1.

USES OF THERMOCYCLERS OTHER THAN PCR

2.

WHAT IS SWOT ANALYSIS AND FIND AN ALTERNATE FOR SWOTANALYSIS WHAT IS ASPM GENES AND THEIR FUNCTIONS

3.

Evolution of the Human ASPM Gene, a Major Determinant of Brain Size

1. Jianzhi Zhang1 + Author Affiliations 1. Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109

1.

1Address for correspondence: Department of Ecology and Evolutionary Biology, University of Michigan, 3003 Natural Science Bldg., 830 N. University Ave., Ann Arbor, MI 48109. E-mail: jianzhi@umich.edu

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Abstract
The size of human brain tripled over a period of 2 million years (MY) that ended 0.20.4 MY ago. This evolutionary expansion is believed to be important to the emergence of human language and other highorder cognitive functions, yet its genetic basis remains unknown. An evolutionary analysis of genes controlling brain development may shed light on it. ASPM (abnormal spindle-like microcephaly associated) is one of such genes, as nonsense mutations lead to primary microcephaly, a human disease characterized by a 70% reduction in brain size. Here I provide evidence suggesting that human ASPM went through an episode of accelerated sequence evolution by positive Darwinian selection after the split of humans and chimpanzees but before the separation of modern non-Africans from Africans. Because positive selection acts on a gene only when the gene function is altered and the organismal fitness is increased, my results suggest that adaptive functional modifications occurred in human ASPM and that it may be a major genetic component underlying the evolution of the human brain. AMONG mammals, humans have an exceptionally big brain relative to their body size. For example, in comparison with chimpanzees, the brain weight of humans is 250% greater while the body is only 20% heavier (MCHENRY 1994). The dramatic evolutionary expansion of the human brain started from an average brain weight of 400450 g 22.5 million years (MY) ago and ended with a weight of 13501450 g 0.20.4 MY ago (MCHENRY 1994; W OOD and COLLARD 1999). This process represents one of the most rapid morphological changes in evolution. It is generally believed that the brain expansion set the stage for the emergence of human language and other high-order cognitive functions and that it was caused by adaptive selection (DECAN 1992), yet the genetic basis of the expansion remains elusive. A study of human mutations that result in unusually small brains may help identify the genetic modifications that contributed to the human brain expansion. In this regard, primary microcephaly (small head) is of particular interest (MOCHIDA and W ALSH 2001; BONDet al. 2002; KUMARet al. 2002). Microcephaly is an autosomal recessive genetic disease with an incidence of 440 per million live births in western countries (MOCHIDA and W ALSH 2001; KUMARet al. 2002). It is defined as a head circumference >3 standard deviations below the population age-related mean, but with no associated malfunctions other than mild-to-moderate mental retardation (MOCHIDA and W ALSH 2001; KUMARet al. 2002). The reduction in head circumference correlates with a markedly reduced brain size. Microcephaly is genetically heterogeneous, associated with mutations in at least five loci (MOCHIDA and W ALSH 2001; KUMARet al. 2002), one of which was recently identified and named ASPM (abnormal spindle-like microcephaly associated; BONDet al. 2002). Four different homozygous mutations in ASPM introducing premature stop codons were found to cosegregate with the disease in four respective families, while none of these mutations were found in 200 normal human chromosomes (BONDet al. 2002). Because the brain size of a typical microcephaly patient (430 g; MOCHIDA and W ALSH 2001; KUMARet al. 2002) is comparable with those of early hominids such as the 2.3- to 3.0MY-old Australopithecus africanus (420 g; MCHENRY 1994; W OOD and COLLARD 1999), I hypothesize that

ASPM may be one of the genetic components underlying the human brain expansion. Signatures of accelerated evolution of ASPM under positive selection during human origins would strongly support my hypothesis, because the action of positive selection indicates a modification in gene function resulting in elevated organismal fitness (ZHANGet al. 2002). Below I provide population genetic and molecular evolutionary evidence for the operation of such adaptive selection on ASPM. Previous SectionNext Section

MATERIALS AND METHODS

Sequencing of ASPM: The human ASPM gene has 28 exons. All 28 exons were PCR amplified from genomic DNA samples of 14 human (Homo sapiens) individuals of different geographic origins (2 African Pygmies, 3 African Americans, 4 Europeans, 2 Southeast Asians, 1 Chinese, 1 Pacific islander, and 1 South American), using the high-fidelity Taq of Invitrogen (Carlsbad, CA). The PCR products were then purified and sequenced in both directions. Polymorphisms that appear only once (singletons) were confirmed by a second PCR-sequencing experiment. The human DNA samples were purchased from Coriell (Camden, NJ). I also amplified all 28 exons from one chimpanzee (Pan troglodytes) and one orangutan (Pongo pygmaeus) and sequenced the insert DNAs after the PCR products were cloned into the pCR4TOPO vector (Invitrogen). To trace the evolutionary origin of a large insertion/deletion in exon 18, two segments (Figure 1, I and II) of exon 18 were also amplified and directly sequenced from genomic DNAs of hyrax (Procavia capensis), sea lion (Zalophus californianus), seal (Phoca vitulina), wolverine (Gulo gulo), fox (Alopex lagopus), dog (Canis familiaris), bear (Ursus maritimus), cat (Felis catus), pig (Sus scrofa), cow (Bos taurus), whale (Balaena mysticetus), rhesus monkey (Macaca mulatta), owl monkey (Aotus trivirgatus), and hamster (Cricetulus griseus). Data analysis: The dN/dS ratios (LI 1997; NEI and KUMAR 2000) in the human, chimpanzee, and orangutan branches of the ASPM gene tree (Figure 2) were estimated using the maximum-likelihood method (YANG 1997), under the model of unequal codon frequencies (CodonFreq = 3) and unequal rates of transitions and transversions. This model fits the data significantly better than those with fixed codon frequencies (CodonFreq = 2) or equal rates of transitions and transversions. The hypothesis of dN/dS = 1 for a given branch and the hypothesis of equal dN/dS ratios between two branches were tested using the likelihood-ratio test, as well as a nonlikelihood method based on inferred ancestral sequences (Z HANG et al. 1997, 1998). The ASPM sequence for the common ancestor of humans and chimpanzees was estimated using the Bayesian method (YANG 1997; ZHANG and NEI 1997). Population genetic analysis was conducted using DnaSP3.99 (ROZAS and ROZAS 1999). TAJIMA's (1989), FU and LI's (1993), and FAY and W U's (2000) tests were conducted using coalescent simulations under the assumption of no recombination across the gene. This assumption makes the tests more conservative. When recombination is considered, the P values become lower in Tajima's and Fu and Li's tests, but remain virtually the same in Fay and Wu's test. The recombination rate in the ASPM locus was estimated from KONG et al. (2002) to be 1.8 cM/106 nucleotides. Since the coding sequences analyzed here span 62 103 nucleotides of the genome, the recombination rate for the sequence is r = 1.8 102 0.062 = 1.1 103 recombination/generation. Assuming that the human effective population size is 104 (TAKAHATAet al. 1995; HARPENDINGet al. 1998), the population recombination parameter (4Nr) equals 4 104 1.1 103 = 44. Gene trees of segment I and II sequences from various mammals were reconstructed by the neighbor-joining method (SAITOU and NEI 1987) with KIMURA's (1980) two-parameter distances. Use of other distances or other methods (NEI and KUMAR 2000) does not change the result. Two thousand bootstrap replications (FELSENSTEIN 1985) were used to test the reliability of the trees. The MEGA2 software (KUMARet al. 2001) was used for the phylogenetic analysis. Computer simulation: Computer simulation of ASPM evolution under pure neutrality was conducted following the procedure described in ZHANG and W EBB (2003). The rate that an open reading frame (ORF) becomes disrupted is mainly determined by the sequence of the ORF, the rate of point mutations, and the rate of insertion/deletion (indel) mutations. Here I used a point mutation rate of 1 109/site/year, which was estimated from a genomic comparison between humans and chimpanzees (YIet al. 2002). The relative mutational frequencies among the four nucleotides have only a negligible effect on the simulation result and I assumed that they are equal. I used an indel mutation rate of 1.1 10 10/site/year, which was also estimated from a genomic comparison between the human and chimpanzee (BRITTEN 2002). I assumed that all indels with sizes that are multiples of three nucleotides do not disrupt an ORF. This simplifies the simulation but does not affect the results, because the majority of indels generated by mutations have small sizes (less than or equal to nine nucleotides; BRITTEN 2002). In the above genomic data analyzed, 19% of the total 1019 indels are of sizes that are multiples of three nucleotides. A simulation was then performed for 20,000 replications with the inferred ASPM coding sequence of the common ancestor of humans and chimpanzees. Under no functional constraints, the substitution rate is identical to the mutation rate and mutations are assumed to be random. An ORF is interrupted when an indel of a size that is not a multiple of three nucleotides or a nonsense point mutation occurs. I thus determined the t1/2 for ASPM, or the time

required for an intact ORF to be interrupted in half of the simulation replications. Under the above parameters, t1/2 = 0.48 MY. When point and indel mutation rates are both halved, t1/2 = 0.97 MY. The probability that ASPM retains its ORF after T MY of neutral evolution is ()T/t. This estimation of the rate of pseudogenization is conservative, because other deleterious events such as insertions of transposable elements and null mutations at promoter regions and splicing sites are not considered here. The computer program for the simulation is from ZHANG and W EBB (2003). Previous SectionNext Section

RESULTS

Elevation of dN/dS in the human ASPM lineage: Human ASPM has 28 coding exons, spanning 62 kb in chromosome 1p31 and encoding a huge protein of 3477 amino acids (Figure 1). I determined the entire coding sequences of ASPM from one human, one chimpanzee, and one orangutan, and compared them in the phylogenetic tree of the three species (Figure 2). The orangutan sequence is used as the outgroup for humans and chimpanzees so that nucleotide substitutions on the human and chimpanzee lineages can be separated. I did not sequence the gorilla because the gorilla sequence may not be appropriate as the outgroup due to incomplete lineage sorting (SATTAet al. 2000). Use of orangutan, a slightly more distant outgroup, solves this problem. A commonly used indicator of natural selection at the DNA sequence level is the ratio of the rate of nonsynonymous nucleotide substitution (dN) to that of synonymous substitution (dS). Most functional genes show dN/dS < 1, because a substantial proportion of nonsynonymous mutations are deleterious and are removed by purifying selection, whereas synonymous mutations are more or less neutral and are generally uninfluenced by selection. A gene may occasionally exhibit dN/dS > 1 when a large fraction of nonsynonymous mutations are advantageous and are driven to fixation by positive selection (LI 1997; NEI and KUMAR 2000). I estimated the dN/dS ratio for ASPM in each of the three tree branches (Figure 2), using a maximum-likelihood method, and found that dN/dS is lowest in the orangutan branch (0.43), higher in the chimpanzee branch (0.66), and highest in the human branch (1.03). The hypothesis of dN/dS = 1 is rejected for the orangutan branch (P < 0.001, likelihood-ratio test), but not for the other two branches, suggesting a difference in selection has occurred. Indeed, a test of the difference in dN/dS between the human and orangutan branches gives a marginally significant result (P = 0.064), but the difference between the chimpanzee and orangutan branches is not significant (P = 0.29), nor is the difference between human and chimpanzee branches (P = 0.45). Because the dN/dS ratio between the orangutan and mouse (Mus musculus) is also low (0.29), an increase of dN/dS in humans is more likely than a decrease in orangutans. The mouse sequence (GenBank accession no. AF533752) was not included in the phylogeny-based analysis as it is relatively distantly related to the ape sequences and contains multiple insertions and deletions, which would make the inference less reliable. Similar results are obtained when I first infer the ASPM sequence for the common ancestor of humans and chimpanzees and then estimate the dN/dS ratio by counting the numbers of synonymous and nonsynonymous nucleotide substitutions on each branch. For instance, this approach gives dN/dS = 1.13, 0.84, and 0.52, respectively, for the human, chimpanzee, and orangutan branches.

