Gad Glaser - Academia.edu (original) (raw)

Papers by Gad Glaser

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction en... more DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S. The small genome size (5 x 108 to 1 x 109 daltons) of the * Corresponding author.

Journal of Biological Chemistry, 1992

Journal of Biological Chemistry, 1991

European journal of medicinal chemistry, Dec 1, 2013

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights

Regulation of Macromolecular Synthesis by Low Molecular Weight Mediators, 1979

A sensitive, specific radioimmunoassay for ppGpp has been developed. Rabbits were injected with p... more A sensitive, specific radioimmunoassay for ppGpp has been developed. Rabbits were injected with ppGpp chemically coupled to rabbit gamma globulin. Serum from these rabbits contained antibody directed against ppGpp. Competition binding experiments were performed between the antibody, high specific activity [ 32 P]-labeled ppGpp, and various nucleotides. As little as 0.1 picomole unlabeled ppGpp measurably displaced the [ 32 P]-ppGpp from the antibody. The limit of detection for ppGpp using this assay is about 1,000-fold lower than previous methods.

Molecular Microbiology, 1994

Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the int... more Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth-phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the relA and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth-phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS. Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF-binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF-binding site was found upstream of the himD/hip promoter. All three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.

Journal of Molecular Biology, 2009

MazG is a homodimeric α-helical protein that belongs to the superfamily of all-α NTP pyrophosphat... more MazG is a homodimeric α-helical protein that belongs to the superfamily of all-α NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-α NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual α-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH b 6, it exists in a molten-globulelike state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.

Hemoglobin, 1986

Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as... more Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with beta-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC). The possibility of exploiting these cells instead of bone marrow as a source for nuclear and cytoplasmic RNA for expression studies was investigated. A simple procedure was developed for enrichment for normoblasts in blood samples withdrawn from patients prior to transfusion. Globin transcripts were analyzed in RNA purified from 12 patients. Unspliced precursor beta-mRNA molecules were observed in a patient with beta o-thalassemia, homozygous for a mutation at the 5' IVS2 splice site of the beta-globin gene. Detailed analyses showed that his mature beta-mRNA was larger than normal, and that a cryptic 5' splice site, approximately 50 nucleotides downstream from the normal one, was utilized. We conclude that peripheral blood can be used as a reliable source of RNA for the analysis of the effects of beta-thalassemia mutations on gene expression and the relationship to the clinical condition. Moreover, this procedure facilitates the comparison of in vivo gene expression with the results obtained from DNA transfection experiments with cloned beta-thalassemia genes.

Gene, 1977

The expression of the ribosomal RNA gene carried by the lambda trans-duc~g phage),rifdl8 is shown... more The expression of the ribosomal RNA gene carried by the lambda trans-duc~g phage),rifdl8 is shown to be subject to stringent amino acid control.),r/fU18 DNA was digested with endonuclease EcoRI and ligated to similarly restricted ColE1 plasmid DNA. Selection for expression of lambda integration and excision gene activity carried by the same DNA fragment results in cloning of the promoter oroximal portion of the 16S ribosomal RNA gene. The resulting chimera expresses), integration and excision functions as well as encoding the promoter proximal half of a 16S ribosomal RNA gene.

Frontiers in Microbiology, 2020

One of the most important stress responses in bacteria is the stringent response. The main player... more One of the most important stress responses in bacteria is the stringent response. The main player in this response is the signal molecule (p)ppGpp, which is synthesized by a Rel family protein. In Escherichia coli, RelA is the main synthetase of (p)ppGpp in response to amino acid starvation. Although the synthetic activity of RelA is well-understood, its regulation is not yet fully characterized. The C-terminus domain (CTD) of the E. coli RelA is responsible for the regulation of the protein and for its complete dependency on wild-type (WT) ribosome. The CTD contains three Cysteine residues, positioned in a very conserved order. Together with our previous results, we show in vitro the negative dominant effect of a part of the WT CTD (AA 564-744) named YG4 on RelA synthetic activity. This effect is abolished using mutated YG4 (YG4-638). In vitro and mass spectrometry (MS)-MS analysis of the native RelA and the mutated RelA in Cys-638 (Rel638) in the presence of the native and mutated YG4 (YG4-638) reveals that RelA forms a homodimer via its CTD by the formation of a disulfide bond between the two Cys-638 residues. This supports our previous data which showed, using a two-hybrid system, interactions between RelA proteins via the CTD. Finally, we show in vitro that excess of the native YG4 inhibited RelA synthetic activity but did not affect the amount of RelA bound to the ribosome. Our results suggest that the regulatory mechanism of RelA is by the dimerization of the protein via disulfide bonds in the CTD. Upon amino-acid starvation, the dimer changes its conformation, thus activating the stringent response in the cell.

Current Microbiology, 1995

ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular proce... more ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium. This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis [4], ppGpp participates in coordinating DNA replication and cell division. We studied the effects of ppGpp on the cell division cycle, using ceils containing plasrnid pSMll that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter. In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp. Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition. However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation. As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells. Cellular growth involves three distinct processes: increase in cell mass, DNA replication, and cell division. These processes are regulated coordinately in response to fluctuations of environmental conditions. In eubacteria these variations involve a change in the level of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp), a molecule that can be regarded as signaling shifts in nutritional abundance. This nucleotide is the candidate for a putative signal molecule whose intracellular concentration can be inversely correlated with growth rate and stable RNA transcription (for review see [4]). This presentation deals with the relationship between elevated ppGpp levels, DNA replication, and cell division. The overall rate of DNA replication in Escherichia coli is determined by the or/C activity which, in turn, appears to be determined by the cellular DnaA protein concentration [17]. The DnaA protein concentration seems to be stringently controlled through the

Annales de l'Institut Pasteur / Microbiologie, 1984

The small size of the mollicute genome considerably restricts the amount of genetic information a... more The small size of the mollicute genome considerably restricts the amount of genetic information available to the organisms. This is reflected in the relatively small number of cell proteins synthesized, the lack of many biosynthetic pathways and the marked dependence on exogenous nutrients for growth. The protein synthesizing machinery of mollicutes resembles that of eubacteria and is sensitive to the same antibiotics, except for rifampicin, to which RNA polymerases of mollicutes appear resistant. The mollicute ribosomes are built of 50 S and 30 S subunits and contain about 50 different proteins and 5 S, 16 S and 23 S rRNA, as in eubacteria. However, the 5 S rRNA in mollicutes appears shorter (107-112 nucleotides) than in eubacteria (116-120 nucleotides). We hybridized restriction endonuclease-digested DNA from a variety of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of M. capricolum. The results suggest the presence of only one or two sets (operons) of rRNA genes in the genome of Mollicutes, a number falling considerably below that of the eubacteria examined so far but resembling that found in archaebacteria. Our data also indicate a marked nucleotide sequence homology along the rrnB rRNA operon of E. coli and the rRNA operons of the various mollicutes, indicating that the rRNA genes in mollicutes are linked in the classical prokaryotic fashion 16 S-23 S-5 S. Each mollicute appeared to possess, on its genome, different flanking sequences adjacent to the rRNA operon(s), resulting in species-specific hybridization patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal of Bacteriology, 1988

RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were... more RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods. By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA. This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent. The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli. A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M. pneumoniae transcripts. When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site. The region surrounding this endpoint did not resemble any known promoter sequence. Dot blot hybridization of M. pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54,...