Will someone with a double mutation in the allosomes be normal?

Normally a female human has an X allosome from her father and an X allosome form her mother. What if an double mutation happened, which causes that someone has two X allosomes form her mother and no allosomes form her father? Will this person be a normal female?

Note that it is both possible that there are no allosomes form the father or mother (known as Turners syndrome, (45, X)) and that there are two X allosomes form the mother (known as (47, XXX) and (47, XXY) respectively).

Since the Turner syndrome happens for 1 in 5000 births, and the Triple X syndrome happens for 1 in 1000 births, I estimate that this would happen once in 5 million births.


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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman 2000.

  • By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Hormone Replacement Therapy and the Factor V Leiden Mutation

From the Veterans’ Affairs Boston Healthcare System and Beth Israel Deaconess Medical Center, Harvard Medical School Boston, Mass.

In 1993, individuals with a hereditary predisposition to venous thromboembolism whose plasmas exhibited a poor response to activated protein C (APC) in an activated partial thromboplastin time assay were identified. 1 The molecular basis for this laboratory phenotype of resistance to APC was a guanine to adenine mutation at nucleotide 1691 in the factor V gene. 2 This results in the replacement of arginine (R) at position 506 by glutamine in the resulting protein, a defect which has been termed factor V Leiden. R506 is the first of three sites at which APC normally cleaves and inactivates procoagulant factor Va. The Q506 substitution causes factor Va to be inactivated approximately l0-fold more slowly than normal, thereby making the cofactor relatively resistant to the anticoagulant action of APC. 3 This allows for increased factor Va availability within the prothrombinase complex, thereby enhancing thrombin generation and the development of a hypercoagulable state.

Factor V Leiden is the most common inherited risk factor for venous thromboembolism, increasing the risk of venous thrombosis by 4- to 10-fold in heterozygotes and 50- to 100-fold in homozygotes. 4,5 Heterozygosity can be identified in 12% to 20% of unselected white patients presenting with venous thrombosis and 40% to 50% of patients with a strong positive family history. Approximately 3% to 7% of normal white patients are heterozygous carriers of factor V Leiden, but the mutation is rare in native African and Asian populations.

Soon after the identification of the factor V Leiden, it was recognized that the presence of the mutation greatly increases the risk of venous thrombosis associated with oral contraceptive use (reviewed by Vandenbroucke et al 6 ). Among women taking oral contraceptives, the risk is increased 35-fold among heterozygous carriers of the mutation compared with a 4-fold increase among noncarriers. This indicates that the combination of these two risk factors has a supra-additive, rather than a merely additive, effect on overall thrombotic risk. The risk of venous thrombosis is greatest in the first year of oral contraceptive use. Third-generation progestins, such as desogestrel and gestodene, in low-estrogen oral contraceptive preparations are associated with greater thrombogenicity. 6,7

The mechanism by which oral contraceptives are prothrombotic is complex. Prothrombotic effects include modest increases in the levels of procoagulant factors (factor VII, factor VIII, factor X, prothrombin, fibrinogen) and decreases in the levels of anticoagulant proteins (antithrombin, protein S). With a thrombin generation assay, it has been shown that women taking oral contraceptives develop acquired APC resistance. Though the molecular basis for this phenomenon is unknown, it provides a plausible explanation for the greatly increased thrombotic risk among oral contraceptive users who are carriers of the factor V Leiden mutation (reviewed by Vandenbroucke et al 6 and Rosendaal et al 8 ).

The thrombogenicity of oral contraceptives was recognized shortly after their introduction in the 1960s, 6 but convincing evidence that the lower estrogen dose used for hormone replacement therapy is associated with an increased risk of venous thromboembolism was only conclusively reported in 1996. 8 The estrogens commonly prescribed for hormone replacement are chemically different from those in oral contraceptives, but they are considered to have substantially lower biologic potency. Venous thrombotic risk associated with hormone replacement therapy is increased 2- to 4-fold, an effect that is similar in magnitude to oral contraceptives. However the use of hormone replacement therapy leads to a considerably larger number of excess cases as the result of an overall age-related increase in the incidence of thrombosis.

Based on the interactions between oral contraceptives and factor V Leiden, it was anticipated that carriers of the mutation receiving hormone replacement therapy would have a significantly increased risk of venous thromboembolism. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Herrington et al 9 report on a nested case-control study of women enrolled in the Heart and Estrogen Replacement Study (HERS) and the Estrogen Replacement and Atherosclerosis Trial. Both were randomized trials of hormone replacement therapy versus placebo in women with clinical evidence of coronary artery disease. The factor V Leiden mutation was found in 17% of venous thromboembolism cases and 6% of controls yielding an odds ratio of 3.3. Hormone replacement therapy carried an odds ratio of 4.5, but users with factor V Leiden had an odds ratio of 14.1 compared with noncarriers receiving placebo. Based on the incidence data from the trials, the authors estimate the risk of venous thrombosis in heterozygous carriers and noncarriers of factor V Leiden to be 15.2 and 5.8 per 1000 patient-years in women on hormone replacement, respectively, compared with 2.0 per 1000 patient-years in noncarriers taking placebo. They estimate that the number of women with coronary disease needed to screen to prevent one episode of venous thromboembolism is 374.

The results of Herrington et al 9 are very similar to those of a recently published study from the United Kingdom. 10 This case-control study of women aged 45 to 64 years with a first episode of venous thromboembolism found a 15-fold increased risk of venous thromboembolism in women on hormone replacement with the factor V Leiden mutation. Analogous to the data in women with the factor V Leiden mutation on oral contraceptives, this odds ratio was greater than the expected odds ratio for the combination of the two risk factors, and the risk was highest in the first year of use.

Estrogen in hormone replacement therapy has effects on the hemostatic system that are similar to those of oral contraceptives. Hormone replacement therapy leads to a dose-dependent increase in markers of prothrombin activation and fibrin generation, while enhancing fibrinolysis and decreasing the plasma levels of plasminogen activator inhibitor-I. 11–13

It is clear that hormone replacement therapy should seldom be prescribed to women if they had a previous venous thrombotic event. 14 For women known to have coronary heart disease and the factor V Leiden mutation without a personal history of venous thrombosis, Herrington and colleagues 9 point out that the risk of venous thromboembolism will far exceed any potential benefits of hormone replacement. 9 This is especially true given that hormone replacement therapy has not been shown to slow the progression of atherosclerotic lesions and may be associated with an increase in coronary events in several secondary-prevention trials including HERS. 15,16 The administration of oral contraceptives or hormone replacement therapy to women with prothrombotic mutations and an established cardiac risk factor may be associated with an increased risk of myocardial infarction. 17–19

Millions of women are prescribed hormone replacement therapy which can be associated with serious side effects. Venous thromboembolism is by no means restricted to those with identifiable prothrombotic defects. As prospective trials have not yet shown that hormone replacement therapy reduces the risk of arterial thrombotic complications, it can be strongly argued that physicians should refrain from prescribing the drug for this purpose rather than screening women for mutations such as factor V Leiden to eliminate women at high risk for venous thrombotic complications.

Supported in part by the Medical Research Service of the Department of Veterans Affairs.