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FIGURE 1. Nucleotide polymorphisms and substitutions in the human ASPM gene. The exons are shown by solid boxes and introns by lines. The sizes of exons, but not introns, are drawn to scale. The thick horizontal line above exon 18 shows the 867-nucleotide fragment that is not found in the mouse Aspm, with the two thin lines above it showing the two segments amplified in various mammals (see text and Figure 3). Fixed nucleotide substitutions in the human lineage after the human-chimpanzee split are shown by circles, whereas common and rare polymorphisms within humans are shown by squares and triangles, respectively. Nonsynonymous changes are solid symbols and synonymous changes are open symbols. Complete functional relaxation does not adequately explain the elevation of dN/dS: Two hypotheses may explain the increase in dN/dS to 1.03 during the evolution of human ASPM. First, the functional constraints and purifying selection on ASPM may have been completely relaxed and many deleterious nonsynonymous mutations were fixed by random genetic drift. Alternatively, advantageous

nonsynonymous substitutions under positive selection occurred at some sites, while purifying selection acted at some other sites, resulting in an average dN/dS of 1. Under the first hypothesis, ASPM has been under pure neutral evolution since the human-chimpanzee separation 67 MY ago (BRUNETet al. 2002). Using rates of single-nucleotide mutations and insertion/deletion mutations estimated from humanchimpanzee genomic comparisons (BRITTEN 2002; YIet al. 2002), I conducted a computer simulation of neutral evolution of ASPM (see MATERIALS AND METHODS). I found that the probability that ASPM retains its open reading frame after 6 MY of neutral evolution is extremely low (1.7 104). Even when the above two mutation rates are both halved, the probability is still very small (0.014), suggesting that ASPM must have been under purifying selection. The fact that nonsense mutations in ASPM lead to microcephaly also demonstrates the presence of functional constraints on the gene. Thus, the hypothesis of complete relaxation of functional constraints and lack of purifying selection for the past 67 MY of human evolution is inconsistent with the data, and some sites in ASPM must have been subject to purifying selection (dN/dS < 1). This result would imply, although not prove, that some other sites are under positive selection (dN/dS > 1), so that the average dN/dS across the entire protein is 1. However, it is difficult to rule out the possibility of an incomplete functional relaxation in human ASPM, which can lead to a dN/dS ratio of 1 when the number of substitutions is relatively small. A population genetic study may help resolve this question.

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FIGURE 2. The dN/dS ratios for the entire ASPM coding sequence in the human, chimpanzee, and orangutan lineages. The values were estimated from a likelihood analysis. Signatures of purifying selection from population genetic data: The entire coding sequence of ASPM is determined from 14 human individuals of different geographic origins. A total of 33 single-nucleotide polymorphisms are found (Tables 1 and 2). The derived and ancestral alleles are inferred using the chimpanzee and orangutan sequences as outgroups. TAJIMA's (1989) and FU and LI's (1993) tests reveal slight departure of the data from the Wright-Fisher model of neutrality (D =1.29, P = 0.081; F =1.76, P = 0.074; Table 2). But FAY and W U's (2000) test, which is designed to detect recent selective sweeps, does not show a significant result (H = 2.08, P = 0.21). Thus, the negative D and F likely reflect recent population expansions and/or purifying background selection. A recent study suggested that negative D values may also be found under certain sampling schemes if there is fine-scale population differentiation (PTAK and PRZEWORSKI 2002). When the synonymous and nonsynonymous sites were analyzed separately, I detected significant negative D and F values at nonsynonymous sites (P < 0.05; Table 2), but not at synonymous sites. H is not significant at either type of site. These results suggest that the nonsynonymous sites in human ASPM are subject to purifying selection. It should be mentioned that the recombination rate in the ASPM region is 1.8 cM/106 nucleotides (KONGet al. 2002), which translates into 1.1 103 recombination/meiosis for the sequences analyzed here. This relatively high recombination rate localizes signatures of selection to a small region surrounding the selected sites. This might in part explain the above differences in the test results between synonymous and nonsynonymous sites.
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TABLE 1 DNA sequence variations in the ASPM gene of humans

Population genetic theory predicts that deleterious mutations do not reach high frequencies in populations, while neutral and advantageous mutations do. A comparison between rare and common polymorphisms may detect purifying selection of deleterious mutations (FAYet al. 2001). Fay et al. recommended a frequency of 10% for the derived allele as a cutoff between rare and common polymorphisms (FAY et al. 2001, 2002). In the present sample of 28 chromosomes, derived alleles that appear one or two times are regarded as rare polymorphisms, and the rest are common. Because of the limited sample size, a truly rare allele may inadvertently appear more than twice in our sample and a truly common allele may inadvertently be regarded as rare. Using probability theory, I computed that the probability of the former error is <5% for an allele with frequency <3% and the probability of the latter error is <5% for an allele with frequency >20%. Thus, the present classification of rare and common alleles is expected to be relatively accurate. I observed that nR = 15 nonsynonymous and sR = 5 synonymous rare polymorphisms and nC = 5 nonsynonymous and sC = 8 synonymous common polymorphisms from the present data (Table 2; Figure 1). The ratio of nC to nR (5/15 = 0.333) is significantly lower than that of sC to sR (8/5 = 1.6; 2 = 4.41, P < 0.05; Table 2). Since synonymous mutations are more or less neutral, the observed deficit of common nonsynonymous polymorphisms suggests that purifying selection has prevented the spread of nonsynonymous deleterious mutations. It is estimated by the likelihood method that there are N = 7459 and S = 2972 potentially nonsynonymous and synonymous sites in ASPM, respectively. Thus, for rare polymorphisms, there are nR/N = 15/7459 = 2.01 103 polymorphisms/nonsynonymous site and sR/S = 5/2972 = 1.68 10 3 /synonymous site. Their difference is statistically insignificant (2 = 0.09, P > 0.5). In contrast, for common polymorphisms, the number is significantly smaller per nonsynonymous site (nC/N = 5/7459 = 0.67 103) than per synonymous site (sC/S = 8/2972 = 2.69 103; 2 = 6.98, P < 0.01), confirming that purifying selection has reduced the number of common nonsynonymous polymorphisms. This result also suggests the absence or rareness of advantageous nonsynonymous polymorphisms of ASPM that are currently segregating in humans, as such polymorphisms would predominantly show up as common polymorphisms and render nC/N higher. This is consistent with the above result from Fay and Wu's test. The proportion of nonsynonymous polymorphisms not under purifying selection may be estimated by (nC/N)/(sC/S) = (0.67 103)/(2.69 103) = 0.25 or by (nC/sC)/(nR/sR) = (5/8)/(15/5) = 0.21. The two estimates are close to each other and to the dN/dS ratio between the mouse and orangutan (0.29). This indicates that human ASPM is currently under relatively strong purifying selection, and the strength of selection is comparable to or even greater than that in the long-term evolution of mammalian ASPM.
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TABLE 2 Intra- and interspecific variations of the human ASPM gene Comparison of polymorphism and divergence suggests past positive selection: Because both the synonymous and nonsynonymous common polymorphisms are largely neutral, comparing them with the fixed substitutions between humans and chimpanzees can reveal the signature of selection that has influenced the substitution processes (MCDONALD and KREITMAN 1991; FAY et al. 2001, 2002; SMITH and EYRE-W ALKER 2002). This comparison shows a significant excess of fixed nonsynonymous substitutions (2 = 3.88, P < 0.05, Table 2), suggesting that some nonsynonymous substitutions were fixed by positive selection. Because the expansion of brain size occurred in the human lineage after the human-chimpanzee split, it is more relevant to examine whether the human branch exhibits an excess of nonsynonymous substitutions. For this, the ASPM sequence of the common ancestor of humans and chimpanzees was inferred by the Bayesian method. Because the sequences considered are closely related, this inference is reliable, with the average posterior probability >0.999. Comparing the ancestral sequence with the polymorphic human sequences, I identified 16 nonsynonymous and 6 synonymous mutations that have been fixed in the human lineage (Table 2; Figure 1). Their ratio (16/6 = 2.67) is significantly greater than that for common polymorphisms (nC/sC = 5/8 = 0.63; 2 = 4.00, P < 0.05). The number of neutral nonsynonymous substitutions may be estimated from the number of synonymous substitutions multiplied by nC/sC, which yielded 6 (5/8) = 3.75 (FAY et al. 2001, 2002; SMITH and EYRE-W ALKER 2002). The number of nonsynonymous substitutions unexplainable by neutral evolution is 16 3.75 = 12, which may have been fixed by positive selection. It should be noted that a recent population expansion can cause an overestimate of the number of adaptive substitutions when slightly deleterious mutations are present. However, such overestimation is unlikely in the present case because the current effective population size of humans, even after the recent expansion, is still smaller than the long-term effective population size separating humans and chimpanzees and the effective population size of the common ancestor of humans and chimpanzees (TAKAHATAet al. 1995; CHEN and LI 2001; KAESSMANNet al. 2001; EYRE-W ALKER 2002).

It is interesting that there is no significant excess of nonsynonymous substitutions for either the chimpanzee or orangutan branches when the common polymorphisms and substitutions are compared (P > 0.05).

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FIGURE 3. Amplification of segments I and II of the 867-nucleotide IQ-repeat-containing fragment in exon 18 of orthologous ASPM genes of mammals. The two segments are shown in Figure 1. + represents a successful amplification and sequencing and represents no amplification. Note that the entire coding sequence of the gene is known for human, chimpanzee, orangutan, and mouse. The phylogenetic relationships of the mammalian orders are depicted following MURPHY et al. (2001). IQ repeats and brain size variation: Human ASPM contains multiple calmodulin-binding IQ repeats (BONDet al. 2002). In a comparison of putative orthologous ASPM genes from the human, mouse, fruit fly (Drosophila melanogaster), and nematode (Caenorhabditis elegans), BOND et al. (2002) noticed that organisms with larger brains have more IQ repeats, implying a possible relation of IQ repeats and brain size. In particular, the predominant difference between the human and mouse ASPM genes is a large IQrepeat-encoding insertion of 867 nucleotides at the end of exon 18. However, my data showed no difference in the number of IQ repeats between human and chimpanzee ASPM sequences. To trace the origin of the large insertion in human ASPM, I amplified and sequenced from several mammals two DNA segments that cover most of the insertion (Figure 1). Segment I is of 212 nucleotides and segment II is of 706 nucleotides. One or both segments were obtained from species belonging to primates, Cetartiodactyla, Carnivora, and Hyracoidea, but not from mouse or hamster (Figure 3). Phylogenetic analyses were conducted to confirm that the obtained sequences are orthologous to the human sequence (Figure 4). While nonamplification of a sequence does not prove its nonexistence, the amplification of the orthologous sequence indicates its presence. From the recently established mammalian phylogeny (MURPHYet al. 2001), it can be inferred that the large human insertion was already present in the common ancestor of most placental mammals, but was deleted in mouse and possibly in other rodents (Figure 3). Thus, this IQrepeat-containing sequence does not explain the brain size variation among many nonrodent mammals.

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FIGURE 4. Neighbor-joining trees of (A) segment I and (B) segment II sequences from ASPM exon 18 (see Figure 1). The aligned sequences of segment I have 155 nucleotide sites after the removal of primer-encoded regions, and those of segment II have 651 sites. Kimura's two-parameter distances are used. Bootstrap percentages from 2000 replications are shown on interior nodes. Previous SectionNext Section

DISCUSSION

In the above, I provided evidence that advantageous amino acid substitutions unrelated to IQ repeats have been fixed by adaptive selection in human ASPM after the human-chimpanzee split, which strongly suggests that ASPM might be an important genetic component in the evolutionary expansion of human brain. The episode of positive selection on ASPM appears to have ended some time ago, as there is no evidence for positive selection on ASPM in current human populations; rather, relatively strong purifying selection is detected. Roughly, selective sweeps occurring in the past 0.5N generations may be detected (FAY and W U 2000), where N is the effective population size of humans and is thought to be 10,000 (TAKAHATAet al. 1995; HARPENDINGet al. 1998). That is, the positive selection detected in ASPM occurred some time between 67 and 0.1 MY ago (0.5 10,000 generations 20 years/generation). The latter date coincides with the suggested time of migration of modern humans out of Africa (reviewed in CAVALLISFORZA and FELDMAN 2003). It is also interesting to note that although the precise time when positive selection acted on ASPM is difficult to pinpoint, my estimate is consistent with the current understanding that the human brain expansion took place between 22.5 and 0.20.4 MY ago (MCHENRY 1994; W OOD and COLLARD 1999). Furthermore, a selective sweep in human FOXP2, a gene involved in speech and language development, has been detected (ENARDet al. 2002; ZHANGet al. 2002). This sweep was estimated to have occurred no earlier than 0.10.2 MY ago (ENARDet al. 2002; ZHANGet al. 2002). That is, the adaptive evolution of FOXP2 postdated that of ASPM, consistent with the common belief that a big brain may be a prerequisite for language (DECAN 1992). Studies of ASPM in model organisms can help us understand how it impacts brain size. The mouse Aspm is highly expressed in the embryonic brain, particularly during cerebral cortical neurogenesis (BONDet al. 2002). The fruit fly ortholog asp is involved in organizing and binding together microtubules at the spindle poles and in forming the central mitotic spindle (GONZALEZet al. 1990; W AKEFIELDet al. 2001). Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development (W AKEFIELDet al. 2001). The amino acid substitutions in human ASPM are located in exons 3, 18, 20, 21, and 22 (Figure 1), which encode a putative microtubule-binding domain and an IQ calmodulinbinding domain (BONDet al. 2002). These features suggest that the adaptive substitutions in human ASPM might be related to the regulation of mitosis in the nervous system, which can be tested in the future by functional assays of human ASPM as well as a laboratory-reconstructed ASPM protein of the common ancestor of humans and chimpanzees. Previous SectionNext Section

Acknowledgments
I thank David Webb for technical assistance and Douglas Futuyma, Priscilla Tucker, and David Webb for valuable comments on an earlier version of the manuscript. This work was supported by a start-up fund of the University of Michigan and a research grant from the National Institutes of Health (GM67030). Previous SectionNext Section

Footnotes

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY36706587. Communicating editor: S. YOKOYAMA Received July 7, 2003. Accepted August 20, 2003. Copyright 2003 by the Genetics Society of America Previous Section

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, 1994 Tempo and mode in human evolution. Proc. Natl. Acad. Sci. USA 91: 67806786. Abstract/FREE Full Text 24. 1. 2. Mochida G. H., Walsh C. A.

