PubMed Health⌕ Search

Biomedical subjects

L Frontali

Publications and source records attributed to L Frontali.

At least 37 records · Page 2Linked to original sources
PubMed1997

Additional copies of the mitochondrial Ef-Tu and aspartyl-tRNA synthetase genes can compensate for a mutation affecting the maturation of the mitochondrial tRNAAsp.

In an attempt to identify new nuclear genes involved in the synthesis and processing of mitochondrial tRNAs, we utilized a multicopy nuclear library to suppress the heat-sensitive phenotype of a Saccharomyces cerevisiae mitochondrial mutant strain. This strain (Ts 932) is defective in the 3'-end processing of the mitochondrial tRNAAsp transcript. The nuclear genes coding for the mitochondrial elongation factor Tuf M and for the mitochondrial aspartyl-tRNA synthetase have been found to restore the temperature-resistant phenotype and to correct the RNA processing defect. Suppression was effective even when the genes were present on a centromeric plasmid.

Aspartate-tRNA Ligase↗
PubMed1996

The 'petite-negative' yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity.

We cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcA delta strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low-affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.

Amino Acid Sequence↗
PubMed1996

Identification of an essential core element and stimulatory sequences in a Kluyveromyces lactis ARS element, KARS101.

A Kluyveromyces lactis chromosomal sequence of 913 bp is sufficient for replication in Saccharomyces cerevisiae and K. lactis. This fragment contains a 12 bp sequence 5'-ATTTATTGTTTT-3' that is related to the S. cerevisiae ACS (ARS consensus sequence). This dodecamer was removed by site-directed mutagenesis and the effect on K. lactis and S. cerevisiae ARS (autonomous replicating sequence) activity was determined. The dodecamer is essential for S. cerevisiae ARS function but only contributes to K. lactis ARS activity; therefore, its role in K. lactis is unlikely to be the same as that of the essential S. cerevisiae ACS. A 103 bp subclone was found to retain ARS activity in both yeasts, but the plasmid was very unstable in S. cerevisiae. Deletion and linker substitution mutagenesis of this fragment was undertaken to define the DNA sequence required for K. lactis ARS function and to test whether the sequence required for ARS activity in K. lactis and S. cerevisiae coincide. We found a 39 bp core region essential for K. lactis ARS function flanked by sequences that contribute to ARS efficiency. The instability of the plasmid in S. cerevisiae made a fine-structure analysis of the S. cerevisiae ARS element impossible. However, the sequences that promote high-frequency transformation in S. cerevisiae overlap the essential core of the K. lactis ARS element but have different end-points.

Base Sequence↗
PubMed1995

A Saccharomyces cerevisiae gene essential for viability has been conserved in evolution.

To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt) tRNA transcripts in Saccharomyces cerevisiae, nuclear mutations able to complement a mt mutant (Ts932) defective for this process were isolated and analyzed. One of these mutants exhibited a growth defect both on respiratory and fermentable media. Complementation of this phenotype with a S. cerevisiae centrometric wild-type genomic library has allowed us to identify a new essential S. cerevisiae gene strongly conserved in various eukaryotic organisms.

Amino Acid Sequence↗
PubMed1994

Ordered processing of the polygenic transcripts from a mitochondrial tRNA gene cluster in K. lactis.

In Saccharomyces cerevisiae, transcription of the mitochondrial genome starts at multiple initiation sites and is followed by the processing of multigenic transcripts at the 5' and 3' termini of tRNA sequences and in some intergenic regions. We have used a comparative approach to investigate the structure and function of the latter processing sites. We present here an analysis of the transcripts of a cluster of tRNA genes from the mitochondrial genome of Kluyveromyces lactis. The gene order of this cluster is the same as that of the cluster in S. cerevisiae but the sequence of the intergenic regions is different. A detailed analysis of transcripts has been performed using S1 mapping and primer extension techniques. The results can be summarized as follows: (1) transcription of the cluster very probably starts at initiation sites having the nonanucleotide sequence TTATAAGTA (which acts as a promoter in S. cerevisiae) and yields polygenic transcripts; (2) processing of these transcripts seems to occur through an ordered pathway of endonucleolytic events in which some tRNA sequences are preferentially excised and some endonucleolytic cuts occur more readily than others; (3) in two intergenic regions, strong signals indicate the existence of processing events. The sequences around these sites are similar in sequence and localization to S. cerevisiae intergenic processing sites, indicating a possible functional importance in maintaining a conserved order of tRNA genes in different species of yeasts.

Amino Acid Sequence↗
PubMed1994

Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3' end-processing.

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA(Asp) gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3' end-processing of the tRNA(Asp). Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3' end-processing. One such suppressor mutation was further characterized: it restores tRNA(Asp) maturation and growth at 36 degrees C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.

Base Sequence↗
PubMed1992

A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae.

We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditions. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.

Amino Acid Sequence↗
PubMed1992

The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames.

As part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABP1, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).

Amino Acid Sequence↗
PubMed1991

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Adenosine Triphosphatases↗
PubMed1991

Plasmid functions involved in the stable propagation of the pKD1 circular plasmid in Kluyveromyces lactis.

