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Biomedical subjects

A Zamir

Publications and source records attributed to A Zamir.

At least 37 records · Page 2Linked to original sources
PubMed1991

A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina.

Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M(r) cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface.

Journal Article↗
PubMed1991

Involvement of GroEL in nif gene regulation and nitrogenase assembly.

Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system.

Bacterial Proteins↗
PubMed1990

Photoinduction of Massive beta-Carotene Accumulation by the Alga Dunaliella bardawil: Kinetics and Dependence on Gene Activation.

The massive accumulation of beta-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of beta-carotene accumulation, cells continuously grown under white light of approximately 27 microeinsteins per square meter per second were exposed to light of approximately 1650 microeinsteins per square meter per second. The exposed cells accumulate beta-carotene in two stages: the first stage, lasting for 24 hours, starts shortly after exposure, whereas the second stage starts concomitantly with the onset of the stationary phase and persists until the cells collapse. Actinomycin D, chloramphenicol, or cycloheximide added to low-illuminated cultures abolish the subsequent induction of beta-carotene accumulation by high light intensity. These results, together with the early exponential kinetics of accumulation, point to the role of gene activation in the process. In vivo labeling of proteins and in vitro translation of poly(A)(+) mRNA revealed several pronounced differences between low-illuminated and high-illuminated cells. A strongly light-induced protein of approximately 55 kilodaltons, as well as other light-induced proteins could possibly fulfill a carotenogenic function.

Journal Article↗
PubMed1989

Outreading promoters are located at both ends of the gamma-delta transposon.

Two plasmids were isolated containing oppositely oriented gamma-delta insertions between the wild-type transcription initiation site of the nifHDKY operon and the nifH coding sequence. The nifHDKY promoter of Klebsiella pneumoniae, similar to other nitrogen fixation (nif) promoters, normally requires the products of ntrA and nifA for activity. Mutations that allowed constitutive expression of the nifHDKY operon were searched for by transforming a plasmid, containing the regulatory region of this operon followed by an in-frame nifH'-'lacZ fusion, into a Lac- Escherichia coli strain (which contains no nifA) and screening for Lac+ derivatives. The plasmids described here were isolated from such derivatives and directed the constitutive expression of beta-galactosidase. Deletion analysis indicated that gamma-delta promoters other than those transcribing tnpA and tnpR were involved in this expression. Nuclease S1 mapping revealed outward-reading transcription initiation sites in both the gamma end and the delta end of the transposon. Most interestingly, one initiation site on each end was located in corresponding positions within the terminal inverted repeats. The sites were in the center of the longest sequence, of 12 bp, contiguously conserved between the terminal inverted repeats of gamma-delta and the related transposon Tn3. In gamma-delta and Tn3, this sequence has been recently implicated in transposase binding. These results suggest a possible interrelationship between transcription from the "end" promoters and transposition.

Base Sequence↗
PubMed1989

Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.

Amino Acid Sequence↗
PubMed1988

Selection of in vivo deletions in Saccharomyces cerevisiae.

A general screening procedure has been devised for the selection of in vivo-generated deletions in haploid Saccharomyces cerevisiae cells. It is based on the introduction into a cyh2 host (resistant to the drug cycloheximide) of a tandemly duplicated CYH2 gene (a dominant allele, conferring sensitivity to cycloheximide), and subsequent selection for Cyhr derivatives. The duplicated CYH2 gene has been introduced on CEN ARS plasmids or integrated into chromosome II. A variable but significant proportion of the Cyhr derivatives of such transformants were deletion mutants in which both CYH2 copies had suffered deletions. Some of the deletions extended into sequences outside the tandemly duplicated CYH2 gene. A total of 61 independently selected deletions ranged in length from 3.1 to over 20 kilobases and had no obvious preferred endpoints. Restriction analysis showed that other frequently isolated Cyhr derivatives appeared to retain one of the two CYH2 copies. Such single-copy derivatives of CEN ARS plasmids did not contain a functional CYH2 gene. The frequency of true deletions in CEN ARS plasmids, of approximately 10(-7) per viable cell, was comparable in RAD52 and rad52 strains. Chromosomal deletions, which occurred at a frequency of approximately 10(-8) per viable cell, were observed only in rad52 hosts. Derivatives exhibiting an additional altered phenotype, such as the inactivation of a neighboring gene or, less frequently, the transcriptional activation of a previously silent gene, were isolated by screening deletion mutants. These results show that the method described can be used for in vivo deletion mapping or for the generation of gene fusions.

Chromosome Deletion↗
PubMed1987

A quantitative approach to sequence comparisons of nitrogenase MoFe protein alpha- and beta-subunits including the newly sequenced nifK gene from Klebsiella pneumoniae.