, 2001 Molecular genetics of human microcephaly. Curr. Opin. Neurol. 14: 151156. CrossRefMedline 25. 1. 2. 3. 4. 5. 6. Murphy W. J., Eizirik E., OBrien S. J., Madsen O., Scally M., et al

., 2001 Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294: 23482351. Abstract/FREE Full Text 26. 1. 2. Nei M., Kumar S.

, 2000 Molecular Evolution and Phylogenetics. Oxford University Press, New York. 27. 1. 2. Ptak S. E., Przeworski M.

, 2002 Evidence for population growth in humans is confounded by fine-scale population structure. Trends Genet. 18: 559563. CrossRefMedline 28. 1. 2. Rozas J., Rozas R.

, 1999 DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15: 174175. Abstract/FREE Full Text 29. 1. 2. Saitou N., Nei M.

, 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406425. Abstract 30. 1. 2. 3. Satta Y., Klein J., Takahata N.

, 2000 DNA archives and our nearest relative: the trichotomy problem revisited. Mol. Phylogenet. Evol. 14: 259275. CrossRefMedline 31. 1. 2. Smith N. G., Eyre-Walker A.

, 2002 Adaptive protein evolution in Drosophila. Nature 415: 10221024. CrossRefMedline 32. 1. Tajima F.

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, 1995 Divergence time and population size in the lineage leading to modern humans. Theor. Popul. Biol. 48: 198221. CrossRefMedline 34. 1. 2. 3. Wakefield J. G., Bonaccorsi S., Gatti M.

, 2001 The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis. J. Cell Biol. 153: 637648. Abstract/FREE Full Text 35. 1. 2. Wood B., Collard M.

, 1999 The human genus. Science 284: 6571. Abstract/FREE Full Text 36. 1. Yang Z.

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Medline 41. 1. 2. 3. Zhang J., Rosenberg H. F., Nei M.

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ASPM (gene)
From Wikipedia, the free encyclopedia

Jump to: navigation, search

Asp (abnormal spindle) homolog, microcephaly associated (Drosophila)

Identifiers

Symbols ASPM; ASP; Calmbp1; MCPH5

External OMIM: 605481 MGI: 1334448 HomoloGene: IDs 7650 GeneCards: ASPM Gene

[show]Gene Ontology

Orthologs

Species

Human

Mouse

Entrez

259266

12316

Ensembl ENSG00000066279 ENSMUSG00000033952

UniProt Q8IZT6

Q8CJ27

RefSeq (mRNA)

NM_001206846.1

NM_009791.4

RefSeq (protein)

NP_001193775.1

NP_033921.3

Location Chr 1: (UCSC) 197.05 197.12 Mb

Chr 1: 139.45 139.49 Mb

PubMed [1] search

[2]

This box:

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Abnormal spindle-like microcephaly-associated protein also known as abnormal spindle protein homolog or Asp homolog is a protein that in humans is encoded by the ASPM gene.[1] ASPM is located on chromosome 1, band q31 (1q31).[2] Defective forms of the ASPM gene are associated with autosomal recessive primary microcephaly.[1] "ASPM" is an acronym for "Abnormal Spindle-like, Microcephaly-associated", which reflects its being an ortholog to the Drosophila melanogaster "abnormal spindle" (asp) gene. The expressed protein product of the asp gene is essential for normal mitotic spindle function in embryonic neuroblasts.[2] The mouse gene, Aspm, is expressed in the primary sites of prenatal cerebral cortical neurogenesis. The difference between Aspm and ASPM is a single, large insertion coding for so-called IQ domains.[3] Studies in mice also suggest a role of the expressed Aspm gene product in mitotic spindle regulation.[4]

Contents
[hide]

1 Evolution 2 Diversity 3 See also 4 External links 5 References

[edit] Evolution
A new allele (version) of ASPM appeared sometime between 14,100 and 500 years ago with a mean estimate of 5,800 years ago. The new allele has a frequency of about 50% in populations of the Middle East and Europe, it is less frequent in East Asia, and has low frequencies among Sub-Saharan African populations.[5] It is also found with an unusually high percentage among the people of Papua New Guinea, with a 59.4% occurrence.[6] The mean estimated age of the ASPM allele of 5,800 years ago, roughly correlates with the development of written language, spread of agriculture and development of cities.[7] Currently, two alleles of this gene exist: the older (pre-5,800 years ago) and the newer (post5,800 years ago). About 10% of humans have two copies of the new ASPM allele, while about 50% have two copies of the old allele. The other 40% of humans have one copy of each. Of those with an instance of the new allele, 50% of them are an identical copy[8] suggesting a highly rapid spread from the original mutation. According to a hypothesis called a "selective sweep", the rapid spread of a mutation (such as the new ASPM) through the population indicates that the mutation is somehow advantageous to the individual.[6][9] Testing the IQ of those with and without new ASPM allele has shown no difference in average IQ, providing no evidence to support the notion that the gene increases intelligence.[9][10][11] However statistical analysis has shown that the older forms of the gene are found more heavily in populations that speak tonal languages like Chinese or many SubSaharan African languages.[12]

[edit] Diversity
The DAB1 gene, involved in organizing cell layers in the cerebral cortex, appears to have come under selection in the Chinese. The SV2B gene, which encodes a synaptic vesicle protein, likewise appears to have undergone a selective sweep among AfricanAmericans.[13][14]

4.

WHICH IS THE RNA DEPENDENT DNA POLYMEASE

The telomere is a special functional complex at the end of linear eukaryotic chromosomes, consisting of tandem repeat DNA sequences and associated proteins. It is essential for

maintaining the integrity and stability of linear eukaryotic genomes. Telomere length regulation and maintenance contribute to normal human cellular aging and human diseases. The synthesis of telomeres is mainly achieved by the cellular reverse transcriptase telomerase, an RNAdependent DNA polymerase that adds telomeric DNA to telomeres. Expression of telomerase is usually required for cell immortalization and long-term tumor growth. In humans, telomerase activity is tightly regulated during development and oncogenesis. The modulation of telomerase activity may therefore have important implications in antiaging and anticancer therapy. This review describes the currently known components of the telomerase complex and attempts to provide an update on the molecular mechanisms of human telomerase regulation.

Telomerase
From Wikipedia, the free encyclopedia Jump to: navigation, search

RNA-directed DNA polymerase

A conceptual diagram showing the protein component of telomerase (TERT) in grey and the RNA component (TR) in yellow Identifiers EC number CAS number 2.7.7.49 9068-38-6 Databases

IntEnz BRENDA ExPASy KEGG MetaCyc PRIAM PDB structures Gene Ontology

IntEnz view BRENDA entry NiceZyme view KEGG entry metabolic pathway profile

RCSB PDB PDBe PDBsum

AmiGO / EGO

[show]Search

Telomerase is a ribonucleoprotein that is an enzyme which adds DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. This region of repeated nucleotide called telomeres contains noncoding DNA and hinders the loss of important DNA from chromosome ends. As a result, every time the chromosome is copied only 100200 nucleotides are lost, which causes no damage to the organism's DNA. Telomerase is a reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres, which are shortened after each replication cycle. The existence of a compensatory mechanism for telomere shortening was first predicted by Soviet biologist Alexey Olovnikov in 1973,[1] who also suggested the telomere hypothesis of aging and the telomere's connections to cancer. Telomerase was discovered by Carol W. Greider and Elizabeth Blackburn in 1984 in the ciliate Tetrahymena.[2] Together with Jack W. Szostak, Greider and Blackburn were awarded the 2009 Nobel Prize in Physiology or Medicine for their discovery.[3]

Contents
[hide]

1 Structure 2 Function 3 Clinical implications o 3.1 Aging o 3.2 Cancer o 3.3 Additional roles in cancer, heart disease, diabetes and quality of life o 3.4 Role in other human diseases o 3.5 Telomerase as a potential drug target 4 See also 5 References 6 External links

[edit] Structure
Human telomerase consists of two molecules each of human telomerase reverse transcriptase (TERT), telomerase RNA (TR or TERC), and dyskerin (DKC1).[4] The genes of telomerase subunits, which are TERT,[5] TERC,[6] DKC1,[7] and TEP1[8] etc, are located on different chromosomes in the human genome. Human TERT gene (hTERT) is translated into a protein of 1132 amino acids.[9] TERT proteins from many eukaryotes have been sequenced.[10] TERT polypeptide folds with TERC, a non-coding RNA (451 nucleotides long in human). TERT has a 'mitten' structure that allows it to wrap around the chromosome to add single-stranded telomere repeats. TERT is a reverse transcriptase, which is a class of enzyme that creates single-stranded DNA using single-stranded RNA as a template. Enzymes of this class (not TERT specifically, but the ones isolated from viruses) are utilized by scientists in the molecular biological process of reverse transcriptase PCR (RT-PCR), which allows the creation of several DNA copies of a target sequence using RNA as a template. As stated above, TERT carries its own template around, TERC.

An image illustrating how telomerase elongates telomere ends progressively.

The protein composition of human telomerase was identified in 2007 by Scott Cohen and his team at the Children's Medical Research Institute in Australia.[4] The high-resolution protein structure of the Tribolium castaneum catalytic subunit of telomerase TERT was decoded in 2008 by Emmanuel Skordalakes and his team at The Wistar Institute in Philadelphia.[11] The structure revealed that the protein consists of four conserved domains (RNA-Binding Domain (TRBD), fingers, palm and thumb), organized into a ring configuration that shares common features with retroviral reverse transcriptases, viral RNA polymerases and bacteriophage Bfamily DNA polymerases.

[edit] Function
By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-TTAGGG (in all vertebrates, the sequence differs in other organisms) to the 3' strand of chromosomes. These TTAGGG repeats (with their various protein binding partners) are called telomeres. The template region of TERC is 3'-CAAUCCCAAUC-5'.[12] This way, telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a

new telomere repeat (5'-GGTTAG-3') sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. (For an explanation on why this elongation is necessary see Telomere shortening.)

[edit] Clinical implications

[edit] Aging

The enzyme telomerase allows for replacement of short bits of DNA known as telomeres, which are otherwise shortened when a cell divides via mitosis. In normal circumstances, without the presence of telomerase, if a cell divides recursively, at some point all the progeny will reach their Hayflick limit.[13] With the presence of telomerase, each dividing cell can replace the lost bit of DNA, and any single cell can then divide unbounded. While this unbounded growth property has excited many researchers, caution is warranted in exploiting this property, as exactly this same unbounded growth is a crucial step in enabling cancerous growth. Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is highly expressed in cells that need to divide regularly (e.g., in the immune system), whereas most somatic cells express it only at very low levels in a cell-cycle-dependent manner.[citation needed] A variety of premature aging syndromes are associated with short telomeres.[14] These include Werner syndrome, Ataxia telangiectasia, Ataxia-telangiectasia like disorder, Bloom syndrome, Fanconi anemia, and Nijmegen breakage syndrome. The genes that have been mutated in these diseases all have roles in the repair of DNA damage, and their precise roles in maintaining telomere length are an active area of investigation. While it is currently unknown to what extent telomere erosion contributes to the normal aging process, maintenance of DNA in general, and telomeric DNA specifically, has emerged as a major consideration in aging theory. Dr. Michael Fossel has suggested in an interview that telomerase therapies may be used not only to combat cancer but also to actually get around human aging and extend lifespan significantly. He believes human trials of telomerase-based therapies for extending lifespan will occur within the next 10 years. This timeline is significant because it coincides with the retirement of Baby Boomers in the United States and Europe.[citation needed] Some experiments have raised questions on whether telomerase can be used as an anti-aging therapy, namely, the fact that mice with elevated levels of telomerase have higher cancer incidence and hence do not live longer. Certain premature aging syndromes have been associated with telomere shortening. But, telomerase also favors tumorogenesis, which leads to questions about its potential as an anti-aging therapy.[15] On the other hand, one study showed that activating telomerase in cancer-resistant mice by overexpressing its catalytic subunit extended lifespan.[16] The potential remains for telomerase activators to contribute to the development of cancer.

