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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Significance of connective tissue proliferation in the breakdown of cartilage: a novel in vivo model.

The implantation of homologous femoral head cartilage in subcutaneous tissues of random bred Wistar rats results in both subchondral and articular surfaces becoming overlaid by an adherent granulation tissue comprising predominantly fibroblast-like cells. The response of the tissue to cartilage encapsulated with cotton fibres was also similar but erosions, mainly subchondral, were more evident and proteoglycan loss markedly greater. The connective tissue response to cotton was the progressive formation of a foreign body granuloma comprising mononuclear cells, multinucleated giant cells, and fibroblasts with very few polymorphonuclear leucocytes.

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.

Adjuvant polyarthritis and the response of air pouch lining cells.

The influence of adjuvant polyarthritis on subcutaneous air pouches in rats was examined in the light of reports of the resemblance of their cavity lining to normal synovium. Marked macroscopic and microscopic changes were observed. These included thickening of air-pouch wall, hyperplasia of lining cells, infiltration of inflammatory cells, fibrosis, and the production of an effusion in the cavity; such changes are comparable to the proliferative synovitis reported in arthritic joints of diseased animals.

Cloning and expression of the Escherichia coli rho gene in a plasmid vector.

In order to further elucidate the role of Rho protein on transcription termination and cells growth control, we have subcloned by two steps the rho+ structural gene of Escherichia coli from Lambda rho+524 into a plasmid vector. The resulting plasmid pEG25 contains a 2.9 kbp insert which is able to complement several different rho mutations and to express a functional Rho protein in U.

Sites of allosteric shift in the structure of the cyclic AMP receptor protein.

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression.

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA.