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S K Brahmachari

Publications and source records attributed to S K Brahmachari.

At least 19 recordsLinked to original sources
PubMed1998

Correlation of clinical profile of myotonic dystrophy with CTG repeats in the myotonin protein kinase gene.

The molecular genetic analyses (PCR and Southern hybridization) of Indian patients with myotonic dystrophy (DM) were carried out to determine the degree of repeat expansion and an attempt was made to correlate the repeat number with disease severity. A scoring system based on the salient clinical features was devised to objectively assess the disease severity. The repeat expansion was seen in 11 of 12 patients examined and showed an inverse correlation with the age of onset confirming the phenomenon of anticipation. This was further established in the two pedigrees studied, clearly demonstrating both clinical and genetic anticipation. The clinical severity score, however, did not correlate well with the repeat number. Nonetheless, such molecular genetic analyses may have immense value as a screening procedure to identify premutations as well as in prenatal diagnoses.

Adolescent↗
PubMed1997

Polypurine/polypyrimidine sequences as cis-acting transcriptional regulators.

Genome sequence information has generated increasing evidence for the claim that repetitive DNA sequences present within and around genes could play a important role in the regulation of gene expression. Polypurine/polypyrimidine sequences [poly(Pu/Py)] have been observed in the vicinity of promoters and within the transcribed regions of many genes. To understand whether such sequences influence the level of gene expression, we constructed several prokaryotic and eukaryotic expression vectors incorporating poly(Pu/Py) repeats both within and upstream of a reporter gene, lacZ (encoding beta-galactosidase), and studied its expression in vivo. We find that, in contrast to the situation in Escherichia coli, the presence of poly(Pu/Py) sequences within the gene does not significantly inhibit gene expression in mammalian cells. On the other hand, the presence of such sequences upstream of lacZ leads to a several-fold reduction of gene expression in mammalian cells. Similar down-regulation was observed when a structural cassette containing poly(Pu/Py) sequences upstream of lacZ was integrated into yeast chromosome V. Sequence analysis of the nine totally sequenced yeast chromosomes shows that a large number of such sequences occur upstream of ORFs. On the basis of our experimental results and DNA sequence analysis, we propose that these sequences can function as cis-acting transcriptional regulators.

Animals↗
PubMed1997

Positional preferences of polypurine/polypyrimidine tracts in Saccharomyces cerevisiae genome: implications for cis regulation of gene expression.

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Chromosome Mapping↗
PubMed1995

Simple repetitive sequences in the genome: structure and functional significance.

The current explosion of DNA sequence information has generated increasing evidence for the claim that noncoding repetitive DNA sequences present within and around different genes could play an important role in genetic control processes, although the precise role and mechanism by which these sequences function are poorly understood. Several of the simple repetitive sequences which occur in a large number of loci throughout the human and other eukaryotic genomes satisfy the sequence criteria for forming non-B DNA structures in vitro. We have summarized some of the features of three different types of simple repeats that highlight the importance of repetitive DNA in the control of gene expression and chromatin organization. (i) (TG/CA)n repeats are widespread and conserved in many loci. These sequences are associated with nucleosomes of varying linker length and may play a role in chromatin organization. These Z-potential sequences can help absorb superhelical stress during transcription and aid in recombination. (ii) Human telomeric repeat (TTAGGG)n adopts a novel quadruplex structure and exhibits unusual chromatin organization. This unusual structural motif could explain chromosome pairing and stability. (iii) Intragenic amplification of (CTG)n/(CAG)n trinucleotide repeat, which is now known to be associated with several genetic disorders, could down-regulate gene expression in vivo. The overall implications of these findings vis-à-vis repetitive sequences in the genome are summarized.

Animals↗
PubMed1995

Intra- and interloop interactions in the folded G quartet structure of Oxytricha telomeric sequence.

The antiparallel intramolecular G quartet structure for the 3.5 copy Oxytricha telomeric sequence d(G4T4)3G4 has been established using a combination of spectroscopic and chemical probing methods. In the presence of Na+ ions, this sequence exhibits a circular dichroism spectrum with a positive band at 295 nm and a negative band around 265 nm, characteristic of an antiparallel G quartet structure. Further, we show that d(G4T4)3G4 adopts an antiparallel intramolecular G quartet structure even in K+ unlike d(G4T4G4). KMnO4 probing experiments indicated the existence of intra and interloop interactions in the Na+ induced structure. We have found that K+ not only increases the thermal stability of G quartet structure but also binds to the loop region and disrupts stacking and interloop interactions. Biological consequences of such cation-dependent conformational micro-heterogeneity in the loop region of G quartet structures is also discussed.

Animals↗
PubMed1994

Structure and stability of human telomeric sequence.

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T2AG3)4, d(G3T2A)3G3, and d(G3T2AG3) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G3T2A)3G3 and d(T2AG3)4 assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G3T2AG3) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide d(G3T2AG3) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T2AG3)4 suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Circular Dichroism↗
PubMed1994

CA/TG sequence at the 5' end of oligo(A)-tracts strongly modulates DNA curvature.

