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The MLGS funds a Research Laboratory at the hospital. The results of the research at Musgrove are published in international journals, increasing worldwide knowledge of the leukaemic process. Having this research based locally has the additional advantage of increasing the range of tests available to consultants at Musgrove to help guide them with the treatment of current patients with all types of leukaemia and lymphoma. A brief description of the current leukaemia research project funded by MLGS Blood is made up of many different types of cells all designed to carry out a particular function. Red blood cells carry oxygen around the body and platelets help with blood clotting. The white blood cells or leucocytes can be divided into 3 main groups, granulocytes, monocytes and lymphocytes. The Blymphocytes and T-lymphocytes are involved with fighting infections. During a person's lifetime many billions of these blood cells are produced to replace cells that have reached the end of their lifespan.In fact 3 million red blood cells and 120,000 white blood cells are produced every second. All these different blood cells come from very rare primitive cells called 'stem cells'. These stem cells are not only capable of maturing into the different types of blood cells but are also capable of 'self-renewal' and are therefore able to remain as stem cells. In healthy individuals the numbers and distribution of the different types of blood cells shows very little variation. This would indicate that the rate at which new blood cells are produced and old blood cells are destroyed is under tight regulation. However, if someone has leukaemia, this regulation has broken down and too many cells of one type are present. If the abnormal cells are immature the leukaemia will be an acute form such as Acute Myeloid Leukaemia, whereas if the cells are mature the leukaemia will be a chronic form such as Chronic Lymphocytic Leukaemia. A hallmark of all cancers, including leukaemia, is the capacity for cells to undergo unlimited self-renewal, like stem cells. It has been proposed that either leukaemias could arise from 'faulty' blood stem cells or alternatively

from a more mature, differentiated blood cell that has re-acquired stem cell characteristics. The scientists at Musgrove Park Hospital are currently investigating the possibility that in Chronic Lymphocytic Leukaemia one of the genetic pathways involved in self-renewal of stem cells has been 'switched back on' in mature B-lymphocytes. The factors that control the process of stem cell self-renewal are still being discovered but some of the key elements are already known. We are looking at these elements in leukaemic cells and comparing them to normal blood cells. A pilot study funded by the MLGS in 2004/05 has already yielded some interesting results in this area and the Research Laboratory is continuing this work in 2005/06 in order to gain further insight into the regulation of this important pathway in low-grade B cell malignancies. The Research Laboratory has now taken delivery of a Polymerase Chain Reaction (PCR) machine, funded by the MLGS. Polymerase Chain Reaction is a rapid way to identify leukaemia and lymphoma cells in patients. Real Time PCR uses fluorescent dye chemistry to accurately monitor the reaction in its early stages. Real Time PCR assesses the level of the disease still present after chemotherapy and the technology allows a greater sensitivity of detection and the ability to monitor whether the level of the leukaemic clone is increasing or decreasing. Real Time PCR will greatly help the service provided to patients at Musgrove Park Hospital.

Exterior of the Claire Milroy Leukaemia Research Building funded by the MLGS

Interior view of part of the Research Laboratory

PCR Machine in use in the Research Laboratory

Publications
Howe D, Hopkins J, Johnson S and Phillips M (1993). Simultaneous analysis of cell surface antigens and DNA content by flow cytometry. Clinical and Laboratory Haematology, 15, 113-118. Johnson S, Richardson D, Hopkins J, Howe D and Phillips M (1993). Complete remission after fludarabine for chronic lymphocytic leukaemia. Blood, 81, 560.

Richardson D, Johnson S, Hopkins J, Howe D and Phillips M (1994). Absence of minimal residual disease detectable by FACS, Southern blot or PCR in patients with chronic lymphocytic leukaemia treated with fludarabine. ActaOncologica, 33 (6), 627-30. Cull G, Howe D, Stack-Dunne M, Phillips. M. J and Johnson, 5 (1995). Tetrasomy of chromosome 8 in a patient with acute myeloid leukaemia. Leukemia and Lymphoma, 19, 355-358. Cull G, Richardson D, Howe D, Hopkins J, Johnson S and Phillips, M (1995) Molecular complete response in a patient with chronic lymphocytic leukaemia treated with 2Chlorodeoxyadenosine. ActaOncologica, 34(4), 531-537. Lynas C, Howe D, Sweeny M and Wagstaff, M (1995). t(8;2 1) detection by RT-PCR: an improved primer set convenient for the routine laboratory. British Journal of Haematology, 91, 924-926. Bromidge T, Howe D, Johnson S and Phillips M (1995). Adaptation of the TdT assay for semi-quantitative flow cytometric detection of DNA strand breaks. Cytometry, 20, 257-260. Lynas C, Howe D, Copplestone J, Johnson S and Phillips M (1995). A rapid and reliable PCR method for detecting clonal T-ce1l populations. Journal of Clinical Pathology: Molecular Pathology. 48, M101-M104. Lynas C, Howe D, Sweeny M and Wagstaff M (1996). Further improvement of the t(8;2 1) detection by RT-PCR. British Journal of Haematology, 94, 422. Lynas C and Howe D (1997). Additional TCRVbeta primers and minor method modifications improve detection of clonal T-cell populations by RT-PCR. Molecular Pathology, 50 (1), 53-55. Bromidge T, Howe D, Johnson S and Rule S (1997). Heterogeneity of clonal lymphocytes with regard to bcl-2 protein concentration and cell size. Molecular Pathology, 50 (6), 326-328. Howe D, Bromidge T, Stack-Dunne M, Davies S, Paxton A, Phillips M, Rule S and Johnson S (1997). Trisomy 12 is a rare event in cases of CLL with typical immunophenotype and morphology. Hematology, 2 , 373-378. Lynas C and Howe D (1998). Simple, reliable detection of T-cell clones by PCR-LISSSCP analysis of TCR gamma rearrangement. Molecular and Cellular Probes, 12, 4148. Macey M, Hou L, Milne T, Parameswarren V, Howe D, Cavenagh J, Howells G and Newland A (1998) A CD4+ proliferation of large granular lymphocytes expresses the protease activated receptor-1. British Journal of Haematology,101, 78-81.