The nucleotide sequence was determined for part of the Klebsiella pneumoniae nif gene cluster containing the 3' end of the nifD gene and the entire length of the nifK gene (encoding the alpha- and beta-subunits of the nitrogenase MoFe protein respectively), as well as the putative start of the nifY gene, a gene of as yet unknown function. A broad-based comparison of a number of MoFe protein alpha-subunits, beta-subunits and alpha-versus beta-subunits was carried out by the use of a computer program that simultaneously aligns three protein sequences according to the mutation data matrix of Dayhoff. A new kind of quantitative statistical measure of the similarity between the aligned sequences was obtained by calculating and plotting standardized similarity scores for overlapping segments along the aligned proteins. This calculation determines if a test sequence is similar to the consensus sequence of two other proteins that are known to be related to each other. The different beta-subunits compared were found to be significantly similar along most of their sequence, with the exception of two relatively short regions centred around residues 225 and 300, which contain insertions/deletions. The overall pattern of similarity between different alpha-subunits exhibits resemblance to the overall pattern of similarity between different beta-subunits, including regions of low similarity centred around residues 225 and 340. Comparison of alpha-subunits with beta-subunits showed that a region of significant similarity between the two types of subunits was located approximately between residues 120 and 180 in both subunits, but other parts of the proteins were only marginally similar. These results provide insights into likely tertiary structural features of the MoFe protein subunits.

Amino Acid Sequence↗
PubMed1987

Nitrogenase MoFe protein subunits from Klebsiella pneumoniae expressed in foreign hosts. Characteristics and interactions.

The expression of selected nitrogen fixation (nif) genes from Klebsiella pneumoniae in foreign hosts provides an approach to determine the pathway, minimal genetic requirements, and host dependence of nitrogenase assembly. In this study, we investigated the assembly of the alpha 2 beta 2 MoFe protein, responsible for substrate binding and reduction, by introducing nifD and nifK (encoding respectively, the alpha and beta subunits) into Escherichia coli and the yeast Saccharomyces cerevisiae. In E. coli, both genes were expressed from the nifHDKY operon; in yeast, the genes, separately fused to the yeast ADH1 promoter, were introduced on two different plasmids. Denaturing immunoblot analyses demonstrated the presence of significant amounts of NifD and NifK in both hosts. In E. coli, the level or perhaps modification of NifD depended on the growth medium of the bacteria. Nondenaturing, anaerobic immunoblot assays revealed in E. coli, nif-specific antigens of lower electrophoretic mobility than Kp1, which may represent assembly intermediates. In yeast, no putative assembled products were evident, and the predominant antigens corresponded to the monomeric forms of the polypeptides. These results indicate that, unlike NifH, the Fe protein subunit (Berman, J., Gershoni, J. M., and Zamir, A. (1985) J. Biol. Chem. 260, 5240-5243), NifD and NifK are insufficient for the assembly of an electrophoretically Kp1-like structure. Homodimerization of nifK and probably of nifD primary gene products does not appear to occur spontaneously and hence is unlikely to represent the initial step in the assembly. The difference between the two hosts suggests that the cellular environment or mode of expression could affect the interaction between the two subunits.

Escherichia coli↗
PubMed1987

Suppressors suaC109 and suaA101 of Aspergillus nidulans alter the ribosomal phenotype in vitro.

A new homologous, cell-free system for protein synthesis has been devised for use with ribosomes and elongation factors from Aspergillus nidulans. Ribosome preparations from strains with either the suaA101 or suaC109 mutations have a higher misreading ratio (non-cognate:cognate amino acid incorporation) in the presence of hygromycin than controls. They can be classed as fidelity mutants. These results also prove that the mutations must be in genes coding for ribosomal proteins or enzymes which modify ribosomal proteins post-translationally. Alternatively, the genes could code for translation factors.

Aspergillus nidulans↗
PubMed1986

Sequence rearrangements may alter the in vivo superhelicity of recombinant plasmids.

Electrophoretic resolution of topoisomers was used to compare the in vivo superhelicity of recombinant plasmids containing a fragment of cDNA for an immunoglobulin light chain, cloned in the two possible orientations into the BamHI site of pBR313 or pBR322. Previously, frequent transpositions of IS1 or IS5 were observed into the sequence upstream to the cloned fragment in recombinants in one orientation [(+) plasmids] but not in recombinants in the opposite, (-) orientation [(1982) Nucleic Acids Res. 10, 4525-4542]. The results of the present analyses show that, on average, (-) plasmids are less negatively supercoiled than (+) plasmids, or pBR322. These results suggest that primary sequence rearrangements in plasmids could affect their in vivo topological state, and consequently, perhaps, their effectiveness as recipients of transposable elements.

