Analysis of CAG/CTG triplet repeats in the human genome: implication in transcription factor gene regulation

Instability and polymorphism at several CAG/CTG trinucleotide repeat loci have been associated with human genetic disorders. In an attempt to identify novel sites that may be possible loci for expansion of CAG/CTG repeats, we searched all human sequences in the EMBL nucleotide sequence database for (CAG)5 and (CTG)5 repeats. We have identified 121 human DNA sequences of known and unknown functions that contain stretches of five or more CAG or CTG repeats. Many repeat stretches were interrupted by variant triplets, a significant number of which differ from the repeat triplet only by a single base, suggesting that these evolved from the parent triplet by point mutations. A large number of human transcription factor genes were found to contain CAG repeats within their coding sequences. Analysis of the EMBL transcription factors database showed that many transcription factor genes of other eukaryotes, including genes involved inDrosophila embryo development, possess these repeats. Interestingly, CAG repeats are absent from prokaryotic transcription factors. Different sequence entries for the human TATA box binding protein showed a polymorphism in the length of the CAG repeat in this gene, suggesting that loci other than those already known to be associated with genetic diseases may be possible sites for repeat instability related disorders. On the basis of our findings in this database analysis, we propose a role for CAG repeats as cisacting regulatory elements involved in fine-tuning gene expression

DNA chip the what's, the why's and the how's

This article presents a brief overview of DNA chips, or microarrays. Microarrays are a very significant technological development in molecular biology, and are perhaps the most efficient tool available for functional genomics today

Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae

BACKGROUND: Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. RESULTS: A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg). Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. CONCLUSION: We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells

(TG/CA)(n )repeats in human gene families: abundance and selective patterns of distribution according to function and gene length

BACKGROUND: Creation of human gene families was facilitated significantly by gene duplication and diversification. The (TG/CA)(n )repeats exhibit length variability, display genome-wide distribution, and are abundant in the human genome. Accumulation of evidences for their multiple functional roles including regulation of transcription and stimulation of recombination and splicing elect them as functional elements. Here, we report analysis of the distribution of (TG/CA)(n )repeats in human gene families. RESULTS: The 1,317 human gene families were classified into six functional classes. Distribution of (TG/CA)(n )repeats were analyzed both from a global perspective and from a stratified perspective based on their biological properties. The number of genes with repeats decreased with increasing repeat length and several genes (53%) had repeats of multiple types in various combinations. Repeats were positively associated with the class of Signaling and communication whereas, they were negatively associated with the classes of Immune and related functions and of Information. The proportion of genes with (TG/CA)(n )repeats in each class was proportional to the corresponding average gene length. The repeat distribution pattern in large gene families generally mirrored the global distribution pattern but differed particularly for Collagen gene family, which was rich in repeats. The position and flanking sequences of the repeats of Collagen genes showed high conservation in the Chimpanzee genome. However the majority of these repeats displayed length polymorphism. CONCLUSION: Positive association of repeats with genes of Signaling and communication points to their role in modulation of transcription. Negative association of repeats in genes of Information relates to the smaller gene length, higher expression and fundamental role in cellular physiology. In genes of Immune and related functions negative association of repeats perhaps relates to the smaller gene length and the directional nature of the recombinogenic processes to generate immune diversity. Thus, multiple factors including gene length, function and directionality of recombinogenic processes steered the observed distribution of (TG/CA)(n )repeats. Furthermore, the distribution of repeat patterns is consistent with the current model that long repeats tend to contract more than expand whereas, the reverse dynamics operates in short repeats

dbSMR: a novel resource of genome-wide SNPs affecting microRNA mediated regulation