Bromidge TD Turner D Howe, D Johnson S and Rule S(1998). In vitro chemosensitivity of chronic lymphocytic leukaemia to purine analogues - correlation with clinical course. Leukemia, 12, 1230-1235. Smith P, Cavenagh JD, Milne T, Howe D, Wilkes SJ, Sinnott P, Forster GE &Helbert M. Benign monoclonal expansion of CD8+ lymphocytes in HIV infection. Journal of Clinical Pathology 2000; 53 (3): 177-181. Bromidge J & Howe D. Screening of the entire coding region of p53 in low grade lymphoproliferative disorders. Journal of Clinical Pathology: Molecular Pathology, 2000; 53 (4): 216-218. Howe D &Lynas C. The cyclin D1 alternative transcripts [a] and [b] are expressed in normal and malignant lymphocytes and their relative levels are influenced by the polymorphism at codon 241. Haematologica, 2001; 86 (6): 563-569. Bromidge T, Lynas C. Relative levels of alternative transcripts of the ING1 gene and lack of mutations of p33/ING1 in haematological malignancies. Leukemia Research 2002, 26(7): 631-5. Bromidge T, Lowe C, Prentice A, Johnson S. p53 intronic point mutation, aberrant splicing and telomeric associations in a case of B-chronic lymphocytic leukaemia. British Journal of Haematology 2000, 111 (1): 223-229.

USE PCR and a single hair to produce a DNA fingerprint

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The Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes
By: Leslie A. Pray, Ph.D. 2008 Nature Education Citation: Pray, L. (2008) The biotechnology revolution: PCR and the use of reverse transcriptase to clone expressed genes. Nature Education 1(1)

Gene cloning and PCR allow scientists to make a large amount of DNA from only a small fragment. How do these technologies work? The cloning of expressed genes and the polymerase chain reaction (PCR), two biotechnological breakthroughs of the 1970s and 1980s, continue to play significant roles in science today. Both technologies give researchers the means to make more DNA, but they do so in different ways. In particular, cloning involves the synthesis of DNA from mRNA using an enzyme called reverse transcriptase. Although this method reverses the flow of genetic information as described by the central dogma, it effectively mimics the process by which RNA viruses "flip" the direction of transcription in their host cells, thereby causing these cells to manufacture viral DNA even though the viruses themselves contain only RNA. In contrast, the polymerase chain reaction does not involve the use of an initial mRNA template to manufacture DNA. Rather, PCR involves the synthesis of multiple copies of specific DNA fragments using an enzyme known as DNA polymerase. This method allows for the creation of literally billions of DNA molecules within a matter of hours, making it much more efficient than the cloning of expressed genes. However, cloning remains the go-to method for researchers when only the mRNA template (and not the DNA template) of a sequence of interest is available.

Making DNA from RNA: Reversal of the Central Dogma

"The central dogma, enunciated by Crick in 1958 and the keystone of molecular biology ever since, is likely to prove a considerable over-simplification. That is the heretical but inescapable conclusion stemming from experiments done in the past few months in two laboratories in the United States." --"Central Dogma Reversed," Nature, June 27, 1970 The so-called central dogma of molecular biology states that all genetic information flows in one direction: from DNA to RNA through the process of transcription, and then from RNA to protein through the process of translation (Crick, 1958). For over a decade, the central dogma was thought to be a universal truth--in other words, researchers believed that genetic information always flowed in this order, otherwise it could not be passed along. In 1970, however, the two experiments mentioned in the Nature quote--one conducted by David Baltimore, then of the California Institute of Technology in Pasadena, and the other by Howard Temlin and Satoshi Mizutani, then of the University of Wisconsin in Madison--called this belief into question. Specifically, these researchers independently published scientific papers demonstrating that RNA tumor viruses contain enzymes that use viral RNA as a template for the synthesis of DNA, thereby reversing the direction of transcription (Baltimore, 1970; Temin &Mizutani, 1970). Not only did these two experiments challenge the validity of the central dogma, but they also laid the foundation for a series of technological developments that eventually earned reverse transcription and the synthesis of complementary DNA, or cDNA, central places in the molecular biologist's toolbox.

