Finding a Gene on the Chromosome Map

As a scientist, you've noticed that a genetic disorder runs in families, and you want to find the gene responsible for it.

First, you identify a large family, in which some individuals have the disorder, and others don't. After enlisting the family's support and collecting DNA samples from all family members, you're ready to begin looking for the gene. Where do you go from here?

Here's one way to think about genes

What if the genetic information in each family member were like a jigsaw puzzle? Each puzzle piece would represent a set of genes organized in a specific way, similar to a chromosome. Because all humans have the same set of genes, arranged in the same order, every family member would have the same basic set of puzzle pieces. A generic human jigsaw puzzle might look like the picture at the right

But the information carried in genes differs slightly from person to person. This is what makes each of us unique. As a result, the colors of the puzzle pieces would be different between family members. While some relatives might share puzzle pieces of a certain color, other pieces would be different. Only identical twins share the exact same combination of colors and shapes.

What might a family's puzzles look like?

Look at the family of jigsaw puzzles below. Can you see how some of the child's genes are derived from one parent and some from the other parent?

The child receives exactly half of its genetic information from the mother and exactly half from the father.

Looking at things this way, can you see how you might identify a genetic link in paternity suits, where a genetic connection is sought between a child and a possible father? Half of the child's puzzle pieces must be the same as the father's.

http://learn.genetics.utah.edu/units/disorders/pedigree/

A Little Telomerase Isn’t Enough

Study Links Length Of Chromosome Ends To A Rare Disease Of Stem Cells

Chromosome ends, or telomeres, are repetitive stretches of DNA that protect chromosomes in much the same way as plastic tips on shoelaces prevent the fabric from fraying. Each time a cell divides, its chromosome ends get a little shorter, and eventually the cell can no longer divide because its critical genetic information is exposed. In stem cells, however, a protein called telomerase normally maintains the telomeres’ length, allowing the cells to divide indefinitely.

Now, the Hopkins researchers report that mice engineered to have just half the normal amount of telomerase can’t maintain their stem cells’ chromosome ends, showing that a little telomerase isn’t enough. In these “half-telomerase” mice, their telomeres shortened over time, bringing an early demise to stem cells that replenish the blood supply, immune system and intestine, the researchers report. Moreover, offspring of these mice bred to have normal levels of telomerase still exhibited early loss of stem cells, the researchers report in the Dec. 16 issue of Cell.

“These offspring have what we have called ‘occult’ genetic disease — their genetic make-up is perfectly normal, but they still have the physical problems of their parents,” says Carol Greider, Ph.D., director and professor of molecular biology and genetics in the Johns Hopkins Institute of Basic Biomedical Sciences. “This phenomenon could complicate the hunt for disease genes.”

Scientists generally figure that inherited disease accompanies an inherited mutation in one or more genes. In the case of the genetically normal offspring of two half-telomerase parents, however, the disease is still present. The problem in these animals turns out to be the animal’s inherited telomere length, not the status of the telomerase gene, says Greider.

“If you were to search for the genetic mutation behind this mouse’s disease, you wouldn’t find it — there isn’t one,” says Greider. “These mice develop disease only because their telomeres are short, and having telomerase doesn’t lengthen them right away.”

http://biosingularity.wordpress.com/2005/12/24/a-little-telomerase-isnt-enough-study-links-length-of-chromosome-ends-to-a-rare-disease-of-stem-cells/

Telomeres

Telomeres, together with the enzyme Telomerase are mentioned a lot lately in association with both aging and with cancer. It seems that their gradual shortening during the lifetime of a cell eventually results in damage to DNA and thus the genes of which they are a part. This has led to a great deal of research into Telomeres and Telomerase, some of which will be outlined in this section of the site. First of all however, let us get some of the basic definitions out of the way.

Definition of a Telomere

A telomere is a region of highly repetitive DNA at the end of a linear chromosome that functions as a disposable buffer. Every time linear eukaryotic chromosomes are replicated during late S-phase the DNA polymerase complex is incapable of replicating all the way to the end of the chromosome; if it were not for telomeres, this would quickly result in the loss of useful genetic information, which is needed to sustain a cell’s activities. http://en.wikipedia.org/wiki/Telomere

Definition of Telomerase

Telomerase is the reverse transcriptase responsible for the extension of telomeric repeat sequences in most species studied. If telomerase activity is diminished or absent, telomeres will shorten. Shortened telomeres appear to lead to cell senescence. Eventually telomeric sequences can shorten to the point where they are not long enough to support the telomere-protein complex protecting the ends and the chromosomes become unstable. These shortened ends become 'sticky' and promote chromosome rearrangements. Some rearrangements may contribute to the development of cancers. http://www.genlink.wustl.edu/teldb/tel.html

Role, Function and Compesition of Telomeres

In the majority of Prokaryotes, the chromosomes are circular. This means that they do not have ends that are prone to damage or ‘premature replication termination.’ Note that a small number of bacteria, Borrelia and Streptomyces for example, do possess linear chromosomes (together with circular plasmids). These chromosomes are very different however, from those which are found in Eukaryotic cells (such as those in humans.