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAAC-CCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

Base Sequence↗
PubMed1993

Polypurine-polypyrimidine sequences adopt unwound structure in pBR322 form V DNA as probed by single-hit analysis of HpaII sites.

Structure at the polypurine-polypyrimidine sequences flanking the HpaII sites (CCGG) in pBR322 form V DNA was probed employing single-hit analysis using HpaII restriction endonuclease. Reduced cleavage efficiency of HpaII sites flanked by polypurine-polypyrimidine sequences suggested that under high torsional stress these sequences adopt unwound structures rendering these sites insensitive to restriction enzyme cleavage. In addition to polypurine-polypyrimidine sequences. HpaII sites flanked by alternating purine-pyrimidine sequence, a potential motif of left handed Z-DNA, were also found to be resistant to HpaII cleavage. Results obtained from various studies implicating structure sensitivity of restriction endonucleases and methylases were compiled and a direct correlation was observed between the occurrence of altered sites in a domain and its G/C content in pBR322 form V DNA.

Base Sequence↗
PubMed1992

Intramolecular triplex potential sequence within a gene down regulates its expression in vivo.

Polypurine/polypyrimidine sequences have been shown to adopt intramolecular triple helix structures under torsional stress and/or at low pH. Such sequences have been observed within the the regulatory as well as the coding regions of several genes and the involvement of triple helical structure adopted by these sequences in transcriptional control has been speculated. Taking advantage of codon degeneracy we have engineered a 38 bp long intramolecular triple helix potential polypurine/polypyrimidine sequence motif between the 37th and 50th codons of beta-galactosidase gene in the plasmid pBluescriptIISK+ to investigate whether in vivo E.coli RNA polymerase would transcribe sequence motifs adopting triple helix structure, when present within the coding region of the gene. E.coli JM109 cells transformed with this construct pSBT1, exhibited 80% inhibition of beta-galactosidase expression compared to another construct pSBmT12 made using less preferred codons for identical amino acid sequence, but lacking the polypurine/polypyrimidine sequence motif. Truncated beta-galactosidase transcripts were observed for pSBT1 but not for pSBmT12. Here we report that a putative triple helix potential sequence within a gene can down regulate its expression by partially blocking the transcription elongation in vivo.

Amino Acid Sequence↗
PubMed1992

Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.

Animals↗
PubMed1991

Distribution of simple repetitive (TG/CA)n and (CT/AG)n sequences in human and rodent genomes.

Sixteen million nucleotide sequence of genome of various organisms have been analysed to detect and study the extent of occurrence of simple repetitive sequences. Two sequence motifs (TG/CA)n and (CT/AG)n capable of adopting unusual DNA structures, left handed Z-conformation and triple-helical conformation respectively, are found to be abundant in rodent and human genomes, but almost completely absent in bacterial genome. (TG/CA)n and (CT/AG)n sequences are present mostly in the intron or 5'/3' flanking regions of the genes. The presence of such repeat motifs in genomic sequence of higher eukaryotes has been correlated with their possible functional significance in nucleosome organization, recombination and gene expression.

Animals↗
PubMed1991

Synthetic gene design to investigate the role of cis-acting DNA structural elements in regulation of gene expression in vivo.

To delineate the DNA structural elements responsible for transcriptional control in vivo, we have developed a novel approach taking advantage of the degeneracy of the genetic code. Using synthetic oligonucleotides as structural cassettes we have been able to replace, within a gene, segments of DNA coding for the same amino acid sequence but capable of adopting unusual DNA structures and monitor the effect of such structural elements on gene expression in vivo. We find that the presence of an inverted repeat sequence, with a potential to adopt cruciform structure, within the beta-galactosidase gene down regulates its expression in vivo.

Amino Acid Sequence↗
PubMed1990

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.

Base Sequence↗
PubMed1990

Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe.

The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHI, PstI, AvaI and HindIII could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as NarI and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences.

Base Sequence↗
PubMed1989

DNA duplex with the potential to change handedness after every half a turn.

Polymorphic forms of the DNA duplex with long stretches of structural monotony are known. Several alternating purine-pyrimidine sequences have been shown to adopt left-handed Z-conformation. We report a DNA sequence d(CGCGCGATCGAT)n exhibiting alternating right-handed B and left-handed Z helical conformation after every half a turn. Further, this unusual conformation with change in handedness after every six base pairs was induced at physiological superhelical density.

Base Sequence↗
PubMed1989

Zintrons in rat alpha-lactalbumin gene.

The eukaryotic genome is characterised by the occurrence of a large amount of repetitive DNA which exists within and around coding sequences. Random repeats of (TG)n sequences have been observed in several introns. In this paper we show that (TG)14 and (TG)24 sequences present in the third intron of rat alpha-lactalbumin gene adopt left-handed Z-helices under varying superhelical densities. The overall sequence of the parent plasmid further influenced the level of supercoiling at which the B to Z transition could be induced in (TG)n sequences. Such Z-potential intervening sequences (zintrons) could act as buffers to maintain desired levels of supercoiling near the transcribed sequences.

Animals↗