Biomedical subjects

C Grandchamp

Publications and source records attributed to C Grandchamp.

15 recordsLinked to original sources

PubMed1992

Interactions between genes involved in exocytotic membrane fusion in paramecium.

Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9a was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the trichocyst membrane to the plasma membrane and in assembly of the "rosette," a complex array of intramembranous particles in the plasma membrane at the trichocyst insertion sites. No revertants were obtained but two new mutants deficient for rosette assembly were identified, nd16b and nd18, whose gene products appear to interact with that of nd9. Indeed, the double mutants grown at 18 degrees, a permissive temperature for each of the single mutants, are characterized by a deficiency in exocytosis and in rosette assembly, as are also double mutants combining other allelic forms of the same genes. Moreover, aberrant dominance relationships among alleles of nd9 and of nd16 indicate the existence of interactions between identical subunits, which most likely assemble into multimeric structures. The nd16 gene product was shown by microinjection experiments to be a cytosolic factor, as is the nd9 gene product. It is therefore tempting to propose that the nd16 gene product also belongs to the connecting material and is involved in rosette assembly, in cooperation with nd9 and nd18.

Animals↗

PubMed1984

Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b.

bI1 RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II-MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand-specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon-intron fragment and labeling it by the same procedure; mRNA and pre-mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm + 20 degrees C). Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self-complementarity of the RNA. A pre-mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe. The amount of the non-sense bI1 RNA strand is very low, in cells from two wild-type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression. bI1 RNA is resistant to RNase. Very little self-complementarity is seen by computer analysis of the sequence. Purified bI1 RNA is seen by electron microscopy under non-denaturing conditions as a mixture of double-stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single-stranded circular and linear molecules is seen as reported previously (Cell, 19, 321-329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules. The ratio of non-sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1981

A low-molecular-weight RNA species in yeast mitochondria arising from a 3' end trimming of cytochrome b pre-mRNA.

Immobilization of yeast mitochondrial RNA on nitrocellulose filters without prior alkali treatment revealed a low-MW RNA species (integral of 300 bases) which hybridizes specifically to the RNA coding strand of a DNA fragment BglII-HinfI at the 3' end of the COB-BOX gene. This RNA species (often a doublet) was found in several independent preparations of wild-type mtRNA and even in box- mutants blocked in the earliest steps of mRNA maturation (e.g. box 8-1). It may, therefore, result from an endonucleolytic cut similar to that which precedes the addition of a poly-A tail in other systems.

Cytochrome b Group↗

PubMed1980

A pathway of cytochrome b mRNA processing in yeast mitochondria: specific splicing steps and an intron-derived circular DNA.

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Base Sequence↗

PubMed1979

Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 micron in contour length; Mt DNA isolated from purified mitochondria contained 2--4% of these 31 micron circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.

Ascomycota↗

PubMed1979

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 mu) were present; instead, smaller circles characteristic for each mutant studied were found. Eco R1 digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type. The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 mu. These circles ranged in size from 0.89 mu to greater than 20 mu; only one molecule out of some 200 molecules was thought to be of full length (31 mu). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm3) and a second at 1.699 g/cm3. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23 x 10(6) daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20 x 10(6) daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length. These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho- mutation in the yeast, S. cerevisiae.

Ascomycota↗

PubMed1979

Genetic and physical characterization of a segment of yeast mitochondrial DNA involved in the control of genetic recombination.

Genetic recombination between the 3 RIB (ribosomal) loci of yeast mitochondrial DNA is under the control of a mitochondrial locus named omega (with alleles omega+ and omega-) which is tightly linked to the RIBI locus. We have attempted to elucidate the molecular mechanisms(s) involved by using rho- mutants with similar (RIBI+ RIB2+ RIB3(0) genotype but different recombination properties in rho- x rho+ crosses. These were obtained through pedigree analysis and their mitochondrial DNAs were mapped on a high resolution physical map of the RIB section that had been built by analysis of thermal denaturation profiles and electron microscopy of partially denatured molecules. By comparison of physical and genetic data it can be shown that possession of the omega+ allele by the rho- cell is not sufficient for its expression in crosses, some additional DNA segments(s) in the ribosomal region being needed. This result and several features of the rho+ x rho- crosses are discussed in the light of current concepts in mitochondrial genetics of yeast and the recently discovered fact that omega+ and omega- strains differ by the presence of a 1000 base pairs insertion in the former.