Speaking to FIT for a different article, Dr Shahid Jameel, too, has talked about how variants and mutations are a natural process that occurs in viruses, and are not a cause for concern in most cases.

"Viruses will often mutate without any consequence. But those mutations that are able to equally or more efficiently infect people, are the ones that we single out as specific variants," says Dr Mishra.

Variants become 'variants of concern' under two circumstances,

  • When it spreads faster and starts making up more than 5 percent or 10 percent of the infections.
  • If there are mutations that we think may create problems in terms of symptoms or vaccines.

He says, "in the case of variants that are spreading faster but don't have any other different consequence, they are known as 'variants of interest' and will be continued to be monitored for any other changes."

When it comes to the variants of concern, Dr Mishra says,

A normal DNA repair process can become a major source of mutations in cancer

Hypermutation is an unusual occurence that can lead to many nearby mutations at once, severely damaging our genetic material and potentially causing cancer. The best known type of local hypermutation, called a mutation shower or thunderstorm, is quite uncommon and it leads to many mutations accumulated in a small area, e.g. a single gene.

Researchers from IRB Barcelona's Genome Data Science Lab, led by the ICREA researcher Fran Supek, have discovered a new type of hypermutation called mutation fog, which can generate hundreds of mutations in every cell. Such mutations are widely distributed, but accumulate in the most important regions of the genome, where genes reside (the so-called euchromatin). The fact that these mutations are spread around explains why they have remained undetected until now.

Surprisingly, the scientists have also identified that the newly discovered hypermutation type is related to a normal DNA repair process. When cells sense a mismatch in their DNA, they undergo a DNA repair reaction, in order to preserve genetic information. Remarkably, this reaction can become coupled to the APOBEC enzyme-typically used by human cells to defend against viruses and having an important role in fighting hepatitis and HIV. The work by the Genome Data Science Lab indicates that, in some cases, when both the APOBEC enzymes and the DNA repair process are active at the same time, APOBEC hijacks the DNA repair, generating the mutation fog.

"We think that this APOBEC-driven mutation fog has a mutagenic potential that matches or even exceeds that of well-known strong carcinogens, such as tobacco smoke or ultraviolet radiation," Fran Supek explains. Recent work by other research groups suggests that the process appears to be more active in late-stage metastatic cancers: it helps the cancer evolve, enabling it to resist drugs and radiation. "This finding makes APOBEC an attractive target for treating cancer, removing its ability to evolve and to become more aggressive," adds Supek.

The origin of a half of the mutations in some lung and breast cancers

A thorough analysis of more than 6,000 human cancer genomes, including lung tumours, breast tumours and melanomas, among others, led to the finding that the mutation fog is a common phenomenon. "More than half of all APOBEC mutations in some lung or breast cancers are generated by the hypermutation mechanism that we have found," says David Mas-Ponte, first author of the study and PhD student in the Genome Data Lab.

Some types of cancer, such as cervical or some head-and-neck cancers, are known to be due to viruses. However, this study has found mutations caused by this APOBEC system not only in these tumours but also in cancers that are not currently known to be virus-related. Further work should clarify what triggers the APOBEC system. "Understanding APOBEC better could have broad implications for cancer treatment," adds Mas-Ponte.

The HyperClust statistical method

Mas-Ponte and Supek designed a statistical method, called HyperClust, that can rapidly analyse large amounts of human genomic data to find unusual mutational processes that can lead to simultaneous mutations, such as these cases of mutation fog. This statistical method is described in the article, which has been published in Nature Genetics, and is also available as an open-source software in a Github repository.

This work has been funded by the ERC Starting Grant "HYPER-INSIGHT" awarded to Fran Supek ICREA reaearcher and EMBO Young Investigator and the Severo Ochoa grant awarded to IRB Barcelona. David Mas-Ponte was the recipient of an FPI-SO fellowship.

Decoding mutations and variants of evolving COVID-19 virus

Ashes to ashes: A mass cremation of people died of Covid-19 at Channenahalli village near Bengaluru | Bhanu Prakash Chandra

Mea culpa, rues Anurag Agarwal. Head of the CSIR-Institute for Genomics and Integrative Biology (CSIR-IGIB) in New Delhi, Agarwal coined the term ‘double mutant’, which has become the buzzword in India now. But he says he never intended it to be part of the common parlance.

“We were writing a scientific note, where we were describing a new Variant of Concern (VoC) that had been found,” says Agarwal. This variant of the SARS-CoV-2 virus, which they first identified in Maharashtra, had several mutations, as is the normal case with newer generations of viruses. However, among those mutations, two were of particular interest, since they showed “immune escape” in vitro. This means that when geneticists cultured virus genomes in the lab and subjected them to extreme antibody pressure, which should effectively neutralise the virus, some mutations still survived.

Using a method of nomenclature called Pango (there are multiple nomenclature methods, which cause much confusion even among the scientific community, forget laypersons), they identified this variant by a most uninteresting sounding name called B.1.617, flagging two of the various mutations. These mutations are E484Q and L452R, which are simply codes for the point on the genome at which a particular amino acid (indicated by the letter) is replaced by another. “While writing the paper, we had to repeatedly talk about the variant, and I referred to it at some point as the double mutant,” explains Agarwal.

This catchy word was first heard in public on March 24, when Union health secretary Rajesh Bhushan used it in his briefing. And before they knew it, the term was bandied around by just about everyone. It also caused a measure of concern, with people beginning to mistakenly believe that the double mutation made the virus a worse enemy than the ancestral Wuhan strand.

A few weeks later, two developments happened. Some scientists noted that there was another mutation, P681R, on the B.1.617 variant that was “interesting” because this mutation “probably helps the virus enter the host cell more easily,” in the words of virologist Saumitra Das, who heads the National Institute of Biomedical Genomics (NIBMG), Kalyani, West Bengal. So, people began talking about the double mutant actually being a “triple mutant” and thereby even more fearsome. “It is the same variant—B.1.617,” says Agarwal. “We were studying for immune escape, so we flagged the two mutations we connected to that trait. We had also noticed P681R, but in our story, it was only a sidekick the protagonists were the first two. For someone else, who was looking for another characteristic, P681R became the star of the narrative.”

But even before this confusion could be clarified, the NIBMG flagged yet another variant, B.1.618, which is prevailing in West Bengal. As it identified three mutations here, this, too, got referred to as the triple mutant, again, attributing to it triple the power for destruction. And so, there are now two triple mutants doing the rounds of the country, the Maharashtra double which is a triple, and the Bengal triple! 618, as it is best to call this variant, is still under study. It is not even classified as a VoC, says Das. It has not shown any potential to spread more virulently or attack more lethally.

The prevailing confusion is only natural, given the complicated, and not standardised, system for naming viruses and their variants. “The general public is usually not interested in virus names, and the existing system of numbers and letters work well for the scientific community, since we all know the codes, and can immediately identify the mutations by their names,” says Agarwal in defence. Right from the ancestral genome (the Adam or Eve genome), the phylogenetic tree of a virus can be a mindboggling alphabet and numerical soup, as subsequent generations are clumped into clades and lineages and variants. Unsurprisingly, terms like double mutant or UK strain enter our vocabulary, all of which are so misleading.