Plasmid factors involved in the stable propagation of pKD1-derived vectors in Kluyveromyces lactis transformants have been identified. Three genes (A, B and C) have been found to be present in pKD1: the interruption of the B and C genes led to high plasmid instability. Stability could be restored in trans when host cells contained pKD1 as the resident plasmid (pKD1+ strains). The A gene, which codes for a site-specific recombinase, did not affect plasmid partitioning. Vectors bearing only the pKD1 replication origin (or a chromosomal ARS), and no other pKD1 sequence, were very unstable both in the presence and absence of the resident plasmid in host cells. These vectors could be stabilized in pKD1+ strains, but not in pKD1 degree strains, by the insertion of a 200 bp-long pKD1 sequence. This sequence, called the cis-acting stability locus (CSL), together with the products of the B and C genes, ensured plasmid partitioning at cell division. Possible hairpin structures and direct repeats were regularly spaced within the CSL. This region, and the corresponding cis-acting stabilizing elements of other yeast plasmids, did not have sequence homology but shared some structural regularities.

Base Sequence↗
PubMed1991

Mitochondrial genome of Saccharomyces douglasii: genes coding for components of the protein synthetic apparatus.

Mitochondrial genes coding for some components of the protein synthetic apparatus in S. douglasii have been studies in detail. A region containing stretches of high homology to the S. cerevisiae tRNA synthesis locus (TSL) and the tRNA(fmet) gene has been identified and sequenced. The organization of this region was very similar to that present in S. cerevisiae, including the presence of a possible transcription starting signal. The S. douglasii TSL gene is shorter due to several deletions which, however, do not involve the regions coding for RNA domains know to be required for the catalytic activity of mitochondrial RNAse P. The S. douglasii LSU rRNA gene has been shown to contain a typical group I intron highly homologous to its S. cerevisiae counterpart, except for the absence of the open reading frame which in S. cerevisiae codes for I-SceI endonuclease.

Base Sequence↗
PubMed1989

A point mutation in a mitochondrial tRNA gene abolishes its 3' end processing.

A temperature sensitive mutation mapping in the tRNA region of the mitochondrial genome of S. cerevisiae has been found to abolish 3' processing of tRNA(asp). Mutant cells grown for a few generations at the non-permissive temperature were found to specifically lack mature tRNA(asp) and to accumulate 3' unprocessed precursors of this tRNA. The accumulation of precursors of other mitochondrial tRNAs was also observed under the same conditions. After longer incubation times, a generalized decrease of mitochondrial transcripts could be observed. The mutation was genetically mapped in a limited region surrounding the tRNA(asp) gene and found, by sequencing, to consist of a C- greater than T transition at position 61 of the tRNA(asp) gene.

Base Sequence↗
PubMed1988

A gene-cloning system for Kluyveromyces lactis and isolation of a chromosomal gene required for killer toxin production.

A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyces lactis. The principle is essentially equivalent to that of the 2 microns/Saccharomyces cerevisiae transformation system. The main features of the system are presented. Using a pKD1-based DNA bank of K. lactis, the KEX1 gene involved in the killer system was isolated by complementation.

Cloning, Molecular↗
PubMed1987

Analysis of transcripts of the major cluster of tRNA genes in the mitochondrial genome of S. cerevisiae.

The transcripts of a 6Kbp region of the mitochondrial genome of S. cerevisiae, localized in the 21S rRNA-OXI1 span and including 12 tRNA genes (from tRNA(thr) to tRNA(ala)) and several G+C clusters, have been studied by analysis of in vitro capped primary transcripts and by fine mapping of the 5' ends of transcripts. The study was performed in the w.t. strain D273-10B and in several rho- mutants retaining different, partially overlapping portions of the studied region; the mutants accumulate incompletely-processed precursors of tRNAs due to the absence of the tRNA synthesis locus. Results show the presence in the region of four sites at which initiation occurs at a consensus nonanucleotide ATTATAAGTA (or a minor variant of the same); however different initiation sites are used in different strains, and several differences as compared to initiation in vitro can also be observed. Termini arising by processing are often localized at AATATAA or AATATATTTT sequences localized immediately adjacent to a G+C cluster or a tRNA sequence.

Base Sequence↗
PubMed1986

Analysis of a 1.6-micron circular plasmid from the yeast Kluyveromyces drosophilarum: structure and molecular dimorphism.

A new plasmid has been found in the yeast Kluyveromyces drosophilarum. It is a double-stranded circular DNA, 1.6 micron in length (4.8 kilobase pairs). As in the case of Saccharomyces 2 mu circles, this plasmid occurs in two isomeric forms corresponding to the inversion of a segment between two 346-bp-long inverted repeats within the molecule. Each form has been separately cloned into bacterial plasmids. The new yeast plasmid, called pKD1, contains sequences that allow its replication in Saccharomyces cerevisiae.

Base Sequence↗
PubMed1985

Mitochondrial transcription and processing of transcripts during release from glucose repression in 'resting cells' of Saccharomyces cerevisiae.

Mitochondrial transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae. Transcripts were identified by hybridization with nick-translated or terminally labelled gene-specific probes. This allowed the determination of the steady-state levels of individual transcripts in the mitochondrial RNA population. Results showed different gene-specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady-state levels of all transcripts (cob); a very strong increase in the processing of the high-molecular-mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3). As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.

Autoradiography↗
PubMed1984

Initiation of transcription of a mitochondrial tRNA gene cluster in S. cerevisiae.

In Saccharomyces cerevisiae most mitochondrial tRNA genes are clustered in a 9 kbp region between the cap and oxil genes. Polygenic transcripts of this region have been previously identified. A transcriptional initiation site at a TTATAAGTA box, located upstream from the tRNAcys gene, has now been detected by S1 mapping experiments and by the capping of primary transcripts. Results are consistent with the hypothesis that this box represents the initiation site for transcription of a cluster of tRNA genes, while the adjacent tRNA2thr is cotranscribed with the 21S rRNA. Results obtained with various strains are compared, and the efficiency of this sequence as a transcriptional initiation site in different mitochondrial contexts is discussed.

Base Sequence↗