Isolation and characterisation of the linked genes, FPS1 and QCR8, coding for farnesyl-diphosphate synthase and the 11 kDa subunit VIII of the mitochondrial bc1-complex in the yeast Kluyveromyces lactis.

The KlQCR8 gene of the yeast Kluyveromyces lactis encoding subunit VIII of the mitochondrial bc1 complex is 70.2% identical to its counterpart in Saccharomyces cerevisiae (ScQCR8). As in S. cerevisiae, chromosomal linkage between the K. lactis QCR8 and FPS1 genes is conserved, the two genes being separated by only 292 bp. Disruption of the KlQCR8 gene results in a respiratory-deficient phenotype. Compared with S. cerevisiae, expression of the KlQCR8 gene in glucose-grown cells is relatively high, yet is significantly induced when the cells are grown on non-fermentable carbon sources. The QCR8 promoters regions of the two yeasts lack overall DNA sequence similarity, but share DNA-binding sites for the transcription factors ABF1, CPF1 and HAP2/3/4. Deletion from the KlQCR8 promoter of a 93 bp region containing these sites significantly lowers mRNA levels during growth on either glucose or ethanol/glycerol, with a consequent reduction of growth rate on ethanol/glycerol.

Isolation and characterisation of the linked genes APA2 and QCR7, coding for Ap4A phosphorylase II and the 14 kDa subunit VII of the mitochondrial bc1-complex in the yeast Kluyveromyces lactis.

We report the isolation and characterization of the KlQCR7 gene encoding subunit VII of the mitochondrial bc1 complex of the yeast Kluyveromyces lactis. The coding region is 69.3% identical to its counterpart in Saccharomyces cerevisiae (ScQCR7). Like the KlQCR8 gene (Mulder et al., accompanying paper) expression of the KlQCR7 gene during growth on glucose is high and can be further induced when cells are grown on non-fermentable carbon sources. The chromosomal linkage of the APA2 and QCR7 genes is conserved between S. cerevisiae and K. lactis. The intergenic regions containing the QCR7 promoters of the two yeasts, differ significantly in length and lack overall DNA sequence similarity, but they do share a binding site for the transcription factor complex HAP2/3/4. The KlQCR7 promoter contains, in addition, a CPF1 consensus binding site which is absent from ScQCR7. Deletion of a 35 bp region containing these two sites severely lowers the mRNA expression during growth on both glucose and ethanol/glycerol, but growth rate on both carbon sources is only mildly affected. Interestingly, in respect to the KlQCR7 gene, KlCPF1 seems to act as an important transcriptional activator, thus contrasting the proposed repressor function of ScCPF1 for the ScQCR8 gene of S. cerevisiae.

Yeast artificial chromosome cloning of the Xq13.3-q21.31 region and fine mapping of a deletion associated with choroideremia and nonspecific mental retardation.

Microscopically detectable deletions and X;autosome translocations have previously facilitated the construction of a high-resolution interval map of the Xq21 region. Here, we have generated three yeast artificial chromosome contigs spanning approximately 7 megabases of the Xq13.3-q21.31 region. In addition, a novel deletion associated with choroideremia and mental retardation was identified and mapped in detail. The proximal deletion endpoint was positioned between the loci DXS995 and DXS232, which enabled us to confirm the critical region for a locus involved in mental retardation. The distal deletion endpoint is situated in the Xq21.33 band, which allowed us to refine the order of several markers in this region.

Carbon catabolite regulation of transcription of nuclear genes coding for mitochondrial proteins in the yeast Kluyveromyces lactis.

Promoter regions of the KlQCR7, KlQCR8 and KlCYC1 genes, coding for subunits of the bc1-complex and cytochrome c respectively, in the short-term Crabtree-negative yeast Kluyveromyces lactis differ markedly in sequence from their Saccharomyces cerevisiae counterparts. They have, however, conserved very similar configurations of binding-site motifs for various transcription factors known to be involved in global and carbon-source regulation in S. cerevisiae. To investigate the carbon source-dependent expression of these genes in K. lactis, we have carried out medium-shift experiments and determined transcript levels during the shifts. In sharp contrast to the situation in S. cerevisiae, the level of expression in K. lactis is not affected when glucose is added to a non-fermentable carbon-source medium. However, the genes are not constitutively expressed, but become significantly induced when the cells are shifted from glucose to a non-fermentable carbon source. Finally, induction of transcriptional activation does not occur in media containing both glucose and non-fermentable carbon sources.

Distinct transcriptional regulation of a gene coding for a mitochondrial protein in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis despite similar promoter structures.

