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Biomedical subjects

R Haselkorn

Publications and source records attributed to R Haselkorn.

At least 109 records · Page 6Linked to original sources
PubMed1982

Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense.

Homology was detected between the structural genes for the nitrogenase complex of K. pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains. Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A. brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K. pneumoniae nif probe. Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K. pneumoniae nif genes was found to be approximately 5 kb. Proteins encoded by the AbRI fragment were examined after infection of E. coli minicells.

Cloning, Molecular↗
PubMed1982

Regulation of glutamine synthetase activity and synthesis in free-living and symbiotic Anabaena spp.

Regulation of the synthesis and activity of glutamine synthetase (GS) in the cyanobacterium Anabaena sp. strain 7120 was studied by determining GS transferase activity and GS antigen concentration under a variety of conditions. Extracts prepared from cells growing exponentially on a medium supplemented with combined nitrogen had a GS activity of 17 mumol of gamma-glutamyl transferase activity per min per mg of protein at 37 degrees C. This activity doubled in 12 h after transfer of cells to a nitrogen-free medium, corresponding to the time required for heterocyst differentiation and the start of nitrogen fixation. Addition of NH3 to a culture 11 h after an inducing transfer immediately blocked the increase in GS activity. In the Enterobacteriaceae, addition of NH3 after induction results in the covalent modification of GS by adenylylation. The GS of Anabaena is not adenylylated by such a protocol, as shown by the resistance of the transferase activity of the enzyme to inhibition by Mg2+ and by the failure of the enzyme to incorporate 32P after NH3 upshift. Methionine sulfoximine inhibited Anabaena GS activity rapidly and irreversibly in vivo. After the addition of methionine sulfoximine to Anabaena, the level of GS antigen neither increased nor decreased, indicating that Glutamine cannot be the only small molecule capable of regulating GS synthesis. Methionine sulfoximine permitted heterocyst differentiation and nitrogenase induction to escape repression by NH3. Nitrogen-fixing cultures treated with methionine sulfoximine excreted NH3. The fern Azolla caroliniana contains an Anabaena species living in symbiotic association. The Anabaena species carries out nitrogen fixation sufficient to satisfy all of the combined nitrogen requirements of the host fern. Experiments by other workers have shown that the activity of GS in the symbiont is significantly lower than the activity of GS in free-living Anabaena. Using a sensitive radioimmune assay and a normalization procedure based on the content of diaminopimelic acid, a component unique to the symbiont, we found that the level of GS antigen in the symbiont was about 5% of the level in free-living Anabaena cells. Thus, the host fern appears to repress synthesis of Anabaena GS in the symbiotic association.

Cyanobacteria↗
PubMed1981

Purification, physical characterization, and NH2-terminal sequence of glutamine synthetase from the cyanobacterium Anabaena 7120.

A procedure for the complete purification of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from the cyanobacterium (blue-green alga) Anabaena 7120 is described. The enzyme has structural characteristics in common with glutamine synthetases from other sources: a subunit molecular weight of approximately 50,000 and a native structure, determined by low dose exposure electron microscopy, consisting of a two-layered regular hexagon made up of 12 subunits. Sequence analysis suggests that the subunits are identical. There was no indication that the enzyme from cyanobacteria is adenylylated, a feature shared with the glutamine synthetase from Bacillus subtilis but not with the enzyme from Escherichia coli. NH2-terminal sequence analysis and the predicted conformation of the NH2-terminal regions showed definite homology among the enzymes from all three sources. The limited analysis suggested a stronger structural homology between the enzyme from Anabaena 7120 and that from E. coli.

Amino Acid Sequence↗
PubMed1981

Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120.

A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined. Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 mM, respectively; that for ammonia was found to be below 20 microM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase.

Amino Acids↗
PubMed1981

A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated.

The coding sequence for Anabaena 7120 glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.1] are shown to be contained within a 7.5-kilobase-pair (kbp) HindIII fragment that has been cloned by plaque hybridization. The hybridization probe for the cyanobacterial gene was a recombinant plasmid containing the glnA gene from Escherichia coli K-12. Evidence that the cloned Anabaena fragment contains the glnA gene includes complementation of a glnA deletion mutant of E. coli and immunological identity of the enzyme produced by the cloned Anabaena fragment in E. coli with glutamine synthetase purified from Anabaena 7120. Heteroduplex analysis reveals 0.65 kbp of homology between the 7.5-kbp Anabaena 7120 fragment and an 11-kbp E. coli fragment that codes for E. coli glutamine synthetase. Studies of Anabaena glnA gene activity in E. coli suggest that the cyanobacterial gene is not repressible and that the Anabaena 7120 glutamine synthetase is not adenylylated in E. coli.

Adenosine Monophosphate↗
PubMed1980

Displacement of parental RNA strands during in vitro transcription by bacteriophage phi 6 nucleocapsids.

We have studied the mode of transcription of the three double-stranded RNA segments found in bacteriophage phi 6. Stable transcription intermediates, isolated following in vitro incorporation of nucleoside triphosphates by phi 6 nucleocapsids, were examined by electron microscopy. Specimens were either spread and shadowed or deposited on polylysine film and stained. In either case, branched molecules with one or more single-stranded arms were seen. The single-stranded arm, in all molecules observed, has about half the contour length of one double-stranded arm. The branched molecules are stable in high salt or hot phenol, resistant to proteinase K, but sensitive to RNAase A in high salt, yielding fragments of double-stranded RNA. These results are consistent with a transcription mechanism in which each new transcript displaces one of the parental RNA strands. From the rate of movement of the branch point, we found transcription rates in vitro of similar to or approximately 25 nucleotides per sec at 30 degrees C and 19 nucleotides per sec at 25 degrees C. Based on the spacing between branches in multiply branched molecules, initiation occurs approximately once every 40 sec at 30 degrees C on M or S RNA templates and about 6 times less frequently on L RNA.

