Biomedical subjects

L Colleaux

Publications and source records attributed to L Colleaux.

44 records · Page 3Linked to original sources

PubMed1992

The sequence of a 9.3 kb segment located on the left arm of the yeast chromosome XI reveals five open reading frames including the CCE1 gene and putative products related to MYO2 and to the ribosomal protein L10.

We report here the sequence of a 9.3 kb DNA segment of chromosome XI of Saccharomyces cerevisiae, located between the MAK11 locus and the centromere. This sequence contains four long open reading frames (ORFs), YKL160, YKL162, YKL164, YKL165 and part of another ORF, YKL166, covering altogether 90% of the entire sequence. One of these ORFs, YKL164, corresponds to CCE1. Translation products of two other ORFs, YKL160 and YKL165, exhibit homology with previously known S. cerevisiae proteins: the ribosomal protein L10, and the MYO2 gene product, respectively.

Amino Acid Sequence↗

PubMed1992

Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii.

Eight respiratory-deficient mutants of Chlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternal mt- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochrome c oxido-reductase) and complex IV (cytochrome c oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochrome b (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses. An in vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.

Animals↗

PubMed1990

Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron.

Group I intron encoded proteins represent a novel class of site specific double strand endonucleases. The endonuclease activity of this class of proteins has been first demonstrated in vivo for I-Sce I which is encoded by a mitochondrial intron of Saccharomyces cerevisiae. Assays using crude cell extracts have shown that I-Sce I can be used in vitro as a restriction endonuclease potentially useful for recombinant DNA technology owing to its large recognition sequence (18 nucleotides). We report here the purification and the first detailed analysis of the in vitro activity and properties of I-Sce I.

Base Sequence↗

PubMed1990

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the alpha insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the alpha insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the alpha insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob.1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.

Amino Acid Sequence↗

PubMed1988

Recognition and cleavage site of the intron-encoded omega transposase.

The optional group I intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae contains a 235-codon-long open reading frame the translation product of which (the omega transposase) catalyzes the formation of a double-strand break within the intron-minus (omega-) copies of the same gene. Purified omega transposase generates in vitro a 4-base-pair staggered cut with 3' hydroxyl overhangs at the exact position where the intron eventually inserts in the gene. Using randomly mutagenized synthetic oligonucleotides, single-base mutants were produced at 21 positions around the cleavage site. Experiments with these oligonucleotides show that the recognition site extends over an 18-base pair-long sequence within which minimal sequence degeneracy is tolerated. The intron-encoded omega transposase is, therefore, one of the most specific restriction endonucleases known to date.

Binding Sites↗

PubMed1986

Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease.

The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (r1 intron) possesses a 235 codon long internal open reading frame (r1 ORF) whose translation product determines the duplicative transposition of that intron during crosses between intron-plus strains (omega+) and intron-minus ones (omega-). Using site-directed mutagenesis, we have constructed a universal code equivalent of the r1 ORF that, under appropriate promoter control, allows the overexpression in E. coli of a protein identical to the mitochondrial intron encoded "transposase". This protein exhibits a double strand endonuclease activity specific for the omega- site. This finding demonstrates, for the first time, the enzymatic activity of an intron encoded protein whose function is to promote the spreading of that intron by generating double strand breaks at a specific sequence within a gene.

Amino Acid Sequence↗

PubMed1986

Mitochondrial introns as mobile genetic elements: the role of intron-encoded proteins.

Introns of organelle genes share distinctive RNA secondary structures that allow their classification into two known families. These structures are believed to play an essential role in splicing, and members of both structural classes have recently been shown to perform self-splicing reactions in vitro. In lower eukaryotes, many structured introns also contain long internal open reading frames (ORFs), which are able to code for hydrophilic proteins. Several properties of self-splicing structured introns suggest that they resemble mobile genetic elements, even though no actual transposition event involving these introns has yet been found. We report here on the characterization of two intron-encoded proteins that strongly support this attractive idea. First, we show that the class I intron of the 21S ribosomal RNA (rRNA) gene of Saccharomyces cerevisiae omega+ strains (rl intron) encodes a specific transposase. This protein has been partially purified from Escherichia coli cells that overexpress it from an artificial universal code equivalent to the rl intronic ORF. The omega transposase shows a double-strand endonuclease activity in vitro. This activity creates a 4-bp staggered cut with 3' OH overhangs within a specific sequence of the 21S rRNA gene of omega- strains. It is precisely within this sequence that the rl intron inserts by a duplicative transposition. Second, we report on the synthesis, in E. coli, of a putative reverse transcriptase encoded by the class II intron of the cytochrome b gene of Schizosaccharomyces pombe. This synthesis was obtained from E. coli expression vectors, using the class II intronic ORF linked to an artificial initiator sequence. As further support of the idea that structured introns are mobile, we show, from a systematic screening of introns in various yeast species, that the rl intron has transposed into the ATPase subunit 9 gene of Kluyveromyces fragilis. Structural features observed at the new intron homing site may be relevant to the transposition event.

Adenosine Triphosphatases↗

PubMedDate not supplied

[A preliminary study to assess the value of the DNA chips SpectralChip to detect subtle constitutional chromosome imbalances].

Comparative genomic hybridization on a microarray (microarray-CGH) allows to detect genomic chromosome imbalances. In order to assess its value to detect small chromosome imbalances observed in a clinical setting, using a DNA chip available commercially (Spectral Genomics, Houston, Texas, USA), we studied the DNA of 9 patients carrying a well characterized chromosome imbalance and the DNA of 11 patients where cytogenetic techniques such as high resolution banding karyotype, FISH using subtelomeric probes and comparative genomic hybridization on metaphase chromosomes conclude to a normal and/or balanced karyotype. A result was obtained for 19/20 patients. Failure of hybridization was observed for one patient. For all the other cases the sex of patients was correctly identified. Microarray-CGH was able to correctly diagnose the chromosome imbalance in 6/8 patients carrying such a defect i.e 9/11 imbalances (deletion or duplication) were detected. No chromosome imbalance was observed in 11 patients considered normal and/or balanced using cytogenetic techniques. Several clones were found to be polymorphic and required FISH studies to eliminate duplication or deletion. In conclusion, we think that this commercially available DNA chip might be useful to screen for chromosome imbalances. However, technical improvements are still necessary before using it in a clinical setting. Also, further studies are necessary to assess its sensitivity and specificity.

Chromosome Aberrations↗