Discovering Reverse Transcription

During the late 1960s, Baltimore, Temlin, and Mizutani were each driven by unanswered questions about how RNA viruses transformed healthy cells into tumor cells. They knew that transformation ensued when healthy cells incorporated DNA from the external environment (in this case, RNA tumor virus DNA) into their genomes. But how could a eukaryotic cell incorporate DNA from a virus that didn't have any DNA? Howard Temlin had hypothesized the existence of an enzyme capable of making DNA from RNA as early as 1964 ("Central Dogma Reversed," 1970). But, as is the case with all scientific hypotheses, the research community remained skeptical of this proposal until the 1970 publications wiped that skepticism away. At that point, the race was on to identify the enzyme responsible for the creation of DNA from RNA. Today, that enzyme is known as reverse transcriptase. Interestingly, in their groundbreaking papers, the two sets of scientists didn't actually identify reverse transcriptase, but they did provide clear and conclusive evidence of the existence of an enzyme that utilized viral RNA as a template for DNA synthesis. The experiments supporting the existence of this DNA polymerase produced data that revealed the following: The DNA polymerase only incorporated deoxyribonucleotides, not ribonucleotides, into its product. The product itself "behaved" like DNA--in other words, it was sensitive to treatment by deoxyribonucleases but not ribonucleases. The RNA itself was the template, as shown by the fact that treatment of virions with ribonucleases destroyed the ability of the polymerase to incorporate radioactively labeled nucleotides. Although the motivation for both studies was to better understand the role of viruses in some cancers, there is also some suggestion in the papers that the scientists were aware, at least on an intuitive level, that there were far greater implications to their findings. As Temin and Mizutani (1970) wrote, "This result would have strong implications for theories of viral carcinogenesis and, possibly, for theories of information transfer in other biological systems." It did not take long for scientists to isolate the reverse transcriptase responsible for Baltimore's findings (Verma et al., 1972). Another team (Bank et al., 1972) then used the enzyme to synthesize DNA from mRNA in a test tube for the first time. (The so-called complementary DNA that results is referred to as cDNA.) Both teams used globin mRNAs, or mRNAs that encode blood hemoglobin polypeptides, to demonstrate that reverse transcriptase does in fact synthesize DNA from mRNA templates. Moreover, the teams also found that the reaction works best in the abundance of short sequences composed entirely of thymine nucleotides known as oligo(dT) primers. Knowing that most eukaryotic mRNAs have a string of adenine nucleotides--also known as a poly(A) tail--at their 3 end, the scientists had predicted that cDNA synthesis would require oligo(dT) primers, or that it would at least be made more efficient by the presence of these primers.

y y y

How Reverse Transcriptase Works

Figure 1 So how exactly does reverse transcriptase work? As shown in Figure 1, the production of cDNA involves two basic steps: separating mRNA molecules from other cellular RNA, and then using reverse transcriptase to copy the mRNA molecules into cDNA. The first step relies on the fact that most eukaryotic mRNAs have poly(A) tails at their 3 ends. The poly(A) tails serve as "hooks" during the separation process. This process involves pouring all of the cellular RNA (tRNA, rRNA, snRNA, mRNA, etc.) into what is known as an elution column of short DNA pieces, mostly thymine nucleotides. As the RNA mixture moves downward through the column, the poly(A) tails of the mRNA molecules bind to the thymine nucleotides. The rest of the RNA molecules--those without poly(A) tails, in other words--run right through. Afterward, the column is washed with a solution that breaks the hydrogen A-T bonds, and the released mRNA molecules are collected.

The second step, the copying of the now-isolated mRNA molecules into cDNA, involves adding oligo(dT) primers to the mRNA collection. These primers pair with the poly(A) tails of the mRNA molecules, again at the 3 end, providing the exposed 3 -OH group required for the initiation of DNA synthesis. At this point, the reverse transcriptase enzyme is added, and this enzyme proceeds to utilize the mRNA strand as a template for the synthesis of a complementary DNA strand. The enzyme adds nucleotides, one by one, to the 3 end of the new strand, with each newly added nucleotide a complement to its template pair just as in DNA replication, with the exception that RNA contains Us in place of Ts. Thus, Cs are paired with Gs and vice versa, Ts are paired with As, and As are paired with Us. Scientists then use several different methods to convert the RNA-DNA hybrids into double-stranded cDNA molecules, such as enzymatic digestion of the RNA strand followed by DNA synthesis utilizing, this time, the cDNA strand as the template.

