Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry.

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes.

The tubulin gene family of Paramecium: characterization and expression of the alpha PT1 and alpha PT2 genes which code for alpha-tubulins with unusual C-terminal amino acids, GLY and ALA.

The ciliated protozoan Paramecium harbours a particularly large diversity of microtubule networks, ranging from the elaborate and stable ciliary axonemes and basal bodies to very dynamic cytoplasmic, cortical or intranuclear arrays. Their organization and individual cycle of assembly/disassembly are well known and extensive immunocytochemical studies of the post-translational modifications in the various microtubule systems have been reported. However, in order to better understand the biogenesis of these multiple and diverse microtubule arrays, it seemed necessary to characterize the tubulin gene family.

Tissue-specific splicing of two mutually exclusive exons of the chicken beta-tropomyosin pre-mRNA: positive and negative regulations.

Alternative splicing of premessenger RNA (pre-mRNA) is a widespread process used in higher eucaryotes to regulate gene expression. A single primary transcript can generate multiple proteins with distinct functions in a tissue- and/or developmental-specific manner. A central question in alternative splicing concerns the selection of splice sites in different cell environments.

Artificial antisense RNA regulation of YBR1012 (YBR136w), an essential gene from Saccharomyces cerevisiae which is important for progression through G1/S.

YBR1012 (YBR136w) is an essential gene from Saccharomyces cerevisiae identified during the systematic sequencing of part of the right arm of chromosome II. We previously constructed a conditional allele of YBR1012 based on antisense RNA, by inserting a small fragment of this gene downstream from the inducible UASGAL10-CYC1 promoter. Several other antisense RNA constructions have since been made and their activity tested.

A T to G mutation in the polypyrimidine tract of the second intron of the human beta-globin gene reduces in vitro splicing efficiency: evidence for an increased hnRNP C interaction.

In a patient with a beta-thalassemia intermedia, a mutation was identified in the second intron of the human beta-globin gene. The U-->G mutation is located within the polypyrimidine tract at position -8 upstream of the 3' splice site. In vivo, this mutation leads to decreased levels of the hemoglobin protein.

DosDNA occurs along yeast chromosomes, regardless of functional significance of the sequence.

Complex genomes contain numerous simple sequence repeats, the biological significance of which remains obscure. Recently it has been shown that several human diseases are the result of changes in such sequences. Thus it has become urgent to undertake a systematic study of their properties.

Mutations in the mitochondrial split gene COXI are preferentially located in exons: a mapping study of 170 mutants.

We have analysed the precise location of a large number (170) of mutations affecting the structural gene for subunit I of the cytochrome c oxidase complex. This gene, COXI, is 12.9 kb long and the major part of the sequence (i.

Cloning of a human gene involved in cytochrome oxidase assembly by functional complementation of an oxa1- mutation in Saccharomyces cerevisiae.

The yeast nuclear gene OXA1 is essential for cytochrome oxidase assembly, so that a null mutation in the OXA1 gene leads to complete respiratory deficiency. We have cloned by genetic selection a human OXA1 (OXA1Hs) cDNA that complements the respiratory defect of yeast oxa1 mutants. The deduced sequence of the human protein shares 33% identity with the yeast OXA1 protein.

MRG1-1, a dominant allele that confers methomyl resistance in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize.

We have previously described a eukaryotic heterologous expression system, with the urf13TW gene in yeast, which mimics the disease susceptibility associated with the Texas cytoplasmic male sterility in maize. This yeast model was used to isolate yeast nuclear mutants conferring methomyl resistance. The genetic strategy we have developed focused on screening for nuclear dominant yeast mutations which restore methomyl resistance.

The sequence of 12.5 kb from the right arm of chromosome II predicts a new N-terminal sequence for the IRA1 protein and reveals two new genes, one of which is a DEAD-box helicase.

We have determined the complete nucleotide sequence of a 12.5 kb segment from the right arm of chromosome II carried by the cosmid alpha 20. The sequence encodes the 5' end of the IRA1 gene.

OXA1, a Saccharomyces cerevisiae nuclear gene whose sequence is conserved from prokaryotes to eukaryotes controls cytochrome oxidase biogenesis.