A telomere consists of repeating base sequences (in humans this is a repeating string of TTAGGG bases between 3 to 20 kilobases in length). In addition to the telomere itself, there is a 100-300 kilobase sequence associated with the telomere, which is located between the telomere and the rest of the chromosome.

As replication of DNA occurs during the lifetime of an organism, the telomeres gradually shorten. This shortening eventually results in damage to the chromosomes themselves and to the genes that they contain. The results of this are either a reduction in the cells ability to express its genes and a descent into cellular senescence (cellular dormancy) or in some cases the over or under-expression of specific genes. When this chromosomal damage leads to either an over or under-expression of certain genes, then cellular functionality can be compromised. In some cases the organisms survival can also be compromised as cellular replication can become unchecked. These cells thus become cancerous and can lead to the death of the organism.

Role, Function and Compesition of Telomeres

During DNA replication, The DNA unzips and a complimentary strand is formed against the unzipped sections. Telomeres shorten during this process due to the ‘lagging strand’ phenomenon.

Basically DNA replication does not begin at the end of the DNA, but in the centre. DNA Helicase unzips the DNA forming Replication bubbles. An RNA Primer or Primase then attaches to each DNA strand and replication begins in the 3-5 direction, thus forming a new strand in the 5 -3 direction.

DNA polymerases move and replicate the DNA in the 3 to the 5 direction (thus making a replica strand in the 5 to 3 direction). Note that 3 refers to the 3 OH group of the sugar and the 5 refers to the 5 phosphate group of the nucleotide.

The leading strand of DNA is the strand that is oriented in the 3-5 direction. The primer of complementary molecules that binds to the first few of the exposed bases ends with a 3 sugar group. The phosphate of a new nucleotide can be attached here by DNA polymerase. DNA polymerase then continues along the strand, synthesising a new strand as it goes. An animation of this process can be seen below, provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/replication_ani.html.

The lagging strand however, faces more problems when it comes to replication. Because DNA polymerase can only attach to the 3 sugar group and thus move in the 3 to the 5 direction, it needs therefore to move in small jumps (in the opposite direction to overall replication) to replicate the lagging strand. See below animation. This is again provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/lagging_ani.html

Note that the small segments of DNA and primers which are produced on the lagging strand are called Okazaki fragments. Another DNA polymerase enzyme is then recruited to remove the primers and to replace them with DNA. Finally DNA Ligase seals the gaps between the Okazaki fragments.

DNA Replication

The problem with Telomere shortening occurs because, in order to change the RNA primers into DNA, there must be another DNA segment in front of the primer. There is a DNA segment ahead of the primer at every section of the strand, except where the last primer attaches (the end of the telomere). This means that this final primer cannot be replaced with DNA. It does get removed by various enzymes however, but in the process, the telomere shortens.

In human blood cells, the range of lengths of the telomeres varies between 8000 base pairs at birth, to 1,500 base pairs in the elderly. During cellular division, an average of 30 to 200 base pairs are removed from the ends of the telomeres.

In normal cases, the cells of a human can divide between 50 to 70 times, with the telomeres shortening with each division, until the cells either commit suicide through a process known as Apoptosis, become Senescent (dormant) or transform to cancerous cells due to genetic damage.

The process of Cellular senescence due to Telomere Shortening

As a cells telomeres shorten during multiple cellular divisions, DNA damage occurs and the cell, recognising this damage shuts itself down. Below is a simplified diagram of how this occurs.

Telomere shortening and aging

DNA damage results in an activation of the p53 gene. p53 then activates p21, which blocks the actions of a number of CDK’s (Cycline Dependent Kinases). Note that CDK’s are involved in the regulation of the cell cycle, transcription and mRNA processing. The blocking of certain CDK’s prevents the phosphorylation of pRb. This lack of hyperphosphorylated pRb results in a failure of expression of several critical genes, which are involved in cellular division. Cellular division then stops.