Using Reverse Transcriptase to Clone Expressed Genes

Following the development of this method, the use of reverse transcriptase to clone expressed genes grew for several decades. However, there were limits to this practice. For example, most cDNA molecules that were synthesized in a single reaction were incomplete, with the 5 end of the mRNA not represented in the final cDNA. Therefore, the cDNA did not contain a complete copy of the amino acid-encoding region of the gene. Eventually, in the late 1990s, PieroCarninci and his colleagues at the Genome Science Laboratory in Ibaraki, Japan, devised a series of methods to get around this and other problems. In particular, these researchers developed a new technique for selecting full-length cDNA molecules. This process is known as biotin capping, and it involves capping the 5 end of the mRNA with a biotin group and then washing the cDNAs with an RNA digestion enzyme, like RNAse I. When washed with a solution containing a cap-binding protein, all of the cDNA-mRNA hybrids with only partial cDNA copies are carried away, thus only leaving behind the hybrids with full-length cDNA molecules. In fact, the researchers demonstrated that biotin capping yielded about 95% full-length cDNA clones (Carninci et al., 1996). Today, scientists continue to build and utilize what are known as cDNA libraries, or collections of cDNAs from particular tissues gathered at particular times during an organism's life cycle. The synthesis of cDNA molecules is referred to as cloning, because the cDNA molecules are matching copies of the DNA responsible for encoding the mRNA template. Scientists often generate cDNA libraries as a way to find genes of interest. They screen these libraries using what are known as probes--complementary pieces of DNA that hybridize to the cDNA molecules. They also use cDNA libraries to identify genes that are expressed differently in different types of tissues or at different developmental stages. Libraries of cDNA molecules provide snapshots of gene activity, because only those genes that are actually expressed and transcribed into mRNA molecules can be cloned. For example, one would expect a cDNA library compiled from mRNA isolated during a stage of prenatal development to be very different from a cDNA library generated from sequences transcribed during adulthood.

Making Copies via Polymerase Chain Reaction

Arguably, the polymerase chain reaction (PCR) machine has recently become as indispensible to biological research as the light microscope was some 100 years ago. Like gene expression and cloning, the idea of PCR was born only in the early 1970s (Swaminathan, 2007). It would take more than a decade before American biochemist Kary Mullis turned the idea into reality (Mullis &Faloona, 1987). Until Mullis's success with this method, the only way biologists could make copies of whatever gene they were interested in was by the relatively laborious and time-consuming process of identifying and isolating the gene--in other words, through constructing and screening a cDNA library, as described earlier--and then inserting that gene into living cells that replicated the target DNA along with their own DNA. In contrast, PCR enables the production, or amplification, of billions of copies of an original piece of DNA in a test tube within minutes or hours, not days.

How PCR Works

Basically, PCR is DNA replication on a grander scale. The polymerase chain reaction relies on the use of several essential chemical ingredients, including the following:

y y y y y

A DNA polymerase A small amount of DNA to serve as the initial template The four deoxyribonucleotides to serve as the substrates for the DNA polymerase and the raw ingredients of the new DNA molecules A few necessary ions and salts A pair of primers with exposed 3 -OH groups that will bind to the particular sequence of interest in the DNA template As previously mentioned, the DNA polymerases can only add new nucleotides to the 3 -OH end of a growing strand. They therefore require the presence of a primer to get started, because they cannot begin synthesis de novo. In fact, two primers are required--one to initiate replication of each of the two DNA strands.

A single PCR reaction involves three temperature-dependent steps, described as follows: 1. 2. The starting solution is heated, usually to between 90 and 100C. The high temperatures break the hydrogen bonds between the two strands of the original DNA double helix, providing the necessary single-stranded templates. After just a couple of minutes at that temperature, the reaction mixture is quickly cooled, usually to somewhere between 30 and 65C. It is then held for less than a minute at this lower temperature--which is enough time for the primers to bind to their complementary sequences on the single-stranded templates. The sample is next heated to 60 to 75C for less than a minute, during which time the DNA polymerase adds nucleotides to the primer, synthesizing a new DNA strand using only the template sequences that bind the primers (Figure 2).

3.

Figure 2: The polymerase chain reaction is used to amplify even very small samples of DNA. Description Used with permission. 2005 by W. H. Freeman and Company. All rights reserved.

Figure 3: Over the course of 30 cycles in PCR amplification, an enormous number of copies can be made from a small DNA fragment.

Used with permission. 2005 by W. H. Freeman and Company. All rights reserved. Within just a few minutes, the chain reaction doubles the number of DNA molecules for the specific sequence of interest. The reaction--and the doubling--are repeated every few minutes, as long as the PCR is allowed to proceed. This leads to the creation of a large amount of DNA in a relatively short period. For example, even if the process were started with just a single DNA molecule, it would result in more than 1 billion molecules at the end of just 30 PCR reactions or cycles (Figure 3). The many changes in temperature required during multiple PCR cycles are carried out in a thermocycler, also known as a PCR machine. After PCR cycling is complete, the amplification products can be subjected to cloning, sequencing, or analysis via gel electrophoresis.

Advances in PCR Technology

As with all genetic technologies, of course, scientists have improved and refined the original PCR process described by Mullis and Faloona in 1987. For example, one of the major limitations of early PCR methods was that fresh DNA polymerase had to be added during every cycle. This repetitive step was not just tedious, but it also greatly increased the likelihood of error. Mullis and colleagues addressed this deficiency just a year later when they demonstrated how a particular type of DNA polymerase, a heat-resistant enzyme isolated from the bacterium Thermusaquaticus, eliminated the need to add fresh polymerase during every cycle. Thermusaquaticus--often referred to by its popular nickname "Taq polymerase"--is a thermophilic bacterium that can survive temperatures up to 95C. In fact, its natural habitat is the hot spring ecosystem of Yellowstone National Park. This innovation greatly improved the quantity and quality of PCR products (Saiki et al., 1988). More recently, another major PCR innovation was the development of real-time PCR. This refinement involves the use of dyes or fluorescent probes that eliminate the need for post-PCR electrophoresis. In real-time PCR, the fluorescence that is associated with the accumulation of newly amplified DNA is measured through the use of an optical sensing system.

