Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Interaction of G-Quadruplexes with Nonintercalating Duplex-DNA Minor Groove Binding Ligands

The enzyme telomerase synthesizes the G-rich DNA strands of the telomere and its activity is often associated with cancer. The telomerase may be therefore responsible for the ability of a cancer cell-to escape apoptosis. The G-rich DNA sequences often adopt tetra-stranded structure, known as the G-quadruplex DNA (G4-DNA). The stabilization of the telomeric DNA into the G4-DNA structures by small molecules has been the focus of many researchers for the design and development of new anticancer agents. The compounds which stabilize the G-quadruplex in the telomere inhibit the telomerase activity. Besides telomeres, the G4-DNA forming sequences are present in the genomic regions of biological significance including the transcriptional regulatory and promoter regions of several oncogenes. Inducing a G-quadruplex structure within the G-rich promoter sequences is a potential way of achieving selective gene regulation. Several G-quadruplex stabilizing ligands are known. Minor groove binding ligands (MGBLs) interact with the double-helical DNA through the minor grooves sequence-specifically and interfere with several DNA associated processes. These MGBLs when suitably modified switch their preference sometimes from the duplex DNA to G4-DNA and stabilize the G4-DNA as well. Herein, we focus on the recent advances in understanding the G-quadruplex structures, particularly made by the human telomeric ends, and review the results of various investigations of the interaction of designed organic ligands with the G-quadruplex DNA while highlighting the importance of MGBL-G-quadruplex interactions.

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein. (C) 2011 Elsevier B.V. All rights reserved.

Features of a unique intronless cluster of class I small heat shock protein genes in tandem with box C/D snoRNA genes on chromosome 6 in tomato (Solanum lycopersicum)

Physical clustering of genes has been shown in plants; however, little is known about gene clusters that have different functions, particularly those expressed in the tomato fruit. A class I 17.6 small heat shock protein (Sl17.6 shsp) gene was cloned and used as a probe to screen a tomato (Solanum lycopersicum) genomic library. An 8.3-kb genomic fragment was isolated and its DNA sequence determined. Analysis of the genomic fragment identified intronless open reading frames of three class I shsp genes (Sl17.6, Sl20.0, and Sl20.1), the Sl17.6 gene flanked by Sl20.1 and Sl20.0, with complete 5' and 3' UTRs. Upstream of the Sl20.0 shsp, and within the shsp gene cluster, resides a box C/D snoRNA cluster made of SlsnoR12.1 and SlU24a. Characteristic C and D, and C' and D', boxes are conserved in SlsnoR12.1 and SlU24a while the upstream flanking region of SlsnoR12.1 carries TATA box 1, homol-E and homol-D box-like cis sequences, TM6 promoter, and an uncharacterized tomato EST. Molecular phylogenetic analysis revealed that this particular arrangement of shsps is conserved in tomato genome but is distinct from other species. The intronless genomic sequence is decorated with cis elements previously shown to be responsive to cues from plant hormones, dehydration, cold, heat, and MYC/MYB and WRKY71 transcription factors. Chromosomal mapping localized the tomato genomic sequence on the short arm of chromosome 6 in the introgression line (IL) 6-3. Quantitative polymerase chain reaction analysis of gene cluster members revealed differential expression during ripening of tomato fruit, and relatively different abundances in other plant parts.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development. (C) 2012 Elsevier Ltd. All rights reserved.

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3 ` and 5 ` End Repair and Role of Polyprotein Processing in Viral Replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.

Recent Developments in the Chemistry and Biology of G-Quadruplexes with Reference to the DNA Groove Binders

DNA is the chemotherapeutic target for treating diseases of genetic origin. Besides well-known double-helical structures (A, B, Z, parallel stranded-DNA etc.), DNA is capable of forming several multi-stranded structures (triplex, tetraplex, i-motif etc.) which have unique biological significance. The G-rich 3'-ends of chromosomes, called telomeres, are synthesized by telomerase, a ribonucleoprotein, and over-expression of telomerase is associated with cancer. The activity of telomerase is suppressed if the G-rich region is folded into the four stranded structures, called G-quadruplexes (G4-DNAs) using small synthetic ligands. Thus design and synthesis of new G4-DNA ligands is an attractive strategy to combat cancer. G4-DNA forming sequences are also prevalent in other genomic regions of biological significance including promoter regions of several oncogenes. Effective gene regulation may be achieved by inducing a G4-DNA structure within the G-rich promoter sequences. To date, several G4-DNA stabilizing ligands are known. DNA groove binders interact with the duplex B-DNA through the grooves (major and minor groove) in a sequence-specific manner. Some of the groove binders are known to stabilize the G4-DNA. However, this is a relatively under explored field of research. In this review, we focus on the recent advances in the understanding of the G4-DNA structures, particularly made from the human telomeric DNA stretches. We summarize the results of various investigations of the interaction of various organic ligands with the G4-DNA while highlighting the importance of groove binder-G4-DNA interactions.

A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer. (C) 2012 Elsevier Inc. All rights reserved.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Unconventional initiator tRNAs sustain Escherichia coli

Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT Escherichia coli cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of Mycoplasma and Rhizobium, is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in E. coli is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that E. coli can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed.

Sequence and structural basis for chromosomal fragility during translocations in cancer

Chromosomal aberration is considered to be one of the major characteristic features in many cancers. Chromosomal translocation, one type of genomic abnormality, can lead to deregulation of critical genes involved in regulating important physiological functions such as cell proliferation and DNA repair. Although chromosomal translocations were thought to be random events, recent findings suggest that certain regions in the human genome are more susceptible to breakage than others. The possibility of deviation from the usual B-DNA conformation in such fragile regions has been an active area of investigation. This review summarizes the factors that contribute towards the fragility of these regions in the chromosomes, such as DNA sequences and the role of different forms of DNA structures. Proteins responsible for chromosomal fragility, and their mechanism of action are also discussed. The effect of positioning of chromosomes within the nucleus favoring chromosomal translocations and the role of repair mechanisms are also addressed.

Suite of tools for statistical N-gram language modeling for pattern mining in whole genome sequences

Genome sequences contain a number of patterns that have biomedical significance. Repetitive sequences of various kinds are a primary component of most of the genomic sequence patterns. We extended the suffix-array based Biological Language Modeling Toolkit to compute n-gram frequencies as well as n-gram language-model based perplexity in windows over the whole genome sequence to find biologically relevant patterns. We present the suite of tools and their application for analysis on whole human genome sequence.

Establishment of an in vitro transcription system for Peste des petits ruminant virus

Background: Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. Results: RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. Conclusion: This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.