. (B) Shape-complementarity method to construct higher assembled DNA structure. (Top) DNA structure binds to its paired structure (top right) in a way that RNase P recognizes its substrate tRNA (top left). (Middle) Upon two motifs engagement, nucleobase stacking interactions occur at the double helical interface of the complementary shapes, but only upon correct fit of the helices. (Bottom) Shape-complementary interaction is highly affected by ion concentration, therefore higher assembled DNA structure, for example, nanorobot of 15 MDa, can be reversibly transformed in three different conformation states: disassembled, assembled with open arms, and assembled with closed arms, respectively, by changing the Mg 2+ concentration.

Part A: adapted from Iinuma et al. (2014). 71 Part B: adapted from Gerling et al. (2015). 72


The DNA double helix

DNA is deoxyribonucleic acid, which is the molecule of inheritance that contains all of our genes. The DNA molecule consists of two strands of nucleotides which are joined together by hydrogen bonds which form between the nitrogen bases.

The backbone of the molecule consists of deoxyribose sugars that alternate with and are attached to phosphate groups by covalent phosphodiester bonds.

The monomer of the DNA molecule is a nucleotide which consists of a sugar molecule, a phosphate group, and a nitrogen base. The base is one of four possible molecules, adenine, thymine, cytosine or guanine.

The deoxyribose is a pentose sugar which means it has five carbons present, which are numbered from 1’ to 5’. The nitrogen base links to the 1’ carbon atom of the sugar molecule and the type of bond between the two structures is a glycosidic bond.

Two nucleotides can link together by a bond which forms between the phosphate group of one and the 3’ carbon of the sugar of the second nucleotide. Several nucleotides linked together form a chain known as a polynucleotide.

The entire molecule of DNA twists to form a helical three-dimensional structure and several molecules of the nucleic acid attach to histones and are organized into chromatin material in the nucleus.

The nitrogen bases of the nucleotide

It is the sequence of the bases that make up the genes of a cell. However, some of the bases do not code for particular proteins.

The thymine and cytosine are classified as pyrimidines because they consist of a single ring structure. The purines are the adenine and guanine, which have two rings in each case.

The nitrogen bases only bond in a specific manner, namely adenine with thymine and cytosine with guanine. These are known as Chargaff’s rules, named for the scientist who first discovered that there were equal quantities of the complementary bases.

Three bonds form the connection between the cytosine and the guanine. By comparison, only two bonds form between thymine and adenine bases.

What all these bonds have in common is that they are relatively weak and break easily. This is very important because the two strands of DNA have to be able to separate for such processes as when the DNA is copied in replication, and when the transcription stage of protein synthesis takes place.

DNA repair

The DNA strand can become damaged but often the cell is able to repair the error. In some cases, cells with damaged DNA simply self-destruct through the process of apoptosis.

Damage can be due to environmental factors including UV radiation or chemicals. Various excision repair methods have been discovered that allow cells to correct mismatch errors and replace any nucleotides or bases that may be wrong.

Repair of the DNA by excision involves enzymes actually cutting out the pieces of the strand that are erroneous. Other enzymes such as polymerase can then act to replace the missing pieces, and ligase can function to fill in any other gaps that may remain in the DNA molecule.


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How DNA Works

­DNA carries the information for making all of the cell's proteins. These pro­teins implement all of the functions of a living organism and determine the organism'­s characteristics. When the cell reproduces, it has to pass all of this information on to the daughter cells.

Before a cell can reproduce, it must first replicate, or make a copy of, its DNA. Where DNA replication occurs depends upon whether the cells is a prokaryotic or a eukaryote (see the RNA sidebar on the previous page for more about the types of cells). DNA replication occurs in the cytoplasm of prokaryotes and in the nucleus of eukaryotes. Regardless of where DNA replication occurs, the basic process is the same.

The structure of DNA lends itself easily to DNA replication. Each side of the double helix runs in opposite (anti-parallel) directions. The beauty of this structure is that it can unzip down the middle and each side can serve as a pattern or template for the other side (called semi-conservative replication). However, DNA does not unzip entirely. It unzips in a small area called a replication fork, which then moves down the entire length of the molecule.

  1. An enzyme called DNA gyrase makes a nick in the double helix and each side separates
  2. An enzyme called helicase unwinds the double-stranded DNA
  3. Several small proteins called single strand binding proteins (SSB) temporarily bind to each side and keep them separated
  4. An enzyme complex called DNA polymerase "walks" down the DNA strands and adds new nucleotides to each strand. The nucleotides pair with the complementary nucleotides on the existing stand (A with T, G with C).
  5. A subunit of the DNA polymerase proofreads the new DNA
  6. An enzyme called DNA ligase seals up the fragments into one long continuous strand
  7. The new copies automatically wind up again

Different types of cells replicated their DNA at different rates. Some cells constantly divide, like those in your hair and fingernails and bone marrow cells. Other cells go through several rounds of cell division and stop (including specialized cells, like those in your brain, muscle and heart). Finally, some cells stop dividing, but can be induced to divide to repair injury (such as skin cells and liver cells). In cells that do not constantly divide, the cues for DNA replication/cell division come in the form of chemicals. These chemicals can come from other parts of the body (hormones) or from the environment.

The DNA of all living organisms has the same structure and code, although some viruses use RNA as the information carrier instead of DNA. Most animals have two copies of each chromosome. In contrast, plants may have more than two copies of several chromosomes, which usually arise from errors in the distribution of the chromosomes during cell reproduction. In animals, this type of error usually causes genetic diseases that are usually fatal. For some unknown reasons, this type of error is not as devastating to plants.