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Biomedical subjects

M E Gottesman

Publications and source records attributed to M E Gottesman.

At least 73 records · Page 4Linked to original sources
PubMed1991

An Escherichia coli rpoB mutation that inhibits transcription of catabolite-sensitive operons.

The Escherichia coli rpoB636 mutant is defective in the transcription of lac and other catabolite-sensitive operons. The lac promoter variant, UV5, which is independent of cyclic AMP and the cyclic AMP receptor protein, CRP, was also defective in rpoB636 mutants. The activity of the lac UV5 promoter was restored to wild-type levels by deletion of cya (adenylate cyclase) or crp. Cyclic AMP and CRP apparently act as inhibitors of the rpoB636 RNA polymerase.

Bacteriophage lambda↗
PubMed1991

Escherichia coli mutations that block transcription termination by phage HK022 Nun protein.

The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage lambda nut sites. Termination by Nun and antitermination by lambda N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at lambda nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase beta' subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by lambda N function. The mutations antagonized Nun only at lambda nutR; termination at lambda nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the beta' subunit of RNP may be Nun-specific.

Bacteriophage lambda↗
PubMed1991

Reversible inhibition of a thyroid-specific trans-acting factor by Ras.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

Animals↗
PubMed1991

A replica filter assay for expression of ion transport proteins.

In thyroid cells, iodide is accumulated intracellularly via a Na+-I-cotransporter. In this report we show that it is possible to detect diffusible 125I-concentrated in thyroid cell colonies that have been replicated onto nylon filters. Using the replica filter assay, we demonstrate that the iodide transport 1) is restricted to thyroid cells, 2) is Na+ dependent and electrogenic, 3) is inhibited by ClO4- and SCN-, and 4) is adenosine 3',5'-cyclic monophosphate dependent. These are all characteristics of thyroidal iodide transport. This technique can, in principle, detect the expression of any transport system that results in the intracellular accumulation of a diffusible molecule. Moreover, the filter assay can be used to screen for colonies carrying structural or functional mutations affecting such transport systems.

Animals↗
PubMed1990

Renaturation of denatured lambda repressor requires heat shock proteins.

The temperature-sensitive bacteriophage lambda cI857 repressor protein rapidly renatures after thermal inactivation. E. coli mutants in the heat shock protein genes dnaK, dnaJ, and grpE do not efficiently reactivate heat-denatured repressor. Our results suggest that protein refolding is promoted by heat shock proteins and that such a process is the basis of the homeostatic role played by these proteins in the heat shock response.

Bacteriophage lambda↗
PubMed1990

Lambda nutR mutations convert HK022 Nun protein from a transcription termination factor to a suppressor of termination.

The Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.

Bacteriophage lambda↗
PubMed1990

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli.

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.

Alleles↗
PubMed1990

Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon.

Two genes, secE and nusG, situated between the tufB and ribosomal protein rplKAJL operons in the rif region at 90 min on the Escherichia coli chromosome, have been sequenced and characterized. The secE gene encodes a 127-amino-acid-long polypeptide, which is an integral membrane protein essential for protein export (P. J. Schatz, P. D. Riggs, A. Jacq, M. J. Fath, and J. Beckwith, Genes Dev. 3:1035-1044, 1989). The nusG gene encodes a 181-amino-acid-long polypeptide and is involved in transcription antitermination. The protein product of nusG is essential for bacterial viability. The secE-nusG genes are cotranscribed, with transcripts initiated at the PEG promoter and terminated at the Rho-independent terminator in the region of the rplK promoter. The majority of transcripts are processed at a number of sites in the 5' untranslated leader region by RNase III and are possibly also processed by a second unidentified nuclease. The role of transcript processing in the regulation of secE and nusG has not yet been established. The juxtaposition and coregulation of a protein export factor and a transcriptional factor raise questions concerning a functional connection between the two processes.

Amino Acid Sequence↗
PubMed1989

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine.

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39 degrees C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39 degrees C. Restoration of Ras activity at 33 degrees C leads to the rapid loss of TgTF1 and G418 resistance.

Animals↗
PubMed1989

Repression of the lambda pcin promoter by integrative host factor.

The cin-1 mutation creates a new promoter (pcin) in the tR1 region of bacteriophage lambda. The pcin promoter transcribes the cI repressor gene constitutively. lambda cin-1 does not propagate on Escherichia coli mutants lacking the integrative host factor (IHF). lambda cI- cin-1 grows normally in IHF- mutants, indicating that repressor overproduction from pcin blocks lytic growth. The presence of an IHF binding site which overlaps the pcin promoter led us to the hypothesis that IHF functions as a repressor of pcin transcription. We find that the pcin promoter is fivefold more active in a host lacking IHF than in wild-type cells. In vitro studies show that IHF directly inhibits transcription initiation at pcin. Abortive initiation and gel retardation assays demonstrate that IHF interferes with the binding of RNA polymerase to the pcin promoter. RNA polymerase bound in an open promoter complex is resistant to IHF. We propose that IHF binding to the pcin promoter region blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure.

Bacterial Proteins↗
PubMed1989

Structure and function of the nun gene and the immunity region of the lambdoid phage HK022.