Yeast cells carrying a mutation in the OXA1 nuclear gene are respiratory deficient and lack cytochrome oxidase activity. We successively examined the different steps in the expression of the mitochondrial genes encoding the cytochrome oxidase subunits and apocytochrome b in strains carrying the oxa1-79 mutation. The ox1-79 strains exhibit a total absence of cytochrome aa3 and a decrease in cytochrome b, even in a strain devoid of mitochondrial introns, in which cox1 and cytb mRNAs normally accumulate.

The sequence of 29.7 kb from the right arm of chromosome II reveals 13 complete open reading frames, of which ten correspond to new genes.

We have determined the complete nucleotide sequence of a 29.7 kb segment from the right arm of chromosome II carried by the cosmid alpha 61. The sequence encodes the 3' region of the IRA1 gene and 13 complete open reading frames, of which ten correspond to new genes and three (CIF1, ATPsv and CKS1) have been sequenced previously.

Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability.

RNA14 and RNA15 were originally identified by temperature-sensitive mutations that cause a rapid decrease in poly(A)-tail length and overall mRNA levels at the restrictive temperature. We have raised antibodies to the RNA14 and RNA15 proteins, and used subcellular fractionation and immunofluorescence to localize these proteins within the yeast cell. RNA14p is a 73 kDa protein found in both the nucleus and the cytoplasm, whilst RNA15p is a 42 kDa protein detected only in the nucleus.

Two homologous introns from related Saccharomyces species differ in their mobility.

We have studied gene conversion initiated by the ai3 intron of the Saccharomyces cerevisiae mitochondrial (mt) COXI gene and its homologous intron (S.cap.ai1) from Saccharomyces capensis.

Multiple group II self-splicing introns in mobile DNA from Escherichia coli.

By PCR (polymerase chain reaction) amplification and cloning, we have identified four group II self-splicing introns encoding proteins related to reverse transcriptases in natural Escherichia coli isolates belonging to the ECOR collection. One intron, IntD, interrupts a DNA sequence virtually identical to that of the previously described IS3411 Insertion Sequence. A second intron, IntC, is located within an open reading frame that is closely related to a reading frame in the T-DNA of Agrobacterium tumefaciens.

Automatic identification of group I intron cores in genomic DNA sequences.

Automatic identification of the ribozyme core of group I catalytic introns in genomic sequences is shown to be feasible in spite of the scarcity of strictly conserved features in the sequence and secondary structure of group I introns. An algorithm is described that successfully identified 132 out of the 143 currently reported group I cores with a false positive rate of only 10(-6) per nucleotide. The recognition process consists in generating and rating large sets of potential local solutions which are gradually combined into more complex structures until an entire core (six to seven pairings, six connecting segments, three terminal loops) has been assembled.

Multipurpose vectors designed for the fast generation of N- or C-terminal epitope-tagged proteins.

In this paper are described a set of new high-copy-number yeast vectors, which are specially designed for the conditional expression of epitope-tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction-amplified open reading frames to be automatically fused in frame with the epitope-coding sequence, avoiding longer procedures such as site-directed mutagenesis. This heterologous construction can be realized either at the 5'-end of the coding sequence, in the pYeF1 vector, or at its 3'-end, in pYeF2, generating N- or C-terminal tagged proteins, respectively.

Identification of a new nuclear gene (CEM1) encoding a protein homologous to a beta-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae.

We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with beta-keto-acyl synthases or condensing enzymes.

Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme-dependent transcriptional regulation in the yeast Saccharomyces cerevisiae.

Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae. This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid). In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence.

Sequence of the mitochondrial gene encoding subunit I of cytochrome oxidase in Saccharomyces douglasii.

We have determined the complete sequence of the mitochondrial (mt) gene (COXI) coding for cytochrome oxidase subunit I of Saccharomyces douglasii. This gene is 7238 bp long and includes four introns. The salient feature of the S.

Mitochondrial dysfunction in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize: analysis at the population and individual cell level.

The urf13TW gene, which is derived from the mitochondrial T-urf13 gene responsible for Texas cytoplasmic male sterility in maize, was expressed in Saccharomyces cerevisiae by targeting its translation product into mitochondria. Analysis by oxygraphy at the population level revealed that in the presence of methomyl the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory substrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrate oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in significant inhibition of oxygen uptake.