Friday 12 June 2009

Microhomology-mediated End Joining

So I wrote a new article on Wikipedia. It's a little brief but with some collaboration from other internet users it will get better in time.

Microhomology-mediated End Joining

Microhomology-mediated End Joining (MMEJ) is one of the pathways for repairing double-strand breaks in DNA. Two other well known means of double-strand breakage repair are Non-homologous end joining (NHEJ) and Homologous recombination (HR). What distinguishes MMEJ from the other repair mechanisms is the use of 5 - 25 base pair microhomologous sequences to align the broken strands before joining, MMEJ repair is different to NHEJ because it uses a Ku protein and DNA-PK independent repair mechanism and repair occurs during the S phase of the cell cycle as a pose to the G0/G1 and early S phases in NHEJ and late S to G2 phase in HR.

MMEJ works by ligating the mismatched hanging strands of DNA, removing overhanging nucleotides and filling in the missing base pairs. When a break occurs a homology of 5 - 25 complimentary base pairs on both strands is identified and used as a basis for which to align the strands with mismatched ends. Once aligned, any overhanging bases (flaps) and mismatched bases on the strands are removed and any missing nucleotides are inserted. As this method's only way of identifying if the two strands are related is based on microhomology down/up-stream from the site of breakage, it does not identify any missing base pairs which may have been lost during the break and even removes nucleotides (flaps) in order to ligate the strand. MMEJ ligates the DNA strands without checking for consistency and causes deletions since it removes base pairs (flaps) on the strand in order to align the two pieces.

MMEJ is an error-prone method of repair and results in deletion mutations in the genetic code which may initiate the creation of oncogenes that could lead to the development of cancer. In most cases a cell uses MMEJ only when the NHEJ method is unavailable or unsuitable due to the disadvantage posed by introducing deletions into the genetic code.

References

1. http://www.cell.com/trends/genetics/abstract/S0168-9525(08)00229-1 MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings
2. http://nar.oxfordjournals.org/cgi/content/abstract/32/17/5249 DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated end-joining
3. http://dx.doi.org/10.1016/j.mrfmmm.2007.08.016 Distinctive differences in DNA double-strand break repair between normal urothelial and urothelial carcinoma cells

Tuesday 2 June 2009

Green-Glowing Marmoset Monkeys

Genetically modified primates that glow green and pass the trait on to their offspring could aid the fight against human disease.

Though primates that make a glowing protein have been created before, these are the first to keep the change in their bloodlines.

Future modifications could lead to treatments for a range of diseases.

The "transgenic" marmosets, created by a Japanese team, have been described in the journal Nature.

The work raises a number of ethical questions about deliberately exposing a bloodline of animals to such diseases.

Scientists have managed to modify the genes of many living organisms in recent years, ranging from bacteria to mice.

Mice have been particularly useful experimental models for studying a wide range of human diseases as modified genes are passed on from parents to progeny.

However, mice are not useful for some human diseases because they are not sufficiently similar to produce effects that are meaningful to human disease. Studies of mice with Alzheimer's disease, for example, were stymied simply because their brains were too small to scan at sufficient resolution.

Jellyfish gene

Now, Erika Sasaki of the Central Institute for Experimental Animals in Japan, and her colleagues, have introduced a gene into marmoset embryos that allows them to build green fluorescent protein (GFP) in their tissues.

The protein is so-called because it glows green in a process known as fluorescence.

GFP was originally isolated from the jellyfish Aequorea victoria, which glows green when exposed to blue light.

The protein has become a standard in biology and genetic engineering, and its discovery even warranted a Nobel prize.

Glowing mouse
Glowing mice have already been created in the lab

From 91 embryos, a total of five GFP-enabled transgenic marmosets were born, including twins Kei and Kou ("keikou" is Japanese for "fluorescence").

Crucially, the team was able to show that their method is maintained in the family - or germline.

They used the sperm from a member of the first generation of transgenic marmosets to fertilise an egg from a normal animal. A significant proportion of the resulting offspring also glowed under ultraviolet light.

Until now, efforts to establish transgenic lines of primates have been unsuccessful. In 2001, a team at the Oregon Regional Primate Research Center, US, succeeded in creating a rhesus macaque that expressed GFP.

Last year, a team at Yerkes National Primate Research Center, Atlanta, US, created rhesus macaque monkeys with Huntington's disease. Four of those are still awaiting puberty, and the researchers hope that they will produce a second generation of macaques with the disease.

Fitting in

The new method improves on previous work using so-called "retroviruses".

