DNA stability and DNA binding proteins.
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Amino Acid Sequence↗
Biomedical subjects
K Sandman
Publications and source records attributed to K Sandman.
Histones and chromatin structure in hyperthermophilic Archaea.
HMf is a histone from the hyperthermophile Methanothermus fervidus. It is the archetype and most studied member of a family of archaeal histones that have primary sequences and three-dimensional structures in common with the eukaryal nucleosome core histones and that bind and compact DNA molecules into nucleosome-like structures (NLS). HMf preparations are mixtures of two similar, small (approximately 7.5 kDa) polypeptides designated HMfA and HMfB that in vivo form both homodimers and heterodimers. HMfA synthesis predominates during exponential growth but the relative amount of HMfB increases as M. fervidus cells enter the stationary growth phase. Analyses of homogeneous preparations of recombinant (r) (HMfA)2 and (rHMfB)2 have demonstrated that these proteins have different DNA-binding and compaction properties in vitro, consistent with different roles in vivo for the (HMfA)2, (HMfB)2 and HMfA. HmfB dimers, and for the NLS that they form, in regulating gene expression and in genome compaction and stability.
Amino Acid Sequence↗
Improved N-terminal processing of recombinant proteins synthesized in Escherichia coli.
Preparations of rHMfA (recombinant histone A from Methanothermus fervidus) synthesized in E. coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMfA molecules, approximately 40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), approximately 50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only approximately 10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMfA molecules synthesized in Mt. fervidus. Expression of the hmfA gene in E. coli cells grown in the presence of trimethoprim and thymidine, coupled with the concurrent over-expression of a methionine aminopeptidase-encoding map gene, has been shown to overcome this N-terminal heterogeneity problem and to result in rHMfA preparations in which > 85% of the molecules have the fully processed, native N-terminal sequence. This procedure should be generally useful for ensuring N-terminal processing of recombinant proteins synthesized in E. coli.
Archaea↗
Methanobacterium formicicum, a mesophilic methanogen, contains three HFo histones.
The mesophilic methanogen Methanobacterium formicicum JF-1 has been shown to contain three members of the HMf family of archaeal histones, designated HFoA1, HFoA2, and HFoB, and their encodinig genes (hfoA1, hfoA2, and hfoB) have been cloned and sequenced. The HFo histones have primary sequences that are 75 to 82% identical to the HMf sequences and appear to share ancestry with the core histones that form the eukaryal nucleosome. The HFo proteins bind and compact DNA molecules into nucleosome-like structures apparently identical to those formed by the HMf proteins, but, in contrast to the HMf proteins, this activity of the HFo proteins is lost after incubation at 95 degrees C for 5 h.
Amino Acid Sequence↗
Growth-phase-dependent synthesis of histones in the archaeon Methanothermus fervidus.
Histone preparations from Methanothermus fervidus (HMf) contain two small polypeptides, HMfA and HMfB, which in solution are dimers and compact DNA to form nucleosome-like structures. These archaeal nucleosome-like structures constrain positive DNA supercoils, in contrast to the negatively supercoiled DNA in eukaryal nucleosomes. HMfA has been found to make up as much as 80% of HMf preparations synthesized by M. fervidus cells during the exponential growth phase of batch cultures but to decrease to approximately 50% as cultures enter the stationary phase. By using a nondenaturing polyacrylamide gel system at pH 6.1, we have demonstrated that HMf preparations contain HMfA homodimers, HMfB homodimers, and HMfA-HMfB heterodimers and that heating a mixture of recombinant HMfA and HMfB homodimers at 95 degrees C for 5 min generates HMfA-HMfB heterodimers. Circular dichroism spectroscopy indicates that HMfA and HMfB have very similar secondary structures, but based on agarose gel electrophoretic mobility shifts, DNA topology assays, and electron microscopy, they have different DNA binding properties. HMfA binding to DNA could be detected at lower protein/DNA ratios than HMfB, but HMfB binding resulted in more extensive DNA compaction. The increased HMfB synthesized in cells approaching the stationary phase and the highly compacted state of HMfB-bound DNA are consistent with preparations for the impending period of limited genome activity.
Archaea↗
Histone-encoding genes from Pyrococcus: evidence for members of the HMf family of archaeal histones in a non-methanogenic Archaeon.
Two genes, designated hpyA1 and hpyA2, have been cloned and sequenced from Pyrococcus strain GB-3a. They are predicted to encode proteins (HPyA1 and HPyA2, respectively) that are approx. 60% identical to the histones HMf and HMt, characterized from methanogenic Archaea. These archaeal histones also contain the amino-acid sequences, conserved in eukaryotic H4 histones, that are thought to interact directly with DNA.
Amino Acid Sequence↗
Transcription in vivo and in vitro of the histone-encoding gene hmfB from the hyperthermophilic archaeon Methanothermus fervidus.
