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S Adhya

Publications and source records attributed to S Adhya.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1988

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity.

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.

ADP Ribose Transferases↗
PubMed1988

Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain.

Pseudomonas exotoxin (PE) contains three domains whose functions are cell recognition, membrane translocation, and ADP ribosylation of elongation factor 2. PE40 is a form of PE which is missing the cell recognition domain. To study the properties of PE40, it was expressed in Escherichia coli using a vector which contains a T7 phage promoter, an OmpA signal sequence, and that portion of the PE gene encoding PE40. Upon induction with isopropyl-1-thio-beta-D-galactopyranoside, large amounts of PE40 were secreted, and highly purified PE40 was prepared from the culture medium. PE40 was chemically coupled to different monoclonal antibodies, and protein synthesis inhibition activities of these immunotoxins was assessed on various cell lines. These activities were compared with the activities of the corresponding immunotoxins made with native PE. These data indicate that PE40 may be useful in the construction of certain immunotoxins.

ADP Ribose Transferases↗
PubMed1988

Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase.

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. In the absence of cGMP, CRP*598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP+-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection ("foot-printing") indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [32P]lacP+ fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding. In contrast, the weakly active unliganded CRP*598 can be shifted to a functional state not only by cAMP but also by cGMP and RNA polymerase.

Cyclic AMP↗
PubMed1988

Interaction of spatially separated protein-DNA complexes for control of gene expression: operator conversions.

Two operators, spatially separated from each other and from the promoters, repress the gal operon when bound to Gal repressor. Conversion of either gal operator to a lac operator results in derepression, although both Gal and Lac repressors are present, suggesting that mere occupation of operator sites is not sufficient to cause repression. Conversion of both operators to lac operators restores normal repression in the presence of Lac repressor protein. We propose that normal repression requires interaction between operator-bound like repressor molecules; this generates a DNA loop, which is part of a higher order structure. RNA polymerase and cyclic AMP receptor protein are present in this complex but unable to initiate transcription because of the higher order structure. Such higher order DNA-multiprotein complexes could occur in a variety of genetic regulatory systems that are controlled from distal sites by regulatory proteins.

Base Sequence↗
PubMed1988

Cytotoxic activity of an interleukin 2-Pseudomonas exotoxin chimeric protein produced in Escherichia coli.

A cDNA clone for human interleukin 2 (IL-2) has been fused to the 5' end of a modified Pseudomonas exotoxin (PE) gene that lacks the sequences encoding the cell recognition domain. The chimeric protein IL-2-PE40 was produced in Escherichia coli. It was extremely toxic to IL-2 receptor-positive cells but had no measurable effect on cells lacking the IL-2 receptor. IL-2-PE40 might be a useful cytotoxic agent in the treatment of diseases involving IL-2 receptor-positive cells and in the treatment of allograft rejection.

Cell Line↗
PubMed1988

Role of domain II of Pseudomonas exotoxin in the secretion of proteins into the periplasm and medium by Escherichia coli.

Pseudomonas exotoxin (PE) is composed of structural domains I, II, and III; when interacting with mammalian cells the function of domain I is cell recognition, the function of domain II is membrane translocation, and domain III functions in ADP ribosylation. PE is secreted by Pseudomonas aeruginosa into its growth medium. The domain responsible for secretion has been examined by expressing modified PE genes in Escherichia coli under the control of a T7 promoter. Without a signal sequence, PE accumulates within the cell, but PE is secreted into the periplasm when part or all of domain I is removed. PE appears in the periplasm and medium when domain I and part of domain II are removed. Domain II alone is secreted into the periplasm, whereas domain III alone remains within the cell. Addition of an OmpA signal sequence results in secretion of mature PE into the periplasm and secretion of domains II-III into the medium. A protein composed of transforming growth factor alpha fused to the amino terminus of domains II-III is secreted into the periplasm without a signal sequence and into the medium with a signal sequence. A protein composed of domain(s) II or II-III fused to the amino terminus of alkaline phosphatase is secreted into the periplasm and the medium with or without a signal sequence. We conclude that domain II contains important information for protein secretion.