The B.1.1.7 lineage, referred to as the UK variant, got its name because it was first identified in the UK it does not mean it originated in the UK. Despite the stigma such names give to a place, they do become common. We have seen it with the Spanish Flu, Ebola, Nipha and Zika viruses and a host of diseases. Despite efforts of the scientific community to not associate the pandemic with China, the Wuhan connection remains indelible. The World Health Organization is working out a standardised nomenclature system, but while that may ease confusion among scientists, for the laypersons, it will still mostly be gobbledygook.

618 was first also referred to as the Indian variant, even though Bhushan insisted there is no such term. While his attempt was to ensure India does not get stigmatised, the fact is that given India's expanse, there cannot be one single Indian variant. Already, 617 is being called the Maharashtra variant, while 618 as the Bengal variant. But if yet another variant is reported from these states, what will that be called, asks Agarwal. “Right now, given the large number of cases, India must be having the largest pool of variants, too,” notes Rakesh Mishra, head of the Hyderabad-based Centre for Cellular and Molecular Biology, adding that mutation is the nature of viruses. Only if a mutation is associated with a more severe infection or antibody resistance does it become a matter of concern.

In fact, over the past year, the virus map of the country has changed dynamically. In the initial weeks, the Wuhan strand came to India, but quickly enough, a variant called D614G established itself all over India, says Mishra. It remained dominant till winter, when the UK variant came over by flights. This variant is known for higher infectivity or spread, but not necessarily higher morbidity in patients.

Immeasurable loss: Umar Farooq of Srinagar measures a grave dug for his mother, who died of Covid-19 | AFP

By December, the variant map of India began changing remarkably. The UK variant, despite being a VoC, was not contained properly, especially in north India. In Punjab, it now accounts for 90 per cent of the cases in Delhi, for half the cases sampled, followed by the 617 variant. On the other hand, despite the international flights to Mumbai and Hyderabad, identification and quarantine of international passengers carrying this infection has meant that the UK variant is less established in the peninsula. While in Maharashtra, 617 is the most prevalent variant, elsewhere in the south, N440K is more commonly seen. “It is of no real consequence as it is not leading to any greater infectivity,'' says Mishra. The Brazil and South African variants are also seen in India, but in very low titre.

In West Bengal, 618 and 617 are vying for supremacy, and scientists feel that the more mobile 617 might become dominant and even edge out 618. The Uttar Pradesh surge is still a study in progress. While it is possible that due to proximity, Uttar Pradesh might have a similar variant pattern as Delhi, Sujeet Singh, head of National Centre for Disease Control, which is analysing samples from north India, says the picture will get clear once their analysis is done. Bengaluru's surge, say geneticists, is not dominated by any particular variant. Overall, in the country, the UK variant is the dominant one, largely because of the north Indian surge.

What does an understanding of these variants mean for the people? Nothing, say scientists. The spread of the virus remains the same, irrespective of the variant. So far, so is the treatment. Thus, the existing protocol for prevention works. “In Punjab, for instance, the UK variant is dominant, but any variant would have done the same job, when people were gathering in droves for protests and parties,” points out Mishra.

These genomic studies are more for the understanding of researchers in epidemiological studies. A dynamic variant map will show how the infection is spreading, and therefore can be effectively used in epidemiological management, by physically cutting off the region from where it is spreading. The identification of 617 in the UK and Canada has shown the path of its travel.

Genomic studies also help in creating target medicines and vaccines. So far, there is no proven medicine for treating Covid-19. But studies show that existing treatment protocol and vaccines work as effectively against all the existing variants. This is because mutations are only at a particular point, but the vaccine targets several points on the virus. Thus, even if one particular site shows an “immune escape” under laboratory conditions, the vaccine would still be able to target the virus as a whole. For the present, at least. Again, none of the mutations have changed the way the virus has spread, so mask, social distance and hand hygiene work against all. This is for the present. As for the future of infection, que sera sera (what will be, will be).

Variant of Interest show changes in binding to receptor. They could have potential for reduced neutralisation by antibodies generated by a previous bout of infection or vaccination, potential diagnostic impact or predicted increase in disease transmissibility or severity. B.1.617 is mostly considered a VoI, B.1.618 may not even qualify as a VoI.

Variant of Concern is a variant for which there is evidence of increased transmissibility and severity of disease, significant reduction in neutralisation by antibodies from previous infection or vaccination, reduced effectiveness of treatment and diagnostic detective failures. The UK variant is a VoC.

DNA Double Take

From biology class to “C.S.I.,” we are told again and again that our genome is at the heart of our identity. Read the sequences in the chromosomes of a single cell, and learn everything about a person’s genetic information — or, as 23andme, a prominent genetic testing company, says on its Web site, “The more you know about your DNA, the more you know about yourself.”

But scientists are discovering that — to a surprising degree — we contain genetic multitudes. Not long ago, researchers had thought it was rare for the cells in a single healthy person to differ genetically in a significant way. But scientists are finding that it’s quite common for an individual to have multiple genomes. Some people, for example, have groups of cells with mutations that are not found in the rest of the body. Some have genomes that came from other people.

“There have been whispers in the matrix about this for years, even decades, but only in a very hypothetical sense,” said Alexander Urban, a geneticist at Stanford University. Even three years ago, suggesting that there was widespread genetic variation in a single body would have been met with skepticism, he said. “You would have just run against the wall.”

But a series of recent papers by Dr. Urban and others has demonstrated that those whispers were not just hypothetical. The variation in the genomes found in a single person is too large to be ignored. “We now know it’s there,” Dr. Urban said. “Now we’re mapping this new continent.”

Dr. James R. Lupski, a leading expert on the human genome at Baylor College of Medicine, wrote in a recent review in the journal Science that the existence of multiple genomes in an individual could have a tremendous impact on the practice of medicine. “It’s changed the way I think,” he said in an interview.

Scientists are finding links from multiple genomes to certain rare diseases, and now they’re beginning to investigate genetic variations to shed light on more common disorders.

Science’s changing view is also raising questions about how forensic scientists should use DNA evidence to identify people. It’s also posing challenges for genetic counselors, who can’t assume that the genetic information from one cell can tell them about the DNA throughout a person’s body.

Human Blueprint

When an egg and sperm combine their DNA, the genome they produce contains all the necessary information for building a new human. As the egg divides to form an embryo, it produces new copies of that original genome.

For decades, geneticists have explored how an embryo can use the instructions in a single genome to develop muscles, nerves and the many other parts of the human body. They also use sequencing to understand genetic variations that can raise the risk of certain diseases. Genetic counselors can look at the results of genetic screenings to help patients and their families cope with these diseases — altering their diet, for example, if they lack a gene for a crucial enzyme.

The cost of sequencing an entire genome has fallen so drastically in the past 20 years — now a few thousand dollars, down from an estimated $3 billion for the public-private partnership that sequenced the first human genome — that doctors are beginning to sequence the entire genomes of some patients. (Sequencing can be done in as little as 50 hours.) And they’re identifying links between mutations and diseases that have never been seen before.

Yet all these powerful tests are based on the assumption that, inside our body, a genome is a genome is a genome. Scientists believed that they could look at the genome from cells taken in a cheek swab and be able to learn about the genomes of cells in the brain or the liver or anywhere else in the body.

