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Biomedical subjects

H Giladi

Publications and source records attributed to H Giladi.

At least 19 recordsLinked to original sources
PubMed2000

Use of transcriptional regulatory sequences of telomerase (hTER and hTERT) for selective killing of cancer cells.

Telomerase (hTER and hTERT) plays a crucial role in cellular immortalization and carcinogenesis. Telomerase activity can be detected in about 85% of different malignant tumors, but is absent in most normal cells. In situ hybridization analysis showed that high levels of hTER and hTERT expression are present in bladder cancer, while no signal was detected in normal tissue. Therefore, in this work we propose to use hTER and hTERT transcriptional regulatory sequences to control the expression of a cytotoxic gene in bladder tumor cells, resulting in the selective destruction of this cell population. Expression vectors containing the diphtheria toxin A-chain (DT-A) gene were linked to hTER and hTERT transcriptional regulatory sequences, respectively. Inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells transfected with the plasmids containing the DT-A gene under the control of the hTER or hTERT promoters in correlation with their activity. These studies support the feasibility of using hTER and hTERT transcriptional regulatory sequences for targeted patient-oriented gene therapy of human cancer.

Base Sequence↗
PubMed1998

Participation of IHF and a distant UP element in the stimulation of the phage lambda PL promoter.

We have previously identified a UP element in the phage lambda PL promoter, centred at position -90 from the transcription start site. Integration host factor (IHF), a heterodimeric DNA-binding and -bending protein, binds upstream of the lambda PL promoter in a region overlapping the UP element. Stimulation of transcription by IHF requires an intact alphaCTD and affects the initial binding of RNA polymerase to the promoter. We propose a model for the stimulation of PL by IHF in which IHF bends the DNA to bring the distal UP sequence in closer proximity to the promoter core sequences to allow the docking of the alphaCTD of RNA polymerase. Furthermore, IHF may also participate in protein-protein interactions with the alphaCTD. In support of this model, we found that alanine substitutions in alphaCTD at positions 265, 268, 270 and 275 reduced PL promoter activity. Mutations in the IHF DNA binding site, as well as IHF mutant proteins exhibiting a decreased ability to bend the DNA, were both defective in stimulating the PL promoter. In addition, some of the mutated IHF residues are clustered at a protein surface that interacts with the UP DNA sequence. These residues may also participate in protein-protein interactions with the alphaCTD.

Bacterial Proteins↗
PubMed1997

Impaired lysogenisation of the Escherichia coli rpoA341 mutant by bacteriophage lambda is due to the inability of CII to act as a transcriptional activator.

The C-terminus of the alpha subunit of Escherichia coli RNA polymerase is known to function in transcriptional activation at certain promoters. This region was previously shown to be necessary for full activation of the pE promoter by the phage lambda CII protein in vitro. In this work we investigated the inability of phage lambda to follow the lysogenic pathway in cells carrying the point mutation rpoA341 (a change of lysine 271 to glutamic acid). We found that neither overexpression of the cII gene nor stabilisation of the CII protein by the can1 mutation or by cIII gene overexpression was able to suppress the block in lysogenisation. In contrast, the lambda cin1 phage, which carries a CII-independent promoter for the expression of the cI gene, was able to efficiently lysogenise the rpoA341 mutant strain. Furthermore, the rpoA341 mutation prevented the activation of pE-lacZ and pI-lacZ transcriptional fusions by CII. Therefore we conclude that transcriptional activation by the cII gene product is abolished by the rpoA341 mutation, most probably due to impaired interaction between the CII activator and mutant RNA polymerase. The inability of RNA polymerase to respond to CII results in the impairment of lysogenisation of the rpoA341 mutant by phage lambda.

Bacteriophage lambda↗
PubMed1997

Stability of CII is a key element in the cold stress response of bacteriophage lambda infection.

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

Bacteriophage lambda↗
PubMed1996

Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage lambda.

An UP element defines a supplementary promoter element located upstream of the -35 region that stimulates transcription by interacting with the C-terminal domain of the RNA polymerase alpha subunit (alpha CTD). The alpha CTD also responds to various transcription activators, including the integration host factor protein, IHF, in the stimulation of the bacteriophage lambda PL promoter. PL consists of the tandem PL1-PL2 promoters where PL1 is stimulated and PL2 is repressed by IHF. We identified a functional UP element that binds the alpha subunit of RNA polymerase and is located in the region from -36 to -60 relative to the PL2 start site. PL2 expression requires the presence of the UP element and requires an intact alpha CTD. The UP element is nested within the DNA region protected by IHF against DNase I digestion. We used mutational analysis to identify the IHF recognition sequence which was found to be located downstream of the UP element, overlapping the -35 region of PL2. The possible function of the complex structure of the PL promoter is discussed.

Amino Acid Sequence↗
PubMed1996

Analysis of the shut-off of ribosomal RNA promoters in Escherichia coli upon entering the stationary phase of growth.

Most bacterial RNA consists of stable RNA which is composed of rRNA and tRNA. We have followed by primer extension analysis the level of ribosomal RNA synthesis along the growth phases of a cell culture. A sharp drop in rRNA synthesis was observed upon the transition from the exponential to the stationary phase of growth. Our results demonstrate that an effective shut-off of rRNA synthesis occurs also in the absence of ppGpp. Mutations in the host factors Fis and H-NS, which are known to regulate rrn P1 promoters, did not affect the shut-off process of ribosomal RNA promoters. We also tested the effect of RpoS, the sigma factor known to induce a number of genes in the stationary phase. It was shown that the host factors Fis, H-NS and RpoS do not play a major role in the regulation of the shut-off process of rRNA synthesis. The results presented demonstrate that the rate of rRNA synthesis provides a sensitive measure of the growth phase of the bacterial culture.