Immediately upstream of the hmfB gene, in a DNA fragment cloned from Methanothermus fervidus, are two identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'TTTATATA, which conforms precisely to the consensus TATA box element proposed for methanogen promoters. By using this duplicated region as the template DNA and a cell-free transcription system derived from Methanococcus thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp apart, each 25 bp downstream from a TATA box, thus providing strong evidence for the functional conservation of this transcriptional signal in two phylogenetically very diverse methanogens. Transcription of the hmfB gene in vivo in M. fervidus was found to occur at only one of these sites, and consistent with this observation, recloning and sequencing of this intergenic region after its amplification by the polymerase chain reaction demonstrated that the genome of M. fervidus contains only one copy of the 73-bp sequence upstream of the hmfB gene. Since the second copy of the 73-bp sequence, presumably generated artifactually during the original hmfB cloning, functioned equally well as a promoter in the M. thermolithotrophicus transcription system, all information needed by the heterologous RNA polymerase to initiate transcription accurately in vitro must be present within this sequence. The hmfB gene encodes HMf-2, one of the two subunits of HMf, an abundant DNA binding protein in M. fervidus which binds to DNA molecules in vitro, forming nucleosomelike structures. Cell-free transcription was inhibited by adding HMf or eucaryotic core histones at protein-to-DNA mass ratios of 0.3:1 and 1:1, respectively, whereas the archael histonelike protein HTa from Thermoplasma acidophilum inhibited transcription in vitro only at much higher protein-to-DNA mass ratios and the bacterial histonelike protein HU from Escherichia coli had no detectable effect on transcription.
Archaeal Proteins↗
HMf, a histone-related protein from the hyperthermophilic archaeon Methanothermus fervidus, binds preferentially to DNA containing phased tracts of adenines.
HMf, a histone-related protein from Methanothermus fervidus, was found to bind preferentially to a DNA that is intrinsically bent as a result of the presence of phased oligo(dA) tracts. The intergenic regions in M. fervidus DNA are A+T rich and frequently contain oligo(dA) tracts, some of which may have the size and phasing required to create a net bending in one direction. The binding of HMf to bent DNA could play a direct role in gene expression and stabilization of the genome of this organism.
Archaeal Proteins↗
HMf, a DNA-binding protein isolated from the hyperthermophilic archaeon Methanothermus fervidus, is most closely related to histones.
Methanothermus fervidus grows optimally at 83 degrees C. A protein designated HMf (histone M. fervidus) has been isolated from this archaeal hyperthermophile that binds to double-stranded DNA molecules and increases their resistance to thermal denaturation. HMf binding to linear double-stranded DNA molecules of greater than 2 kilobase pairs also increases their electrophoretic mobilities through agarose gels. Visualization of this compaction process by electron microscopy has demonstrated the formation of quasispherical, macromolecular HMf-DNA complexes. HMf is a mixture of approximately equal amounts of two very similar polypeptides designated HMf-1 and HMf-2. Determination of the DNA sequence of the gene encoding HMf-2 (hmfB) has revealed that over 30% of the amino acid residues in HMf-2 are conserved in the consensus sequences derived for eucaryal histones H2A, H2B, H3, and H4. These archaeal polypeptides and eucaryal histones appear therefore to have evolved from a common ancestor and are likely to have related structures and functions.
Amino Acid Sequence↗
Identification of the promoter for a spore coat protein gene in Bacillus subtilis and studies on the regulation of its induction at a late stage of sporulation.
The cotA (pig) gene of Bacillus subtilis encodes a 65,000 Mr protein that is a component of the spore coat and is responsible for the brown pigment characteristic of colonies in which cells are undergoing sporulation. To study developmental regulation of the cotA gene, we identified its promoter and studied its transcription in a large number of mutants blocked at various stages of sporulation and germination. Deletion analysis showed that induction and efficient transcription of cotA required DNA sequences extending no more than 55 base pairs (bp) upstream (and no more than 130 bp downstream) from the 5' terminus of cotA mRNA. Transcription from the cotA promoter was found to be switched on at approximately the time (4 to 5 h after the onset of sporulation) of spore coat synthesis and deposition. Strikingly, this transcription was substantially inhibited in almost all asporogenous mutants blocked prior to the developmental stage (V) of spore coat formation. cotA transcription was also impaired in several stage V mutants but not in other stage V mutants or in mutants blocked in germination. The germination mutant gerE caused a several-fold overexpression of cotA. The dependence of cotA expression on so many genes required at early to intermediate stages of sporulation suggests that transcription of this spore coat gene is somehow coupled (directly or through several intervening steps) to a morphological or physiological feature(s) of the developing sporangium.
Amino Acid Sequence↗
The promoter for a sporulation gene in the spoIVC locus of Bacillus subtilis and its use in studies of temporal and spatial control of gene expression.