Exposure of T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity.[17] A study that focused on Ashkenazi Jews found that those that live the longest inherit a hyperactive version of telomerase that rebuilds telomeres.[18] Mice engineered to block the gene that produces telomerase, unless they are given a certain drug, aged at a much faster rate and died at about six months, instead of reaching the average mouse lifespan of about three years. Administering the drug at 6 months turned on telomerase production and caused their organs to be "rejuvenated," restored fertility, and normalized their ability to detect or process odors. The finding raises hope for treatment of conditions such as progeria and other accelerated aging disorders, as well as possible organ regeneration therapies, such as repair of liver damage due to hepatitis or alcoholism.[19] A study published in the journal Nature in January 2011 found that Telomerase reactivation reversed tissue degeneration in older telomerase-deficient mice.[20][21]
[edit] Cancer

When cells are approaching the Hayflick limit in cell cultures, the time to senescence can be extended by the inactivation of the tumor suppressor proteins - p53 and Retinoblastoma protein (pRb)[citation needed]. Cells that have been so-altered will eventually undergo an event termed a "crisis" when the majority of the cells in the culture die. Sometimes, a cell does not stop dividing once it reaches crisis. In a typical situation, the telomeres are lost,[citation needed] and the integrity of the chromosomes declines with every subsequent cell division. Exposed chromosome ends are interpreted as double-stranded breaks (DSB) in DNA; such damage is usually repaired by reattaching (religating) the broken ends together. When the cell does this due to telomere-shortening, the ends of different chromosomes can be attached together. This temporarily solves the problem of lacking telomeres; but, during anaphase of cell division, the fused chromosomes are randomly ripped apart, causing many mutations and chromosomal abnormalities. As this process continues, the cell's genome becomes unstable. Eventually, either sufficient damage will be done to the cell's chromosomes such that cell dies (via programmed cell death, apoptosis), or an additional mutation that activates telomerase will take place.[citation needed] With the activation of telomerase, some types of cells and their offspring become immortal, that is, their chromosomes will not become unstable no matter how many cell divisions they undergo (they bypass the Hayflick limit), thus avoiding cell death as long as the conditions for their duplication are met. Many cancer cells are considered 'immortal' because telomerase activity allows them to divide virtually forever, which is why they can form tumors. A good example of cancer cells' immortality is HeLa cells, which have been used in laboratories as a model cell line since 1951. While this method of modeling human cancer in cell culture is effective and has been used for many years by scientists, it is also very imprecise. The exact changes that allow for the formation of the tumorigenic clones in the above-described experiment are not clear. Scientists have subsequently been able to address this question by the serial introduction of several mutations present in a variety of human cancers. This has led to the understanding of several combinations of mutations that are sufficient for the formation of tumorigenic cells, in

a variety of cell types. While the combination varies depending on the cell type, a common theme is that the following alterations are required: activation of TERT, loss of p53 pathway function, loss of pRb pathway function, activation of the Ras or myc proto-oncogenes, and aberration of the PP2A protein phosphatase.[citation needed] That is to say, the cell has an activated telomerase, eliminating the process of death by chromosome instability or loss, absence of apoptosis-induction pathways, and continued activation of mitosis. This model of cancer in cell culture accurately describes the role of telomerase in actual human tumors. Telomerase activation has been observed in ~90% of all human tumors,[22] suggesting that the immortality conferred by telomerase plays a key role in cancer development. Of the tumors that have not activated TERT,[23] most have found a separate pathway to maintain telomere length termed ALT (Alternative Lengthening of Telomeres).[24] The exact mechanism behind telomere maintenance in the ALT pathway has not been made clear, but likely involves multiple recombination events at the telomere.
[edit] Additional roles in cancer, heart disease, diabetes and quality of life

Additional roles for telomerase per work by Elizabeth Blackburn et al., include the upregulation of 70 genes known or suspected in cancers' growth and spread through the body, and the activation of glycolysis, which enables cancer cells to rapidly use sugar to facilitate their programmed growth rate (roughly the growth rate of a fetus).[25] E. V. Gostjeva et al. (MIT) recently imaged colon cancer stem cells and compared them to fetal colon stem cells trying to make a new colon; they were the same.[citation needed] Elizabeth Blackburn et al. UCSF has shown work that reveals that mothers caring for their very sick children have shorter telomeres when they report that their emotional stress is at the greatest point. She also found telomerase active at the site of blockages in coronary artery tissue. This could be why heart attacks can come on so suddenly: Telomerase is driving the growth of the blockage. In 2009, it was shown that the amount of telomerase activity significantly increased due to psychological stress. Across the sample of patients telomerase activity increased by 18% one hour after the end of the stress. Telomerase activity was examined in peripheral blood mononuclear cells.[26] A study in 2010 found that there was "significantly greater" telomerase activity in participants than controls after a three-month meditation retreat.[27] According to a 2007 study, there is no correlation between socio-economic status and telomere length.[28] Telomerase deficiency has been linked to diabetes mellitus and impaired insulin secretion in mice, due to loss of insulin-producing cells in the pancreas[29]. Blackburn and the two other co-discoverers of telomerase won the Lasker Award (2006), and the Nobel Prize (2009) for the discovery of telomerase and subsequent work on telomerase. Blackburn also won the 2006 Gruber Genetics Prize for same.

[edit] Role in other human diseases

Mutations in TERT have been implicated in predisposing patients to aplastic anemia, a disorder in which the bone marrow fails to produce blood cells, in 2005.[30] Cri du chat Syndrome (CdCS) is a complex disorder involving the loss of the distal portion of the short arm of chromosome 5. TERT is located in the deleted region, and loss of one copy of TERT has been suggested as a cause or contributing factor of this disease.[31] Dyskeratosis congenita (DC) is a disease of the bone marrow that can be caused by some mutations in the telomerase subunits.[32] In the DC cases, about 35% cases are X-linkedrecessive on the DKC1 locus[33] and 5% cases are autosomal dominant on the TERT[34] and TERC[35] loci. Patients with DC have severe bone marrow failure manifesting as abnormal skin pigmentation, leucoplakia (a white thickening of the oral mucosa), and nail dystrophy, as well as a variety of other symptoms. Individuals with either TERC or DKC1 mutations have shorter telomeres and defective telomerase activity in vitro than other individuals of the same age.[36] There has also been one family in which autosomal dominant DC has been linked to a heterozygous mutation in TERT.[37] These patients also exhibited an increased rate of telomere-shortening, and genetic anticipation (i.e., the DC phenotype worsened with each generation).
[edit] Telomerase as a potential drug target

Cancer is a very difficult disease to fight because the immune system has trouble recognizing it. Telomerase is necessary for the immortality of so many cancer types, and so it is thought to be a potential drug target. If a drug can be used to turn off telomerase in cancer cells, the above process of telomere-shortening will resumetelomere length will be lost as the cells continue to divide, mutations will occur and cell stability will decrease. Experimental drug and vaccine therapies targeting active telomerase have been tested in mouse models, and some have now entered early clinical trials. Geron Corporation is currently conducting four human clinical trials involving telomerase inhibition and telomerase vaccination. Merck, as a licensee of Geron, has recent approval of an IND for one vaccine type. The vaccine platform is being tested (and now jointly with Merck) using three different approaches. One vaccine is adenovirus/plasmid based (Merck IND). The second is an autologous dendritic cell based vaccine (GRNVAC1), formerly called TVAX when tested in Phase I clinical trials in Prostate Cancer, and it showed significant PSA doubling times as well as T-cell response. Geron's embryonic stem cell derived dendritic cell vaccine targeting telomerase is the third approach and is currently at the pre-clinical stage. These vaccine methods attempt to teach the human immune system to attack cancer cells expressing telomerase. Geron's telomerase inhibitor drug (GRN163L) attempts to stop cancer cell proliferation by inhibiting telomerase and it is in three separate early stage human clinical trials. Indeed, telomerase inhibition in many types of cancer cells grown in culture has led to the massive death of the cell population. However, a variety of caveats, including the presence of the ALT pathway,[23][24] complicate such therapies. Some have reported ALT

methods of telomere maintenance and storage of DNA in cancer stem cells, however Geron claims to have killed cancer stem cells with their telomerase inhibitor GRN163L at Johns Hopkins. GRN163L binds directly to the RNA template of telomerase. Even a mutation of the RNA template of telomerase would render the telomerase unable to extend telomeres, and therefore not be able to grant replicative immortality to cancer, not allow glycolysis to be inititated, and not upregulate Blackburn's 70 cancer genes. Since Blackburn has shown that most of the harmful cancer-related effects of telomerase are dependent on an intact RNA template, it seems a very worthwhile target for drug development. If indeed some cancer stem cells use an alternative method of telomere maintenance, it should be noted that they are still killed when the RNA template of telomerase is blocked. According to Blackburn's opinion at most of her lectures, it is a big mistake to think that telomerase is involved with only extending telomeres. Stopping glycolysis in cancer stem cells and preventing the upregulation of 70 bad genes is probably what is killing cancer stem cells if they are using alternative methods. The following section will teach you the basics of telomeres and telomerase. It will also introduce you to the potential applications of current telomerase research. Words in italics are defined in the glossary. At the end of some paragraphs, you may view a short animation that will help describe what you just read.(Flash Player is required to view the animations.)

What are telomeres and telomerase?

To better understand telomeres and telomerase, let's first review some basic principles of biology and genetics. The human body is an organism formed by adding many organ systems together. Those organ systems are made of individual organs. Each organ contains tissues designed for specific functions like absorption and secretion. Tissues are made of cells that have joined together to perform those special functions. Each cell is then

made of smaller components called organelles, one of which is called the nucleus. The nucleus contains structures called chromosomes that are actually "packages" of all the genetic information that is passed from parents to their children. The genetic information, or "genes", are really just a series of bases called Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). These base pairs make up our cellular alphabet and create the sequences, or instructions needed to form our bodies. In order to grow and age, our bodies must duplicate their cells. This process is called mitosis. Mitosis is a process that allows one "parent" cell to divide into two new "daughter" cells. During mitosis, cells make copies of their genetic material. Half of the genetic material goes to each new daughter cell. To make sure that information is successfully passed from one generation to the next, each chromosome has a special protective cap called a telomere located at the end of its "arms". Telomeres are controlled by the presence of the enzyme telomerase. Now that we have covered some basics, let's explore telomeres, telomerase, and their importance to you! (view animation) A telomere is a repeating DNA sequence (for example, TTAGGG) at the end of the body's chromosomes. The telomere can reach a length of 15,000 base pairs. Telomeres function by preventing

chromosomes from losing base pair sequences at their ends. They also stop chromosomes from fusing to each other. However, each time a cell divides, some of the telomere is lost (usually 25-200 base pairs per division). When the telomere becomes too short, the chromosome reaches a "critical length" and can no longer replicate. This means that a cell becomes "old" and dies by a process called apoptosis. Telomere activity is controlled by two mechanisms: erosion and addition. Erosion, as mentioned, occurs each time a cell divides. Addition is determined by the activity of telomerase. (view animation) Telomerase, also called telomere terminal transferase, is an enzyme made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes. Telomerase is found in fetal tissues, adult germ cells, and also tumor cells. Telomerase activity is regulated during development and has a very low, almost undetectable activity in somatic (body) cells. Because these somatic cells do not regularly use telomerase, they age. The result of aging cells is an aging body. If telomerase is activated in a cell, the cell will continue to grow and divide. This "immortal cell" theory is important in two areas of research: aging and cancer. Cellular aging, or senescence, is the process by which a cell becomes old and dies. It is due

to the shortening of chromosomal telomeres to the point that the chromosome reaches a critical length. Cellular aging is analogous to a wind up clock. If the clock stays wound, a cell becomes immortal and constantly produces new cells. If the clock winds down, the cell stops producing new cells and dies. Our cells are constantly aging. Being able to make the body's cells live forever certainly creates some exciting possibilities. Telomerase research could therefore yield important discoveries related to the aging process. Cancer cells are a type of malignant cell. The malignant cells multiply until they form a tumor that grows uncontrollably. Telomerase has been detected in human cancer cells and is found to be 10-20 times more active than in normal body cells. This provides a selective growth advantage to many types of tumors. If telomerase activity was to be turned off, then telomeres in cancer cells would shorten, just like they do in normal body cells. This would prevent the cancer cells from dividing uncontrollably in their early stages of development. In the event that a tumor has already thoroughly developed, it may be removed and antitelomerase therapy could be administered to prevent relapse. In essence, preventing telomerase from performing its function would change cancer cells from "immortal" to "mortal".(view animation)

Knowing what we have just learned about telomeres and telomerase, it can be said that scientists are on the verge of discovering many of telomerase's secrets. In the future, their research in the area of telomerase could uncover valuable information to combat aging, fight cancer, and even improve the quality of medical treatment in other areas such as skin grafts for burn victims, bone marrow transplants, and heart disease. Who knows how far this could go?