Bacteriophages↗
PubMed1980

Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes.

In the filamentous blue-green alga Anabaena 7120, aerobic nitrogen fixation is linked to the differentiation of specialized cells called heterocysts. In order to study control of heterocyst development and nitrogen fixation in Anabaena, we have used cloned fragments of the Klebsiella pneumoniae nitrogen fixation (nif) genes as probes in DNA.DNA hybridizations with restriction endonuclease fragments of Anabaena DNA. Using this technique, we were able to identify and clone Anabaena nif genes, demonstrating the feasibility of using heterologous probes to identify genes for which no traditional genetic selection exists. From the patterns of hybridization observed, we deduced that although DNA sequence homology has been retained between some of the nif genes of these divergent organisms, the nif gene order has been rearranged.

Journal Article↗
PubMed1980

Nucleotide sequence of a cyanobacterial nifH gene coding for nitrogenase reductase.

The nucleotide sequence of nifH, the structural gene for nitrogenase reductase (component II or Fe protein of nitrogenase) from the cyanobacterium Anabaena 7120 has been determined. Also reported are 194 bases of the 5'-flanking sequence and 170 bases of the 3'-flanking sequence. The predicted amino acid sequence was compared with that determined for the complete nitrogenase reductase of Clostridium pasteurianum and the cysteine-containing peptides of the protein from Azotobacter vinelandii. Amino acid sequences around five cysteines, located in the NH(2)-terminal two-thirds of the protein, are highly conserved in all three species. Codon usage in the first gene from a cyanobacterium to be sequenced shows striking asymmetries for eight amino acids.

Journal Article↗
PubMed1980

Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena.

Phycobiliprotein degradation can be initiated in cultures of the cyanobacterium Anabaena by removal of combined nitrogen from the medium. Certain strains of Anabaena differentiate cells specialized for aerobic nitrogen fixation (heterocysts) under such conditions. We describe here a procedure for the preparation of extracts from heterocysts or vegetative cells that contain an activity capable of degrading only the phycobiliproteins in a mixture of soluble Anabaena proteins in vitro. This activity increased under nitrogen starvation conditions or in ammonia-replete cultures treated with the glutamine synthetase inhibitor methionine sulfoximine. The increase in activity induced by nitrogen starvation was prevented by chloramphenicol or by carbon starvation. Under all these conditions, phycobiliprotein degradative activity assayed in vitro was correlated with the loss of phycobiliprotein absorbance in vivo. Finally, starvation of a met auxotroph of Anabaena for methionine (in the presence of ammonia) did not induce phycobiliprotein degradation in vivo or the increase in proteinase activity. Together with direct measurements of ppGpp, these results indicate that proteolysis in Anabaena is not controlled by compounds associated with the stringent response in Escherichia coli. Since the increase in proteinase activity appears to be regulated by the same variables that control heterocyst differentiation, the activity should provide a useful biochemical marker for the early events of differentiation.

Adenosine Triphosphate↗
PubMed1978

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.

Ammonia↗
PubMed1976

Counting the genes for stabel RNA in the nucleus and chloroplasts of Euglena.

Improved procedures for counting the genes for ribosomal and 4 S RNA in the nucleus and chloroplasts of Euglena are described. These procedures exploit (a) the chloroplast-free strain ZHB, (b) the flotation method for purifying chloroplasts, (c) Hg2+/Cs2SO4 density gradients to fractionate large quantities of DNA and (d) more efficient conditions of DNA - RNA hybridization particularly for 4 S RNA. We find that the average Euglena cell in an exponentially growing culture contains 800 nuclear genes for each cytoplasmic rRNA and 800-880 chloroplast genes for chloroplast rRNA. Each chloroplast chromosome of molecular weight 92 - 10(6) contains two genes for each rRNA. The number of nuclear genes for 4 S RNA is approx. 760. The total number of chloroplast genes for 4S RNA is approx. 10 000, or 22-25 on each chloroplast chromosome.

Animals↗
PubMed1976

Cyanobacterial DNA-binding protein related to Escherichia coli HU.

A DNA-binding protein has been isolated from two cyanobacteria (blue-green algae): Anabaena sp and Aphanocapsa sp. It has a molecular weight as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of about 10,000, is rich in lysine and arginine, and lacks tyrosine, tryptophan, and cysteine. The proteins from both strains show immunological identity with a similar DNA-binding protein from Escherichia coli, when tested by immunodiffusion with an antiserum prepared against the E. coli protein. Both the Aphanocapsa and E. coli proteins form compact, rapidly sedimenting complexes with cyanophage or bacteriophage DNA. The similarities between the proteins from cyanobacteria and E. coli suggest a degree of evolutionary conservation comparable to that of the histones of eukaryotes.

Amino Acids↗
PubMed1975

Ribosomal RNA genes in the nucleus and chloroplast of Euglena.

Centrifugation of DNA from Euglena gracilis in Hg2+-CS2SO4 equilibrium gradients allows the chloroplast DNA to be separated clearly from the nuclear DNA. Cytoplasmic ribosomal RNA isolated from a heat-bleached strain hybridizes only to the nuclear DNA. The ribosomal RNA cistrons in the chloroplast DNA are not related to those in the nuclear DNA. A possible origin for the chloroplast genome is suggested by the observation that roughly one-fourth of the ribosomal RNA complementary sequences in chloroplast DNA anneal with RNA from the blue-green alga Anacystis nidulans.

Binding Sites↗