In Saccharomyces cerevisiae transcription of QCR8, encoding subunit VIII of the mitochondrial ubiquinol cytochrome c oxidoreductase, is subject to glucose repression, whereas in the distantly related yeast Kluyveromyces lactis it is not. The homologous promoter regions lack overall DNA-sequence identity, but do share binding sites for the transcription factors Abf1p, Cpf1p and Hap2/3/4p. For S. cerevisiae it has been established that these factors are involved in growth and carbon source control of nuclear genes encoding mitochondrial proteins. Here we present experimental evidence that K. lactis counterparts of Abf1p and Cpf1p bind independently to the KIQCR8 promoter. The presence of the KIHap2/3/4p binding site enhances binding of KIAbf1p, strongly suggesting that the KIHap2/3/4p complex stabilizes binding of KIAbf1p. In reciprocal exchanges of the QCR8 genes between S. cerevisiae and K. lactis, overall regulation of transcription was found to be species specific. In contrast to S. cerevisiae, additional elements and factors in K. lactis, distinct from Abf1p, Cpf1p and Hap2/3/4p, are able to activate transcription of the QCR8 gene during both fermentative and non-fermentative growth, as well as to induce transcription during growth on non-fermentable carbon sources. In both yeasts, Abf1p is involved in transcriptional activation under both fermentative and non-fermentative growth conditions. Hap2/3/4p plays a minor activational role during fermentative growth, but is mainly responsible for transcriptional induction under non-fermentative growth conditions. Under these latter conditions Abf1p and Hap2/3/4p activate transcription synergistically.

Sequence of the HAP3 transcription factor of Kluyveromyces lactis predicts the presence of a novel 4-cysteine zinc-finger motif.

The Kluyveromyces lactis homologue of the Saccharomyces cerevisiae HAP3 gene was isolated by functional complementation of the respiratory-deficient phenotype of the S. cerevisiae hap3::HIS4 strain SHY40. The KlHAP3 gene encodes a protein of 205 amino acids, of which the central B-domain of 90 residues is highly homologous to HAP3 counterparts of S. cerevisiae and higher eukaryotes. The protein contains a novel 4-cysteine zinc-finger motif and we propose by analogy that all other homologous HAP3 proteins contain the same motif, with the position containing the third cysteine being occupied by a serine residue. In contrast to the situation in S. cerevisiae, disruption of the KlHAP3 gene in K. lactis does not result in a respiratory-deficient phenotype and the growth of the null strain is indistinguishable from wild type. There is also no effect on the expression of the carbon source-regulated KlCYC1 gene, suggesting either a different role for the HAP2/3/4 complex, or the existence of a different mechanism of carbon source regulation. Sequence verification of the S. cerevisiae HAP3 locus reveals that, just as in K. lactis, a long open reading frame (ORF) is present upstream of the HAP3 gene. These highly homologous ORFs are predicted to have at least eight membrane-spanning fragments, but do not show significant homology to any known sequence present in databases. The ScORFX gene is transcribed in the opposite direction to ScHAP3, but, in contrast to an earlier report by Hahn et al. (1988), the transcripts of the two genes do not overlap. The model proposed by these authors, in which the ScHAP3 gene is regulated by an anti-sense non-coding mRNA, is therefore not correct.

Cloning and characterization of DXS6673E, a candidate gene for X-linked mental retardation in Xq13.1.

In several families with non-specific X-linked mental retardation (XLMR) linkage analyses have assigned the underlying gene defect to the pericentromeric region of the X chromosome, but none of these genes have been isolated so far. Here, we report on the cloning and characterization of a novel gene, DXS6673E, that maps to Xq13.1, is subject to X-inactivation and is disrupted in the 5' untranslated region by a balanced X;13 translocation in a mentally retarded female. The DXS6673E gene is highly conserved among vertebrates and its expression is most abundant in brain. It encodes a hydrophilic protein of 1358 amino acids (aa) that does not show sequence homology to other known proteins. A segment of this protein consisting of neutral and hydrophobic aa with a proline residue in every second position may represent a transmembrane domain. Almost complete sequence identity was found between the 3' end of the DXS6673E gene and two expressed sequence tags (ESTs) and between the 5' end of the DXS6673E gene and a third EST. Moreover, weaker sequence similarity was observed between coding regions and two other ESTs.

Centromere promoter factors (CPF1) of the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are functionally exchangeable, despite low overall homology.

The KlCPF1 gene, coding for the centromere and promoter factor CPF1 from Kluyveromyces lactis, has been cloned by functional complementation of the methionine auxotrophic phenotype of a Saccharomyces cerevisiae mutant lacking ScCPF1. The amino-acid sequences of both CPF1 proteins show a relatively-low overall identity (31%), but a highly-homologous C-terminal domain (86%). This region constitutes the DNA-binding domain with basic-helix-loop-helix and leucine-zipper motifs, features common to the myc-related transcription factor family. The N-terminal two-thirds of the CPF1 proteins show no significant similarity, although the presence of acidic regions is a shared feature. In KlCPF1, the acidic region is a prominent stretch of approximately 40 consecutive aspartate and glutamate residues, suggesting that this part might be involved in transcriptional activation. In-vitro mobility-shift experiments were used to establish that both CPF1 proteins bind to the consensus binding site RTCACRTG (CDEI element). In contrast to S. cerevisiae, CPF1 gene-disruption is lethal in K. lactis. The homologous CPF1 genes were transformed to both S. cerevisiae and K. lactis cpf1-null strains. Indistinguishable phenotypes were observed, indicating that, not withstanding the long nonconserved N-terminal region, the proteins are sufficiently homologous to overcome the phenotypes associated with cpf1 gene-disruption.