Telomerase reverses ageing process

Dramatic rejuvenation of prematurely aged mice hints at potential therapy. Ewen Callaway

Protecting chromosome tips doesn't just prevent ageing. It can reverse it.Peter Lansdorp/Visuals Unlimited/Corbis
Premature ageing can be

reversed by reactivating an enzyme that protects the tips of chromosomes, a study in mice suggests. Mice engineered to lack the enzyme, called telomerase, become prematurely decrepit. But they bounced back to health when the enzyme was replaced. The finding, published online today in Nature1, hints that some disorders characterized by early ageing could be treated by boosting telomerase activity. It also offers the possibility that normal human ageing could be slowed by reawakening the enzyme in cells where it has stopped working, says Ronald DePinho, a cancer geneticist at the Dana-Farber Cancer Institute and Harvard Medical School in Boston, Massachusetts, who led the new study. "This has implications for thinking about telomerase as a serious antiageing intervention." Other scientists, however, point out that mice lacking telomerase are a poor stand-in for the normal ageing process. Moreover, ramping up telomerase in humans could potentially encourage the growth of tumours. Eternal youth After its discovery in the 1980s, telomerase gained a reputation as a fountain of youth. Chromosomes have caps of repetitive DNA called telomeres at their ends. Every time cells divide, their telomeres shorten, which eventually prompts them to stop dividing and die.

Telomerase prevents this decline in some kinds of cells, including stem cells, by lengthening telomeres, and the hope was that activating the enzyme could slow cellular ageing. Two decades on, researchers are realizing that telomerase's role in ageing is far more nuanced than first thought. Some studies have uncovered an association between short telomeres and early death, whereas others have failed to back up this link. People with rare diseases characterized by shortened telomeres or telomerase mutations seem to age prematurely, although some tissues are more affected than others.

They are not studying normal ageing, but ageing in mice made grossly abnormal.
David Harrison Jackson Laboratory, Bar Harbor, Maine When mice are engineered to lack telomerase completely, their telomeres progressively shorten over several generations. These animals age much faster than normal mice they are barely fertile and suffer from agerelated conditions such as osteoporosis, diabetes and neurodegeneration. They also die young. "If you look at all those data together, you walk away with the idea that the loss of telomerase could be a very important instigator of the ageing process," says DePinho.

To find out if these dramatic effects are reversible, DePinho's team engineered mice such that the inactivated telomerase could be switched back on by feeding the mice a chemical called 4OHT. The researchers allowed the mice to grow to adulthood without the enzyme, then reactivated it for a month. They assessed the health of the mice another month later. "What really caught us by surprise was the dramatic reversal of the effects we saw in these animals," says DePinho. He describes the outcome as "a near 'Ponce de Leon' effect" a reference to the Spanish explorer Juan Ponce de Leon, who went in search of the mythical Fountain of Youth. Shrivelled testes grew back to normal and the animals regained their fertility. Other organs, such as the spleen, liver and intestines, recuperated from their degenerated state. The one-month pulse of telomerase also reversed effects of ageing in the brain. Mice with restored telomerase activity had noticeably larger brains than animals still lacking the enzyme, and neural progenitor cells, which produce new neurons and supporting brain cells, started working again. "It gives us a sense that there's a point of return for ageassociated disorders," says DePinho. Drugs that ramp up telomerase activity are worth pursuing as a potential treatment for rare disorders characterized by premature

ageing, he says, and perhaps even for more common agerelated conditions. Cancer link The downside is that telomerase is often mutated in human cancers, and seems to help existing tumours grow faster. But DePinho argues that telomerase should prevent healthy cells from becoming cancerous in the first place by preventing DNA damage. David Sinclair, a molecular biologist at Harvard Medical School in Boston, agrees there is evidence that activating telomerase might prevent tumours. If the treatment can be made safe, he adds, "it could lead to breakthroughs in restoring organ function in the elderly and treating a variety of diseases of aging." Other researchers are less confident that telomerase can be safely harnessed. "Telomere rejuvenation is potentially very dangerous unless you make sure that it does not stimulate cancer," says David Harrison, who researches ageing at the Jackson Laboratory in Bar Harbor, Maine. Harrison also questions whether mice lacking telomerase are a good model for human ageing. "They are not studying normal ageing, but ageing in mice made grossly abnormal," he says. Tom Kirkwood, who directs the Institute for Ageing and Health at Newcastle University, UK, agrees, pointing out that telomere erosion "is surely not

the only, or even dominant, cause" of ageing in humans. DePinho says he recognizes that there is more to ageing than shortened telomeres, particularly late in life, but argues that telomerase therapy could one day be combined with other therapies that target the biochemical pathways of ageing. "This may be one of several things you need to do in order to extend lifespan and extend healthy living," he says.

5.

WHAT IS C0T CORE ANALYSIS ANF APPLICATION

A C0t curve describes the sequential complexity of a DNA sample. It describes the kinetics of reassociation and reannealing of a sample of double-stranded DNA that has been (1) fragmented to small pieces, approximately 400 nucleotides, (2) denatured to single strands, and (3) then permitted to reanneal. The extent to which two complementary DNA strands reassociate is proportional to t. C0 is the original molar concentration of the complementary strands of DNA, and t is the

incubation time reannealing reaction. If the

is the value of ^t required for completing half of the

sequences of all initial DNA fragments were identical, complementary fragments encounter each other readily and the mixture reanneals readily at a low value of ^t. At the other extreme, reannealing is very slow and occurs only at high ^t values if the original DNA contains many different sequences, so that the concentrations of complementary strands are very low and they encounter each other only infrequently. The value of ^t^ is a measure of the sequential complexity of the original DNA. Such studies on genomic DNA were one of the first indications that genomes often contain repetitive DNA. Such DNA segments are present in higher concentrations than those segments present in only one copy per genome, and this fraction of the DNA reanneals at a correspondingly lower t value. The results indicate that different sequences are present in widely varying frequencies throughout the genome, and only a fraction occurs only once per genome.

MEASURING GENOME SIZE AND COMPLEXITY USING C0T CURVES

Rate of reassociation
Protocol: 1. Shear DNA to ca. 400bp (eg. blender,sonicator, passage through a small orifice). 2. Denature fragments by boiling. 3. Reassociate in 0.18M salt 4. Measure A260 or, if radioactively labeled, amount bound to HAP (hydroxyapatite) column. HAP preferentially binds dsDNA at 0.18M salt

DEMO: kinetic_class_demo.obj
The reassociation of DNA molecules in solution is described by the relation:

-dC
where

kC2 = --dt

C ::= [ssDNA] (mole L-1) dC ::= change in [ssDNA]/sec. t ::= time (sec.) k ::= 2nd order rate constant, dependent on temperature and ionic conditions and complexity of DNA (L mole-1 sec-1)
This equation can be transformed into the more convenient expression,

C --C0
where

1 = ----------1 + k C0t

C0 ::= the initial[ssDNA] at time 0. C0t ::= the value of C0t at which annealing has proceeded to half completion (C/C0=0.5).

Redrawn from Russel, P.J. (1986) Genetics Figure 7.24. Ideal time course for the renaturation of DNA as seen in a Cot (initial DNA concentration x time) plot. In the initial state the DNA is single-stranded, and in the final state it is all double-stranded. Note that 80 percent of the renaturation occurs over a 2 log Cot interval.

C0t = Complexity

1 --k

The complexity of a sequence is defined as the longest non-repetitive sequence that can be derived from a sequence

sequence

complexity

AAAAAAAAAA TTTTTTTTTT ATATATATAT TATATATATA

1 2

ATGATGATG TACTACTAC ATGCATGC TACGTACG ATGCCATGCC TACGGTACGG

Demo: kinetic_class_demo.obj

3 4 5

The complexity (X) of a population of uniformly-sized DNA molecules can be measured as follows:

X= KC0t
where K has been determined under standard conditions (0.18M cations {eg. Na + }, 400 nucleotide fragmentsize) as approximately 5 x 10 5 L bp mole-1 sec-1 .
Rearranging the equation, we see that C 0t= X/K. Therefore, C0t increases with the complexity of the DNA. We can use this relation to measure genome sizeas shown in the figure below:

Redrawn from Russel,P.J. (1986)Genetics Fig. 7-25a. Cot plots showing the renaturation of DNAs from organisms with small genomes: the bacterium E.coli, the bacterial viruses T2 and Lambda, and the animal virus SV40.

Redrawn from Russel,P.J. (1986 )Genetics Fig. 725b. Kinetics of renaturation of DNAfrom calf thymus and E. coli as seen in a Cot plot. The E.coli DNA consists almost entirely of unique sequences. However, the shape of the Cot curve for the calf DNA is very different from that of E. coli and indicates that there are some sequences (toward the right of the curve) that renature much more slowly and some (toward the left of the curve) that renature much more quickly than the bacterial DNA sequences.

The C0t curves for Calf thymus and E.coli DNA indicate that, while E.coli DNA anneals at a relatively sharp inflection point, Calf DNA contains three major kinetic classes: highly repetitive, which reanneals very early in the reaction, middle repetitive, which anneals over more than 3 log C0t, and single copy, which anneals at very high C 0 t values. The term "single-copy" is a bit misleading, in that it refers to 1 - 10 copies per haploid genome. In fact, any distinction between single copy and middle repetitive forces you to draw an arbitrary line. They are useful concepts because they bring out something of the content of genomes, but the definitions of highly repetitive, middle repetitive and single-copy shouldn't be pushed too far. Another point to mention is that although most protein coding genes are found in the single copy fraction, not all of the single copy fraction is protein coding genes. There appears to be a lot of non-coding single copy "junk" in many eukaryotic genomes.

Calculation of complexity of a single kinetic class

Let f represent the genome fraction of a kinetic class of DNA. For example, the genome fraction f for the highly-repetitive fraction of Calf DNA is about 0.15, or 15% of the total genome. We can calculate the C0t for a genome fraction that is part of a mixture (ie. a complex genome) by the following equation

(pure) = fC0t (mixture) This value can be plugged into the equation for complexity:

C0t

X = KC0t (pure)
By combining data for the different kinetic classes, the complexity and size for a wide range of genomes has been measured, as shown in the table:

Table 1 from Okamuro & Goldberg p8 in The Biochemistry of Plants Vol. 15

TABLE 1. Plant and animal genome sizeand genome complexity. Species Arabidopsis thaliana Cotton (Gossypium hirsutum) Flax (Linum usitatissimum) Maize (Zea mays) Mung bean (Vigna radiata) Parsley (Petroselinum sativum) Pea (Pisum sativum) Pearl millet (Pennisetum americanum) Soybean (Glycine max) Tobacco (Nicotiana tabacum) Genome size Genome complexity (kb) (kb) 7.0 x 104 7.2 x 105 1.5 x 105 5.7 x 106 4.7 x 105 3.8 x 106 4.5 x 106 3.8 x 105 1.3 x 106 1.8 x 106 1.5 x 106 5.5 x 104 5.1 x 105 6.8 x 104 2.3 x 106 2.6 x 105 1.3 x 106 1.3 x 106 1.0 x 105 6.9 x 105 7.3 x 105 6.4 x 105

2.4 x 106 Wheat (Triticum aestivum) Man (Homo sapiens) Mouse (Mus musculus) Fruit fly (Drosophilia melanogaster) 5.2 x 106 3.0 x 106 1.6 x 106 1.5 x 105

1.0 x 106 6.2 x 105 1.0 x 106 9.1 x 105 1.1 x 105 7.0 x 104 3.4 x 104 4.2 x 103

Nematode worm (Caernorhabditis elegans ) 8.0 x 104 Water mold (Achyla bisexualis) Escherichia coli 4.2 x 104 4.2 x 103

6.

WHAT IS HIGH RESOLUTION MELTING(HRM)

High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples. It was discovered and developed by Idaho Technology and the University of Utah.[1] It has advantages over other genotyping technologies, namely:

It is cost effective vs. other genotyping technologies such as sequencing and Taqman SNP typing. This makes it ideal for large scale genotyping projects. It is fast and powerful thus able to accurately genotype many samples rapidly. It is simple. With a good quality HRM assay, powerful genotyping can be performed by nongeneticists in any laboratory with access to an HRM capable real-time PCR machine.

