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Biomedical subjects

S Scherer

Publications and source records attributed to S Scherer.

At least 163 records · Page 9Linked to original sources

Transmembrane electron transport and the neutral theory of evolution.

Based on the concept of "pairs of basic functional states" the evolution of the first chemiosmotic mechanism of energy conversion is discussed in terms of point mutations, gene duplications and of the neutral theory of evolution. A model for estimating the overall probability of the evolutionary step in question is presented, both for the "selectionist" and "neutralist" position. It is concluded that, concerning the present stage of knowledge, the evolution of transmembrane electron transport is an unsolved problem in evolutionary biology.

Adenosine Triphosphatases↗

Oxygen-dependent proton efflux in cyanobacteria (blue-green algae).

The oxygen-dependent proton efflux (in the dark) of intact cells of Anabaena variabilis and four other cyanobacteria (blue-green algae) was investigated. In contrast to bacteria and isolated mitochondria, an H+/e ratio (= protons translocated per electron transported) of only 0.23 to 0.35 and a P/e ratio of 0.8 to 1.5 were observed, indicative of respiratory electron transport being localized essentially on the thylakoids, not on the cytoplasmic membrane. Oxygen-induced acidification of the medium was sensitive to cyanide and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Inhibitors such as 2,6-dinitrophenol and vanadate exhibited a significant decrease in the H+/e ratio. After the oxygen pulse, electron transport started immediately, but proton efflux lagged 40 to 60 s behind, a period also needed before maximum ATP pool levels were attained. We suggest that proton efflux in A. variabilis is due to a proton-translocating ATP hydrolase (ATP-consuming ATPase) rather than to respiratory electron transport located on the cytoplasmic membrane.

Adenosine Diphosphate↗

Reversion of a promoter deletion in yeast.

Promoter function in yeast has been examined by obtaining revertants of a his3 promoter deletion in vivo. Events which provide a new promoter for the his3 gene include insertion of the transposable element Ty1, rearrangements of the plasmid vector, and chromosomal mutations. A role for dicentric chromosomes as a source of the plasmid rearrangements is discussed.

Base Sequence↗

Cloning of bacterial DNA replication genes in bacteriophage lambda.

Recombinant lambda phages containing the genes for dnaZ protein (the gamma subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichia coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.

Bacterial Proteins↗

Recombination of dispersed repeated DNA sequences in yeast.

Yeast transformation can be used to insert new sequence arrangements into a variety of chromosomal locations by homologous recombination. These newly inserted sequences can recombine with similar sequences located on other chromosomes. In these events, information is duplicated without being lost at the site from which it is derived. Similar mechanisms might be utilized by cells to provide new functions during development or differentiation.

Base Sequence↗

Regulatory effect of hydrogen on nitrogenase activity of the blue-green alga (cyanobacterium) Nostoc muscorum.

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under an atmosphere of argon plus acetylene in the light led to a greater than fourfold increase of light-induced hydrogen evolution and to a 50% increase of acetylene reduction, as compared to cells that had not been preconditioned. The basic and the increased hydrogen evolution were both due to nitrogenase activity. Furthermore, after preincubation the hydrogen uptake, usually observed with unconditional cells, was abolished. Nostoc preincubated under acetylene evolved hydrogen in the light even in the presence of nitrogen for at least 2 h, with a 15-fold increase as compared to the unconditioned cells. These acetylene effects could be completely abolished by the presence of hydrogen during acetylene preincubation. These findings indicate that the hydrogen concentration in N. muscorum cells plays a role in regulation of nitrogenase activity.

Acetylene↗

Increase of nitrogenase activity in the blue-green alga Nostoc muscorum (Cyanobacterium).

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under hydrogen or argon (nongrowing conditions, neither CO(2) nor N(2) or bound nitrogen present) in the light resulted in a two- to fourfold increase of light-induced hydrogen evolution and a 30% increase of acetylene reduction. Preincubation under the same gases in the dark led to a decrease of both activities. Cultivation of algae under a hydrogen-containing atmosphere (N(2), H(2), CO(2)) increased neither hydrogen nor ethylene evolution by the cells. Formation of both ethylene and hydrogen is due to nitrogenase activity, which apparently was induced by the absence of N(2) or bound nitrogen and not by the presence of hydrogen. Inhibitors of protein biosynthesis prevented the increase of nitrogenase activity. Hydrogen uptake by the cells was almost unaffected under all of these conditions. With either ammonia or chloramphenicol present, nitrogenase activity decreased under growing conditions (i.e., an atmosphere of N(2) and CO(2)). The kinetics of decrease were the same with ammonia or chloramphenicol, which was interpreted as being due to rapid protein breakdown with a half-life of approximately 4 h. The decay of nitrogenase activity caused by chloramphenicol could be counteracted by nitrogenase-inducing conditions, i.e., by the absence of N(2) or bound nitrogen. A cell-free system from preconditioned algae with an adenosine 5'-triphosphate-generating system exhibited the same increase or decrease of nitrogenase activity as the intact cell filaments, indicating that this effect resided in the nitrogenase complex only. We tentatively assume that not the whole nitrogenase complex, but merely a subunit or a special protein with regulatory function, is susceptible to fast turnover.

Acetylene↗

Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments.

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.

Containment of Biohazards↗

Replacement of chromosome segments with altered DNA sequences constructed in vitro.

We have developed a method that may be of general application for the stable introduction of foreign sequences or deletions, constructed in vitro, into the chromosomes of Saccharomyces cerevisiae. No vector sequences are present in the final strains. Ability to transform cells with DNA, availability of a single selective marker, and integration of the transforming DNA by homologous recombination into the chromosomes are the requirements of the system. Any isolated gene can be deleted or altered and then be used to replace the wild-type chromosomal copy. An internal deletion mutant of the his3 gene and a transposition of a galactose-inducible region into chromosome XV have been generated by using the ura3 gene as the selective marker.

Chromosomes↗

High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.

A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.

DNA Replication↗

Occurrence and structure-function relationship of pentameric short sequence repeats in microbial genomes.

It is suggested that genomes found in any form of cellular life contain potentially size-variable repetitive DNA moieties. In eukaryotes, large proportions of the multi-chromosomal genome consist of various classes of repetitive DNA. Also in archaeal genomes, repetitive DNA is encountered and, as is the case for the eukaryotes as well, little or no function is at present attributable to most of it. For prokaryotes, elegant experiments have highlighted so-called slipped strand nucleotide mispairing (SSM) as a basic and causal mechanism, giving rise to repeat unit number variation at a distinct locus. Illegitimate base pairing in regions of repetitive DNA during replication, in association with defective DNA repair and enhanced nuclease susceptibility of replication intermediates, in the end gives rise to deletion or addition of repeat units. Prokaryotic short sequence repeats (SSRs) harbour arrays of short repeat units, between one and approximately 20 nucleotides in length. SSRs are involved in various mechanisms of microbial gene expression regulation. Promoter strength can be affected by altering the spacing between important structural domains as can the integrity of open reading frames. In the present communication the literature on microbial SSRs harbouring repeat units that are five nucleotides in length will be briefly reviewed. Examples of these SSRs with discrete functionality are encountered in bacterial species such as Haemophilus influenzae, Neisseria gonorrhoeae, and Pasteurella haemolytica. In addition, several of the currently known bacterial and archaeal whole genome sequences were scanned for the presence of novel examples of potential five-nucleotide SSRs (and others) in order to gather additional knowledge on the propensity and putative functions of this type of potential genetic switch.

Base Sequence↗