In the mid-1900s, scientists began to get clues that this was not always true. In 1953, for example, a British woman donated a pint of blood. It turned out that some of her blood was Type O and some was Type A. The scientists who studied her concluded that she had acquired some of her blood from her twin brother in the womb, including his genomes in his blood cells.

Chimerism, as such conditions came to be known, seemed for many years to be a rarity. But “it can be commoner than we realized,” said Dr. Linda Randolph, a pediatrician at Children’s Hospital in Los Angeles who is an author of a review of chimerism published in The American Journal of Medical Genetics in July.

Twins can end up with a mixed supply of blood when they get nutrients in the womb through the same set of blood vessels. In other cases, two fertilized eggs may fuse together. These so-called embryonic chimeras may go through life blissfully unaware of their origins.

One woman discovered she was a chimera as late as age 52. In need of a kidney transplant, she was tested so that she might find a match. The results indicated that she was not the mother of two of her three biological children. It turned out that she had originated from two genomes. One genome gave rise to her blood and some of her eggs other eggs carried a separate genome.

Women can also gain genomes from their children. After a baby is born, it may leave some fetal cells behind in its mother’s body, where they can travel to different organs and be absorbed into those tissues. “It’s pretty likely that any woman who has been pregnant is a chimera,” Dr. Randolph said.

Everywhere You Look

As scientists begin to search for chimeras systematically — rather than waiting for them to turn up in puzzling medical tests — they’re finding them in a remarkably high fraction of people. In 2012, Canadian scientists performed autopsies on the brains of 59 women. They found neurons with Y chromosomes in 63 percent of them. The neurons likely developed from cells originating in their sons.

In The International Journal of Cancer in August, Eugen Dhimolea of the Dana-Farber Cancer Institute in Boston and colleagues reported that male cells can also infiltrate breast tissue. When they looked for Y chromosomes in samples of breast tissue, they found it in 56 percent of the women they investigated.

A century ago, geneticists discovered one way in which people might acquire new genomes. They were studying “mosaic animals,” rare creatures with oddly-colored patches of fur. The animals didn’t inherit the genes for these patches from their parents. Instead, while embryos, they acquired a mutation in a skin cell that divided to produce a colored patch.

Mosaicism, as this condition came to be known, was difficult to study in humans before the age of DNA sequencing. Scientists could only discover instances in which the mutations and the effects were big.

In 1960, researchers found that a form of leukemia is a result of mosaicism. A blood cell spontaneously mutates as it divides, moving a big chunk of one chromosome to another.

Later studies added support to the idea that cancer is a result of mutations in specific cells. But scientists had little idea of how common cases of mosaicism were beyond cancer.

“We didn’t have the technology to systematically think about them,” said Dr. Christopher Walsh, a geneticist at Children’s Hospital in Boston who recently published a review on mosaicism and disease in Science. “Now we’re in the midst of a revolution.”

Benign Differences

The latest findings make it clear that mosaicism is quite common — even in healthy cells.

Dr. Urban and his colleagues, for example, investigated mutations in cells called fibroblasts, which are found in connective tissue. They searched in particular for cases in which a segment of DNA was accidentally duplicated or deleted. As they reported last year, 30 percent of the fibroblasts carried at least one such mutation.

Michael Snyder of Stanford University and his colleagues searched for mosaicism by performing autopsies on six people who had died of causes other than cancer. In five of the six people they autopsied, the scientists reported last October, they found cells in different organs with stretches of DNA that had accidentally been duplicated or deleted.

Now that scientists are beginning to appreciate how common chimerism and mosaicism are, they’re investigating the effects of these conditions on our health. “That’s still open really, because these are still early days,” Dr. Urban said.

Nevertheless, said Dr. Walsh, “it’s safe to say that a large proportion of those mutations will be benign.” Recent studies on chimeras suggest that these extra genomes can even be beneficial. Chimeric cells from fetuses appear to seek out damaged tissue and help heal it, for example.

But scientists are also starting to find cases in which mutations in specific cells help give rise to diseases other than cancer. Dr. Walsh, for example, studies a childhood disorder of the brain called hemimegalencephaly, in which one side of the brain grows larger than the other, leading to devastating seizures.

“The kids have no chance for a normal life without desperate surgery to take out half of their brain,” he said.

Dr. Walsh has studied the genomes of neurons removed during those surgeries. He and his colleagues discovered that some neurons in the overgrown hemisphere have mutations to one gene. Two other teams of scientists have identified mutations on other genes, all of which help to control the growth of neurons. “We can get our hands on the mechanism of the disease,” said Dr. Walsh.

Other researchers are now investigating whether mosaicism is a factor in more common diseases, like schizophrenia. “This will play itself out over the next 5 or 10 years,” said Dr. Urban, who with his colleagues is studying it.

Moving Cautiously

Medical researchers aren’t the only scientists interested in our multitudes of personal genomes. So are forensic scientists. When they attempt to identify criminals or murder victims by matching DNA, they want to avoid being misled by the variety of genomes inside a single person.

Last year, for example, forensic scientists at the Washington State Patrol Crime Laboratory Division described how a saliva sample and a sperm sample from the same suspect in a sexual assault case didn’t match.

Bone marrow transplants can also confound forensic scientists. Researchers at Innsbruck Medical University in Austria took cheek swabs from 77 people who had received transplants up to nine years earlier. In 74 percent of the samples, they found a mix of genomes — both their own and those from the marrow donors, the scientists reported this year. The transplanted stem cells hadn’t just replaced blood cells, but had also become cells lining the cheek.

While the risk of confusion is real, it is manageable, experts said. “This should not be much of a concern for forensics,” said Manfred Kayser, a professor of Forensic Molecular Biology at Erasmus University in Rotterdam. In the cases where mosaicism or chimerism causes confusion, forensic scientists can clear it up by other means. In the Austrian study, for example, the scientists found no marrow donor genomes in the hair of the recipients.

For genetic counselors helping clients make sense of DNA tests, our many genomes pose more serious challenges. A DNA test that uses blood cells may miss disease-causing mutations in the cells of other organs. “We can’t tell you what else is going on,” said Nancy B. Spinner, a geneticist at the University of Pennsylvania, who published a review about the implications of mosaicism for genetic counseling in the May issue of Nature Reviews Genetics.

That may change as scientists develop more powerful ways to investigate our different genomes and learn more about their links to diseases. “It’s not tomorrow that you’re going to walk into your doctor’s office and they’re going to think this way,” said Dr. Lupski. “It’s going to take time.”

Project Report on DNA

An exclusive project report on DNA. This project report will help you to learn about: 1. Introduction to DNA 2. Constituents of DNA 3. Molecular Structure 4. Forms 5. DNA as the Genetic Material 6. DNA Content 7. Unique and Repetitive DNA.

  1. Project Report on Introduction to DNA
  2. Project Report on the Constituents of DNA
  3. Project Report on the Molecular Structure of DNA
  4. Project Report on the Forms of DNA
  5. Project Report on DNA as the Genetic Material
  6. Project Report on DNA Content
  7. Project Report on Unique and Repetitive DNA

Project Report # 1. Introduction to DNA:

The chromosomes are made up of two types of macromolecules — proteins and nucleic acids. The nucleic acids are of two types, viz. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA are chain-like macro-molecules that function in the storage and trans­fer of genetic information.