These virus "vectors" were added to a soup of sugary solution and this was in turn injected into the monkey embryos.

Although the work demonstrates the principle that a gene can be introduced into a primate bloodline, study co-author Hideyuki Okano of the Keio University School of Medicine said it may not be suitable for studying all diseases.

"We can just introduce genes by virus vectors," he told BBC News, "so the limitation comes from the sizes of genes that can be carried by the retroviruses."

That limitation is about 10,000 bases, or letters, of the genetic code. That upper bound will constrain the diseases that can be studied.

Genes implicated in Parkinson's disease and amyotrophic lateral sclerosis (ALS, a form of motor neurone disease) may well be suitable.

However, genetic regions implicated in Huntington's disease might not fit into a retrovirus.

GM marmosets
Two of the first transgenic marmosets did their own genetic trick: they are twins

The work has raised a number of ethical questions about the use of primates in disease research.

Marmosets are New World monkeys and therefore more distantly related to humans than, for example, chimpanzees. But they are particularly useful for the study of disease because they reproduce often and from a young age.

Jarrod Bailey, science consultant to the British Union for the Abolition of Vivisection (BUAV), says he is "disappointed" both ethically and scientifically with the results.

"This sort of research on animals as sentient as monkeys and apes does not have public support," he told BBC News.

Furthermore, he thinks the underlying science is flawed. Animal researchers, he said, "have failed in research to find treatments for Aids, for hepatitis, for malaria, for strokes. Many treatments for strokes work in monkeys but don't work in humans."

"Monkeys do not predict human response and do not tell us about human disease," he added.

However, scientists argue that, because primates are more similar to humans than mice, they present a more refined model of human disease. This would allow scientists to test treatments more effectively, meaning that fewer animals need be experimented on in the long run.

"This experiment is reminiscent of the exciting early days of transgenic research where it was initially difficult to fully know what the potential applications and future research directions might be," said Mark Hill, a cell biologist at the University of New South Wales in Australia.

"As always in this area of research, there needs to be a close linkage between the scientific work, ethical issues and regulatory guidelines."

http://news.bbc.co.uk/1/hi/sci/tech/8070252.stm

Sequencing of Mouse Genome completed

Scientists have finished sequencing the mouse genome after a 10-year effort.

The humble mouse is the experimental workhorse in laboratories worldwide, so this high-quality genome sequence will aid in the fight against human disease.

The search for novel treatments could benefit from a greater understanding of the mouse genetic code, which is about 75% similar to our own.

An international team of researchers have published details of the work in the open-access journal PLoS Biology.

The sequence comprises the full complement of genetic material in the nucleus of a cell. It is effectively the genetic "instruction booklet" for a living animal.

The mouse (Mus musculus) becomes only the second mammal after humans to have its complete genome laid bare.

But draft sequences have been published for the chimp, dog, rat, cat, macaque and even the duck-billed platypus

The mouse is the animal most often used to better understand human illnesses and how they develop.

Research carried out using mice has led to advances in the treatment of cancer, diabetes, heart disease and countless other conditions.

Good model

Co-author Professor Chris Ponting, from the University of Oxford, told BBC News the work confirmed that the mouse was an excellent experimental model for human disease.

"Completion of the genome is extremely important in helping us to identify the genes that underpin biology that is the same across all mammals," he said.

But he said it was also important to separate the genes humans shared with mice from those which differed between them.

About 75% of mouse genes have a single equivalent in humans. But some 5,000 genes arose after the ancestors of mice and humans went their separate evolutionary ways.

"In retrospect, our previous picture of the mouse genome was incomplete," said Dr Leo Goodstadt from the University of Oxford.

"Only when all the missing pieces of the genomic puzzle had been filled in did we realise that we had been missing large numbers of genes found only in mice, and not in humans."

The mouse genome sequencing effort began in 1999, and a draft sequence was published in 2002.

The cost, borne by US and UK sequencing centres, is estimated to exceed $100m (£62m).

Some groups oppose animal experimentation, campaigning to ban or limit the animals used.

In the UK, growth in the use of genetically modified (GM) animals - mainly mice - is largely responsible for a steady rise in the numbers of animals used in experiments since 1997.

Professor Ponting, from the Medical Research Council's (MRC) Functional Genomics Unit at Oxford, said the complete genome could provide insights into the evolution of mammals.

Humans and mice share a remarkable level of similarity, despite having evolved independently for the last 90 million years.

http://news.bbc.co.uk/1/hi/sci/tech/8069235.stm