Summary
Thus, both cloning of expressed genes and PCR continue to serve as essential tools for genetic researchers. Cloning--which involves the creation of DNA from mRNA and thus represents a reversal of the central dogma--is particularly useful when scientists aren't able to isolate the DNA template of a sequence of interest. In addition, because this method relies on mRNA rather than DNA, it provides an excellent means for studying the differences ingene expression in different cells at different

points in development. PCR, on the other hand, is more akin to "traditional" DNA synthesis in that it requires the presence of an initial DNA (rather than RNA) template. The primary advantage of PCR is its speed--even if researchers begin with only a single segment of DNA, they can produce literally billions of molecules within a matter of hours. As with all technologies, scientists continue to improve both PCR and the cloning process, thereby ensuring that these methods will play a role in genetic breakthroughs for years to come.

References and Recommended Reading

Baltimore, D. Viral RNA-dependent DNA polymerase: RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226, 12091211 (1970) doi:10.1038/2261209a0 (link to article) Bank, A., et al. In vitro synthesis of DNA components of human genes for globins. Nature New Biology 235, 167169 (1972) Carninci, P., et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327336 (1996) Central dogma reversed. Nature 226, 11981199 (1970) doi:10.1038/2261198a0 (link to article) Crick, F. On protein synthesis. The Biological Replication of Macromolecules: Symposium for the Society of Experimental Biology 12, 138162 (1958) Mullis, K. B., &Faloona, F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology 155, 335350 (1987) Saiki, R. K., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487 491 (1988) doi:10.1126/science.2448875 Swaminathan, S. Milestone 11: Chain reaction. Nature.com, (2007) (accessed July 3, 2008) Temin, H. M., &Mizutani, S. Viral RNA-dependent DNA polymerase: RNA-dependent DNA polymerase in virions of rous sarcoma virus. Nature 226, 12111213 (1970) doi:10.1038/2261211a0 (link to article)

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Tandem Repeats and Morphological Variation

By: P. Z. Myers, Ph.D. (University of Minnesota, Morris) 2007 Nature Education Citation: Myers, P. (2007) Tandem repeats and morphological variation. Nature Education 1(1), http://scienceblogs.com/pharyngula/2007/10/tandem_repeats_and_morphologic.php

All mammals have basically the same set of genes, yet there are obviously some significant differences that distinguish the various species. Recent research suggests that one such difference involves tandem repeats, or short lengths of DNA that are repeated multiple times within a gene. But what, if anything, does having a different number of tandem repeats do to an organism? All mammals have basically the same set of genes, yet there are obviously some significant differences that distinguish the various species. Researchers currently think that much of mammalian morphological diversity involves cis regulatory regions, or stretches of DNA outside the actual coding region of a gene that are responsible for switching the gene on and off. However, an important paper by Fondon and Garner (2004) suggests that there is yet another source of variation: tandem repeats. Tandem repeats are short lengths of DNA that are repeated multiple times within a gene, anywhere from a handful of times to more than a hundred. These sequences are also called VNTRs, or variable number tandem repeats, because different individuals within a population may have different numbers of repeats. VNTRs are relatively easy to detect with molecular tools, and researchers know that populations (humans included) may carry a large reservoir of different numbers of repeats. For example, one person might carry three tandem repeats in a particular gene, while another person might bear 15, with no obvious differences between the two individuals that can be traced to that particular gene. So, the question is, what, if anything, does having a different number of tandem repeats do to an organism?

The Basic Premise of Fondon and Garner's Study

In their 2004 study, researchers John Fondon and Harold Garner set out to answer this question by first looking for populations that exhibited large and obvious morphological differences between individuals, and then looking within these individuals' genomes to see whether those differences could be correlated with the number of tandem repeats present. The duo decided to use domestic dogs as their population; after all, not only are dogs diverse, but dog breeders are notoriously picky about shape and character, and purebred dogs have been under intense selection for specific attributes for many years. Once a range of morphologies in a particular trait, such as snout shape, has been identified, one can ask whether this range is reflected in the number of repeats in any genes. Thus, Fondon and Garner examined 142 dogs from 92 different breeds, and they looked at 37 different tandem repeats in 17 genes in each dog. The genes selected were those that encoded transcription factors that were at least suspected of playing a role in the formation of specific morphologies during development. Fifteen of the 17 genes turned out to have multiple alleles that varied in the number of copies of repeats they contained.