They are major com­ponents of all cells. DNA is found predominant­ly in the nucleus while RNA is predominant in cytoplasm. DNA is the genetic material of most organisms including many viruses. Some viruses,’ however, have RNA as their genetic material.

Deoxyribonucleic acid (DNA) is found in the cells of living organisms except plant viruses. DNA is double stranded but in some cases like bacteriophages (e.g., φ x 174) the DNA is single stranded and remains coiled and enclosed in a protein coat. DNA may be circular in bacteria, spirally coiled and un-branched threads in mito­chondria and plastids of eukaryotic cells.

Project Report # 2. Constituents of DNA:

Deoxyribonucleic acid is a long chain poly­mer (polynucleotide) composed of monomeric units, called nucleotides. Each nucleotide is composed of a nucleoside (a sugar and a base) and a phosphate group.

Chemical analysis of highly purified DNA have shown that it is made of four kinds of monomeric building blocks each of which con­tains three types of molecules :

The phosphoric acid (H3PO4) in the nucleic acid is called phosphate (Fig 4.1). Phosphoric acid has three reactive hydroxyl (-OH) groups of which two are involved in forming sugar phosphate backbone of DNA. The phosphate makes a nucleotide negatively charged.

DNA contains a five carbon sugar, hence it is a pentose sugar (Fig. 4.1). Since one oxygen atom at the 2′ carbon is missing, hence it gets its name 2′-deoxyribose. Four of the five carbon atoms plus a single oxy­gen atom forms a five-membered ring. The fifth carbon atom is outside the ring and forms a part of a -CH2 group.

Different types of hetero­cyclic nitrogen containing ring compounds are found in the structure of DNA. They are called simply as bases because they can combine with H^ in acidic solution. They are also referred to as nitrogenous bases due to presence of nitrogen.

The bases are of two types mainly — Pyrimidine and Purine.

Pyrimidine bases are made up of a six-membered pyrimidine ring which is similar to the benzene ring except that it contains nitrogen in place of carbon at the positions 1 and 3. Pyrimidine bases are of 2 types — thymine and cytosine (Fig. 4.1), commonly abbreviated as T and C respectively.

Purine is a derivative of pyrimidine. It consists of a pyrimidine ring and a five- membered imidazole ring (having nitrogen at 7 and 9 positions) which are fused together at 5 and 4 positions. There are two purine com­pounds namely – adenine (A) and guanine (G) (Fig. 4.1).

In addition to the four common bases (A, T, G, C), certain other unusual bases of purine and pyrimidine derivatives, called rare or minor bases, occur in small amounts in DNA of some organisms.

In some viruses, uracil occurs in place of thymine in DNA. The T-even phages contain 5-hydroxy- methyl-cytosine in place of cytosine. These modifications protect the viral DNA from degra­dation by the host cell endonuclease. Other rare bases in DNA are 5-methyl-cytosine, N 6 -methyl- adenine, N 2 -methyl-guanine, etc.

Molar Ratio of Nitrogenous Bases in DNA (Chargaff Rules, 1955):

(i) The purine and pyrimidine components occur in equal amounts in a DNA molecule.

(ii) The amounts of adenine (A) is equivalent to the amount of thymine (T) and the amount of cytosine is equivalent to that of guanine (G).

(iii) In DNA, A + G / T + C value is always one or nearly one.

(iv) The base ratio A +T/G + C may vary in the DNA of different groups of organisms but is constant for particular species. Therefore, this ratio has been used to identify the DNA from a particular species.

Project Report # 3. Molecular Structure of DNA (Watson and Crick’s Model):

In 1953 Watson and Crick postulated a three dimensional working model of DNA, i.e., double helix structure of DNA based on the X-ray data of Wilkins and base-equivalence observed by Chargaff.

A base combined with a sugar molecule is called a nucleoside. When deoxy­ribose sugar binds with base, it makes deoxyribonucleoside. Obviously, in DNA four different nucleosides are found.

A nucleotide is derived from a nucleoside by addition of a molecule of phosphoric acid. The phosphate molecule is linked with sugar molecule at carbon number 5 or at carbon number 3 (Fig. 4.2).

The nucleotides in DNA are of 4 types:

(iv) Deoxyguanylic acid (Fig. 4.3, Table 4.1).

A number of deoxyribonucleotides are covalently linked one by one to form a polynucleotide chain, i.e., deoxyribonucleotide monomer units are united through the formation of phosphodiester bonds (a diester bond is one which involves two ester bonds). Fig. 4.4.

Watson and Crick suggested that in a DNA molecule, there are two such polynucleotide chains which are coiled about one another in a spiral.

The two polynucleotide strands are held together in their helical configu­ration by hydrogen bonding between bases in opposing strands, the resulting base pairs being stacked between the two chains perpendicular to the axis of the molecule like the steps of a spiral stair case (Fig. 4.5).

The base-pairing is specific adenine is always paired with thymine, and guanine is always paired with cytosine (Fig. 4.6). Thus, all base-pairs consist of one purine and one pyrimidine.

The specificity of base-pairing results from the hydrogen-bonding capacities of the bases in their normal configurations. In their most common structural configurations, adenine and thymine form two hydrogen bonds, guanine and cytosine form three hydrogen bonds (Fig. 4.7).

The two strands of DNA double helix are thus said to be complementary (not identical). This property, that is complementarity of the two strands, makes DNA uniquely suited to store and transmit genetic information. The base-pairs in DNA are stacked 3.4 Å apart with 10 base-pairs per turn (360°) of the double-helix.

The sugar- phosphate backbones of the two complementary strands are antiparallel, that is, they have oppo­site chemical polarity.

As one moves unidirectionally along a DNA double helix, the phos­phodiester bonds in one strand go from a 3′ car­bon of one nucleotide to a 5′ carbon of the adjacent nucleotide, whereas those in complemen­tary strand go from a 5′ carbon to a 3′ carbon. This opposite polarity of the complementary strand is very important in considering the mechanism of replication of DNA.

The high degree of stability of DNA double helices results in part from the large number of hydrogen bonds between the base-pairs and in part from the hydrophobic bonding between the stacked base-pairs. The planar sides of them are relatively nonpolar and thus tend to be water insoluble (hydrophobic).

This hydrophobic core of stacked base-pairs con- tributes considerable stability to DNA molecules present in the aqueous protoplasm’s of living cells.

Project Report # 4. Forms of DNA:

The DNA molecules exhibit a considerable amount of conformational flexi­bility. It can exist in A, B, C, D and Z forms (Table 4.2). B form (B-DNA) is the structure proposed by Watson & Crick and is the native conformation of DNA in solution.

It consists of a right-handed antiparallel double helix of sugar-phosphate backbone, with purine-pyrimidine base-pairs roughly perpendicular to the axis of the helix. The tilt of base-pairs of the helix is 6.3°. One turn of the helix consists of 10 base-pairs. The rise of the helix per base pair is 3.37 Å.

A form (A-DNA) has 11 base pairs. The base pairs are considerably tilted from the axis of the helix. The axial rise is 2.56Å. The helix observed under conditions of dehydration and high con­centrations of salt is wider and shorter than B- helix, the distinction between the major and minor grooves are reduced.

