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A Zamir

Publications and source records attributed to A Zamir.

At least 55 records · Page 3Linked to original sources
PubMed1983

Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon.

The nifHDKY operon of Klebsiella pneumoniae encodes for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the nifHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in phase to lacZ. beta-Galactosidase activity served as a measure for nifH expression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, beta-galactosidase activity expressed from the mutant plasmids in the absence of nifA was 6-30% of the nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. In K. pneumoniae, IS2-containing plasmids expressed low levels of beta-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.

Bacterial Proteins↗
PubMed1982

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids.

Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.

Animals↗
PubMed1982

Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3' end of tRNA.

A derivative of tRNAPhe carrying at its 3' end a photolyzable group was chemically synthesized by coupling p-azidobenzoylglycylhydrazide to periodate-oxidized tRNA. The reaction converts the 3'-terminal ribofuranoside residue into a six-membered ring. The binding of p-azidobenzoylglycylhydrazide-tRNA to 70-S ribosomes resembles the binding of Phe-tRNA in its requirement for poly(U) and Mg2+ dependence. Irradiation at wavelengths greater than 300 nm of complexes of p-azidobenzoylglycylhydrazide-tRNA and 70-S ribosomes results in the covalent binding of approximately 20% of the reversibly bound tRNA to the ribosomes. The labeling occurs predominantly at a single site in 23-S rRNA within the sequence included in the 18-S fragment. To identify the modified sequence, an RNase A digest of 23-S rRNA was fractionated on a hydrophobic matrix (Porapak Q) and adsorbed and non-adsorbed fractions were compared by fingerprint analysis. A relatively intense spot was observed in the expected region in the fingerprint of the adsorbed fraction. The presence of an unusual nucleotide in the sequence of this oligonucleotide was demonstrated by a mobility-shift analysis of a partial nuclease P1 digest. The sequence derived is G*-A-A-G-C (the asterisk denoting the site of modification). Sequences at six positions in the 23-S rRNA are compatible with the sequence of the oligonucleotide determined. Of the six positions five are located within nucleotides 1467-1887. This region of approximately 400 nucleotides is likely to include the sequence interacting with the 3' end of tRNA and possibly forming part of the peptidyl-transferase center.

Affinity Labels↗
PubMed1982

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid.

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogeneous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.

Cloning, Molecular↗
PubMed1981

Stable chromosomal integration of the entire nitrogen fixation gene cluster from Klebsiella pneumoniae in yeast.

A bacterial plasmid containing the entire nitrogen fixation (nif) gene cluster (consisting of at least 15 genes) from Klebsiella pneumoniae was used in conjunction with an Escherichia coli-yeast shuttle plasmid containing the yeast his4 gene cluster to cotransform a his4- recipient strain of Saccharomyces cerevisiae. Of 87 histidine-independent clones screened, 2 contained nif DNA. Restriction and hybridization analyses showed that two copies of the nif plasmid (46 kilobases each) are integrated in tandem in the recipient chromosome by recombination between homologous regions in the transforming plasmids. Chromosomal integration was also verified by tetrad analysis, showing that the nif DNA behaved in meiosis like a Mendelian element. During mitotic growth, one of the two copies of the nif region is frequently lost. The remaining copy of nif is stable, even after 40 generations in nonselective medium.

DNA Restriction Enzymes↗
PubMed1980

Synthesis of part of a mouse immunoglobulin light chain in a bacterial clone.

We have cloned double stranded cDNA sequences encoding a mouse immunoglobulin light chain (L-321) into the PstI site of the beta-lactamase gene of plasmid pBR322 by the oligo (dG)-oligo (dC) tailing procedure. Escherichia coli X1776 transformed by the recombinant plasmids were screened for the expression of L-321 antigenic determinants by a newly developed in situ radio-immunoassay. One out of seven transformants screened was found to synthesize an L-chain like protein. Each bacterial cell produces about 550 molecules of the L-chain sequence. Preferential segregation of the L-chain sequence to the periplasmic space suggest covalent attachment of the L-chain sequence to the N-terminal portion of beta-lactamase. Restriction mapping of the plasmid DNA isolated from the positive clone indicated the presence of a DNA sequence coding for the entire constant region and extending into the variable region for a length corresponding to about 40 amino acid residues. The orientation of the cloned cDNA with respect to the plasmid DNA is compatible with the formation of a fused beta-lactamase-L-321 peptide.

Animals↗
PubMed1977

Mapping of 23-S rRNA at the ribosomal peptidyl-transferase center by photo-affinity labeling.

Photo-sensitive peptidyl-tRNA's were used to scan the environment of the peptidyl-transferase center of the ribosome. The specificity of the previously described labeling in the 18-S fragment of 23-S rRNA by Boc-Phe(N3)-Phe-tRNA (4-azido-N-t-butoxycarbonyl-phenylalanyl-phenylalanyl-tRNA) was demonstrated by the ability of the covalently anchored molecule to serve as donor substrate in peptide bond formation. Labeling patterns were also obtained with Boc-Phe(N3)-Phe-Phe-tRNA bound at the acceptor site and with Boc-Phe(N3)-(Gly)n-Phe-tRNA (n = 2,4). The results indicate that subsequences within the 18-S fragment of 23-S rRNA are located close to the acceptor site as well as along the path where the peptide moiety adheres to the ribosome. Identification of the labeled sequences is expected to shed light on the spatial arrangement as well as functional role of rRNA in the peptidyl transferase center.

Acyltransferases↗
PubMed1975

The effect of tRNA derivatives bound with natural or synthetic mRNA on the interaction of Escherichia coli ribosomes with colicin E3.

Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site. The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis. The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site. The poly(U) directed complex of AcPhe-tRNA and the T4-mRNA-directed complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes. Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the binding complexes. Thus, poly(U)-translating ribosomes were less affected than ribosomes translating the viral mRNA. The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo.

Colicins↗
PubMed1975

The use of cellulose xanthate for the immobilisation of biological molecules.

The mercapto groups of cellulose xanthate can reversibly form disulphide bridges with L-cysteine. This property has been utilised for the immobilisation of a protein and an enzyme. These macromolecules, as polythiol derivatives, formed disulphide linkages with the matrix without serious disturbance of their active sites, became firmly bound to the xanthate, and were not eluted by normal washing conditions. Cellulose xanthate is a cheap, easily prepared matrix which permits a simple coupling reaction. The immobilisation process is selectively reversible.

Binding Sites↗
PubMed1975

Identification of a region in 23S rRNA located at the peptidyl transferase center.

A photolyzable derivative of dipeptidyl-tRNA, p-azido-N-tBoc-Phe-[3H]Phe-tRNA, bound reversibly to 70S ribosomes in the presence of poly(U), becomes, when irradiated, covalently attached to components of the 50S ribosomal subunit. Most of the reaction occurs within the 23S RNA, while ribosomal proteins are only weakly labeled. Reversible binding as well as the covalent reaction are reduced in the absence of poly(U) or in the presence of several antibiotics specific for the 50S ribosomal subunit. There are apparently few sites (or perhaps a single site) of reaction on the 23S RNA that are exclusively located within the 2000 nucleotides from the 3 terminus of the molecule. Part of the 23S RNA within this region must therefore be closely associated with the peptidyl transferase center of the ribosome.

Binding Sites↗