Background: MicroRNAs (miRNAs) regulate several biological processes through post-transcriptional gene silencing. The efficiency of binding of miRNAs to target transcripts depends on the sequence as well as intramolecular structure of the transcript. Single Nucleotide Polymorphisms (SNPs) can contribute to alterations in the structure of regions flanking them, thereby influencing the accessibility for miRNA binding. Description: The entire human genome was analyzed for SNPs in and around predicted miRNA target sites. Polymorphisms within 200 nucleotides that could alter the intramolecular structure at the target site, thereby altering regulation were annotated. Collated information was ported in a MySQL database with a user-friendly interface accessible through the URL: http://miracle.igib.res.in/ dbSMR. Conclusion: The database has a user-friendly interface where the information can be queried using either the gene name, microRNA name, polymorphism ID or transcript ID. Combination queries using 'AND' or 'OR' is also possible along with specifying the degree of change of intramolecular bonding with and without the polymorphism. Such a resource would enable researchers address questions like the role of regulatory SNPs in the 3' UTRs and population specific regulatory modulations in the context of microRNA targets

Supercoil-induced unusual DNA structures as transcriptional block

The transcriptional activity of pBR322 form V DNA template, a topologically unlinked, highly supercoiled molecule having unusual structures around or within coding regions was studied. Significant transcription was observed in vitro from this template despite high levels of supercoiling. An attenuated transcript, initiated accurately from the P4 promoter of rep gene, was observed which indicated pausing of E. coli RNA polymerase within the gene. This pausing could be removed by relieving the torsional stress implying that a supercoil induced structural alteration within the gene was acting as a transcriptional block. A stabilized unusual structure, most likely a cruciform, was found to be responsible for the elongation block. Absence of initiation from the tetR gene was correlated with the unusual structure present within its promoter region in form V DNA. These in vitro studies show that structural alterations within natural DNA could act as transcriptional blocks both at the level of initiation and elongation

Preparation of megabase DNA from adult insects and mammalian spleen for pulsed-field gel electrophoresis

While standard techniques for obtaining megabase-size DNA from microorganisms and cells in tissue culture are now available, new methods are needed for handling solid tissues and, in the case of small-sized animals, whole organisms. Here we describe a simple and rapid method for preparing large DNA molecules from mammalian spleen, whole insects of Drosophila and Planococcus lilacinus, a mealybug. Briefly the method involves gentle steps for preparing cell suspensions and handling of these cells within agarose blocks for preparing the DNA. In mammals like mice, the spleen, rather than the liver, is the organ of choice because, in the latter, endogenous nuclease activity is high

Genome-wide expression profile of RNA polymerase II subunit mutant of yeast using microarray technology

Rpb4, the non-essential core subunit of RNA polymerase II has been assigned a function of regulating stress response in S. cerevisiae based mainly on phenotypes associated with its deletion. The actual mechanism has been elusive, although various hypotheses have been put forth. We have shown previously that it plays a significant role in activation of a subset of genes, rather than causing generalized defect in transcription. We used the microarray technology to look at the effect of this RNA polymerase subunit on the expression pattern of the entire S. cerevisiae genome. Many surprises emerged when we compared the genome-wide expression patterns of wild type and a mutant lacking the RPB4 gene (rpb4Δ) subjected to heat shock. The initial analysis of genes downregulated in the mutant showed that the co-regulation of genes is not position-dependent, although the locus carrying the deletion had unexpectedly a large cluster of down-regulated genes. We also found that among the known down-regulated genes, a majority is involved in hexose uptake and utilization. We speculate that this could potentially contribute to the slow growth rate of the mutant. Compared to the other components of the transcription machinery, the Rpb4 subunit affects a unique set of genes

An Indian effort towards affordable drugs: "generic to designer drugs"

This review discusses the progress of India from being one of the largest producers of generics to its coming of age and initiating novel drug development programs such as the Open Source Drug Discovery for tuberculosis. A few groups have also begun to emerge which focus their research on rational or structure based drug design. We discuss here some of the ongoing efforts in drug discovery in India primarily in national research institutions and academia

Evolution and distribution of RNA polymerase II regulatory sites from RNA polymerase III dependant mobile Alu elements

BACKGROUND: The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting. RESULTS: We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes. CONCLUSIONS: Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of regulatory elements in the preexisting genes through Alus could thus have contributed to evolution of novel regulatory networks in the primate genomes. With such a wide spectrum of regulatory sites present in Alus, it also becomes imperative to screen for variations in these sites in candidate genes, which are otherwise repeat-masked in studies pertaining to identification of predisposition markers