C form (C-DNA) results by reduction of hydration of the B form below 66% with excess of salt still present. The size of helix of C form DNA is greater than Å type of DNA but is sma­ller than B-DNA. It is about 31 Å. There are 9.33 base pairs per turn. The axial rise of base pairs is 3.32 Å with a tilting of about 7.8°.

D form (D-DNA) and E form (E-DNA) are found rarely as extreme variants. In case of D- form there are 8 base pairs per turn of helix. An axial rise of base pairs is 3.03 Å with tilting of about 16.7°.

In case of E-form, there are 7.5 base pairs per turn of helix. Z form (Z-DNA) is an unique left-handed (Fig. 4.8) double helical form with a zig-zag (Fig. 4.9) sugar-phosphate backbone in antiparallel organization. This DNA has been called Z-DNA.

Project Report # 5. DNA as the Genetic Material:

Transformation Experiment:

Transformation experiment was initially conducted by F. Griffith in 1928 (Fig. 4.21). The injected a mixture of two strains of Pneumococcus (Diplococcus pneumo­niae) into mice. One of these two strains, S III was virulent and other strain R II was non-virulent (causing no infection).

Heat-killed virulent S III strain when injected, showed that infectivity after heat killing is lost. The mice injected with a mixture of R II (living) and S III (heat killed) died and virulent Pneumococcus could be isolated from these mice. This phenomenon was described as transformation.

O. T. Avery, C. M. Macleod and M. McCarthy repeated Griffith’s experiment in an in vitro system in order to identify the transforming prin­ciple responsible for converting non-virulent into virulent type and reported their results in 1944 (Fig. 4.22). Virulence in Pneumococcus depends on a polysaccharide capsule which is present in viru­lent strain S III and is absent in non-virulent strain R II.

The cells of non-capsulated type – Rll were treated with an extract of DNA from capsulated strain S III. A few cells of S III type could be iso­lated from the mixture.

This phenomenon of transferring characters of one strain to another by using a DNA extract of the former is called transformation. When the extract was treated with DNAse (an enzyme which destroys DNA) this transforming ability was lost. Proteases (enzymes which destroy pro­teins) did not affect the transforming ability. These experiments thus indicated that DNA and not the protein, is the genetic material.

Hershey-Chase Experiment:

Additional direct evidence indicating that DNA is the gene­tic material was published in 1952 by A. D. Hershey and M. Chase. The experiment showed that genetic information of a particular bacterial virus (bacteriophage T2) was present in DNA. Bacteriophage T2 infects the common colon bacillus E. coli (Fig. 4.23).

The basis for Hershey-Chase experiment is that DNA contains phosphorus but no sulphur, whereas proteins contain sulphur but no phos­phorus.

Thus, Hershey and Chase were able to specifically label either (1) the phage DNA by growth in a medium containing radioactive iso­tope of phosphorus 32 P, in place of the normal isotope 31 P, or (2) phage protein coats by growth in a medium containing radioactive sulphur 35 S, in place of the normal isotope 35 S (Fig. 4.24).

When T2 phage particles labelled with 35 S were mixed with E. coli cells for a few minutes and were then subjected to shearing forces by placing the infected cells in a warring blender, it was found that most of the radioactivity (and thus the proteins) could be removed from the cells without affecting progeny phage production.

When T2 phage in which DNA was labelled with 32 P were used, essentially all radioactivity was found inside the cells, that is, it was not subjec­ted to removal by shearing in a blender (Fig. 4.25).

The sheared off phage-coats were separated from the infected cells by low-speed centrifugation which pellets (sediments) cells while leaving phage particles suspended. The results indicated that DNA of the virus enters the host cell, where­as protein coat remains outside the cell.

Since progeny viruses are produced inside the cell, Flershy and Chase’s results indicated that the genetic information directing the synthesis of both the DNA molecules and protein coats of the progeny viruses must be present in the parental DNA. Moreover, progeny particles were shown to contain some of the 32 P, but none 35 S.

Project Report # 6. DNA Content:

Large variation in DNA content among species of the same genus as well as among genera of the same family has been observed. This wide range of variation in the nuclear DNA contents among species within a genus may be partly attributed to differences in chromosome numbers.

However, species at the same ploidy level and with the same chromosome number may either show little or ‘no variation (e.g., Hordeum, Avena, etc.) or may exhibit many-fold differences (e.g., Lathyrus, Vicia, Helianthus, Crepis, Allium, etc.). This inter specific variation may be continuous or discontinuous.

Intra- specific variation in DNA content has also been reported in recent years, so that the variation of DNA among genotypes is a rule rather than an exception. This variation has sometimes been used to explain mechanism of evolution of spe­cific groups.

In general, the huge difference in DNA amount not necessarily correlated with the nature and status of the organism, is principally due to the repetitive DNA sequences present in higher amount. In the plant system, nearly 70- 80% of the DNA in the cereals is repetitive and only a fraction of the DNA controls the structural genes for qualitative characters.

In fact, the com­parison of the genomes of different plant species ranging from algae to angiosperms show marked variation in the C value.

The haploid un-replicated DNA content of an individual is described as its C value. C value is nearly of 600-fold difference within the angiosperms alone, ranging from 0.2 pg in the crucifer – Arabidopsis thaliana to 127pg in Fritillaria assavrica, a liliaceous species. This paradoxical situation in C value termed as C value paradox is principally attri­buted to repetitive DNA content.

In human, of the 3 billion base pairs which constitute the entire genome, only about 50000 genes are supposed to be present. More than 40% of the DNA is highly repetitive. In general, only a fraction of the total amount of DNA codes for structural proteins and enzymes, the rest are repeats – noncoding or coding for non-specific-effect.

Project Report # 7. Unique and Repetitive DNA:

The chromo­somes of prokaryotes contain DNA molecule with unique (non-repeated) base-pair sequences, i.e., each gene which is a linear sequence of few thousand base-pairs, present only once in the genome. If prokaryotic chromosomes are broken into many short fragments, each fragment will contain a different sequence of base-pairs.

In higher organisms on the other hand, the unique DNA sequences which are principally responsible for qualitative characters are present in much lower amount than that of the repetitive sequences. Such unique sequences are present in the chromosomes of higher organism in between repetitive sequences, controlling enzyme-protein.

The chromosomes of eukaryotes in general are very complex. Certain base sequences are repeated many times in the haploid chromosome complement, sometimes as many as million times. DNA containing such repeated sequences, called repetitive DNA, often representing a major component of the eukaryotic genome.

Types of Repetitive DNA:

The discovery of multiple copies of similar DNA sequences in chromosomes noted by Crick is a major event in the study of chro­mosome research. These repeats may be highly homogeneous, as in satellite DNA sequences in Xenopus, or may be moderate or minor in nature.

These sequences may also be inverted as in palindromes. In the chromosome structure, the highly homogeneous repeats may be tandem located in one locus in cluster form, whereas minor or moderate repeats may be interspersed located in intercalary positions or terminal. In tandem repeats, each sequence arranged adja­cent to the other forming monomeric unit.

Tandem repeats are of two types — similar repeats and complex combinations of different repeats, interspersed repeats are highly scattered, i.e., dispersed throughout the genome along with other sequences. Dispersed repeats include mobile elements such as long interspersed nucleotide element (LINE), short interspersed nucleotide element (SINE), long terminal repeats (LTR).

