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S Adhya

Publications and source records attributed to S Adhya.

At least 73 records · Page 4Linked to original sources
PubMed1992

Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients.

Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2.5-25 microliters of splenic aspirate or of 50-500 microliters of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2.5 x 10(-7) microliters of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.

Animals↗
PubMed1991

DNA-binding properties of a lac repressor mutant incapable of forming tetramers.

The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. Lac repressor mediates the formation of "DNA loops" by the simultaneous interaction of a single protein tetramer with two DNA-binding sites. The DNA-binding properties of a Lac repressor mutant (LacIadi) deficient in the association of protein dimers to tetramers was investigated. The results of quantitative footprint and gel mobility-shift titrations suggest that the wild-type Lac repressor (LacI+) binds cooperatively to two operator sites separated by 11 helical turns on a linear DNA restriction fragment by the formation of a "looped complex." LacIadi binds to this two-site operator non-cooperatively and without formation of a looped complex. These results demonstrate that the dimer-tetramer association of LacI+ is directly responsible for its cooperative binding and its ability to mediate formation of a looped complex. The Iadi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacIadi to a single site is unchanged relative to the wild-type protein. These results suggest that DNA binding and dimer-tetramer association are functionally unlinked. The similarity of the DNA-binding properties of LacIadi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed.

Autoradiography↗
PubMed1991

Evidence for translational control of beta-tubulin synthesis during differentiation of Leishmania donovani.

Tubulin biosynthesis was rapidly induced during transformation of the mammalian (amastigote) stage of the kinetoplastid parasite Leishmania donovani to flagellated promastigotes. However, transcription of beta-tubulin genes occurred constitutively, as judged by nascent RNA synthesis in isolated nuclei and Northern blotting of steady-state mRNA. Two mRNA species of 2.2 and 2.4 kb were shared by the two cell-types, while a third 2.6 kb species, constituting about 20% of the total, was present in large amounts in promastigotes. RNase protection experiments demonstrated sequence microheterogeneity in the 5'-untranslated region, the pattern of which was identical in promastigotes and amastigotes. By primer extension assays, heterogeneity in the 5'-terminal cap structure of amastigote beta-tubulin mRNA and differential pausing of reverse transcriptase within the mini-exon leader region were detected. These differences correlated with enhanced translational efficiency of tubulin mRNA from promastigotes in a rabbit reticulocyte lysate system. The results indicate that translational control plays a major role in tubulin induction during L. donovani differentiation.

Animals↗
PubMed1991

A mutation defining ultrainduction of the Escherichia coli gal operon.

Tn10 insertion in the galS (ultrainduction factor) gene of Escherichia coli allows the gal operon to be constitutively expressed at a very high level, equal to that seen in a delta galR strain in the presence of an inducer. The insertion has been mapped by criss-cross Hfr matings and by marker rescue into Kohara phages at 46 min on the E. coli chromosome.

DNA Transposable Elements↗
PubMed1991

Further inducibility of a constitutive system: ultrainduction of the gal operon.

In wild-type Escherichia coli, expression of the gal operon is negatively regulated by the Gal repressor and is induced 10- to 15-fold when the repressor is inactivated by an inducer. In strains completely deleted for galR, the gene which encodes the Gal repressor, the operon is derepressed by only 10-fold without an inducer. But this derepression is increased further by threefold during cell growth in the presence of an inducer, D-galactose or D-fucose. This phenomenon of extreme induction in the absence of Gal repressor is termed ultrainduction--a manifestation of further inducibility in a constitutive setup. Construction and characterization of gene and operon fusion strains between galE and lacZ, encoding beta-galactosidase as a reporter gene, show that ultrainduction occurs at the level of transcription and not translation. Transcription of the operon, from both the cyclic AMP-dependent P1 and the cyclic nucleotide-independent P2 promoters, is subject to ultrainduction. The wild-type galR+ gene has an epistatic effect on ultrainducibility: ultrainduction is observed only in cells devoid of Gal repressor protein. Titration experiments show the existence of an ultrainducibility factor that acts like a repressor and functions by binding to DNA segments (operators) to which Gal repressor also binds to repress the operon.

DNA Mutational Analysis↗
PubMed1990

Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of the E. coli cAMP receptor protein.