Tandem Repeats and Mutations

Figure 1: Slipped-strand mispairing. Slipped-strand nucleotide mispairing can generate variation in gene expression. Illegitimate base pairing in regions of repetitive DNA during replication, coupled with inadequate DNA mismatch repair systems, can produce deletions or insertions of repeat units. Bulging in the replicated and template strands gives rise to larger and smaller numbers of repeat units, respectively. The figure shows a strand of DNA (blue) being carried through two rounds of replication. Copyright 2003 Nature Publishing Group, Thompson, N., et al., The value of comparison, Nature Reviews Microbiology 1, 1112 The fact that Fondon and Garner found a substantial amount of genetic variation in tandem repeat number is not at all surprising, because tandem repeats are subject to very high mutation rates. This increased probability ofmutation (up to 100,000 times greater than the probability of a point mutation) exists because tandem repeats are prone to a kind of error called slipped-strand mispairing. Tandem repeats contain many copies of the same short sequence over and over, so it is easy for the two strands of DNA to get misaligned in this local regionthe GTAC sequence on one strand could base-pair with the first CATG in the other strand, or the second, or the third, for example. If the strands are mispaired, then DNA replicating enzymes can err and either clip off some of the repeats or add extra repeats (Figure 1). This represents a special kind of error in that the DNA changes do not occur in random nucleotides, and they produce only different numbers of repeats. Also, note that this lack of fidelity in copying tandem repeats means that such repeats are only found in regions of genes that can tolerate some variability. Interestingly, slipped-strand mispairing can be foiled by point mutations, even to synonymous codons, within a tandem repeat. Here, a small change in theDNA sequence gives the replication machinery a local difference that can be used to properly align the two strands. Over time, however, a stable tandem repeat will accumulate these small changes and lose its repeated character. On the other hand, a deletion caused by slipped-strand mispairing can remove a point difference, and subsequent mispairing can then expand the sequence, producing a repeat free of imperfections. Thus, one measure of how much selection for variation has occurred within a tandem repeat is its purity. If there are few interruptions in the perfection of the repeat, there has been much deletion and expansion going on within the sequence throughout its history. Conversely, if there are multiple deviations from perfect repetition, then the sequence has not undergone much length variation in the recent past.

Repeat Variants in Dogs and Their Association with Morphology

Figure 2 The purity of a tandem repeat sequence is therefore a measure of how much selection for new variants has occurred in an organism's lineage. Based on this principle, Fondon and Garner compared the same repeat loci in humans and dogs, and they found that the dog repeats were purer than the human repeats in 29 of 36 cases; in the other seven cases, the dog and human repeats were of equal purity. This finding strongly suggests that the variations in dogs are not just random, neutral changes, but rather, they are the outcome of recent selection at these loci. Thus, Fondon and Garner determined that there are multiple interesting gene variants in dogs, and these variants have apparently undergone selection. But what effect do the repeats have? Let's consider two specific genesRunx-2 andAlx4as examples.

Runx-2

Figure 3 The Runx-2 (runt-related transcription factor 2) gene is related to the Drosophila pair-rule gene runt, which is involved insegmentation. In vertebrates, one of the functions of Runx-2 is to regulate the differentiation of osteoblasts, which are the cells responsible for forming new bone. Runx-2 contains two repeats, one coding for 1820 glutamines (the poly-Q region) and another coding for 1217 alanines (the poly-A region). A statistical comparison of the total length of both of these repeats (poly-Q + poly-A) with various parameters of canine skull size revealed a correlation with the dogs' midface length, as well as with a property called clinorhynchy, or dorsoventral nose bend. To better understand what clinorhynchy looks like, think about the distinctive, long nose of bull terriers, which features a downward droop (Figure 2). Bull terriers tend to have a short pair of tandem repeats in Runx-2, and they have long midfaces and pronounced downturn of the snout. The breed has been intentionally selected for this trait, and museum specimens over the past 70 years show increased prominence of this feature (Figure 3). In reality, the relationship between Runx-2 and canine morphology is not as simple as short repeat length equals downturned snout. Remember, one of the ways that the activity of transcription factors is regulated is by binding, and chains of amino acids can affect how transcription factors interact and bind with one another. Moreover, it turns out that polyglutamine can increase the rate of transcription in the genes that it regulates, while polyalanine can reduce it. Of course, the Runx-2 protein has both a polyglutamine (poly-Q) and a polyalanine (poly-A) chain. Thus, what might matter more in a situation such as this (where two competing components modulate activity) is the ratio of poly-Q to poly-A. Indeed, this ratio shows an even stronger correlation with clinorhynchy than does the total combined length of the poly-Q and poly-A regions (Figure 4).

Alx-4

Figure 4 A second gene example is Alx-4 (aristaless-like homeobox 4). This gene is also related to a transcription factor found inDrosophila, and knocking out the gene in mice produces individuals with six toes. In Fondon and Garner's study, one specific allele of this gene, Alx-4 51, was found in only one breed of dog, the Great Pyrenees. One peculiarity of this breed is hind limb polydactylypurebreds are supposed to have a double dewclaw, for a total of six digits on each of their hind legs. Thus, it is not surprising that the Alx-4 51 allele features a deletion that knocks out 51 nucleotides from a specific tandem repeat, for a total loss of 17 amino acids. All Great Pyrenees with polydactyly have this particulardeletion (Figure 5); moreover, in Fondon and Garner's study, the only Great Pyrenees that did not have the extra dewclaw carried the full-length tandem repeat.