Base Sequence↗
PubMed1986

Spontaneous amplification of yeast CEN ARS plasmids.

Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-delta 4 allele (as well as the URA3 and TRP1 markers) yielded His+ transformants at 0.1%-50% the frequency of Ura+ Trp+ transformants. Additional His+ derivatives arose on continuous growth of transformants originally scored as His- Ura+ Trp+. In all cases, the His+ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His+ phenotype was lost more readily than the Ura+ Trp+ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura+ Trp+ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.

Alleles↗
PubMed1986

Transcriptional analysis of promoter mutations in the Klebsiella pneumoniae nifHDKY operon.

Previously isolated promoter mutations that allow expression of the Klebsiella pneumoniae nifHDKY operon in the absence of nifA (R. Bitoun, J. Berman, A. Zilberstein, D. Holland, J.B. Cohen, D. Givol, and A. Zamir, Proc. Natl. Acad. Sci. USA, 80:5812-5816, 1983) were further characterized. pRB1 and pRB5, containing, respectively, point and duplication mutations in the nifHDKY regulatory region, were transformed into Escherichia coli and K. pneumoniae hosts with different nifA and ntrA backgrounds. nif transcription start sites were determined by nuclease S1 mapping. The results indicated that nifA-independent expression from both mutants did not require ntrA. Transcription from pBR5 started 3 base pairs (bp) upstream of the start site of nif-regulated transcription and could stem from a canonical promoter sequence generated at the junction between the two copies of the duplicated sequence. In the presence of nifA-ntrA, transcription from pRB5 started predominantly at the site characteristic of the nif-regulated promoter. The site of constitutive transcription initiation in pRB1 was located 33 bp upstream of the point mutation and 40 bp upstream of the start of nifA-ntrA-activated transcription. Low-level transcription from the upstream site was also evident, in the absence of nifA or nifA or both, with the plasmid containing the wild-type nifHDKY regulatory region. However, when nifA and ntrA were present to activate transcription from the major nif promoter, no activity was evident from the upstream site in either pRB1 or the parental plasmid. Thus, the mutation enhanced the activity of a pre-existing constitutive promoter, the activity of which was repressed on nifA-ntrA activation of the major nif promoter.

Base Sequence↗
PubMed1985

Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli.

Animals↗
PubMed1985

Expression of nitrogen fixation genes in foreign hosts. Assembly of nitrogenase Fe protein in Escherichia coli and in yeast.

In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase. Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K. pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide. This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E. coli and in yeast. Immunoblotting methods, as well as 55Fe2- labeling of K. pneumoniae were employed to detect native nitrogenase components in cell lysates. E. coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP. While in E. coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.

Adenosine Triphosphate↗
PubMed1985

Expression of a nitrogen-fixation gene encoding a nitrogenase subunit in yeast.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme complex exclusive to prokaryotes. We used the yeast Saccharomyces cerevisiae to study the synthesis, and subsequently the assembly, of nitrogenase components in a eukaryote. Here, the Klebsiella pneumoniae nifH gene, encoding the subunit of the Fe protein (Kp2) component of nitrogenase, was expressed in S. cerevisiae from the yeast ADHI promoter. The nifH gene product, detected in yeast by immunoblot analysis with anti-Kp2 antibodies, exhibited the same electrophoretic mobility in SDS-polyacrylamide gels as that of the Kp2 subunit synthesized in K. pneumoniae. Estimates of Kp2 antigen and assays of beta-galactosidase activity specified by nifH'-'lacZ fusions showed that the level of nifH product was similar in anaerobically and aerobically grown yeast, but varied with different transforming plasmids and in various haploid and diploid yeast strains. A cistron located downstream to nifH in a transcript resembling the polycistronic mRNA of the nifHDKY operon in K. pneumoniae is not translated in yeast.

Gene Expression Regulation↗
PubMed1984

Secretion and processing of an immunoglobulin light chain in Escherichia coli.

When a cDNA coding for the kappa light chain (L-321) from the mouse MOPC321 myeloma was cloned into Escherichia coli, L-321 antigens were found in both cytoplasmic and periplasmic fractions. In cells synthesizing the intact chain, starting with its signal peptide, the periplasm contained a mature-size immunoglobulin indicating that the eukaryotic signal peptide can initiate secretion and be processed. When the entire cDNA for L-321 (including its signal peptide) was inserted in the gene for bacterial beta-lactamase, processing cleaved only the first bacterial signal sequence of the hybrid protein synthesized. Removal of the beta-lactamase signal peptide was also observed with another beta-lactamase-L-321 hybrid which did not include the immunoglobulin signal peptide and the adjacent part of the variable region. The two hybrid proteins may, however, differ in their mode of secretion.

Cloning, Molecular↗