Wild type cAMP receptor protein (CRP) activates in vitro lac transcription only in the presence of cAMP. In contrast the mutant CRP*598 (Arg-142 to His, Ala-144 to Thr) can activate lac transcription in the absence of cyclic nucleotide or at concentrations of cAMP below that required by CRP. To further characterize the properties of CRP*598, the binding of cAMP and cGMP to CRP and CRP*598 has been determined. The intrinsic binding constant (K) values obtained for cAMP binding are: CRP, 1.9 x 10(4) M-1; CRP*598, 3.8 x 10(5) M-1. The K values obtained for cGMP binding are: CRP, 2.9 x 10(4) M-1; CRP*598, 2.7 x 10(4) M-1. The results indicate that the affinity of CRP and CRP*598 for cGMP is relatively unchanged while the affinity of CRP*598 for cAMP is approximately twenty times greater than that shown by CRP. Binding of cAMP by CRP and cGMP by CRP or CRP*598 exhibits slight negative cooperativity. The major difference seen is that CRP*598 binds cAMP with strong positive cooperativity. The importance of the unsubstituted N6 position of the adenine moiety is also shown by the similar affinity of both forms of CRP for N6-butyryl cAMP. The cAMP binding properties evinced by CRP*598 suggest that its intrinsically altered conformation may be related to that assumed by CRP in a CRP-DNA or a cAMP-CRP-DNA complex.

Adenosine↗
PubMed1990

Positive control.

Explore the source record for details and available documents.

Allosteric Regulation↗
PubMed1990

Interaction of the Escherichia coli Gal repressor protein with its DNA operators in vitro.

The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OI. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 degrees C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, delta GI and delta GE, and a constant describing cooperativity, delta GIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 degrees C was delta GIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. delta GE and delta GI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, delta Cp, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 degrees C yielded values for delta GIE that were consistent with those resolved from the footprint analysis. Unique values of delta GIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that delta GE = delta GI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.

Base Sequence↗
PubMed1990

DNA looping in cellular repression of transcription of the galactose operon.

Communication between distant DNA sites is a central feature of many DNA transactions. Negative regulation of the galactose (gal) operon of Escherichia coli requires repressor binding to two operator sites located on opposite sides of the promoter. The proposed mechanism for regulation involves binding of the repressor to both operator sites, followed by a protein-protein association that loops the intervening promoter DNA (double occupancy plus association). To assess these requirements in vivo, we have previously converted gal operator sites to lac and shown that both operator sites must be occupied by the homologous repressor protein (Lac or Gal) for negative regulation of the gal operon. We have now addressed more directly the need for protein-protein association by the use of the converted operator sites and a mutant Lac repressor defective in association of the DNA-binding dimers. We have compared the biological and biochemical activity of two Lac repressors: the wild-type (tetramer) I+ form, in which the DNA-binding dimer units are tightly associated; and the mutant Iadi repressor, in which the dimer units do not associate effectively. The I+ repressor is an efficient negative regulator of the gal operon in vivo, but the Iadi mutant is an ineffective repressor. Purified I+ repressor efficiently forms DNA loops between operator sites that we have visualized by electron microscopy; the Iadi repressor fails to form DNA loops, although the protein binds effectively to both operator sites. From the clear correlation between looping in vitro and repression in vivo, we conclude that regulation of the gal operon depends on the association of repressor proteins bound to the two operator sites.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗
PubMed1989

Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector.

The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment [Wu and Crothers, Nature 308 (1984) 509-513]. We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations. The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites. The protein-binding sequence is inserted at one of these cloning sites. To investigate the functional significance of bending, we have compared, using pBend2, the cAMP.cAMP-receptor protein (CPR)-induced bending of CRP-binding sites found in five different genes of Escherichia coli. We have also shown that the bacteriophage lambda 0R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.

Bacteriophage lambda↗
PubMed1989

Domain II mutants of Pseudomonas exotoxin deficient in translocation.

Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.

ADP Ribose Transferases↗
PubMed1989

Effect of ethylation of operator-phosphates on Gal repressor binding. DNA contortion by repressor.