The immunity region of the lambdoid phage, HK022, has been sequenced. The HK022 repressor gene, its cognate operators and promoters, and several early phage genes can be discerned. The overall design of the immunity region resembles that of other lambdoid phages. The location of the HK022 nun gene, whose product excludes superinfecting lambda by terminating transcription at (or near) the lambda nut sites, is analogous to that of gene N in lambda. nun is preceded by sequences similar to the lambda nut sites and the lambda pL promoter and is followed by several transcription termination signals. Despite these similarities, Nun is required neither for the lytic nor the lysogenic pathway of phage development. Again, unlike N, Nun is expressed in a prophage, perhaps from a promoter other than pL. We suggest that Nun and N have diverged in evolution and now perform different functions for their respective phages. Although Nun and N compete at the lambda nut sites and interact with the same host Nus proteins, they are only distantly related in predicted amino acid sequence. The presence of transcription terminators in the pL operon suggests that the expression of the HK022 early functions, like those of lambda, entails an antitermination mechanism. However, Nun does not appear to be an essential component of this mechanism. Our most economic model is that the HK022 nutL sequence suppresses pL operon terminators in the absence of a phage-encoded antitermination protein. Striking homologies between the HK022 nutL sequence and related sequences in the Escherichia coli rrn operons support this notion. Alternatively, a phage antitermination gene may be located outside the pL operon.

Bacteriophages↗
PubMed1987

The remarkable specificity of a new transcription termination factor suggests that the mechanisms of termination and antitermination are similar.

E. coli lysogenic for the temperate, lambda-related phage HK022 do not support lambda growth. The exclusion of lambda is caused by the HK022 nun gene product, which blocks the expression of genes located downstream of and in the same transcription unit as the lambda nutL and nutR sequences. Transcripts terminating prematurely at or near nutR have been detected after inactivation of lambda repressor in lambda, HK022 dilysogens. Nun therefore appears to be a transcription termination factor with a remarkable specificity; it converts the lambda nut sequences, which normally interact with lambda N protein to suppress transcription termination, into terminators. These and other similarities between Nun-promoted termination and N-promoted antitermination argue strongly that the mechanisms of the two reactions have steps in common.

Bacteriophage lambda↗
PubMed1986

Reconstitution of an operon from overlapping fragments: use of the lambda SV2 integrative cloning system.

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.

Alleles↗
PubMed1983

Escherichia coli nusB mutations that suppress nusA1 exhibit lambda N specificity.

The bacteriophage lambda N protein regulates phage development by selectively suppressing transcription termination in its host, Escherichia coli. The E. coli nus mutants prevent N activity. To provide additional information on transcription termination, we have isolated pseudo-revertants of the nusA1 mutation that restore lambda N function. One series of pseudo-revertants lie in the E. coli nusB gene, whose product is normally required for lambda N activity. These mutations are N-specific: mutations that restore lambda N activity do not restore the activity of the analogous N protein of phage 21. Similarly, nusB mutations that restore phage 21 N function are deficient for lambda N function. Mapping of the two classes of mutation is consistent with their location in two distinct domains in the nusB protein. We discuss whether nusB is specific for N protein or for some other component of this regulation system, e.g. the phage site (nut) required for N action.

Bacteriophage lambda↗
PubMed1983

lambda mutation in the Escherichia coli rho gene that inhibits the N protein activity of phage lambda.

Certain Escherichia coli rho mutations, exemplified by rho026, block the growth of phage lambda by interfering with phage gene expression. The phage gene N, whose product suppresses transcription termination, appears to be expressed normally in the mutants, and the functional stability of the N protein is not affected. Our data suggest that these rho mutations allow transcription to terminate despite the presence of N. Other E. coli mutants displaying a similar phenotype (Nus(-)) fail to propagate wild-type lambda but permit the growth of the lambda variant lambdanin5, which has undergone a deletion of the lambda terminator t(R2). The phenotype of the rho026 mutant differs: the growth of lambda is only marginally improved by the nin5 deletion. Interestingly, N activity at rho-independent terminators is not inhibited by the mutations, whereas its ability to suppress rho-dependent terminators is markedly reduced. The relevance of this specificity in terms of models of N action is discussed.

Bacteriophage lambda↗
PubMed1982

Suppression of transcription termination by phage lambda.

The bacteriophage lambda N gene product positively controls development by preventing termination of transcription at terminator sites critical to the sequential expression of phage genes. Many host transcription factors, including RNA polymerase, are involved in N gene action. Recent findings have shown that ribosomal proteins are also involved. The current understanding of how the N protein affects transcription termination is reviewed, and a possible model and current problems are discussed.

Bacterial Proteins↗
PubMed1981

The nus mutations affect transcription termination in Escherichia coli.

The nusA1 and nusB5 mutations in a partial suppression of polarity and thus transcription termination in Escherichia coli. As these mutations block the transcription antitermination activity of bacteriophage lambda N gene product, they paradoxically seem to enhance transcription termination at phage termination sites. The rho mutation HDF026 displays almost identical properties. The observations suggest that the nusA and nusB gene products may act as termination factors analogous to rho protein.

Bacterial Proteins↗