Telomerase and Telomere extension

A study was conducted (as so many aging studies are) using the nematode Caenorhabditis elegans (Joeng KS, Song EJ, Lee KJ, Lee J (2004). "Long lifespan in worms with long telomeric DNA". Nature Genetics 36 (6): 607-11.). This study indicates that by lengthening the Telomere, longevity can be increased.

Two distinct groups of Nematodes were engineered. The only difference between the two groups, was the length of the Telomere. The group with the longer telomere’s lived approximately 20 percent longer than the group with the shorter telomeres. Also, it was observed that the Nematode’s with the longer telomeres possessed a greater resistance to the effects of heat exposure.

Telomerase is the natural enzyme which promotes telomere repair. It is however not active in most cells. It certainly is active though in stem cells, germ cells, hair follicles and (worryingly) in 90 percent of cancer cells. Telomerase functions by adding bases to the ends of the telomeres. As a result of this telomerase activity, these cells seem to possess a kind of immortality.

In 1990 a team at Geron Corp in Menlo Park, California led by Serge Lichtsteiner and Andrea Bodnar,in association with the University of texas Southwestern Medical center, managed to activate the production of telomerase in cells that do not usually produce this enzyme. The results were that the telomeres started to lengthen. By the time that they released the results of their study, their cells had divided 20 or more times than would normally be expected before senescence would set in. These cells also seemed to retain their normal gene expression and showed no signs of becoming cancerous.

I will now try to explain in more detail the details and results of the teams study.

Basically, telomerase is present in all cell types, however the human gene for the catalytic protein telomerase transcriptase (hTRT) is present only in immortal cells (such as stem cells or cancerous cells). Scientists from the teams observed that lengthening of telomeres in retinal pigment epithelial cells, foreskin fibroblasts and vascular endothelial cells by introducing the hTRT gene into these cells, results in a resumption of telomerase activity. This resumption resulted in the extended longevity of the cultured cells, as discussed previously.

Varying Telomere decline

One interesting study by Peter Lansdorp of the Terry Fox Laboratory, Canada, observed in human fibroblasts that the rate of telomere shortening as cells divide varies between different telomeres. This variation is between 50 to 150 base pairs per cell division. It is worth noting that the telomeres that are shorter initially. For example in humans the 17p telomere, are not necessarily the ones to be destroyed first! It does seem that it is the shortening of specific telomeres that are linked to a cells decline into senescence, apoptosis or its transformation into a cancer. Martens et al, 1998, observed that the shortening of telomeres 1p, 5p and 22p, but not that of 17p, showed a statistical correlation with the descent into cellular senescence.

Despite all of the above outlined observations, it is a statement of fact that the mean telomere length of a species does not always relate to the longevity of that species. For example Katuo et al, 1999, noted that of all studied primates, humans seem to have both the shortest telomeres and the longest lifespan! Bassham et al, 1998, also observed that the long lived frog Xenopus Laevis displayed a great variation in telomere length and that telomere length could even diminish between parent and offspring, with no detectable consequences.

To summarise, it seems that it has not yet been determined whether the instability of a chromosome and its eventual deterioration is a result of general telomere shortening or the shortening of specific telomeres. It has however been found that by artificially lengthening the telomeres of cells, which do not normally have a mechanism to lengthen their own telomeres, the cells longevity and ability to function normally for longer, does increase. More research therefore needs to be conducted in order to determine the exact significance of telomere length in the aging of an organism and the exact consequences of artificially stimulating telomere repair.

http://www.whyweage.com/node/11

What is DNA?

The work of many scientists paved the way for the exploration of DNA. Way back in 1868, almost a century before the Nobel Prize was awarded to Watson, Crick and Wilkins, a young Swiss physician named Friedrich Miescher, isolated something no one had ever seen before from the nuclei of cells. He called the compound "nuclein." This is today called nucleic acid, the "NA" in DNA (deoxyribo-nucleic-acid) and RNA (ribo-nucleic-acid).

Two years earlier, the Czech monk Gregor Mendel, had finished a series of experiments with peas. His observations turned out to be closely connected to the finding of nuclein. Mendel was able to show that certain traits in the peas, such as their shape or color, were inherited in different packages. These packages are what we now call genes.