Repeats may be microsatellites (simple sequence repeats), minisatellites, satellites.

Size of Repetitive DNA:

The length of repetitive sequences may vary from simple sequence repeats of di-, tri-, tetra- or hexanucleotides to nucleosome repeats of 180 bp and up to 10000 bp or more in rDNA repeats.

Amount of Repetitive DNA:

The demonstration of the repeated sequences accounts, to a great extent, for the huge amount of DNA, noted in the different organisms. In higher organisms only little amount of the DNA represents structural genes contai­ning unique, the rest being amplification of non-coding sequences or repeats.

In the biologi­cal system, as a whole, the nuclear DNA content varies amongst the organisms without any con­comitant increase in the number and structure of genes.

The importance of repetitive DNA sequences can be judged from the very fact that in the human system almost 40% of DNA is repetitive in nature. In several plant species, as a rule 72-75% of DNA is repetitive whereas in Drosophila only 50% constitutes the sequences.

Location of Repetitive DNA:

Repetitive DNA sequences are present throughout the chromosomes including in centromere and telomeres (also may be pericentromeric and sub-telomeric), introns, nucleo­lar organizing regions, segments of heterochro­matin.

In general, in the entire chromosome structure, normally highly repeated or homo­geneous repeats are located in one locus, e.g., secondary constriction or centromere and mode­rate, minor repeats are interspersed throughout. Highly homogeneous repeats have been located in ribosomal RNA (5S, 18S, 5.8S, 25S) gene loci and are mostly AT-rich.

A characteristic repeat (GGGGATT) found at the telomeres. Accessory (B) chromosomes which are heterochromatic in nature, rich in highly repeated sequences. Large portion of cereal genome is with interspersion of short repeats. Presence of repetitive sequence in introns, in transposons and in flanking regions of replicon has been noted.

Origin of Repetitive DNA:

The mechanisms suggested for origin of repeated sequences include salutatory replication, unequal crossing over, transposition including insertion. Repeated sequences are sus­ceptible to change resulting from amplification, translocation, deletion and mutation leading to novel genomic configuration. Sequences in new environments may undergo patterning and amplification, leading to new repeat families.

Functions of Repetitive DNA:

Several non-specific functions involving cell-nuclear size and volume, chromo­some cycle, generation time, duration of meiosis, chromatin folding have often been attributed to repeated sequences.

Role of interspersed repeats has been sug­gested for repair synthesis, regulation of chromo­some structure in folding during pairing, acting as initiation points in replication, gene expre­ssion through methylation, gene conversion and compression.

Function attributed to palindromic repeats at different levels of protein synthesis includes recognition systems, both at DNA and RNA levels, involving deletion and translocation, cleavage sites, termination of transcription, bind­ing of regulatory proteins and attachment of chromosomes with each other for information transfer.

Intron repeats facilitates alternate splicing or reshuffling of exons and inter-genic conversion, permit dispersion and promote genetic diversity through mobility. Accessory chromosome repeats in plants are associated with adaptation in different environmental set up.

Significance of Repetitive DNA:

Some of the simple sequence repeats can serve as good markers. Certain tandem repeated sequences may be unique to a particular species. Their distribution and copy number help in differentiating various linkage groups in the chromosome complement.

DNA fingerprinting and molecular hybridization involving repeats have immense potential in documentation and analysis of biodiversity, and tracing the trends in evolution. The sequence, location and frequency of short tandem repeats (STR), which is common in plants’ genome, have role in determining phylogenetic status of species.

Detection of Repeats Tm and Cot Value:

For detection of repetitive DNA, the double stranded DNA is first denatured by heating into single stranded DNA which is accompanied with increase in optical density (hyperchromicity).

The single stranded DNA is then allowed to cool slowly to cause re-association between the com­plementary sequences into double stranded DNA which accompanies decrease in optical density (hypochromicity). Tm is the temperature at which 50% re-association is achieved.

The formation of double stranded DNA is actually measured over different values of a parameter which is described as Cot value (conc. x time). If solution of different DNA concentrations is to be compared, same Cot value can be achieved by altering the time allowed for re-association.

For DNA sample with high proportion of repeti­tive DNA, re-association will be faster and higher degree of re-association achieved at lower Cot values.

Localization of Repeats:

For localization of repetitive DNA, chromosome banding and molecular in situ hybridization (ISH) techniques are being applied.

Differential banding patterns of chromosomes, usually observed at specific regions on particular levels, were initially developed for the analysis of human chromo­some segments.

These bands are made visible through low and high intensity regions under the fluorescence microscope or as differentially stained areas under the light microscope. The methods were then extended first to different ani­mals and later to plant chromosomes.

The protocol for molecular hybridization, that is, denaturation at the cytological level, if followed by renaturation and staining with diffe­rent dyes, particularly Giemsa, gives intensely positive reaction at similar segments of chromo­somes which otherwise show repetitive DNA.

Obviously such treatment is capable of revealing repetitive segments in chromosomes. This ban­ding, following denaturation-renaturation and Giemsa staining, is termed G-banding.

Earlier, Caspersson and his colleagues had recorded differential fluorescence of different chromosome segments following staining with various fluorochromes (quinacrine) and observa­tion under the ultraviolet microscope and had successfully employed it in formulating a band­ing pattern analysis of the human karyotype.

Such bands were referred to as Q-bands. Other fluorochromes produce banding patterns as well, e.g., Hoechst 33258 similar to Q and ethidium bromide the reverse.

These banding patterns are unique for each chromosome like fingerprints. The bands are generally consistent for a taxon except for minor variations. A number of other chemicals can also produce bands either identical with or different from the fluorescent ones, based on different principles.

Such differential banding patterns, usually observed at specific regions on particular chromosomes, are being increasingly used for the identification of chromosomes.

The chromosome band nomenclature, adopted at the Paris Conference in 1971, recog­nized the following types of banding in human chromosomes (Fig. 4.26A):

By quinacrine staining and fluores­cence.

By staining techniques using Giemsa and related stains after appropriate pre- treatment.

R-bands or banding reverse to Q-bands:

By staining with Giemsa after heating to 87°C.

For constitutive heterochromatin, demonstrated by the denaturation-re-association technique.

Produced by enzymic digestion, as classified by Lejeune (1973), show woolen and shrunken regions corresponding to the dark and faint regions of G-banding. The same nomenclature can be applied to banding patterns observed in other eukaryotic chromosomes.

Other banding patterns of chromosomes include:

The centromeric and telomeric segments show bands after treatment in barium hydroxide, incubation and staining in ‘Stains All’ (4, 5, 4′, 5′-dibenzo-3, 3′-diethyl-9-methyl-thi- acarbocyanine bromide).

At nucleolus-organizing regions, possibly due to acidic proteins.

With orcein staining, mainly for plant chromosomes both intercalary and centro­meric heterochromatin show bands. All the above methods of banding bring out clearly the differentiation of chromosome seg­ments which can easily be analysed under the microscope. This technique permits identifica­tion of chromosome segments with specific molecular complexity such as repeat sequence.