The Benefits and Limitations of an Extreme Example

The good news about these findings is that they demonstrate another mode by which morphological diversity can be added to a population relatively rapidly, as well as another mechanism for fine-tuning evolution. Because tandem repeats are common in the vertebrate genome, these repeats could clearly be a reservoir of variation and a robust and flexible way to add new variations to populations. There are some limitations to Fondon and Garner's results, though. First, this study focused on an extreme case: purebred dogs that have undergone very strong selection for specific and, in some cases, outright deleterious characteristics. Thus, we simply don't know how important this mode of evolutionary change is under less-artificial conditions. Second, this study and others like it have revealed only correlations, not experimental perturbations. While these correlations are convincing, at some point in the future, it would be helpful to see direct manipulation of the poly-Q/poly-A ratio in the Runx-2 gene of a collie, for instance, to give it the downturned nose of a bull terrier. Finally, it would also be beneficial to obtain additional correlative evidence through developmental studies of the patterns of Runx-2 and Alx-4 gene expression in dog embryos to see exactly how these variations play out.

Figure 5: Large magnitude repeat length mutations can result in gross morphological change. (A) Alx-4/ mice exhibit a duplication of their first digit (as indicated by the arrowhead). (B) A radiograph of the rear paw of a Great Pyrenees shows the typical double dewclaw phenotype specified in the breed standard (as indicated by the arrowhead). Copyright 2004 National Academy of Sciences, Fondon, J. W., & Garner, H. R., Molecular origins of rapid and continuous morphological evolution, Proceedings of the National Academy of Sciences 101, 1805818063

References and Recommended Reading

Fondon, J. W., & Garner, H. R. Molecular origins of rapid and continuous morphological evolution. Proceedings of the National Academy of Sciences 101, 1805818063 (2004) Thompson, N., et al. The value of comparison. Nature Reviews Microbiology 1, 11 (2003) (link to article)

Isolating Hereditary Material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase
By: Clare O'Connor, Ph.D. (Biology Department, Boston College) 2008 Nature Education Citation: O'Connor, C. (2008) Isolating hereditary material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase. Nature Education 1(1)

How did scientists determine that DNA is the hereditary material? Groundbreaking experiments by Griffith, Avery, Hershey, and Chase disproved the notion that proteins were genetic material. In the first half of the twentieth century, Gregor Mendel's principles of genetic inheritance became widely accepted, but the chemical nature of the hereditary material remained unknown. Scientists did know that genes were located on chromosomes and that chromosomes consisted of DNA and proteins. At the time, however, proteins seemed to be a better choice for the genetic material, because chemical analyses had shown that proteins are more varied than DNA in their chemical composition, as well as in their physical properties. Therefore, the eventual identification of DNA as the hereditary material came as a surprise to scientists. This breakthrough resulted from a series of experiments with bacteria and bacteriophages, or viruses that infect bacteria. Together, these experiments demonstrated that DNA was transferred between generations and that this molecule had the ability to transform the properties of a cell.

Frederick Griffith Discovers Bacterial Transformation

Figure 1: R variant phenotypes. In this figure the colonies of R variant on the left are grown on agar in the absence of the transforming substance (x 3.5) and on the right are colonies after induction of the transforming substance. Used with Permission from the Journal of Experimental Medicine, Rockefeller University Press. Journal of Experimental Medicine 79, 137 - 158 In the aftermath of the deadly 1918 flu epidemic, governments across the globe rushed to develop vaccines that could stop the spread of infectious diseases. In England, microbiologist Frederick Griffith was studying two strains of Streptococcus pneumoniae that varied dramatically in both their appearance and their virulence, or their ability to cause disease.

Specifically, the highly virulent S strain had a smooth capsule, or outer coat composed of polysaccharides, while the nonvirulent R strain had a rough appearance and lacked a capsule (Figure 1). Mice injected with the S strain died within a few days after injection, while mice injected with the R strain did not die. Through a series of experiments, Griffith established that the virulence of the S strain was destroyed by heating the bacteria. Thus, he was surprised to find that mice died when they were injected with a mixture of heat-killed Sbacteria and living R bacteria (Figure 2), neither of which caused mice to die when they were injected alone. Griffith was able to isolate live bacteria from the hearts of the dead animals that had been injected with the mixed strains, and he observed that these bacteria had the smooth capsules characteristic of the S strain. Based on these observations, Griffith hypothesized that a chemical component from the virulent S cells had somehow transformed the R cells into the more virulent S form (Griffith, 1928). Unfortunately, Griffith was not able to identify the chemical nature of this "transforming principle" beyond the fact that it was able to survive heat treatment. Looking back on Griffith's results, which were published in 1928, the interpretation seems obvious. Today, we know that DNA molecules can renature after heat treatment and that bacteria can take up foreign DNA from the environment by a process that we still refer to as transformation. These facts would not be discovered, however, until other scientists conducted further explorations of the nature and function of DNA.

Figure 2: Genetic Transformation of Nonvirulent Pneumococci. Frederick Griffith's experiments demonstrated that something in the virulent S strain could transform nonvirulent R strain bacteria into a lethal form, even when the S strain bacteria had been killed by high temperatures. FURTHER RESEARCH: How would you show that heat-killed R strain bacteria can transform living S strain bacteria? 2008 by Sinauer Associates, Inc. All rights reserved. Used with permission.