Contents
[hide]

1 Method o 1.1 Spot the difference o 1.2 Wild type, heterozygote or homozygote? 2 Applications of HRM o 2.1 SNP typing/Point mutation detection o 2.2 Zygosity testing o 2.3 Epigenetics

o 2.4 Intercalating dyes 3 See also 4 References 5 Further information

[edit] Method
HRM analysis is performed on double stranded DNA samples. Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. Essentially the PCR process turns a tiny amount of your region of DNA of interest in to a large amount, so you have quantities large enough for better analysis. In the tube there are now many copies of your region of DNA of interest. This region that is amplified is known as the amplicon. After the PCR process the HRM analysis begins. The process is simply a precise warming of the amplicon DNA from around 50C up to around 95C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or melt apart.

The secret of HRM is to monitor this process happening in real-time. This is achieved by using a fluorescent dye. The dyes that are used for HRM are known as intercalating dyes and have a unique property. They bind specifically to double-stranded DNA and when they are bound they fluoresce brightly. In the absence of double stranded DNA they have nothing to bind to and they only fluoresce at a low level. At the beginning of the HRM analysis there is a high level of fluorescence in the sample because of the billions of copies of the amplicon. But as the sample is heated up and the two strands of the DNA melt apart, presence of double stranded DNA decreases and thus fluorescence is reduced. The HRM machine has a camera that watches this process by measuring the fluorescence. The machine then simply plots this data as a graph known as a melt curve, showing the level of fluorescence vs the temperature:

[edit] Spot the difference

The melting temperature of the amplicon at which the two DNA strands come apart is entirely predictable. It is dependent on the sequence of the DNA bases. If you are comparing two samples from two different people, they should give exactly the same shaped melt curve. However if one of the people has a mutation in the DNA region you have amplified, then this will alter the temperature at which the DNA strands melt apart. So now the two melt curves appear different. The difference may only be tiny, perhaps a fraction of a degree, but because the HRM machine has the ability to monitor this process in high resolution, it is possible to accurately document these changes and therefore identify if a mutation is present or not.

[edit] Wild type, heterozygote or homozygote?

Things become slightly more complicated than this because organisms contain two (or more) copies of each gene, known as the two alleles. So, if a sample is taken from a patient and amplified using PCR both copies of the region of DNA (alleles) of interest are amplified. So if we are looking for mutation there are now three possibilities:
1. Neither allele contains a mutation 2. One or other allele contains a mutation 3. Both alleles contain a mutation.

These three scenarios are known as Wild type, Heterozygote or Homozygote respectively. Each gives a melt curve that is slightly different. With a high quality HRM assay it is possible to distinguish between all three of these scenarios.

[edit] Applications of HRM

[edit] SNP typing/Point mutation detection

Conventional SNP typing methods are typically time consuming and expensive, requiring several probe based assays to be multiplexed together or the use of DNA microarrays. HRM is more cost effective and reduces the need to design multiple pairs of primers and the need to purchase expensive probes. The HRM method has been successfully used to detect a single G to A substitution in the gene Vssc (Voltage Sensitive Sodium Channel) which confers resistance to the acaricide permethrin in Scabies mite. This mutation results in a coding change in the protein (G1535D). The analysis of scabies mites collected from suspected permethrin susceptible and tolerant populations by HRM showed distinct melting profiles. The amplicons from the sensitive mites were observed to have a higher melting temperature relative to the tolerant mites, as expected from the higher thermostability of the GC base pair
[2]

In a field more relevant to clinical diagnostics, HRM has been shown to be suitable in principle for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. More than 400 mutations have been identified in these genes. The sequencing of genes is the gold standard for identifying mutations. Sequencing is time consuming and labour intensive and is often preceded by techniques used to identify heteroduplex DNA, which then further amplify these issues. HRM offers a faster and more convenient closed-tube method of assessing the presence of mutations and gives a result which can be further investigated if it is of interest. In a study carried out by Scott et al. in 2006,[3] 3 cell lines harbouring different BRCA mutations were used to assess the HRM methodology. It was found that the melting profiles of the resulting PCR products could be used to distinguish the presence or absence of a mutation in the amplicon. Similarly in 2007 Krypuy et al.[4] showed that the careful design of HRM assays (with regards to primer placement) could be successfully employed to detect mutations in the TP53 gene, which encodes the tumour suppressor protein p53 in clinical samples of breast and ovarian cancer. Both these studies highlighted that fact that changes in the melting profile can be in the form of a shift in the melting temperature or an obvious difference in the shape of the melt curve. Both of these parameters are a function of the amplicon sequence. The overall consensus is

that HRM is a cost efficient method that can be employed as an initial screen for samples suspected of harbouring polymorphisms or mutations. This would reduce the number of samples which need to be investigated further using more conventional methods.
[edit] Zygosity testing

Currently there are many methods used to determine the zygosity status of a gene at a particular locus. These methods include the use of PCR with specifically designed probes to detect the variants of the genes (SNP typing is the simplest case). In cases where longer stretches of variation is implicated post PCR analysis of the amplicons may be required. Changes in enzyme restriction, electrophoretic and chromatographic profiles can be measured. These methods are usually more time consuming and increase the risk of amplicon contamination in the laboratory, due to the need to work with high concentrations of amplicons in the lab post-PCR. The use of HRM reduces the time required for analysis and the risk of contamination. HRM is a more cost effective solution and the high resolution element not only allows the determination of homo and heterozygosity, it also resolves information about the type of homo and heterozygosity, with different gene variants giving rise to differing melt curve shapes. A study by Gundry et al. 2003,[5] showed that fluorescent labelling of one primer (in the pair) has been shown to be favourable over using an intercalating dye such as SYBR green I. However, progress has been made in the development and use of improved intercalating dyes [6] which reduce the issue of PCR inhibition and concerns over non-saturating intercalation of the dye.
[edit] Epigenetics

The HRM methodology has also been exploited to provide a reliable analysis of the methylation status of DNA. This is of significance since changes to the methylation status of tumour suppressor genes, genes that regulate apoptosis and DNA repair, are characteristics of cancers and also have implications for responses to chemotherapy. For example, cancer patients can be more sensitive to treatment with DNA alkylating agents if the promoter of the DNA repair gene MGMT of the patient is methylated. In a study which tested the methylation status of the MGMT promoter on 19 colorectal samples, 8 samples were found to be methylated.[7] Methylated DNA can be treated by bi-sulphite modification, which converts non-methylated cytosines to uracil. Therefore, PCR products resulting from a template that was originally unmethylated will have a lower melting point than those derived from a methylated template. HRM also offers the possibility of determining the proportion of methylation in a given sample, by comparing it to a standard curve which is generated by mixing different ratios of methylated and non-methylated DNA together. This can offer information regarding the degree of methylation that a tumour may have and thus give an indication of the character of the tumour and how far it deviates from what is normal. HRM also is practically advantageous for use in diagnostics, due to its capacity to be adapted to high throughput screening testing, and again it minimises the possibility of amplicon spread and contamination within a laboratory, owing to its closed-tube format.

[edit] Intercalating dyes

To follow the transition of dsDNA (double-stranded) to ssDNA (single-stranded), intercalating dyes are employed. These dyes show differential fluorescence emission dependent on their association with double-stranded or single-stranded DNA. SYBR Green I is a first generation dye for HRM. It fluoresces when intercalated into dsDNA and not ssDNA. Because it may inhibit PCR at high concentrations, it is used at sub-saturating concentrations. Recently, some researchers have discouraged the use of SYBR Green I for HRM,[8] claiming that substantial protocol modifications are required. This is because it is suggested that the lack of accuracy may result from dye jumping, where dye from a melted duplex may get reincorporated into regions of dsDNA which had not yet melted.[5][8] New saturating dyes such as LC Green and LC Green Plus, ResoLight, EvaGreen, Chromofy and SYTO 9 are available on the market and have been used successfully for HRM. However, some groups have successfully used SYBR Green I for HRM with the Corbett Rotorgene instruments [9] and advocate the use of SYBR Green I for HRM applications.

Resolution Melting (HRM) is a novel, homogeneous, close-tube, post-PCR method, enabling genomic researchers to analyze genetic variations (S ations, methylations) in PCR amplicons. It goes beyond the power of classical melting curve analysis by allowing to study the thermal denaturation ble-stranded DNA in much more detail and with much higher information yield than ever before.HRM characterizes nucleic acid samples based on sociation (melting) behavior. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even s changes such as SNPs (single nucleotide polymorphisms) can be readily identified.

most important High Resolution Melting application is gene scanning - the search for the presence of unknown variations in PCR amplicons prior to ternative to sequencing. Mutations in PCR products are detectable by High Resolution Melting because they change the shape of DNA melting curv bination of new-generation DNA dyes, high-end instrumentation and sophisticated analysis software allows to detect these changes and to d mation about the underlying sequence constellation.

M Applications introduction of HRM has renewed interest in the utility of DNA melting for a wide range of uses, including: Mutation discovery (gene scanning) Screening for loss of heterozygosity DNA fingerprinting SNP genotyping Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Somatic acquired mutation ratios HLA compatibility typing Association (case/control) studies Allelic prevalence in a population Identification of candidate predisposition genes

HRM, these and other applications are done using low-cost generic dyes where previously custom labeled probes such as TaqMan or fluoresc nance energy transfer (FRET) probes were required. HRM is thus a simpler and much more cost-effective way to characterize samples.

pedia

olecular biology High Resolution Melt or HRM analysis as it will be referred to herein is a hugely powerful technique for the detection of mutat morphisms and epigenetic differences in double stranded DNA samples. It has advantages over other genotyping technologies. Namely:

It is massively cost effective vs. other genotyping technologies such as sequencing and Taqman SNP typing. This makes it ideal for large genotyping projects. It is fast and powerful thus able to accurately genotype huge numbers of samples in rapid time. It is simple. With a good quality HRM assay powerful genotyping can be performed by non-geneticists in any laboratory with access to an capable real-time PCR machine. http://en.wikipedia.org/wiki/High_Resolution_Melt

Instrumentation

everal years, various researchers and instrument makers have independently investigated the utility of high-resolution DNA dissociation analysis mple, the team at Idaho Technology has done an admirable job of vigorously promoting their research through traditional journal publicat ersely, Corbett Life Science does not pursue publication, but instead relies on the publications of customers to promote the technology. Regard companies have independently advanced the field of high resolution dissociation analysis and successfully introduced what has now become know resolution melt (HRM) ana

o Technology was first to market with an instrument made specifically to do dissociation analysis; the HR-1. The HR-1 was a showpiece for nology with the singular aim of producing the most detailed melt curve possible. As such, it opened the eyes of many to the potential of HRM ains the performance benchmark for the acquisition of an individual melt curve. However the HR-1 is not capable of thermal cycling and can yze a single sample from within a glass capillary per run making data analysis time consuming. http://www.idahotech.com/HR-1/index.

i-well instruments with greater practical utility were introduced to the market very soon after the HR-1. The first multi-well HRM instruments were r-Gene 6000 (Corbett Life Science) and the LightScanner (Idaho Technology) (PDF). These two instruments were introduced at about the same time oyed fundamentally different technical innovations to achieve HRM. The LightScanner uses a modified block-based design available in 96-well or versions. Despite advanced engineering, it still suffers from measurable sample-to-sample thermal and optical variation and is unable to match ormance benchmark set by the original HR-1 instrument. Like the HR-1, the LightScanner is not capable of thermal cyc

Rotor-Gene 6000 was the first of the multi-well instruments capable of both thermal cycling and HRM. This dual capability enables samples to be essed in the one instrument (i.e. pre-amplification and HRM done consecutively in the one run). A major advantage of this is that amplification plots sed to help interpret HRM results since aberrant amplification plots (i.e. those that amplified differently to what was expected) also produce aber data. In this way compromised samples can be easily identified and removed from downstream HRM analysis. The main advantage of the Rotor-G HRM stems from its rotary design, in which samples spin under centrifugal force past a common optical detector. This is seemingly ideal for HR mal or optical variation between samples is insignificant. The result is that the Rotor-Gene HRM performance closely matches the HR-1 benchmark ompromise that samples are not arranged in a conventional array format (as they are in block-based instruments) but are instead arranged around meter of a spinning r

more recently introduced LightCycler 480 (Roche Molecular Systems)is capable of HRM and thermal cycling. The LightCycler 480 is a block-b ument design and it has better thermal uniformity than other block-based instruments, it nevertheless does exhibit measurable thermal and op uniformity.

r instrument providers are now rushing to introduce HRM capability and some are planning to release software upgrades to support HRM analysis er here is that instruments not specifically engineered for HRM will deviate so much from the HR-1 performance benchmark that careful investiga need be done before accepting those instruments as HRM capable.

ample HRM data for each of the multi-well HRM systems discussed here is shown in the figures (A-E) below.

r comparison purposes, similar data for two standard real-time PCR instruments (i.e. not engineered for HRM) is also shown. All data has been enlar hout modification directly from (Herrmann et al 2007)Normalized melting curves of a 110 bp beta-globin amplicon (triplicate HRM data) contai

gle and double SNPs are shown.