The comparison of banding pattern between different genotypes, altered and unaltered, can localize the segments which have undergone alterations. The importance of banding can be judged by the very fact that, R-banding that is the reverse banding has been utilized for mapping of gene sequences in human chromosome.

In Situ Hybridization (ISH: FISH & GISH):

Besides the chromosome banding, for localiza­tion and mapping of different segments of chro­mosomes and gene loci at the microscopic level, application of molecular hybridization in situ is now widely adopted (Fig. 4.26B).

In situ hybri­dization (ISH) principally uses probe sequences, tagged with radioisotopes or fluorescent com­pounds (or a chemical reporter). The initial step is denaturation of the target which is followed by hybridization with probe of the complementary sequences to undergo re-annealing or pairing.

The complementary sequences of the probe bind selectively at the target site. The hybridized sites are localized either through autoradiography or immune-fluorescence as well as counter staining with specific stains detected cytologically. The in situ hybridization technique was developed ini­tially by Pardue and Gall and later modified by different authors.

The fluorescent in situ hybridization (FISH) is the most powerful technique at present, through which the target loci at the chromosome is hybridized with complementary probe sequence, tagged with fluorescent compound. There are two approaches, namely direct or indi­rect, of FISH technique in plant chromosome.

In the indirect method, the probes are tagged with reporter molecule, such as biotin, digoxigenin and finally they are located by fluorochrome conjugated antibodies such as avidin.

The main principle of this method is to make the probe- target sequence as antigenic so that, it can be detected through antibody. The common fluo­rochromes are FITC (fluorescein-isothiocyanate) as well as rhodamine. In the direct method, the probe is directly labelled by fluorochrome- labelled antibodies. The direct labelling of fluorochrome with the probe is the rapid method of ensuring good resolution.

Due to lack of karyomorphological markers, metaphase chromosome analysis cannot distin­guish parental genomes in hybrids. If ISH tech­nique is applied with total genomic probes where the plant has multi-genomic constitution, the parental chromosome can be directly identi­fied in the hybrids, such as Triticum and Secale in Triticale.

Thus method of total genome in situ hybridization is otherwise termed as GISH (genomic in situ hybridization) technique.

Since its first demonstration in identification of parental genomes in hybrid between Hordeum chilense and Secale africanum by Schwarzacher et al., the technique has been extensively applied to elucidate ancestry of hybrids and polyploids. GISH remains a very effective tool in genome identification, their orientation and in establishing genomic relationships between species.

The use of GISH in meiosis helps in understanding inter-genomic homologies as well as in elucidating the possible transfer of chromo­some segments through inter-genomic recombi­nation.

The FISH technique, using different colour combinations by different probes, is now being applied to detect simultaneously different genomes, or chromosome segments by extension of the technique – otherwise termed as multi­-colour FISH. This method has been used to dis­tinguish three genomes in hexaploid wheat and to detect several translocation sites and insertions in polyploid species of Triticum and Aegilops.

The multi-colour FISH technique is now regarded as a powerful tool for gene mapping as well as detection of abnormalities, including insertion and breakage points with chromosome specific or genome specific dispersed probes. The term chromosome painting is used in FISH technique where chromosome specific dispersed probes are used to detect the location of comple­mentary target sequences in the complement.

The FISH technique in recent years has further been modified to locate single copy or tandem sequences, utilizing primer mediated extension and amplification in situ by PGR method. The application of this method lies also in confirma­tion of location of foreign gene at the chromo­some level in transgenic individuals.

Author Affiliations

From the Departments of Neuropediatrics (M.S., C.H.), Pediatric Radiology (T.R.), and Neonatology (W.K.), Charité, University Medical Center Berlin, Berlin the Departments of Neurology (K.R.W.) and Molecular Biology and Genetics (S.-J.L.), Johns Hopkins University School of Medicine, Baltimore the Department of Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge, Mass. (L.E.S., J.F.T.) and the Institute of Physiological Chemistry, Martin Luther University, Halle-Wittenberg, Germany (T.B.).

Address reprint requests to Dr. Schuelke at the Department of Neuropediatrics, Charité, University Medical Center Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany, or at [email protected] .

Scientists find gene mutation linked to exfoliation syndrome, most common cause of glaucoma

A team of researchers from the Agency for Science, Technology and Research's (A*STAR) Genome Institute of Singapore (GIS) and Bioprocessing Technology Institute (BTI), as well as Singapore Eye Research Institute (SERI), have identified a genetic mutation (functionally defective CYP39A1 gene) associated with exfoliation syndrome, the most common cause of glaucoma. The findings could pave the way for future research on the cause of exfoliation syndrome and potential cures. Their research was published in Journal of the American Medical Association (JAMA) on 24 February 2021.

Exfoliation syndrome is a systemic disorder characterised by abnormal protein material that progressively accumulates in the front of the eye. This disorder is the most common cause of glaucoma, and a major cause of irreversible blindness.

In this study, the scientists sequenced all protein encoding genes of more than 20,000 participants from 14 countries across Asia, Europe, and Africa, including more than 1,200 Singaporeans. They observed that people with exfoliation syndrome are twice as likely to carry damaging mutations in the gene encoding for the CYP39A1 protein, an enzyme which plays an important role in the processing of cholesterol. Further extended analyses suggest that defective CYP39A1 function is strongly associated with increased risk of exfoliation syndrome.

Although exfoliation syndrome is the most common cause of glaucoma, its origin is shrouded in mystery because it is not known where the abnormal protein deposits (exfoliative material) originate, and how the disease comes about. Answers to these questions could provide approaches to design and develop an effective treatment. The current findings point to the important role of cholesterol processing in the exfoliation syndrome disease process. As cholesterol is found abundantly in all cells, disruption to how cholesterol is processed due to defective CYP39A1 activity could adversely impact their normal functions. In particular, this study discovered that epithelial cells in the front of the eye responsible for filtering the blood supply to produce the clear fluid known as aqueous humour that bathes and nourishes other cells in the eye, were most affected by the CYP39A1 gene mutation. Disruption to the gene function can compromise the filtering function of epithelial cells and lead to leakage of exfoliative material from the blood into the eye.

Prof Patrick Tan, Executive Director of GIS, said, "This is a ground-breaking study that could facilitate future research efforts aimed at restoring defective CYP39A1 function and inhibiting the formation of exfoliation material in the eye as treatments for exfoliation syndrome and glaucoma."

Prof Aung Tin, Director of SERI and Deputy Medical Director of SNEC, said, "This is a major eye disease, affecting over 70 million people worldwide, which causes a lot of visual morbidity and blindness, not only from glaucoma but also due to complications related to cataract surgery. This study was notable for involving many centres from many different countries around the world, but led from Singapore. The study findings are very exciting as we found a new pathway for the disease which opens up possibilities for new treatments."

Prof David Friedman, the Albert and Diane Kaneb Chair in Ophthalmology at Harvard University and Director of the Glaucoma Service at the Massachusetts Eye and Ear Infirmary, Boston, commented, "Very exciting work. The researchers have identified rare gene variants that results in disrupted cholesterol homeostasis and transport that will open the door to novel therapeutics. Having studied over 20,000 individuals, the study demonstrates the power of studying rare variants to detect disease-causing genes in complex conditions." Prof Friedman was not involved in the study.