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R Haselkorn

Publications and source records attributed to R Haselkorn.

At least 55 records · Page 3Linked to original sources
PubMed1993

Chromosomal structure of Rhodobacter capsulatus strain SB1003: cosmid encyclopedia and high-resolution physical and genetic map.

A combination of cosmid genome walking and pulsed-field gel electrophoresis was used to construct a high-resolution physical and genetic map of the 3.8-megabase (Mb) genome of Rhodobacter capsulatus SB1003. The mapping was done by hybridization of pulsed-field gel blots and by grouping and further mapping of the cosmids and bacteriophages from genomic libraries. Cosmid clones formed two uninterrupted and ordered groups, one corresponding to the chromosome of R. capsulatus, the other to its 134-kb plasmid. Cos site end-labeling and partial EcoRV digestion of cosmids were used to construct a high-resolution EcoRV map of the genome. Overlapping of the cosmids was confirmed by the resemblance of the cosmid restriction maps and by direct end-to-end hybridization with SP6- and T7-specific transcripts. Twenty-three previously cloned genes and eight groups of repeated sequences, revealed in this work, were located in the ordered gene library and mapped with an accuracy of 1-10 kb. Blots of a minimal set of 192 cosmids, covering the chromosome and the plasmid with the known map position of each cosmid, give to R. capsulatus the same advantages that the Kohara phage panel gives to E. coli.

Chromosome Mapping↗
PubMed1993

Wheat acetyl-CoA carboxylase.

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of 14C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.

Acetyl-CoA Carboxylase↗
PubMed1993

Isolation and characterization of the genes encoding allophycocyanin subunits and two linker proteins from Synechocystis 6714.

Genes encoding the phycobilisome core subunits allophycocyanin alpha and beta and a small core linker protein in Synechocystis sp. strain PCC 6714 were cloned and sequenced. These genes form an operon, apcABC, with a single transcription start site and two possible termination sites, one following apcB and the other following apcC. The promoter region, like those of the apcABC operons of other cyanobacteria, does not resemble the consensus promoter sequences of Escherichia coli. However, the apcABC promoters identified in four strains of cyanobacteria have conserved sequences centered at -50 and -10 with respect to the start of transcription. The apcE gene, encoding the protein that links the phycobilisome core to the thylakoid membrane, was also cloned from Synechocystis 6714 and sequenced. It is unlinked to the apcABC operon. As in other Synechocystis strains, the LCM polypeptide encoded by the apcE gene contains three repeats of the basic phycobiliprotein linker domain. The apcE gene promoter sequence bears little resemblance to either the E. coli consensus or the apcABC promoter region, but it is similar to the corresponding regions of other cyanobacterial apcE genes. In these cases, there are conserved sequences centered at -40 and -10 with respect to the transcription start site. These conserved promoter elements from the apcABC and apcE genes were also identified in the corresponding 5'-flanking regions of eleven transcript starts for cpc genes encoding phycocyanin subunits in cyanobacteria and algal chloroplasts. These results suggest that a factor yet to be described participates in transcription of phycobiliprotein genes.

Bacterial Proteins↗
PubMed1993

Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein.

Genes for two subunits of acetyl-coenzyme A carboxylase, biotin carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp. strain PCC 7120. The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively. The sequence of the biotinylation site in Anabaena sp. strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent carboxylases. The amino acid sequence of biotin carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin carboxylase domain of other biotin-dependent carboxylases. Genes for these two subunits of acetyl-coenzyme A carboxylase are not linked in Anabaena sp. strain PCC 7120, contrary to the situation in E. coli, in which they are in one operon.

Acetyl-CoA Carboxylase↗
PubMed1993

The patB gene product, required for growth of the cyanobacterium Anabaena sp. strain PCC 7120 under nitrogen-limiting conditions, contains ferredoxin and helix-turn-helix domains.

A mutant of Anabaena sp. strain PCC 7120, called PAT-2, that grows poorly under nitrogen-fixing conditions, has been isolated. The heterocysts of the mutant strain develop much more slowly than those of the wild type and are spaced more closely in an older culture of the mutant than in the wild type. The wild-type gene that complements the mutation in PAT-2, called patB, was isolated and characterized. The predicted 529-amino-acid PatB protein contains a region very similar to the Fe4S4 bacterial-type ferredoxins near its N terminus and a helix-turn-helix motif near its C terminus. This pattern of domains resembles those of transcriptional regulators in both prokaryotes and eukaryotes. The mutation in strain PAT-2 is the deletion of G at nucleotide 1342 in the patB gene, resulting in the loss of a 62-amino-acid fragment from the C terminus of the PatB protein, including the helix-turn-helix motif.