Base Sequence↗

PubMed1978

[Electron microscopy of mitochondrial DNA in Podospora anserina and the presence of a multimeric range of circular DNA molecules from senescent cultures].

Mitochondrial DNA from young cultures of race s of Podospora anserina was isolated. Its density in Cesium chloride density equilibrium gradients was 1.694 g/cc. Examination by the electron microscope revealed that ca 1% of this DNA consisted of circles, 31 micrometer in contour length; the remaining DNA was composed of linear molecules ranging in length from 2 to 33 micrometer. In DNA of similar density obtained from senescent cultures of the same race s, about 11% of the molecules consisted of a multimeric set of circles ranging in size from 0.9 to 15 micrometer, with most being in the 1.8 and 2.7 micrometer classes. The similarity of these DNA molecules with the mitochondrial DNA from rho(-) yeast mutants is discussed.

Ascomycota↗

PubMed1976

Circular DNA of a yeast episome with two inverted repeats: structural analysis by a restriction enzyme and electron microscopy.

Small circular DNA molecules from genetically characterized clones of Saccharomyces cerevisiae have been studied by restriction endonuclease analysis and electron microscopy. The circular monomers (6000 bases) are shown to contain two inverted repeats of the same sequence (600 bases) situated opposite each other along the perimeter. Four endonuclease EcoRI fragments are obtained in 1:1:1:1 stoichiometry, and their sum gives a length of about 12,000 bases. The two large fragments and the two small ones differ from each other by 200 bases. We propose a model for the structure of the monomer molecule. Two classes of monomers can be generated by intramolecular recombinations within inverted repeats; they differ by the relative orientation of nonrepeated segments. The structure of dimers as predicted by the model is verified by self-renaturation of single-stranded circles. Inverted repeats in circular molecules may be related to the insertion release faculty of II episome in the chromosomes.

Base Sequence↗

PubMed1975

Rearrangement of mitochondrial DNA molecules during the differentiation of mitochondria in yeast. I.-Electron microscopic studies of size and shape.

Size and shape of purified mitochondrial DNA was analyzed by electron microscopy as a function of mitochondrial differentiation. The mitochondrial DNA was extracted at fourth growth stages corresponding to different steps of mitochondria repression and depression. It was heterogeneous both in form and length. The size of linear molecules ranged from 1 mu to 25 mu but most of the molecules could be assigned into four Gaussian subpopulations with mean lengths of 2.2 mu to 4.0 mu, 6.0 mu and 10.0 mu. The circular molecules were all open and sized varied from 0.5 mu to 10 mu. Their length repartition was congruent with a logarithmic Gaussian distribution. The relative proportion of the different classes of molecules changed according to the stage of the growth cycle: during the repression most of the mitochondrial DNA molecules were short: the population of 2.2 mu was predominant. The longest linear molecules were observed during derepression where the populations of 4.0 mu and 10.0 mu were only found as well as the highest proportion of circular molecules. At the stationary phase the mitochondrial DNA became short again and the circles disappeared completely. The mitochondrial DNA extracted from a cytoplasmic "petite" was composed of linear and circular molecules. The linear molecules ranged from 0.1 mu to 32 mu and most of them could be assigned to two subpopulations of 1.3 mu and 4.2 mu. The circular molecules which accounted for 11 percent had contour lengths of 0.7 mu and 1.5 mu. The physiological meaning of the change in the relative proportion of different classes of mitochondrial DNA is discussed.

DNA, Circular↗

PubMed1973