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Biomedical subjects

G Dujardin

Publications and source records attributed to G Dujardin.

At least 55 records · Page 3Linked to original sources
PubMed1992

NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae.

The yeast nuclear gene NAM7 was previously isolated within a genomic fragment of 7.7 kb (1 kb = 10(3) bases or base-pairs), having the ability to suppress mitochondrial intronic mutations defective in RNA splicing. We have identified and sequenced the region on the insert corresponding to the NAM7 gene. A long open reading frame has been revealed which could code for a protein of 971 amino acids. Comparison of the NAM7 putative protein with data libraries did not reveal any strong similarity with known proteins. However, the NAM7 protein contains several motifs typical for proteins interacting with nucleic acids: (1) five motifs diagnostic for a superfamily of helicases appear in the same order and with similar distances; (2) the N-terminal portion possesses potential Zn-ligand structures belonging to the C chi superfamily. Deletion of the chromosomal copy of NAM7 gene leads to a partial impairment in respiratory growth that is particularly striking at low temperature. Southern blot analysis of DNA extracted from a nam7 :: URA3 deleted strain revealed the presence of a second gene whose sequence is related to that of the NAM7 gene and which could participate in the same process.

Amino Acid Sequence↗
PubMed1992

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

We have characterized the nuclear gene NAM8 in Saccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wild-type strains, its overexpression is a prerequisite for the suppressor action. Inactivation of the NAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. The NAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.

Amino Acid Sequence↗
PubMed1991

Participation of the TATA factor in transcription of the yeast U6 gene by RNA polymerase C.

Fractionation of transcription extracts has led to the identification of multiple transcription factors specific for each form of nuclear RNA polymerase. Accurate transcription in vitro of the yeast U6 RNA gene by RNA polymerase C requires at least two factors. One of them was physically and functionally indistinguishable from transcription factor IID (TFIID or BTF1), a pivotal component of polymerase B transcription complexes, which binds to the TATA element. Purified yeast TFIID (yIID) or bacterial extracts that contained recombinant yIID were equally competent to direct specific transcription of the U6 gene by RNA polymerase C. The results suggest the formation of a hybrid transcription machinery, which may imply an evolutionary relation between class B and class C transcription factors.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

ABC1, a novel yeast nuclear gene has a dual function in mitochondria: it suppresses a cytochrome b mRNA translation defect and is essential for the electron transfer in the bc 1 complex.

We have cloned and sequenced a novel yeast nuclear gene ABC1 which suppresses, in multicopy, the cytochrome b mRNA translation defect due to the nuclear mutation cbs2-223. Analysis of the ABC1 gene shows that it is weakly expressed, it could code for a protein of 501 amino acids which has a typical presequence of a protein imported into mitochondria and which does not display a strong similarity to any known protein. Inactivation of the ABC1 gene is not lethal to the cell but leads to a respiratory defect: no oxygen uptake and no growth on non-fermentable media. A total absence of NADH-cytochrome c oxidoreductase and succinate-cytochrome c oxidoreductase activities concomitant with the presence of specific dehydrogenases, suggests a block in the bc 1 segment of the respiratory chain. However, all the cytochromes are spectrally detectable. Cytochrome b is quite efficiently reduced while cytochromes c1 and c are not. The function of ABC1 in the suppression of a defect in apocytochrome b mRNA translation and in the activity of the bc1 complex suggests that the ABC1 protein would be a novel chaperonin involved both in biogenesis and bioenergetics of mitochondria.

Amino Acid Sequence↗
PubMed1990

Two group I mitochondrial introns in the cob-box and coxI genes require the same MRS1/PET157 nuclear gene product for splicing.

We have studied the role of the product of the nuclear gene PET157 in mitochondrial pre-mRNA splicing. Cytoduction experiments show that a mitochondrial genome deleted for the three introns bI3, aI5 and aI6 is able to suppress the pet157-1 mutation: the strain recovers respiratory competency indicating that the product of the PET157 gene is only required for mitochondrial pre-mRNA splicing. Characterization of the high molecular weight pre-mRNAs which accumulate in the pet157 mutant demonstrate that the product of the PET157 gene is required for the excision of two group I introns bI3 and aI6 (corresponding to aI5 beta) located in the cob-box and coxI genes respectively. Furthermore, the pet157 mutant strain accumulates the bI3 maturase in the form of a polypeptide of 50K (p50) previously observed in mitochondrial mutants defective in the excision of bI3. We have shown by restriction analysis and allelism tests that the pet157-1 mutation is allelic to the nuclear mrs1 mutation, previously described as specifically blocking the excision of bI3. Finally, revertants obtained by the deletion of bI3 or aI6 from the mitochondrial DNA were isolated from the MRS1 disrupted allele, confirming the involvement of the product of the MRS1/PET157 gene in the excision of the two introns bI3 and aI6.

Alleles↗
PubMed1989

Novel class of nuclear genes involved in both mRNA splicing and protein synthesis in Saccharomyces cerevisiae mitochondria.