Rotor-Gene: all ur genotypes are arly tinguished

(click figure to enlarge)

LightScanner: y heterozygotes n be tinguished (PDF)

(click figure to enlarge)

LightCycler 0:double terozygote can clearly tinguished

(click figure to enlarge)

AB 7300:double terozygotes can distinguished

(click figure to enlarge)

asterCycler:none the genotypes n be tinguished

(click figure to enlarge)

data normalizationshape& shift are two ways HRM curve the plots shape can of the discriminate melt between curve itself

sam

Shape ,i.e. using detail in Shift; i.e. the thermal offset of a curve from other curves.

re HRM curves are plotted, the raw data is first normalized. Melt curves are normally plotted with fluorescence on the Y axis and temperature on t This is similar to real-time PCR amplification plots but with the substitution of temperature for cycle number. As with real-time PCR plots escence axis of HRM plots is normalized onto a 0 to 100% s

merging trend is to also apply normalization to the temperature (X) axis. This has the desired effect of compensating for well-to-well tempera surement variations between samples. Known astemperature shifting, it was introduced by Idaho Technology and is now also supported by the R Cycler 480. Unfortunately, temperature shifting normalization removes any potential discriminatory power provided by the temperature

ome applications, temperature shifting normalization may be a useful solution but for many routine applications it is actually detrimental. A mple of this is the discrimination of homozygous SNPs. On the one hand, heterozygous samples are often more easily discriminated after tempera ng normalization (because their curves have a complex shape), but the discrimination of homozygous samples is usually made more difficult bec often have a simple and identical curve shape (Figure 1). While homozygous SNP samples have an identical curve shape, they can usuall iminated by HRM analysis by observing a change in their respective Tms. This characteristic means the melt plots of different homozygotes w t one from another thereby allowing them to be readily discriminated (so long as temperature shifting normalization is not applied and the perature data is precise enough). Currently, the only instrument system that does not use temperature shifting normalization and can rel iminate homozygous SNPs is the Rotor-Gene (Corbett Life Science). The Rotor-Gene can discriminate homozygotes because well-to-well the tion is so low on that instrument that the collected temperature data is sufficiently precise (Figure 2).

ure 1: Thermal shifting normalization on the LightCycler 480 (Roche Applied Science)

licate HRM data was captured on a Roche LightCycler 480 for SNP genotyping (Herrmann et al 2007) Normalized melting curves are of a 110 bp beta-globin amplicon. Genotypes are discriminated or as follows; green = homozygous wild type, red = homozygous mutant (20A>T), black = single heterozygous mutant (20A>T), blue = double heterozygous mutant [9C>T; 20A>T]. Plots are sho ore (A) and after (B) temperature shifting normalization. Double normalized melt curves of homozygous genotypes overlay and cannot be discriminated; however, discrimination of heterozyg otypes is improved.

ure 2: Thermal sifting normalization on the Rotor-Gene (Corbett Life Science)

licate HRM data was captured on a Rotor-Gene for SNP genotyping (Herrmann et al 2007) Normalized melting curves are of a 110 bp beta-globin amplicon. Each category of SNP genotype can dily discriminated prior to thermal shifting normalization. However, when curves are thermal shifted the homozygous genotypes overlay precisely and can no longer be discriminated.

h-resolution DNA Melting Analysis

en it comes to genotyping and mutation scanning, high-resolution DNA ting is emerging as the technique of choice because it is inexpensive ple, accurate and rapid. Development of this method of DNA analysis been underway since its introduction in 2002 by a team of researchers m our Pathology Department led by Dr. Carl Wittwer and Dr. Karl lkerding at the University of Utah coupled with collaborative efforts m Idaho Technology. High-resolution melting required new rumentation. The first high-resolution instrument developed, named HR-1, remains the most accurate with the fastest analysis speed, while LightScanner has the highest throughput. In addition to the special rumentation, high-resolution melting uses special saturation dyes that resce only in the presence of double stranded DNA. These dyes are uded in the PCR amplification process. When the sample is heated to temperatures, the DNA denatures and the fluorescent color fades y as the double stranded DNA separates, generating a melting curve. ause different genetic sequences melt at slightly different rates, they be viewed, compared, and detected using these curves. Even a single e change will cause differences in the melting curve. The process can be d for specific genotyping, comparing sequence identity between two A samples, and scanning for any sequence variant between two primers. h-resolution DNA melting is becoming more popular as its accuracy and plicity is recognized. High-res DNA melting makes it possible to quickly accurately determine whether DNA sequences match, providing an resting option for transplantation matching and forensics. Genotyping high-resolution melting is more streamlined and less expensive than hods that use complex probes.

No processing is required, and when combined with the speed of rapid-cylce PCR, has interesting potential for personal DNA diagnostics. For example, the amount of medication a person needs is often dependent on sequence variants in genes that can be determined through high-resolution DNA melting. Hi-res melting can also be used to scan large genes for variation, in many cases greatly reducing or eliminating the need for sequencing. Although high-resolution DNA melting is relatively new, it is expanding and being improved upon by our talented team of scientists in Pathology and we are excited to be at the forefront of such innovative and important technology.

e information at http://www.path.utah.edu/news/hi-res-dna-melting-analysis

software

applica

Resolution Melting (HRM) Software v 2.0 by Applied Biosystems

No Temperature Shift Required. Fast. Accurate. Identify more new variants, quickly and accurately. High Resolution Melting (HRM) analysis is an alternative to dHPLC sequencing screening of new gene variants. The HRM Software is now avai on the Applied Biosystems 7500 Fast System and on the 7900HT Fast Real-Time PCR System. The 7500 Fast Real-Time PCR System delivers pr results with fast thermal cycling in a standard 96-well format. Achieve high-throughput HRM analysis with the 384-well 7900HT, the gold stan high throughput system. The AB HRM application does not require temperature shifting, which results in a greater likelihood of identifying homozygous mutations than methods that require temperature shifting.

Applied Biosystems HRM Software provides an easy and intuitive workflow that: Shortens analysis time by auto-calling genotypes and automatically omitting the no template controls

Minimizes subjective analysis by automatically grouping unknown variant clusters Allows easy data review with customizable plot views, expandable windows, and one-click color assignment to highlight curves of interest Ability to analyze multiple targets (assays) on one plate

ownload the HRM guide B. 7900HT Fast Real-Time PCR System

500 Fast Real-Time PCR System

eterozygote

Homozygous - Wildtype

Homozygous - Variant

re 1 rence plot generated with the Applied Biosystems HRM application on the 7500 Fast & 7900HT Fast Real-Time PCR Systems.

The ability to easily identify new variants is key for successful HRM applications. By eliminating the temperature shift step, the Applied Biosystems HRM solution (Fig 1) was able to clearly distinguish homozygous variant samples from homozygous wildtype samples in 97.5% of the population, whereas the other HRM system from Competitor R (Fig 2) was only able to distinguish them in 10% of the population. All genotypes were auto-called by the respective software packages and were not altered by the operator. Class 1 SNP (A/G), multiple technical replicates of nine DNA samples representing three genotypes: homozygous wildtype (G/G), homozygous mutant (A/A) and heterozygous re 2 Difference plot generated on another plate-based (A/G).

time HRM system.

more info here => http://marketing.appliedbiosystems.com/mk/get/HRM_LANDING

Workflow in the LC 480

e Scanning by High Resolution Melting Curve Analysis generally requires the use of

a special generic DNA dye that works at high, saturating concentrations without inhibiting PCR and therefore leads to homogeneous stainin homo-or heteroduplex DNA an instrument with suitable excitation/emmission wavelengths, high data acquisition rates, and outstanding temperature homogeneity a software algorithm that analyzes the shape of the melting curves and groups those that are similar.

Gene Scanning experiment, sample DNA is first amplified via real-time PCR in the presence of a proprietary saturating DNA dye. A melting curve is ormed using high data acquisition rates, and data are finally analyzed using a Gene Scanning Software, by three basic steps:

1. Normalization: the pre-melt (initial fluorescence) and post-melt (final fluorescence) signals of all samples are set to uniform, relative values
100% to 0%

2. Temperature shifting: the temperature axis of the normalized melting curves is shifted to the point where the entire double-stranded DN

completely denatured. Samples with heterozygous SNPs can then be easily be distinguished from the wild type by the different shapes of melting curves. 3. Difference Plot:the differences in melting curve shape are further analyzed by subtracting the curves from a reference curve. This helps clu samples automatically into groups that have similar melting curves (e.g., those who are heterozygote as opposed to homozygotes). 4. Application Manuals and Technical Guide LightCycler 480 Technical Note No. 1: "High Resolution Melting: Optimization Strategies" (12 pa Enables HRM users to successfully set up and carry out mutation scanning experim Copy available for download => LC480-Technical-Note-01-HRM.pdf

-resolution melting curve analysis on the LightCycler 480 PCR system (presented by Roche Aplied Science) e Applied Sciences LightCycler family of real-time PCR systems offer fast, accurate and versatile platforms for genetic variation research. The -based LightCycler 480 System provides the temperature homogeneity and optical characteristics required for high-performance melting-c ysis (MCA). On the level of data acquisition and available detection channels, this new instrument opens the way to more advanced applications in rging field of gene scanning where amplicons can be screened for unknown sequence variations with low efforts in time and

LightCycler 480 real-time PCR system: a versatile platform for genetic variation rese time PCR is a well established technique for studying genetic variation using various probe-based methods for genotyping as well as high-resolu ysis of whole amplicons melted in the presence of saturating DNA dyes. The latter, relatively new, method allows screening for unknown mutation modifications. The LightCycler 480 real-time PCR system is a multiwell platebased instrument that provides integrated applications for detecting acterizing genetic variation using all these methodological approac

sfering PCRs to HRM-assays on the LightCycler 480 SystemExamples for BR -resolution melting curve analysis (hrMCA) is an attractivetechnique to scan for unknown mutations in genes. To evaluate how easy or difficult it gn hrMCA assays using the LightCycler480 Instrument, we selected 3 different fragments in exon 11 of the BRCA1 gene, designed an MCA assay d its sensitivity to detect known vari

high-throughput

Methylation

analysis

using

the

LightCycler

480

system

(presented

by

Roche

Aplied

Scie

osatellite Analysis of Grapevine Varieties by HRM Analysis (by John Mackay)

Genotyping by High Resolution Melt (Corbett Life Science)

imination of human ACTN3 (R577X) SNP genotypes (C to T substitution) using SYTO 9 intercalation dye (no probes). Homozygous wild type, mutat heterozygote samples are shown on a standard normalized melt curve (A) and a difference plot normalized to mutant samples (B). Amplification an analysis was done using a Rotor-Gene 6000 instrument and genotypes were automatically assigned by the Rotor-Gene software.

M - Assay Design and Analysis (by Corbett Life Science)

ry good explanation of the HRM method !

M - Product information sheet (by Corbett Life Science)

M - Pyrosequencing information sheet (by Qiagen)

ntly, HRM was the subject of a detailed and independent Technology Assessment rt from the National Genetics Reference Laboratory (Wessex, UK). A wide range of ple types were tested, including examples of challenging G to C and A to T single base titutions. The full report is now available for download =>

ation Scanning by High Resolution Melt Analysis. Evaluation of Rotor-Gene 6000 bett Life Science), HR-1 and 384-well LightScanner (Idaho Technology)

e H and Potts G.; National Genetics Reference Laboratory (Wessex, 2006) NHS nology Assessment Report

//www.ngrl.org.uk/Wessex/downloads.htm

elines for Developing Robust and Reproducible High-Resolution Melt Analysis Assays ean Taylor, Rachel Scott, Richard Kurtz, Viresh Patel, and Frank Bizouarn - Bio-Rad Laboratories

//www.bioradiations.com

ifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. Many common disease etes, cancer, osteoporosis, etc.) and clinically relevant phenotypic traits are elicited from the complex interaction between a subset of multiple gen ucts and environmental factors. High resolution melt (HRM) analysis is the quantitative analysis of the melt curve of a DNA fragment following ification by PCR and can be considered the next-generation application of amplicon melting analysis. It is a low-cost, readily accessible technique th ely requires a real-time PCR detection system with excellent thermal stability and sensitivity and HRM-dedicated software. However, careful sample aration and planning of experimental and assay design are crucial for robust and reproducible results. The following guidelines assist in the lopment of such assays.

7. 8.