For a long time the connection between nucleic acid and genes was not known. But in 1944 the American scientist Oswald Avery managed to transfer the ability to cause disease from one strain of bacteria to another. But not only that: the previously harmless bacteria could also pass the trait along to the next generation. What Avery had moved was nucleic acid. This proved that genes were made up of nucleic acid.

Francis Crick and James Watson, 1953. Photo: Cold Spring Harbor Laboratory Archives

Maurice Wilkins.

http://nobelprize.org/educational_games/medicine/dna_double_helix/readmore.html

DNA

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.

Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.

Within cells, DNA is organized into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, and fungi) store their DNA inside the cell nucleus, while in prokaryotes (bacteria and archae) it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

The structure of part of a DNA double helix


http://en.wikipedia.org/wiki/DNA

1,000 Genomes

Gene-sequencing projects keep getting bigger.

In a testament to the steady plummet in sequencing costs, today the National Human Genome Research Institute (NHGRI) announced a massive international collaboration to sequence the genomes of 1,000 people from around the world.

According to the NHGRI statement,

"The 1000 Genomes Project will examine the human genome at a level of detail that no one has done before," said Richard Durbin, Ph.D., of the Wellcome Trust Sanger Institute, who is co-chair of the consortium. "Such a project would have been unthinkable only two years ago. Today, thanks to amazing strides in sequencing technology, bioinformatics and population genomics, it is now within our grasp. So we are moving forward to build a tool that will greatly expand and further accelerate efforts to find more of the genetic factors involved in human health and disease."

The findings should give added power to the recent wave of studies identifying specific genetic risk factors for common health problems, such as diabetes, heart disease, lupus, and others. (See "Genes for Several Common Diseases Found.")

According to NHGRI director Francis Collins,

"This new project will increase the sensitivity of disease discovery efforts across the genome five-fold and within gene regions at least 10-fold. Our existing databases do a reasonably good job of cataloging variations found in at least 10 percent of a population. By harnessing the power of new sequencing technologies and novel computational methods, we hope to give biomedical researchers a genome-wide map of variation down to the 1 percent level. This will change the way we carry out studies of genetic disease."

Like previous international sequencing projects, the data will be made available for analysis in free public databases. Once scientists identify part of the genome associated with a particular disease, they will be able to look up that area of the genome in the database to find a list of gene variants in that region.

The project will be a huge technological feat; to date, only three human genomes have been sequenced.

From NHGRI:

The project depends on large-scale implementation of several new sequencing platforms. Using standard DNA sequencing technologies, the effort would likely cost more than $500 million. However, leaders of the 1000 Genomes Project expect the costs to be far lower--in the range of $30 million to $50 million--because of the project's pioneering efforts to use new sequencing technologies in the most efficient and cost-effective manner.

In the first phase of the 1000 Genomes Project, lasting about a year, researchers will conduct three pilots. The results of the pilots will be used to decide how to most efficiently and cost effectively produce the project's detailed map of human genetic variation.

The first pilot will involve sequencing the genomes of two nuclear families (both parents and an adult child) at deep coverage that averages 20 passes of each genome. This will provide a comprehensive dataset from six people that will help the project figure out how to identify variants using the new sequencing platforms, and serve as a basis for comparison for other parts of the effort.

The second pilot will involve sequencing the genomes of 180 people at low coverage that averages two passes of each genome. This will test the ability to use low-coverage data from new sequencing platforms to identify sequence variants and to put them in their genomic context.

The third pilot will involve sequencing the coding regions, called exons, of about 1,000 genes in about 1,000 people. This is aimed at exploring how best to obtain an even more detailed catalog in the approximately 2 percent of the genome that is comprised of protein-coding genes.

During its two-year production phase, the 1000 Genomes Project will deliver sequence data at an average rate of about 8.2 billion bases per day, the equivalent of more than two human genomes every 24 hours. The volume of data--and the interpretation of those data--will pose a major challenge for leading experts in the fields of bioinformatics and statistical genetics.

The 1,000 volunteers will be selected from those who participated in the HapMap project, a map of common genetic variation (see "A New Map for Health"), and will include:

Yoruba in Ibadan, Nigeria; Japanese in Tokyo; Chinese in Beijing; Utah residents with ancestry from northern and western Europe; Luhya in Webuye, Kenya; Maasai in Kinyawa, Kenya; Toscani in Italy; Gujarati Indians in Houston; Chinese in metropolitan Denver; people of Mexican ancestry in Los Angeles; and people of African ancestry in the southwestern United States.

http://www.technologyreview.com/blog/editors/22007/