We have identified the transcription start site and regulatory region governing the expression of a sporulation gene in the spoIVC locus of Bacillus subtilis. Efficient expression and developmental regulation of this gene was controlled from a promoter region that extended no more than 110 base pairs upstream and no more than 4 base pairs downstream from the start site of transcription, on which basis we infer that spoIVC is regulated at the level of transcription initiation. Using a transcriptional fusion of the spoIVC gene to the lacZ gene of Escherichia coli, we found that spoIVC expression was turned on at the third to fourth hour of sporulation (at about the developmental stage [IV] that its products are required in spore formation) and that this transcription was largely restricted to the mother cell chamber of the sporangium. Mutations in many different spo genes (causing blocks at stages 0 to V) were found to influence (negatively and positively) the level of spoIVC expression. Our results distinguish the mode of spoIVC regulation from that of previously studied sporulation genes and indicate that it is representative of a new regulon of mother cell-specific gene expression.
Bacillus subtilis↗
Emergency stabilization in transport of critically ill neonate.
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Body Temperature Regulation↗
Genes encoding spore coat polypeptides from Bacillus subtilis.
Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell, known as the coat, that consists of a dozen or more different polypeptides. We have cloned structural genes designated cotA, cotB, cotC and cotD that encode spore coat proteins of Mr 65,000, 59,000, 12,000 and 11,000, respectively. These genes were cloned by using as hybridization probes synthetic oligonucleotides that were designed on the basis of partial NH2-terminal sequence determinations of the purified coat proteins. To determine the location of the cot genes on the chromosome and to study their function genetically, we tagged each gene by insertion of a chloramphenicol-resistance determinant (cat) within its coding sequence. We then replaced each wild-type cot gene in the chromosome with the corresponding, insertionally inactivated gene. Genetic mapping experiments showed that cotA, cotB, cotC and cotD were located at 52 degrees, 290 degrees, 168 degrees and 200 degrees, respectively, on the B. subtilis chromosome. None of the cot::cat insertion mutants were Spo-, but spores of the cotD mutant were found to germinate somewhat more slowly than did wild-type spores, and the cotA mutant was found to be blocked in the appearance of the brown pigment characteristic of colonies of wild-type sporulating cells. Physical and genetic experiments established that cotA was identical to a previously identified gene called pig, known to be responsible for sporulation-associated pigment production. Spores from all four insertion mutants exhibited the wild-type pattern of coat polypeptides, except for the absence in each instance of the corresponding product of the cot gene that had been insertionally inactivated.
Amino Acid Sequence↗
Genetic analysis of Bacillus subtilis spo mutations generated by Tn917-mediated insertional mutagenesis.
Mutations that cause sporulation defects (spo mutations) often identify developmentally regulated transcription units or genes whose products are required for the expression of sporulation-specific regulons. We report here the isolation, genetic analysis and phenotypic characterization of spo mutations produced by insertional mutagenesis with transposon Tn917, a form of mutagenesis that facilitates genetic and physical manipulation of mutated genes in many ways. Twenty-four insertional spo mutations were studied in detail. On the basis of transformation-mediated and transduction-mediated linkage analysis and a range of phenotypic tests, these mutations were assigned to 20 distinct loci, at least 9 of which are different from the 40 previously described spo loci. The insertional mutations caused blocks at a variety of different stages of sporulation, and therefore probably identify genes active at different times during sporulation. In addition to increasing substantially the total of known spo loci, we anticipate that this collection will include representatives of many of the temporally regulated sets of genes that comprise the overall program of sporulation-specific gene activation in Bacillus subtilis. Given the kinds of manipulations that are possible with genes disrupted by Tn917 insertions, this should significantly facilitate efforts to understand the regulation of these gene sets.
Bacillus subtilis↗
A promoter whose utilization is temporally regulated during sporulation in Bacillus subtilis.
The formation of endospores in the Gram-positive bacterium Bacillus subtilis proceeds according to a temporally ordered program of gene activation. To investigate timing mechanisms in sporulation gene expression, we have isolated and sequenced the promoter region for a B. subtilis gene known as 0.3 kb whose transcription is switched on at about stage III of development. The 5' terminus of the 0.3 kb mRNA was mapped by the S1 nuclease procedure to a position just upstream from its apparent ribosome binding site and initiation codon and just downstream from the transcription termination site for an adjacent gene. This information enabled us to construct a transcriptional fusion in which the 5' region of the 0.3 kb gene was joined to the lacZ gene of Escherichia coli. When introduced into cells of B. subtilis, the 0.3 kb-lacZ fusion caused the synthesis of a fusion-specified RNA that originated from within the 0.3 kb promoter region and extended into the adjacent E. coli DNA, and the induction of beta-galactosidase synthesis at the third to fourth hour of sporulation. Enzyme synthesis required the 0.3 kb promoter, since a deletion of the 5' region of the 0.3 kb gene in the transcription fusion eliminated the production of beta-galactosidase. Induction of the 0.3 kb-lacZ fusion was under developmental control, since the production of beta-galactosidase was blocked or substantially impaired by chromosomal mutations in the sporulation genes spoOB, spoIIA, spoIIE and spoIIIE, but not by a spoIIC mutation. We conclude that the 0.3 kb gene promoter is subject to a developmental clock, which delays its utilization until an intermediate stage of sporulation, and discuss models for how the timing of gene expression is regulated.
Bacillus subtilis↗