ADP Ribose Transferases↗
PubMed1988

Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations.

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.

Allosteric Site↗
PubMed1987

Probing the structure of gal operator-repressor complexes. Conformation change in DNA.

The gal operon is regulated by binding of Gal repressor to two operator loci, OE and OI, which are separated by 114 base pairs (bp). We have probed the actual operator DNA segments with and without Gal repressor occupation by characterizing the regions protected by repressor from DNase I digestion and dimethyl sulfate methylation. The segments which are protected from DNase I digestion in both OE and OI are about 22 bp long and seem to include 2-3 extra bp on either side of a 16-bp similar sequence containing an approximate dyad symmetry, with a consensus half-symmetry sequence GTG(G/T)AA-C. Repressor occupation hinders the reactivity of the consensus guanines in the four half-symmetry sequences, as shown by retardation of methylation at the N-7 positions by dimethyl sulfate owing to repressor binding. The protected guanines are symmetrically located. Since a dimeric Gal repressor affects symmetrically located bases, it is consistent with the notion that each half-operator is occupied by a repressor subunit. Because the N-7 positions of methylation of guanines lie in the major grooves and the protected guanines are located at positions 1, 3, 8 and the rotational 1', 3', and 8' in the 16-bp dyad symmetry, we suggest that Gal repressor establishes direct contacts with bases at 1, 3, 1', and 3' through two major grooves lying on one face of an operator helix and prevents reactivity of the guanines at 8 and 8' of a third major groove on the opposite face by changing the DNA helical structure at this position. Contacts at other positions are also discussed.

Base Sequence↗
PubMed1987

Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector.

The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.

Amino Acid Sequence↗
PubMed1987

Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli.

Pseudomonas exotoxin A is a single chain toxin with three structural domains that inhibits protein synthesis in eukaryotic cells by catalyzing ADP ribosylation of elongation factor 2. To study the function of these domains, we deleted different portions of the PE structural gene and expressed these constructs in E. coli using an inducible T7 promoter. These studies indicate that structural domain Ia is required for cell recognition, that structural domain II is required to translocate the toxin across a cellular membrane, and that structural domain III and a portion of domain Ib are required for ADP ribosylation activity. Toxin lacking domain Ia is about 100-fold less toxic to mice than intact PE and should be a useful molecule for the construction of immunotoxins.

ADP Ribose Transferases↗
PubMed1987

Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin.

The transforming growth factor type alpha gene has been fused to a modified Pseudomonas toxin gene from which the cell-recognition domain has been deleted. The chimeric gene has been expressed in Escherichia coli, and the chimeric protein, PE40-TGF-alpha, has been highly purified. PE40-TGF-alpha kills cells expressing epidermal growth factor receptors and has little activity against cells with few receptors. This chimeric protein might be useful in treating cancers that contain high numbers of epidermal growth factor receptors.

ADP Ribose Transferases↗
PubMed1986

Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

Adenoviridae↗
PubMed1986

A simple procedure for the preparation of pure kinetoplast DNA network free of nuclear DNA from the kinetoplast hemoflagellate Leishmania donovani.

A simple, inexpensive procedure for preparing pure kinetoplast DNA network from Leishmania donovani is described. L. donovani promastigotes were lysed by incubating with pronase in presence of sodium dodecylsulfate. Crude kinetoplast DNA networks were obtained by centrifugation of the lysate through a 20% sucrose solution. The pellet containing kinetoplast DNA was deproteinized by phenol extraction. Contaminating nuclear DNAs were removed by denaturation with alkali, neutralization, and addition of polyethylene glycol-8000 to a concentration of 10% to facilitate precipitation of kinetoplast DNA. kDNA isolated after centrifugation was deproteinized several times with phenol and finally precipitated with ethanol. The average yield by this procedure is 30-50 micrograms of kDNA per gram of wet cells. By slot-blot hybridization with a nuclear DNA probe, no nuclear DNA contamination of the kDNA networks could be detected.

Animals↗