Bacterial Proteins↗
PubMed1996

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.

Amino Acid Sequence↗
PubMed1995

Cloning and primary sequence of the rpoH gene from Pseudomonas aeruginosa.

A DNA fragment from Pseudomonas aeruginosa containing the rpoH gene encoding the heat-shock sigma factor sigma 32 has been cloned and sequenced. The gene is expressed in Escherichia coli and complements an rpoH- strain. An open reading frame encoding 284 amino acids shows 61% identity and 78% similarity to the RpoH protein of Escherichia coli.

Amino Acid Sequence↗
PubMed1995

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

The response of the early phage lambda PL promoter to temperature was investigated. Experiments with lacZ reporter gene fusions demonstrated that the activity of the phage lambda PL promoter is inversely dependent on temperature. The bacterial DNA-binding protein integration host factor (IHF) further enhances lambda PL promoter activity at low temperature, although no apparent changes in the cellular level of IHF protein were observed at the different temperatures. IHF protein binds DNA in vitro more avidly at low temperatures. In vitro transcription assays further revealed that the temperature response of PL is the result of an intrinsic property of the promoter as well as its activation by IHF.

Bacterial Proteins↗
PubMed1995

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

We found that the activity of the phage lambda PL promoter is inversely dependent on temperature. Both in vivo and in vitro transcription assays revealed that the rather unique temperature response of PL is a sum of an intrinsic property of the promoter and its activation by integration host factor. We also found that at low temperature, phage lambda can lysogenize efficiently but cannot complete the lytic cycle. We hypothesize that by sensing the low environmental temperature the PL promoter plays a role in determining the direction of phage lambda development.

Bacterial Proteins↗
PubMed1995

Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid.

Regulation of the xyl gene operons of the Pseudomonas putida TOL plasmid is mediated by the products of the downstream clustered and divergently oriented xylR and xylS regulatory genes. The xylR-xylS intergenic region contains the xylR and xylS promoters Pr and Ps, respectively. A binding site for the XylR activator protein is located upstream of Ps and overlapping Pr. DNase I footprint experiments showed that one of these sites, which overlaps the recognition site for XylR activator, as well as an AT-rich region comprising the Ps promoter consensus were protected by integration host factor (IHF). IHF was found to act negatively in the in vivo activation of the Ps promoter, since the activity of a Ps promoter::lacZ fusion was elevated in an Escherichia coli mutant lacking IHF. In contrast, no alteration in the synthesis of XylR protein in the E. coli IHF-deficient mutant was detected.

Bacterial Proteins↗
PubMed1994

Genetic and biochemical analysis of IHF/HU hybrid proteins.

Integration host factor (IHF) is a small heterodimeric DNA-binding protein of E coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to DNA at a consensus sequence and bends DNA. HU protein, encoded by the hupA and hupB genes, is similar to IHF except that it does not bind to a specific DNA sequence. To investigate the protein determinants for IHF specificity we exchanged progressively longer segments from the C-terminus of Hip with those of HupA, and followed the activity in vivo and in vitro of four such IHF/HU hybrids. Replacement of 11 residues from the C-terminal alpha helix of Hip by the complementary eight residues of HupA (hybrid 1), had only minor effects on the DNA binding activity of the protein. As progressively longer segments of Hip were replaced by HupA, a precipitous decrease in IHF activity was observed. The hybrid with the longest substitution, hybrid 4, was totally inactive in vivo and could not be purified. None of the hybrid proteins could complement HU activity. Comparing the activities of hybrid 1, hybrid 2 and IHF point mutants, led us to conclude that the structural integrity of the C-terminal alpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity. We favor the hypothesis that alpha helices 3 of both IHF subunits interact with the body of IHF so as to anchor the arms. This interaction stabilizes the arms to permit DNA binding specificity. Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.

Amino Acid Sequence↗
PubMed1994

Expression of the genes coding for the Escherichia coli integration host factor are controlled by growth phase, rpoS, ppGpp and by autoregulation.

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.

Bacterial Proteins↗
PubMed1993

Genetic and biochemical analysis of the integration host factor of Escherichia coli.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to the minor groove at a consensus sequence and bends DNA. We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro. Substitutions at the C-terminal alpha-helix (alpha-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA. These results indicate that the C-terminal region of Hip participates in determining IHF specificity. Alanine substitutions in beta-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA. Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity. This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF. The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

Bacterial Proteins↗
PubMed1992

Stimulation of the phage lambda pL promoter by integration host factor requires the carboxy terminus of the alpha-subunit of RNA polymerase.

Escherichia coli integration host factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda. IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB). We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the alpha subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF. Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.

Bacterial Proteins↗
PubMed1992

Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter.

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

Bacterial Proteins↗
PubMed1991

Genes coding for integration host factor are conserved in gram-negative bacteria.

A genetic system for the selection of clones coding for integration host factor and HU homologs is described. We demonstrate that the himA and hip genes of Serratia marcescens and Aeromonas proteolytica can substitute for the Escherichia coli genes in a variety of biological assays. We find that the sequence and genetic organization of the himA and hip genes of S. marcescens are highly conserved.

Amino Acid Sequence↗