Amino Acid Sequence↗
PubMed1992

The patA gene product, which contains a region similar to CheY of Escherichia coli, controls heterocyst pattern formation in the cyanobacterium Anabaena 7120.

In Anabaena 7120, heterocysts (cells specialized for nitrogen fixation) develop at the ends of filaments and at intervals within each filament. We have isolated a mutant Anabaena strain that develops heterocysts mostly at the ends of filaments. This mutant, PAT-1, grows poorly under nitrogen-fixing conditions. The wild-type gene that complements the mutation in PAT-1, called patA, was cloned and sequenced. The predicted PatA protein contains 379 amino acids distributed among three "domains" based on predictions of hydropathy and flexibility. The carboxyl-terminal domain is very similar to that of CheY and other response regulators in two-component regulatory systems in eubacteria. The patA mutation suppresses the multiheterocyst phenotype produced by extra copies of the wild-type hetR gene described previously, suggesting that PatA and HetR are components of the same environment-sensing regulatory circuit in Anabaena.

Amino Acid Sequence↗
PubMed1992

Multiple evolutionary origins of prochlorophytes, the chlorophyll b-containing prokaryotes.

Prochlorophytes are prokaryotes that carry out oxygenic photosynthesis using chlorophylls a and b, but lack phycobiliproteins as light-harvesting pigments. These characteristics distinguish them from cyanobacteria, which contain phycobiliproteins, but no chlorophyll b. Three prochlorophyte genera have been described: Prochloron, Prochlorothrix and Prochlorococcus. The prochlorophytes share their pigment characteristics with green plant and euglenoid chloroplasts, which has led to a debate on whether these chloroplasts may have arisen from an endosymbiotic prochlorophyte rather than a cyanobacterium. Molecular sequence data, including those presented here based on a fragment of the rpoC1 gene encoding a subunit of DNA-dependent RNA polymerase, indicate that the known prochlorophyte lineages do not include the direct ancestor of chloroplasts. We also show that the prochlorophytes are a highly diverged polyphyletic group. Thus the use of chlorophyll b as a light-harvesting pigment has developed independently several times in evolution. Similar conclusions have been reached in parallel studies using 16S ribosomal RNA sequences.

Amino Acid Sequence↗
PubMed1992

Physical map of the genome of Rhodobacter capsulatus SB 1003.

A map of the chromosome of Rhodobacter capsulatus was constructed by overlapping the large restriction fragments generated by endonucleases AseI and XbaI. The analyses were done by hybridization of single fragments with the restriction fragments blotted from pulsed-field gels and by grouping cosmids of a genomic library of R. capsulatus into contigs, corresponding to the restriction fragments, and further overlapping of the contigs. A technical difficulty due to a repeated sequence made it necessary to use hybridization with cloned genes and prior knowledge of the genetic map in order to close the physical circle in a unique way. In all, 41 restriction sites were mapped on the 3.6-Mb circular genome and 22 genes were positioned at 26 loci of the map. Cosmid clones were grouped in about 80 subcontigs, forming two groups, one corresponding to the chromosome of R. capsulatus and the other corresponding to a 134-kb plasmid. cos site end labeling and partial digestion of cosmids were used to construct a high-resolution EcoRV map of the 134-kb plasmid. The same method can be extended to the entire chromosome. The cosmid clones derived in this work can be used as a hybridization panel for the physical mapping of new genes as soon as they are cloned.

Blotting, Southern↗
PubMed1992

Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes.

The sigA gene of Anabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.

Amino Acid Sequence↗
PubMed1991

Promoter recognition by the RNA polymerase from vegetative cells of the cyanobacterium Anabaena 7120.

The transcription start points (tsp) of seven genes of Anabaena 7120 were previously identified by S1 nuclease protection and primer extension experiments using RNA extracted from cells. In the present work, these tsp were confirmed, with one exception, by in vitro transcription using purified RNA polymerases of Anabaena 7120 and Escherichia coli, and crude extracts of Anabaena 7120 active in transcription. In all cases, the template for transcription consisted of closed circular plasmid DNA in which the putative promoter-containing fragment was cloned in front of a strong terminator, which resulted in defined 'pseudo-runoff' transcripts whose sizes correspond (with one exception) to those expected on the basis of the tsp determined for in vivo RNA. These results, together with others obtained with templates containing bacteriophage T4 or cyanophage N1 promoters, led to the conclusion that the principal Anabaena 7120 RNA polymerase prefers promoters whose sequence and spacing approximate that of the E. coli consensus promoter, and that the Anabaena 7120 genes expressed in vegetative cells, characterized to date, have relatively weak promoters.

