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S Adhya

Publications and source records attributed to S Adhya.

At least 55 records · Page 3Linked to original sources
PubMed1994

Interaction of small ribosomal and transfer RNAs with a protein from Leishmania donovani.

Using synthetic antisense RNA from the 5'-untranslated region of the beta-tubulin gene as probe in gel retardation assays, a heat stable RNA-binding factor was identified in promastigotes of the kinetoplastid protozoan Leishmania donovani. The same or similar factors interact with several small ribosomal RNA (srRNA) species and, more weakly, with tRNA, as shown by binding and competition experiments. Deletion analysis indicated involvement of repeated purine-rich motifs on the antisense RNA, in the reaction. Related, conserved motifs occur on at least two of the srRNAs. By a modified Western blot assay, the RNA-binding species was identified as a single, small polypeptide. The activity is apparently specific for the promastigote stage of the parasite, being undetectable in amastigotes. The properties of this RNA-binding factor suggest that it is a novel, previously uncharacterized protein.

Animals↗
PubMed1993

Promoter resurrection by activators--a minireview.

Frequently, in nature, defective promoters can be resurrected by activator proteins in response to cellular demands. The activators bind to nearby DNA sites for action. Various protein-protein and DNA-protein contacts involving activators, RNA polymerase, and different segments of DNA in and around a defective promoter form a DNA-multiprotein complex (cage) which enhances transcription.

DNA↗
PubMed1993

CRP-binding sites: evidence for two structural classes with 6-bp and 8-bp spacers.

While classifying protein binding DNA sequences of the type GTGNxCAC, based on the size of Nx [Shumilov, Mol. Biologya (Engl. Transl.) 21 (1987) 168-187], we had previously found that the cyclic AMP receptor protein (CRP)-binding sites found in the Escherichia coli genome are of at least two classes: (i) those with a conventional 6-bp spacer (N6) and (ii) those with a potential 8-bp spacer (N8) [Barber and Zhurkin, J. Biomol. Struct. Dyn. 8 (1990) 213-232]. In this paper, we present the first experimental evidence that CRP binds to DNA with an N8 spacer with relatively high affinity, as measured by gel electrophoresis of CRP-DNA complexes. We have tested two types of N8 spacers: A+T-rich and G+C-rich. Compared with the affinity of CRP for a reference site with an N6 spacer, the binding strength of CRP toward an A+T-rich N8 sequence is lower and that toward a G+C-rich N8 site is comparable. Just like DNA sites with N6 spacers, those with N8 spacers utilize both halves of the symmetrical protein recognition sequences, TGTGA and TCACA. Because of the increased number of nucleotides in the N8 spacer, the two recognition sequences in DNA will have an increased distance and a helical twist between them. These would cause displacement of the two recognition sequences with respect to the two symmetrically located alpha-helices of the CRP dimer, if there is no change in the DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

RNA polymerase idling and clearance in gal promoters: use of supercoiled minicircle DNA template made in vivo.

We have developed an in vivo system to engender supercoiled "minicircle" DNA containing a single promoter by using the integrative recombination system of bacteriophage lambda. The resulting minicircle templates allow quantitative analysis of the stages of transcription initiation from a promoter, including synthesis of both full-length and aborted transcripts in the same reactions under physiological conditions. We have used such minicircle DNA templates to study in vitro transcription of the Escherichia coli gal promoter. The full-length transcripts from gal P1 and P2 promoters responded to cAMP-cAMP receptor protein in a manner identical to that observed in vivo. There is a 3.5-fold stimulation of P1 and almost total inhibition of P2 in the presence of cAMP. Thus, the unitary promoter system described here duplicates the in vivo physiology. In spite of the synthesis in equimolar amounts of full-length transcripts from P1 and P2 in the absence of cAMP in vitro, as in vivo, RNA polymerase encountered different rate-limiting steps of transcription initiation at the two promoters.

Bacteriophage lambda↗
PubMed1993

Pivotal role of amino acid at position 138 in the allosteric hinge reorientation of cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli needs cAMP for an allosteric change to regulate gene expression by binding to specific DNA sites. The hinge region connecting the DNA-binding domain to the cAMP-binding domain has been proposed to participate in the cAMP-induced allosteric change necessary to adjust C and D alpha-helices for movement of the DNA-binding F alpha-helix away from the protein surface. The role of the hinge region for a conformation change in CRP was tested by studying the effects of single amino acid substitutions at residue 138 located within the hinge. Physiological studies of wild-type and mutant cells and biochemical analysis of purified wild-type and mutant CRP revealed at least three groups of altered CRPs: (i) CRP that behaves like wild type (CRP+); (ii) CRP that binds cAMP but does not complete the structural changes required for specific DNA binding, proteolytic cleavage, and transcription activation (CRPallo); and (iii) CRP that shows some or all of these conformational changes without cAMP (CRP*). These results show a pivotal role of position 138 from which change emanates and provide further evidence that a hinge reorientation involving residue 138 is involved in the interhelical adjustments.

Allosteric Site↗
PubMed1993

Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani: primers and probes for DNA diagnosis.

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

Animals↗
PubMed1993

The galactose regulon of Escherichia coli.

Galactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by two different galactose-specific transport systems. At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences. These sequences are recognized by at least three regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The negative regulators, GalR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by CRP. Both promoters are strongly repressed by GalR but weakly by GalS. All but one of the constituent promoters of the gal regulon have two operators. The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.

Base Sequence↗
PubMed1993

Control of transcription of gal repressor and isorepressor genes in Escherichia coli.

