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Biomedical subjects

M Schweizer

Publications and source records attributed to M Schweizer.

137 records · Page 8Linked to original sources
PubMed1979

Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA.

An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2(+) gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2(-) mutation and an arom-9(-) mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2(+) and had normal levels of catabolic dehydroquinase. DNA.DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2(+) gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (i) replacement of the qa-2(-) gene by the qa-2(+) gene without any effect on the expression of the other genes in the qa cluster, (ii) linked insertion of a normal qa-2(+) gene accompanied by inactivation of the adjacent qa-4(+) gene, and (iii) insertion of a normal qa-2(+) gene at an unlinked site in the N. crassa genome. This newly integrated qa-2(+) genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2(+) transformants are unlinked to the qa-2(-) gene of the recipient.

DNA Restriction Enzymes↗
PubMed1976

Mutational change of membrane architecture. Mutants of Escherichia coli K12 missing major proteins of the outer cell envelope membrane.

Mutants of Escherichia coli have been analyzed which miss two of the major proteins of the outer cell envelope membrane. The two proteins I and II, normally are present at high concentrations (about 10(5) copies per cell). In such mutants, as compared with wild type, the phospholipid-to-protein ratio in the outer membrane has increased by a factor of 2.3 causing a considerable difference in density between wild type and mutant membranes. The concentrations of two other major components of the outer membrane, lipopolysaccharide and Braun's lipoprotein, did not change. The protein-deficient mutants do not exhibit gross functional defects in vitro. An increased sensitivity to EDTA and a slight such increase to dodecyl sulfate (but not to deoxycholate or Triton X-100) was observed, loss of so-called periplasmic enzymes was not found, and other differences to wild type are marginal. The mutants can grow with normal morphology. It is not possible, however, to prepare "ghosts" (particles of size and shape of the cell without murein, surrounded by a derivative of the outer membrane, and possessing the major proteins of this membrane) from them. This fact confirms our earlier suggestion that the proteins in question are required for the shape maintenance phenomenon in ghosts, and the mutants reject the speculation that these proteins are involved in the expression of the genetic information specifying cellular shape. Freeze-fracturing showed that in mutant cells, and in sharp contrast to wild type, the far predominant fracture plane is within the outer membrane. The concentration of the well known densely packed particles at the outer, concave leaflet of this fracture plane is greatly reduced. It was not possible, however, to clearly establish that one or the other protein is part of these particles because these ultrastructural differences were not apparent in mutants missing either one of the proteins only. The biochemical and ultrastructural data allow the conclusion that the loss of two major proteins and the concomitant increase of phospholipid concentration has changed the architecture of the outer membrane from a highly oriented structure, with a large fraction of protein-protein interaction, to one predominantly exhibiting planar lipid bilayer characteristics. E. coli thus can assemble rather different outer membranes, a fact excluding that outer membrane formation constitutes a highly ordered or strictly sequential assembly-line process.

Cell Membrane↗
PubMedDate not supplied

Mapping of the trifunctional fatty acid synthetase gene FAS2 on chromosome XVI of Saccharomyces cerevisiae.

The trifunctional FAS2 gene encoding subunit alpha of the Saccharomyces cerevisiae fatty acid synthetase complex was mapped on the left arm of chromosome XVI 24 centimorgans proximal to GAL4 and 39 centimorgans distal to PEP4 relative to the centromere. Mapping was achieved by three independent methods: meiotic co-segregation of FAS2 and ARO7 in recombination-deficient spo11-mutants: tetrad analysis of crosses between FAS2, GAL4 and PEP4; and Southern hybridization of purified FAS2 DNA with individual yeast chromosomes separated by pulsed-field gel electrophoresis.

Blotting, Southern↗
PubMedDate not supplied

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+.

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.

Alcohol Oxidoreductases↗
PubMedDate not supplied

The cloning and analysis of the aroD gene of E. coli K-12.

A 5.6-kb PstI fragment containing the structural gene (aroD) for 5-dehydroquinate hydrolyase (DHQase) of Escherichia coli K-12 has been cloned into recombinant plasmid pJKK12. The bacterial fragment contains two Bg/II, one HpaII, one SalI and one XhoI site, but no EcoRI, HindIII or BamHI sites. the DHQase activity extracted from strains harboring pJKK12 had properties identical to those of the enzyme isolated from wild-type E. coli. The native protein appears to be a dimer composed of two 31 500 dalton subunits. aroD6 strains transformed with pJKK12 had an 11-fold and 34-fold increase in activity compared to untransformed wild-type controls grown on L broth and minimal medium, respectively. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains of E. coli. At least four constitutively expressed genes are encoded on the fragment.

Amino Acids↗