Gal repressor inhibits transcription by binding to two operators (OE and OI) in the gal operon. By ethylating DNA, we have identified 23 phosphate groups (11 on OE and 12 in OI) in the DNA backbone of gal operators that when ethylated interfere with repressor binding. By inference, either (1) such a phosphate is contacted or closely approached by Gal repressor, or (2) the structure of DNA generated by ethylation of such a phosphate, although not a site of direct contact, is not compatible with repressor binding. Within an operator, these phosphates are arranged with a perfect symmetry aligned with the operator dyad symmetry, indicating that each half-symmetry is contacted by a subunit of repressor dimer. Unlike in many other similar DNA-protein systems, the same phosphates in the gal operator are distributed around a B-form of DNA helix cylinder covering greater than 180 degrees. Models have been proposed to describe the disposition of the Gal repressor-operator complex, which would explain the layout of the participating phosphate groups around the surface of the DNA helix. We have discussed two ways by which Gal repressor can induce structural changes in DNA.

Bacterial Proteins↗
PubMed1989

Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin.

A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia). The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed. TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors. Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha. In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.

ADP Ribose Transferases↗
PubMed1989

DNA bending by negative regulatory proteins: Gal and Lac repressors.

The ability of two negative regulatory proteins, Gal and Lac repressors, to induce DNA bending was tested by the respective cloning of gal and lac operator DNA sequences into a sensitive vector, pBend2. pBend2 can generate a large number of DNA fragments in each of which a cloned operator is present in circularly permuted positions along the length of the DNA. Gel electrophoresis of these DNA fragments individually complexed to a repressor shows that the Gal repressor bends both of the gal operators, OE and OI. Similarly, the Lac repressor induces a bend to a lac operator DNA. In each case, the center of the average bent segment is located at or close to the dyad symmetry axis of the operator sequence. In view of these findings, we discuss how these negative regulatory proteins may function by a more dynamic mechanism than was perceived previously.

Base Sequence↗
PubMed1989

Cyclic-AMP-dependent switch in initiation of transcription from the two promoters of the Escherichia coli gal operon: identification and assay of 5'-triphosphate ends of mRNA by GTP:RNA guanyltransferase.

We have studied the initiation of transcription of the gal operon in Escherichia coli (i) by analyzing the 5'-triphosphate ends and (ii) by measuring the level of promoter-proximal gal mRNA made in vivo. The 5' termini were identified and quantified by capping with GTP:mRNA guanyltransferase, and the mRNA levels were determined by hybridization of pulse-labeled [32P]RNA with a specific DNA probe. Our results conclusively demonstrate the in vivo activities of two promoters, P1 and P2, with separate initiation sites (S1 and S2) as suggested before from in vitro and in vivo experiments (S. Adhya and W. Miller, Nature [London] 279:492-494, 1979; R. E. Musso, R. DiLauro, S. Adhya, and B. de Crombrugghe, Cell 12:847-854, 1977). We have also studied the effect of cyclic AMP (cAMP) on in vivo gal transcription and found that whereas total gal transcription remains largely unchanged, the relative proportions of the S1 and S2 mRNAs are influenced by the level of cAMP in the cell. In strains devoid of cAMP (cya), transcription initiates equally at S1 and S2; in cAMP-proficient cells (cya+), the S1 initiation increases twofold with a concomitant decrease in S2 initiation. Addition of a saturating amount of exogenous cAMP to cya mutant cells results in a relatively larger switch from S2 to S1. Our results clearly show that while cAMP is an inhibitor of S2, it is not an absolute requirement for transcription initiation at S1, but only acts to increase low-level transcription from the P1 promoter. Using these approaches, we have also studied gal promoter mutants (P211, P18, and P35) which show altered behavior in transcription initiations and in response to cAMP. On the basis of these results, we have discussed models by which transcription initiates at the two overlapping gal promoters (P1 and P2) and discussed how cAMP level modulates the switch between them.

Base Sequence↗
PubMed1988

DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry.

Gal repressor dimer binds to two gal operator sites, OE and OI, which are 16 bp long similar sequences with hyphenated dyad symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1, 3 and 8, and the rotational 1', 3' and 8' of the symmetries. We have shown that Gal repressor binding to OE or OI DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and OE and OI DNA. The CD spectral change was not observed when the central 8,8' G-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1' G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8' base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.

Base Composition↗