DNA Is Identified as the Transforming Principle

Figure 3 The actual identification of DNA as the "transforming principle" was an unexpected outcome of a series of clinical investigations of pneumococcal infections performed over many years (Steinman &Moberg, 1994). At the same time that Griffith was conducting his experiments, researcher Oswald Avery and his colleagues at the Rockefeller University in New York were performing detailed analyses of the pneumococcal cell capsule and the role of this capsule in infections. Modern antibiotics had not yet been discovered, and Avery was convinced that a detailed understanding of the pneumococcal cell was essential to the effective treatment of bacterial pneumonia. Over the years, Avery's group had accumulated considerable biochemical expertise as they established that strains of pneumococci could be distinguished by the polysaccharides in their capsules and that the integrity of the capsule was essential for virulence. Thus, when Griffith's results were published, Avery and his colleagues recognized the importance of these findings, and they decided to use their expertise to identify the specific molecules that could transform a nonencapsulated bacterium into an encapsulated form. In a significant departure from Griffith's procedure, however, Avery's team employed a method for transforming bacteria in cultures rather than in living mice, which gave them better control of their experiments. Avery and his colleagues, including researchers Colin MacLeod and Maclyn McCarty, used a process of elimination to identify the transforming principle (Avery et al., 1944). In their experiments (Figure 3), identical extracts from heat-treated S cells were first treated with hydrolytic enzymes that specifically destroyed protein, RNA, or DNA. After the enzymetreatments, the treated extracts were then mixed with live R cells. Encapsulated S cells appeared in all of the cultures, except those in which the S strain extract had been treated with DNAse, an enzyme that destroys DNA. These results suggested that DNA was the molecule responsible for transformation. Avery and his colleagues provided further confirmation for this hypothesis by chemically isolating DNA from the cellextract and showing that it possessed the same transforming ability as the heat-treated extract. We now consider these experiments, which were published in 1944, as providing definitive proof that DNA is the hereditary material. However, the team's results were not well received at the time, most likely because popular opinion still favored protein as the hereditary material.

Hershey and Chase Prove Protein Is Not the Hereditary Material

Figure 4

Figure 5 Protein was finally excluded as the hereditary material following a series of experiments published by Alfred Hershey and Martha Chase in 1952. These experiments involved the T2 bacteriophage, a virus that infects the E. coli bacterium. At the time, bacteriophages were widely used as experimental models for studying genetic transmission because they reproduce rapidly and can be easily harvested. In fact, during just one infection cycle, bacteriophages multiply so rapidly within their host bacterial cells that they ultimately cause the cells to burst, thus releasing large numbers of new infectious bacteriophages (Figure 4). The T2 bacteriophage used by Hershey and Chase was known to consist of bothprotein and DNA, but the role that each substance played in the growth of the bacteriophage was unclear. Electron micrographs had shown that T2 bacteriophages consist of an icosahedral head, a cylindrical sheath, and a base plate that mediates attachment to the bacterium, shown schematically in Figure 5. After infection, phage particles remain attached to the bacterium, but the heads appear empty, forming "ghosts." To determine the roles that the T2 bacteriophage's DNA and protein play in infection, Hershey and Chase decided to use radioisotopes to trace the fate of the phage's protein and DNA by taking advantage of their chemical differences. Proteins contain sulfur, but DNA does not. Conversely, DNA contains phosphate, but proteins do not. Thus, when infected bacteria are grown in the presence of radioactive forms of phosphate (32P) or sulfur (35S), radioactivity can be selectively incorporated into either DNA or protein. Hershey and Chase employed this method to prepare both 32P-labeled and 35S-labeled bacteriophages, which they then used to infect bacteria. To determine which of the labeled molecules entered the infected bacteria, they detached the phage ghosts from the infected cells by mechanically shearing them off in an ordinary kitchen blender. The ghosts and bacterial cells were then physically separated using a centrifuge. The larger bacterial cells moved rapidly to the bottom of the centrifuge tube, where they formed a pellet. The smaller, lighterphage ghosts remained in the supernatant, where they could be collected and analyzed. During analysis, Hershey and

Chase discovered that almost all of the radioactive sulfur remained with the ghosts, while about one-third of the radioactive phosphate entered the bacterial cells and could later be recovered in the next generation of bacteriophages. From these experiments, Hershey and Chase determined that protein formed a protective coat around the bacteriophagethat functioned in both phage attachment to the bacterium and in the injection of phage DNA into the cell. Interestingly, they did not conclude that DNA was the hereditary material, pointing out that further experiments were required to establish the role that DNA played in phage replication. In fact, Hershey and Chase circumspectly ended their paper with the following statement: "This protein probably has no function in the growth of intracellular phage. The DNA has somefunction. Further chemical inferences should not be drawn from the experiments presented" (Hershey & Chase, 1952). However, a mere one year later,the structure of DNA was determined, and this allowed investigators to put together the pieces in the question of DNA structure and function.

References and Recommended Reading

Avery, O. T., et al. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Journal of Experimental Medicine79, 137157 (1944) Griffith, F. The significance of pneumococcal types. Journal of Hygiene 27, 113159 (1928) Hershey, A. D., & Chase, M. Independent functions of viral protein and nucleic acid in growth of bacteriophage. Journal of General Physiology 36, 3956 (1952) Steinman, R. M., &Moberg, C. L.A triple tribute to the experiment that transformed biology. Journal of Experimental Medicine 179, 379384 (1994)