Biomedical subjects

M Z Gilman

Publications and source records attributed to M Z Gilman.

25 records · Page 2Linked to original sources

PubMed1986

Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression.

We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.

Animals↗

PubMed1985

Bacillus subtilis sigma 28 and Escherichia coli sigma 32 (htpR) are minor sigma factors that display an overlapping promoter specificity.

Bacillus subtilis sigma 28-specific promoters (P28) are utilized by a minor form of B. subtilis RNA polymerase (sigma 28RNA polymerase) and not by the predominant RNA polymerases of B. subtilis (sigma 43 RNA polymerase) or Escherichia coli (sigma 70 RNA polymerase). However B. subtilis P28 are effective promoters in E. coli. This transcription depends on the E. coli htpR+ gene. Similarly, the E. coli rpoD heat shock promoter which is under control of htpR is used in vitro by B. subtilis sigma 28 RNA polymerase. These observations are explained by the fact that E. coli htpR is a minor sigma factor (sigma 32) which shares an overlapping promoter specificity with B. subtilis sigma 28 RNA polymerase. Hence control of bacterial regulons by minor sigma factors is not restricted to Bacilli, or bacteria that carry out a complex differentiation process, but is probably a general, regulatory mechanism in prokaryotes. Transcription from B. subtilis P28 in E. coli does not depend on a heat shock. This suggests that the sequences that control E. coli sigma 32 action are separate from those that control the heat shock regulon. Hence the action of polymerases controlled by minor sigma factors in both B. subtilis and E. coli appears to be controlled by a separate set of regulatory factors.

Bacillus subtilis↗

PubMed1984

Isolation of sigma-28-specific promoters from Bacillus subtilis DNA.

Sigma-28-RNA polymerase is a minor form of RNA polymerase found in vegetative cells of Bacillus subtilis which utilizes promoter sites distinct from those recognized by the major RNA polymerase. We have isolated a collection of cloned B. subtilis DNA segments that contain in vitro promoter sites for sigma 28-RNA polymerase by screening a bacteriophage lambda library of B. subtilis genomic fragments. At least nine new sigma 28-specific promoter sites have been identified in this collection, and four have been partially mapped for further study. Our strategy employed a mix of RNA probes prepared by in vitro transcription with sigma 28-RNA polymerase of total B. subtilis DNA EcoRI and HindIII fragments. Over 70% of the unique clones identified contain sigma 28-specific promoter sites, suggesting that the method may have general application for identification of promoter-containing sequences. The efficiency with which sigma 28-specific promoters are detected is consistent with there being a relatively small number of such sites in the B. subtilis genome of which twelve have been cloned.

Bacillus subtilis↗

PubMed1983

Developmental and genetic regulation of Bacillus subtilis genes transcribed by sigma 28-RNA polymerase.

Sigma-28 RNA polymerase is a minor form of Bacillus subtilis RNA polymerase that is highly specific for transcription from a small number of promoter sites in the B. subtilis genome. We have followed transcription from two of these loci (P28-1 and P28-2) in vivo using a quantitative S1 nuclease mapping procedure. Both promoters are used at a modest rate in vegetatively growing cells (about 10 RNA copies per cell) and transcripts are initiated at the same start sites as found in vitro with the purified sigma 28-RNA polymerase. Transcription from the sigma 28 promoters varies somewhat with growth conditions and is shut off rapidly and almost completely after the first hour of sporulation. Neither sigma 28 transcripts is detected in vegetative cells of certain B. subtilis mutants (spoO classes A, B, E, and F) that are defective in sporulation. Transcription from these promoters is restored in second site revertants that are able to sporulate. Hence the action of sigma 28-RNA polymerase appears to be regulated by the spoO genes and the functions controlled by sigma 28-promoters may be closely tied to the system involved in the initiation of sporulation.

Bacillus subtilis↗

PubMed1981

Nucleotide sequences of two Bacillus subtilis promoters used by Bacillus subtilis sigma-28 RNA polymerase.

RNA polymerase holoenzymes from many bacterial species share a common promoter recognition specificity since they use the same promoter sites on a variety of templates. These promoters generally include sequences homologous to the average sequences -TTGACA- and -TATAATA-, located -35 and -10 base pairs, respectively, upstream of the transcriptional state site. We have isolated a minor form of B. subtilis RNA polymerase in which the normal sigma subunit (sigma 55) is replaced by a smaller polypeptide (sigma 28) and which is highly specific for a class of promoter sites not used by the sigma 55-holoenzyme. Sequencing of two B. subtilis promoter sites used by the sigma 28-holoenzyme reveals identical sequences at -35 and -10 base pairs from the start site, which are -CTAAA- and -CCGATAT-, respectively. These results confirm that sigma subunit plays a major direct role in promoter sequence recognition, and support a model in which sigma interacts sequentially with -35 and -10 regions, respectively.

Bacillus subtilis↗

PubMed1981

Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Preparations of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme II) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) and lacks the normal sigma subunit [Jaehning, J. A., Wiggs, J. L. & Chamberlin, M. J. (1979) Proc. Natl. Acad. Sci. USA 76, 5470-5474]. By heparin-agarose chromatography we have obtained holoenzyme II fractions that have no detectable holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template we have tested. These fractions are far more active with B. subtilis DNA than with T7 DNA or other heterologous templates. This high degree of specificity has allowed identification of plasmids containing cloned fragments of B. subtilis DNA that bear strong promoter sites for holoenzyme II. These promoter sites are not used at all by B. subtilis RNA polymerase holoenzyme I. The specificity of holoenzyme II is dictated by a peptide of Mr 28,000 as judged by copurification of the peptide with specific holoenzyme II activity and by reconstitution of the holoenzyme II promoter specificity when the isolated peptide is added to B. subtilis core polymerase. Hence the 28,000 Mr peptide appears to be a sigma factor that determines a promoter specificity distinct from that of RNA polymerase holoenzyme I and all other known bacterial RNA polymerases.

Bacillus subtilis↗

PubMedDate not supplied

Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay.

Numerous nucleic acid sequence motifs have been identified as transcriptional regulatory elements, and proteins involved in the control of gene replication and transcription have been successfully purified using nucleic acid affinity procedures. We have developed an assay that allows direct characterization of cellular proteins binding to the enhancer element of the human immunodeficiency virus (HIV), which is analogous to immunoprecipitation techniques. In extracts of H9 cells, a human CD4+ lymphoblast line clonally selected for its ability to support HIV replication, we find a class of proteins that interact specifically with the HIV enhancer. Reproducible high-resolution, two-dimensional gel electrophoresis has allowed us to resolve these proteins into two major sets. One set is common to H9 cells, two human B-lymphoblast lines, and a phytohaemagglutinin (PHA)-stimulated human CD4+ lymphoblast line (Jurkat). A second set of proteins is found only in H9 cells. Thus, with this assay we have identified HIV enhancer-binding proteins that are constitutive or inducible and that are cell-type specific.

Base Sequence↗