We have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids. These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII. Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix. Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing. The possible mechanisms by which the NAM1 gene product may function are discussed.

Amino Acid Sequence↗
PubMed1988

The NAM2 proteins from S. cerevisiae and S. douglasii are mitochondrial leucyl-tRNA synthetases, and are involved in mRNA splicing.

We have cloned and sequenced the NAM2 gene of Saccharomyces douglasii, which is a homologue of the NAM2 gene of Saccharomyces cerevisiae. The wild-type S.douglasii gene possesses the suppressor functions of the mutant S. cerevisiae NAM2-1 allele, being able to cure a mitochondrial b14 maturase deficiency. By sequence comparisons and direct measurements we have demonstrated that the NAM2 genes encode mitochondrial leucyl tRNA synthetases (EC 6.1.1.4.). Using a derivative of the NAM2 gene, where the expression of the gene is under the control of the UAS GAL10, we have shown that the processing of the pre-mRNA from the two mosaic genes oxi3 and cob-box is impaired when transcription of the gene is repressed. These results lead us to conclude that the mitochondrial leucyl tRNA synthetase is involved in protein synthesis and mRNA splicing. Sequence comparisons show that the mitochondrial and Escherichia coli leucyl tRNA synthetases are highly homologous; however, significant features which may be important for the splicing functions of the mitochondrial enzymes are absent from the bacterial enzyme.

Amino Acid Sequence↗
PubMed1988

Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: a paradigm of incipient evolution.

We studied the NAM2 genes of Saccharomyces douglasii and Saccharomyces cerevisiae, and showed that they are interchangeable for all the known functions of these genes, both mitochondrial protein synthesis and mitochondrial mRNA splicing. This confirms the prediction that the S. douglasii NAM2D gene encodes the mitochondrial leucyl tRNA synthetase (EC 6.1.1.4.). The observation that these enzymes are interchangeable for their mRNA splicing functions, even though there are significant differences in the intron/exon structure of their mitochondrial genome, suggests that they may have a general role in yeast mitochondrial RNA splicing. A short open reading frame (ORF) precedes the synthetase-encoding ORF, and we showed that at least in S. cerevisiae this is not essential for the expression of the gene; however, it may be involved in a more subtle type of regulation. Sequence comparisons of S. douglasii and S. cerevisiae revealed a particularly interesting situation from the evolutionary point of view. It appears that the two yeasts have diverged relatively recently: there is remarkable nucleotide sequence conservation, with no deletions or insertions, but numerous (albeit non-saturating) silent substitutions resulting from transitions. This applies not only to the NAM2 coding regions, but also to two other ORFs flanking the NAM2 ORF. The regions between the ORFs (believed to be intergenic regions) are much less conserved, with several deletions and insertions. Thus S. douglasii and S. cerevisiae provide an ideal system for the study of molecular evolution, being two yeasts "caught in the act" of speciation.

Alleles↗
PubMed1987

Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene.

Dominant mutations of the nuclear NAM2 gene are able to compensate for a deficiency of the maturase encoded by the fourth intron of the mitochondrial cytochrome b gene. We have determined the complete nucleotide sequence of the NAM2-1 suppressor allele. The results of S1 nuclease protection experiments show that two overlapping poly(A)+ RNAs are transcribed from the gene using different promoters. The longer transcript contains two open reading frames (ORFs), a long ORF which could encode a protein of 894 amino acids, mol. wt 102,000 daltons, and a short ORF of 51 codons which is omitted from the shorter transcript. The wild-type nam2+ and two other suppressor alleles, NAM2-6 and NAM2-7, have been cloned. A comparison of the sequence of the wild-type and the three suppressor alleles shows that on three separate occasions the same codon specifying glycine was mutated (once to serine and twice to cysteine). Finally sequence comparisons identified two regions in the long ORF, distinct from the position of the suppressor mutations, that could correspond to binding domains for a nucleotide and a nucleic acid.

Alleles↗
PubMed1985

The yeast nuclear gene NAM2 is essential for mitochondrial DNA integrity and can cure a mitochondrial RNA-maturase deficiency.

Dominant mutations in the yeast nuclear gene NAM2 cure the RNA splicing deficiency resulting from the inactivation of the bI4 maturase encoded by the fourth intron of the mitochondrial cytochrome b gene. This maturase is required to splice the fourth intron of this gene and to splice the fourth intron of the mitochondrial gene oxi3 encoding cytochrome oxidase subunit I. We have cloned the nuclear gene NAM2, which codes for two overlapping RNAs, 3.2 kb and 3.0 kb long, which are transcribed in the same direction but differ at their 5' ends. NAM2 compensating mutations probably result from point mutations in the structural gene. Integration of the cloned gene occurs at its homologous locus on the right arm of chromosome XII. Inactivation of the NAM2 gene either by transplacement with a deleted copy of the gene, or by disruption, is not lethal to the cell, but leads to the destruction of the mitochondrial genome with the production of 100% cytoplasmic petites.

Cloning, Molecular↗