Anabaena↗
PubMed1991

Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120.

Anabaena 7120 mutant 216 fails to differentiate heterocyst. We previously identified a 2.4-kb wild-type DNA fragment able to complement this mutant. We show here that the sequence of this fragment contains a single open reading frame (hetR), encoding a 299-amino-acid protein. Conjugation of deletion subclones of this fragment into strain 216 showed that the hetR-coding region is both necessary and sufficient for complementation of the Het- phenotype. The mutation in 216 is located at nucleotide 535 in the hetR gene, converting a serine at position 179 in the wild-type protein to an asparagine in the mutant. Interruption of the hetR gene in wild-type cells results in a mutant phenotype identical to that of 216. Both 216 and wild-type cells containing wild-type hetR on a plasmid display increased frequency of heterocysts, even on media containing fixed nitrogen. These results suggest that hetR encodes a product that is not only essential for but also controls heterocyst development. This putative regulatory protein lacks known structural motifs characteristic of transcription factors and probably acts at a level one or more steps removed from its target genes.

Amino Acid Sequence↗
PubMed1991

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120.

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.

Amino Acid Sequence↗
PubMed1991

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.

Anaerobiosis↗
PubMed1991

Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 responds to combined nitrogen deprivation by forming specialized nitrogen-fixing cells at regular intervals along the filament. Genetic and biochemical studies have indicated that regulation of gene expression during differentiation occurs at the transcriptional level. As part of a characterization of RNA polymerase during differentiation, the gene encoding the 52-kDa principal sigma factor of the Anabaena sp. strain PCC 7120 vegetative-cell RNA polymerase was isolated by using an oligonucleotide probe based on the sequence of the N-terminal seven amino acids of the purified protein. sigA codes for a 390-amino-acid polypeptide that has a predicted molecular weight of 45,641. The amino acid sequence of the polypeptide encoded by sigA contains four regions corresponding to conserved domains of the principal RNA polymerase sigma factors of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). Thus, although the subunit composition of cyanobacterial RNA polymerase core differs from that of other eubacteria (G. J. Schneider and R. Haselkorn, J. Bacteriol. 170:4136-4140, 1988), the principal sigma factor of at least one cyanobacterium is typically eubacterial. In contrast to sigma 70 and sigma 43 operon organization, sigA is monocistronic and encodes two transcripts of 1.7 and 2.2 kb. The abundance of the 1.7-kb transcript remains constant under both nitrogen-replete and nitrogen-limiting conditions, whereas the 2.2-kb transcript is induced following the removal of combined nitrogen. Continued or enhanced transcription of sigA under nitrogen starvation conditions is consistent with the observation that the principal RNA polymerase in differentiating cells contains SigA.

Amino Acid Sequence↗
PubMed1991

A vector for analysis of promoters in the cyanobacterium Anabaena sp. strain PCC 7120.

A plasmid vector containing a multiple-cloning site followed by a promoterless chloramphenicol acetyltransferase (cat) gene, protected by transcription terminators and mobilizable by conjugation from Escherichia coli into Anabaena sp. strain PCC 7120, was constructed. The utility of the vector was shown by deletion analysis of the promoter region of the Anabaena psbB gene.

Base Sequence↗
PubMed1990

Cloning of salinity stress-induced genes from the salt-tolerant nitrogen-fixing cyanobacterium Anabaena torulosa.

A subtractive hybridization procedure was used to clone genes of the cyanobacterium Anabaena torulosa expressed in response to salt stress. The method uses total RNA from salt-treated cells, labeled in vitro, as the probe. Hybridization to restriction digests of total DNA was used for interspecies comparison; the procedure was also successful for isolation of cosmids by colony hybridization, semiquantitative dot blots, and cosmid characterization by Southern blotting. Analysis of eleven independent cosmids containing genes whose transcription is abundantly induced by salt suggests that a substantial portion of the A. torulosa genome, probably in excess of 100 kilobases, responds to salt.

Cloning, Molecular↗