Two regulatory proteins, Gal repressor and isorepressor, control the expression of the gal and mgl operons in Escherichia coli. The transcription start sites for galR and galS, the genes for the repressor and isorepressor, were determined by primer extension of in vivo transcripts. Study of the promoter-lacZ gene fusions introduced into the chromosome indicated that galS expression was elevated in cells in which the normal galS gene was interrupted, but not in cells in which the galR gene was deleted. When both genes were disrupted, galS expression was further elevated. Expression from the galS promoter was stimulated by the addition of D-fucose, repressed by glucose, and dependent on cyclic AMP receptor protein (CRP). Expression of a similar gene fusion of the galR promoter to lacZ was unregulated. Both galR and galS genes contain two potential operator sites (OE and OI) and a CRP-binding site. The arrangement of OE, OI, and the CRP-binding site in the galS gene is analogous to the arrangement in the gal and mgl promoters, but the arrangement in galR is atypical. The increased concentration of the isorepressor when inducer is present may facilitate early shutoff of the isorepressor-regulated genes of the gal regulon when inducer (substrate) concentration falls.

Base Sequence↗
PubMed1993

Tuberculous endometritis in hills of Darjeeling: a clinicopathological and bacteriological study.

Combined histological and bacteriological investigations of 800 specimens of nonpregnant endometrial curettings of 15 to 60 years age group of hill women of Darjeeling District were carried out for detection of tuberculous endometritis. The principal complaints were infertility (47.5 per cent), abnormal uterine bleeding (30.75 per cent), amenorrhoea (11.25 per cent), leucorrhoea (6.25 per cent), and miscellaneous conditions (pelvic pain and pyometra) (4.25 per cent) cases. By histological examination alone, only 10.9 per cent cases could be diagnosed while by combined study the incidence rate was 11.8 percent, an increase in the diagnostic acumen by more than 10.3 per cent. Bacteriological study was of greater value in doubtful cases where there was absence of tuberculous granuloma or epithelioid cell but presence of nonspecific inflammatory cells along with variable degree of necrosis of glandular epithelia. The incidence of M.tuberculosis was 97.7 percent while that of atypical mycobacteria was 2.3 per cent. Thus simultaneous use of culture and biopsy yielded better results. Our prevalence is a little higher than other reports from India. In cold weather at a high altitude, the tubercle bacilli survive longer in fomites which serve as important sources of infection in Darjeeling. Women of third decade are more frequently affected (43.2 per cent).

Adolescent↗
PubMed1992

Control of gal transcription through DNA looping: inhibition of the initial transcribing complex.

Involvement of DNA looping between two spatially separated gal operators, OE and OI, in repression of the gal operon has been demonstrated in vivo. An in vitro transcription assay using a minicircle DNA containing the gal promoter region with lac operators was employed to elucidate the molecular mechanism of repression. Wild-type lac repressors (LacI+ protein molecules), which are capable of associating into a tetramer and forming a DNA loop, repressed transcription from promoter sites P1 and P2, whereas a non-looping lac repressor mutant (LacI(adi)) failed to show normal repression of both of the gal promoters. Thus a DNA loop is also required for repression of transcription in vitro. Repression mediated by DNA looping resulted in the inhibition of the synthesis of complete as well as aborted transcripts, demonstrating that the repressive action was on the formation or activity of the initial transcribing complex. Under similar conditions, the gal repressor (GalR protein) did not repress the gal promoters effectively, apparently because it failed to loop DNA containing gal operators in the purified system. The component(s) or conditions that aid GalR in DNA looping remain to be identified.

Base Sequence↗
PubMed1992

Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation.

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.

Allosteric Regulation↗
PubMed1992

A family of bacterial regulators homologous to Gal and Lac repressors.

We describe a family of proteins which regulate transcription of inducible genes in bacteria (GalR-LacI family). An alignment of the proteins in the GalR-LacI family is presented in which these proteins show a very high degree of similarity (60%) throughout the entire sequences. The homology is greatest among the amino-terminal DNA binding domains. Since a portion of the operator sequences occupied by these proteins is also conserved, a similar DNA structure may be required for specific recognition of DNA by members of the GalR-LacI family. Highly conserved motifs involved in effector binding and oligomerization are also identified. This compilation suggests a widespread conservation of these regulators among bacteria, and have strong implications for further study of peptide motifs in domain function, as well as pathways of protein evolution.

Amino Acid Sequence↗
PubMed1992

Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.

Chloramphenicol O-Acetyltransferase↗
PubMed1992

Isorepressor of the gal regulon in Escherichia coli.

Inducible overexpression of the Escherichia coli gal operon in the absence of the Gal repressor is known as ultrainduction. The requirement of induction can be eliminated by mutation of a new locus, galS, resulting in constitutive and ultrainduced levels of gal expression. Characterization of the galS gene and its product has revealed an isorepressor of the gal regulon. The Gal isorepressor is a protein of 346 amino acid residues whose amino acid sequence and cellular function, as described here, are very similar to that of Gal repressor, encoded by the galR gene. Transcription from different promoters of the gal regulon, galP1, galP2 and mglP, was examined by primer extension and reverse transcription of mRNA isolated from strains containing mutations in galR and/or galS. In strains containing a galS mutation, overexpression of gal message occurred only in the presence of inducer, while mgl message was constitutively derepressed. The galS mutation also constitutively derepressed an mglA::lacZ fusion, demonstrating that GalS is the mgl repressor. A potential operator site in the mgl promoter was identified at a position analogous to OE in gal. Thus, the gal and mgl operons constitute a regulon. Crosstalk, temporal action, induction spectrum or heteromer formation between repressor and isorepressor may help co-ordinate high affinity galactose transport and galactose utilization.

Amino Acid Sequence↗