WHAT IS COPY NUMBER WHAT IS RE- ASSOCIATION KINETICS IN C0T CURVES AND WHAT DOES COLLISION NUMBER WHAT IS LIGASE CHAIN REACTION EXPLAIN GENE FAMILIES WHAT IS 7SLRNA WHAT IS ALU SEQUENCE WHAT IS CpG ISLAND WHAT IS PSEUDOGENES AND EXPLAIN THE TYPES OF PSEUDOGENES WHAT IS ALPHA OR ALPHOID SEQUENCES NAME SOME LTRS WHICH ARE CAPABLE OF TRANPOSITION WHAT IS RISC AND EXPLAIN IN DETAIL GIVE A NOTE ON DICER WHICH IS THE BIGGEST PROTEIN IN HUMAN

9. 10. 11. 12. 13. 14.

15. 16.

17. 18. 19.

Titin
From Wikipedia, the free encyclopedia

Jump to: navigation, search

Titin

The three-dimensional structure of a type I module from titin. PDB rendering based on 1bpv.

Available structures PDB Ortholog search: PDBe, RCSB [show]List of PDB id codes

Identifiers

Symbols TTN; CMD1G; CMH9; CMPD4; EOMFC; HMERF; LGMD2J; MYLK5; TMD

External OMIM: 188840 MGI: 98864 HomoloGene: IDs 130650 GeneCards: TTN Gene

EC number

2.7.11.1

[show]Gene Ontology

RNA expression pattern

More reference expression data

Orthologs

Species

Human

Mouse

Entrez

7273

22138

Ensembl ENSG00000155657 ENSMUSG00000051747

UniProt Q8WZ42

A2ASS6

RefSeq (mRNA)

NM_001256850.1

NM_011652.3

RefSeq (protein)

NP_001243779.1

NP_035782.3

Location Chr 2: (UCSC) 179.39 179.7 Mb

Chr 2: 76.7 76.98 Mb

PubMed [1] search

[2]

This box:

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Titin ( /tatn/), also known as connectin, is a protein that in humans is encoded by the TTN gene.[1][2] Titin is a giant protein that functions as a molecular spring which is responsible for the passive elasticity of muscle. It is composed of 244 individually folded protein domains connected by unstructured peptide sequences.[3] These domains unfold when the protein is stretched and refold when the tension is removed.[4]

Titin is the largest known protein.[5] Furthermore the gene for titin contains the largest number of exons (363) discovered in any single gene.[6] Titin is important in the contraction of striated muscle tissues. It connects the Z line to the M line in the sarcomere. The protein contributes to force transmission at the Z line and resting tension in the I band region.[7] It limits the range of motion of the sarcomere in tension, thus contributing to the passive stiffness of muscle. Variations in the sequence of titin between different types of muscle (e.g., cardiac or skeletal) has been correlated with differences in the mechanical properties of these muscles.[1][8]

Contents
[hide]

1 Genomics 2 Isoforms 3 Structure 4 Function 5 Clinical relevance 6 Linguistic significance 7 Interactions 8 References 9 Further reading 10 External links

Genomics
The gene is found on chromosome 2 and has 363 exons.

Isoforms
A number of titin isoforms are produced in different striated muscle tissues as a result of alternative splicing.[9] All but one of these isoforms are in the range of ~27,000 to ~33,000 amino acid residues in length. The exception is the small cardiac novex-3 isoform which is only 5,604 amino acid residues in length. The following table lists the known titin isoforms: Isoform alias/description length Q8WZ42-1 the "canonical" full length sequence 34,350 Q8WZ42-3 small cardiac N2-B 26,926 Q8WZ42-4 soleus 33,445 small cardiac novex-3, Q8WZ42-6 5,604 phosphorylated on Thr-5304 and Ser-5306. Q8WZ42-7 cardiac novex-2 27,118 Q8WZ42-8 cardiac novex-1 27,051

Structure
Titin is the largest known protein; its human variant consists of 34,350 amino acids, with the molecular weight of the mature "canonical" isoform of the protein being approximately 3,816,188.13 Da.[10] Its mouse homologue is even larger, comprising 35,213 amino acids with a MW of 3,906,487.6 Da.[11] It has a theoretical isoelectric point of 6.01.[10] The protein's empirical chemical formula is C169 723H270 464N45 688O52 243S912.[10] It has a theoretical instability index (II) of 42.41, classifying the protein as unstable.[10] The protein's in vivo half-life, the time it takes for half of the amount of protein in a cell to break down after its synthesis in the cell, is predicted to be approximately 30 hours (in mammalian reticulocytes).[9] Titin consists primarily of a linear array of two types of modules (also referred to as protein domains; 244 copies in total): type I (fibronectin type III domain; 132 copies) and type II (immunoglobulin domain; 112 copies).[3] This linear array is further organized into two regions: N-terminal I-band acts as the elastic part of the molecule and is composed mainly of type II modules. More specifically the I-band contains two regions of tandem type II immunoglobulin domains on either side of a PEVK region that is rich in proline, glutamate, valine and lysine. Titin is found between the myosin thick filament and the Z disk.[12] C-terminal A-band is thought to act as a protein-ruler and possesses kinase activity. The A-band is composed of alternating type I and II modules with super-repeat segments. These have been shown to align to the 43 nm axial repeats of myosin thick filaments with immunoglobulin domains correlating to myosin crowns.[13]

Function

Sliding filament model of muscle contraction. (Titin labeled at upper right.)

Titin is a large abundant protein of striated muscle. An N-terminal Z-disc region and a Cterminal M-line region bind to the Z-line and M-line of the sarcomere respectively so that a single titin molecule spans half the length of a sarcomere. Titin also contains binding sites for muscle-associated proteins so it serves as an adhesion template for the assembly of contractile machinery in muscle cells. It has also been identified as a structural protein for chromosomes. Considerable variability exists in the I-band, the M-line and the Z-disc regions of titin. Variability in the I-band region contributes to the differences in elasticity of different titin isoforms and, therefore, to the differences in elasticity of different muscle types. Of the many titin variants identified, five are described with complete transcript information available.[1][2] Titin interacts with many sarcomeric proteins including:[6]

Z line region: telethonin and alpha-actinin I band region: calpain-3 and obscurin M line region: myosin-binding protein C, calmodulin 1, CAPN3, and MURF1

Clinical relevance
Mutations in this gene are associated with familial hypertrophic cardiomyopathy 9[7][14] and tibial muscular dystrophy.[15] Autoantibodies to titin are produced in patients with the autoimmune disease scleroderma.[16]

Linguistic significance
The name titin is derived from the Greek Titan (a giant deity, anything of great size).[17] As the largest known protein, titin also has the longest IUPAC name. The full chemical name, which starts methionyl... and ends ...isoleucine, contains 189,819 letters and is sometimes stated to be the longest word in the English language, or any language.[18][19] However, lexicographers regard generic names of chemical compounds as verbal formulae rather than English words.[20]

Interactions
Titin has been shown to interact with:

ANK1,[21] ANKRD1,[22] ANKRD23[22] CAPN3,[23][24] FHL2,[25] OBSCN,[26] TCAP,[27][28][29][30] and TRIM63.[31]

Defects in Titin, the largest Human Gene, cause Chronic Congestive Heart Failure
A chronically failing human heart, inable to pump an adequate amount of blood, can be the consequence of myocardial infarctions, high blood pressure or cardiac valve disease. However, it can also occur without any obvious cause and a significant number of such cases are due to unknown gene defects. Now, Dr. Brenda Gerull and Prof. Ludwig Thierfelder (both from the Max Delbrck Center for Molecular Medicine, MDC, in Berlin-Buch and the FranzVolhard Clinic, Charit, Humboldt University, Berlin), in collaboration with researchers from Brisbane (Australia), Mannheim (Germany), and Boston (USA)* have shown that defects in the titin gene which carries the blueprint for the largest known human protein can lead to an inherited form of chronic congestive heart failure, a condition called familial dilated cardiomyopathy (DCM). This discovery by the MDC researchers has now been published in the prestigous journal Nature Genetics. Titin is a very large thread-like molecule which is only found in cardiac and skeletal muscle where it performs a number of structural, functional and regulatory roles. Titin consists of a large series of similar protein modules and, interspersed, specific elements. Among its properties, titin also contains spring elements maintaining structural integrity of a stretched (as well as relaxed) muscle. Titin or titin-like molecules are found in all species with striated muscle tissues. The structure of human titin was first described by Prof. Siegfried Labeit and his group at the University of Mannheim. Titins gene sequence is approximately one hundred times longer than that of an average gene. Whether defects in titin cause cardiac diseases has remained unanswered until now. Patients with DCM suffer from chronic heart failure which sometimes even necessitating a cardiac transplant. In addition to chronic congestive heart failure, DCM can cause fatal cardiac arrhythmias and sudden death. DCM is often a genetic disorder affecting an average of 50 per cent of descendants. The research team has genetically mapped one inherited form of DCM in two families to the long arm of human chromosome 2. The titin gene was screened for disease causing mutations and, in patients of both DCM families, specific defects in titin were identified. In one case, the mutation caused the molecule to be shortened to less than half of its original size, and, in the second family, one of the repeat modules was shown to be altered in such a way that normal folding of the repeat module seems impossible. These findings will form the basis of a better understanding of the pathogenesis of inherited forms of chronic heart failure. Many questions however remain: how do titin defects located in different regions of this huge molecule result in identical pathologies. Also, it remains to be shown how the damaged titin molecules cause a cardiac phenotype but no pathology in skeletal muscle.
Abstract

Titin, is definitely the largest protein in the body, with a molecular weight of 3 million Dalton and composed of 27,000 amino acids. Paradoxically, this huge structure was elusive until the last decade but, since it was described in muscle tissue, its importance has rapidly emerged.

Titin constitutes about 10% of muscle mass, to represent the third most abundant protein in the muscle following actin and myosin. It is estimated that titin acts like a "ruler" that controls the relative positioning of the latter 2 muscle proteins, and regulates the flexibility and "springiness" of the contracting muscle. Titin has also been implicated in the condensation of chromosomes during mitosis, while induced mutations in titin caused enhanced fragility of chromosomes. A recent demonstration of a high titer of autoantibodies to titin in sera of patients diagnosed with myasthenia gravis, is interpreted as a prognostic parameter to indicate a severe course of the disease.

20. 21.

WHAT IS GENE ODDITIES NAME THE LEVEL-4 IN INDIA

The National Institute of Virologys bio-safety level 4 (BSL-4) laboratory, which will store highly dangerous viruses, will be commissioned in a few months, Dr V M Katoch, director-general of the Indian Council of Medical Research (ICMR) and secretary, Department of Health Research, said on Thursday. Katoch, who was in Pune to attend the diamond jubilee celebrations of NIV at the institutes microbial containment centre campus at Pashan, told The Indian Express that this would be the first laboratory in the country to have the unique capability of tackling the most dangerous viruses. There has been no delay in commissioning the laboratory and the Rs 55-crore lab will be commissioned in a few months. Highly infectious pathogenic agents of diseases like ebola, anthrax, lassa, haemorrhagic fever and smallpox (variola virus) will be stored in a facility akin to a heavily guarded bank vault to conduct tests, Dr A C Mishra, director of NIV said. A series of precautionary measures have to be taken to isolate dangerous biological agents in an enclosed facility, Mishra said. Prof N K Ganguly, former director-general of ICMR was the key person to push for such a laboratory. He said they faced several crises, when dangerous pathogens like the Severe Acute Respiratory Syndrome (SARS) virus and others were detected. We wanted to make reagents and our requests to BSL-4 laboratories outside the country were not entertained. Thats when we decided to set up our own facility and partnered with the Department of Science and Technology for the effort, Ganguly said. Bio-safety levels measure the extent of containment precautions required to isolate dangerous biological agents in an enclosed facility. The levels of containment range from the lowest, BSL-1, to the highest, BSL-4. Bio-safety suits resembling space suits, with a dedicated oxygen line, have been imported and scientists have been trained on how to operate a BSL-4 lab. This laboratory will not just be the first in the country, but the only one in Southeast Asia, says Mishra.

22. 23. 24.

WHY PCR AMPLIFY ONLY THE AMPLICON ERRORS THAT OCCURS DUE TO MICROPIPETTE GIVE THE WORKING PRICIPLE OF THE FOLLOWING INSTRUMENTS

LAMINAR AIR FLOW PH METER LYOPHILIZERS- OPERON MpS55 CO2 INCUBATOR- NEW BRINSWICK GALAXY170S ULTRA CENTRIFUGE 5430R AND 5418R GEL DOC- UNITEC INVERTED MICROSCOPE-SYMBIONT TECHNOLOGY-ST-ITM-100PH ELSIA PLATE READER- LAB TECH MT 4000 MILLIPORE WATER DISTILLATION PCR/ THERMOCYCLERS- EPPENDERFORF MASTER CYCLER GRADIENT BIOSAFTY CABINATE