Ana María Rodríguez-Torres

Codon usage in Kluyveromyces lactis and in yeast cytochrome c-encoding genes

Codon usage (CU) in Kluyveromyces lactis has been studied. Comparison of CU in highly and lowly expressed genes reveals the existence of 21 optimal codons; 18 of them are also optimal in other yeasts like Saccharomyces cerevisiae or Candida albicans. Codon bias index (CBI) values have been recalculated with reference to the assignment of optimal codons in K. lactis and compared to those previously reported in the literature taking as reference the optimal codons from S. cerevisiae. A new index, the intrinsic codon deviation index (ICDI), is proposed to estimate codon bias of genes from species in which optimal codons are not known; its correlation with other index values, like CBI or effective number of codons (Nc), is high. A comparative analysis of CU in six cytochrome-c-encoding genes (CYC) from five yeasts is also presented and the differences found in the codon bias of these genes are discussed in relation to the metabolic type to which the corresponding yeasts belong. Codon bias in the CYC from K. lactis and S. cerevisiae is correlated to mRNA levels.

Disruption of six novel Saccharomyces cerevisiae genes reveals that YGL129c is necessary for growth in non-fermentable carbon sources, YGL128c for growth at low or high temperatures and YGL125w is implicated in the biosynthesis of methionine.

Six open reading frames (ORFs) from chromosome VII, YGL131c, YGL129c, YGL128c, YGL125w, YGL124c and YGL121c, were disrupted by deletion cassettes with short flanking regions homologous to the target locus (SFH). YGL129c is necessary for growth in non‐fermentable carbon sources, YGL128c for growth at low or high temperatures and YGL125w is implicated in the biosynthesis of methionine. With regard to the other ORFs, basic phenotypic analyses did not reveal any significant clues about their function. Copyright

Involvement of Pta1, Pcf11 and a KlCYC1 AU-rich element in alternative RNA 3'-end processing selection in yeast.

This work reports the involvement of yeast RNA processing factors Pta1 and Pcf11 in alternative 3′‐end RNA processing. The pta1‐1 and pcf11‐2 mutations changed the predominance of KlCYC1 1.14 and 1.5 kb transcript isoforms. Mutation of the KlCYC1 3′‐UTR AU‐rich sequence at positions 679–690 (mutant M1) altered transcript predominance. Moreover, expression of M1 in the yeast mutants partially suppressed their effects in the predominance pattern. The combination of the M1 and M2 (694–698 deletion) mutations abolished the alternative processing. Pta1 involvement in this selection was confirmed using the Pta1‐td degron strain.

IDENTIFICATION OF A PUTATIVE METHYLENETETRAHYDROFOLATE REDUCTASE BY SEQUENCE ANALYSIS OF A 6.8 KB DNA FRAGMENT OF YEAST CHROMOSOME VII

We report the sequence analysis of a 6·8 kb DNA fragment from Saccharomyces cerevisiae chromosome VII. This sequence contains five open reading frames (ORFs) greater than 100 amino acids. There is also an incomplete ORF flanking one of the extremes, G2868, which is the 3′ end of the SCS3 gene (Hosaka et al., 1994). The translated sequence of ORF G2882 shows similarity to the human methylenetetrahydrofolate reductase (Goyette et al., 1994). ORF G2889 shows no significant homologies with the sequences compiled in databases. ORF G2893 corresponds to the gene SUP44, coding for the yeast ribosomal protein S4 (All‐Robin et al., 1990). G2873 and G2896 are internal ORFs. The whole sequence of the fragment is available at the EMBL nucleotide sequence database, GenBank and Data Bank of Japan under the Accession Number X94106.

PICDI, a simple program for codon bias calculation

PICDI is a very simple program designed to calculate the Intrinsic Codon Deviation Index (ICDI). The program is available in Macintosh as well a PC format. Requirements for correct input of the sequences have been kept to a minimum and the analysis of sequences up to 2000 codons is very quick. The ICDI is very useful for estimation of codon bias of genes from species in which optimal codons are not known. The availability of a computer program for its calculation will increase its usefulness in the fields of Molecular Biology and Biotechnology.

A stress response related to the carbon source and the absence of KlHAP2 in Kluyveromyces lactis

The Kluyveromyces lactisHIS4 gene (KlHIS4) is transcriptionally regulated by the carbon source. The promoter region encompassing positions −238 to −139 is responsible for this regulation according to lacZ reporter assays. Electrophoretic Mobility Shift Assay (EMSA) experiments on KlHIS4 promoter (positions −218 to −213, Fragment 6, F6) show a specific gel-shift band, CS1, whose intensity is carbon-source dependent in K. lactishap2 (klhap2) knock-out strains. The klhap3 mutation is not able to cause this effect by itself, but the combination of klhap2 and klhap3 mutations has an enhanced effect on CS1 band formation. Introducing a heat shock element (HSE) at the sequence in the F6 fragment (mutated F6, F6*) increases the binding activity in the klhap2 mutant.KlHIS4 mRNA levels in the klhap2 or the double Klhap2/3p mutant do not correlate with the increase in CS1 binding activity, indicating that the factor causing CS1 is acting and only detectable in vitro. EMSA experiments with K. lactis wild-type cells under temperature stress conditions show a band enhancement (Ts1), similar in size to CS1. Cross-competition experiments between F6 and F6* show that F6* competes more efficiently than F6 for both CS1 and Ts1 formation, indicating the involvement of the HSE in the formation of the specific gel-shift bands. Moreover, the similar gel-shift patterns suggest that both bands are caused by the same heat shock-like factor under different stress conditions. Therefore, the enhancement of the CS1 band signal in the klhap2 (and klhap2/3) mutants is due to the increase in heat shock-like factors in the protein extracts from these mutant cells grown in a non-fermentable carbon source. This Klhap2-dependent stress effect was not previously described in K. lactis.

Sequence Analysis of a 10 kb DNA Fragment from Yeast Chromosome VII Reveals a Novel Member of the DnaJ Family

We report the sequence analysis of a 10 kb DNA fragment of Saccharomyces cerevisiae chromosome VII. This sequence contains four complete open reading frames (ORFs) of greater than 100 amino acids. There are also two incomplete ORFs flanking the extremes: one of these, G2868, is the 5′ part of the SCS3 gene (Hosaka et al., 1994). ORFs G2853 and G2856 correspond to the genes CEG1, coding for the alfa subunit of the mRNA guanylyl transferase and a 3′ gene of unknown function previously sequenced (Shibagaki et al., 1992). G2864 is identical to SOH1 also reported (Fan and Klein, 1994). The translated sequence of G2861 is similar to the human dnaJ homolog. The nucleotide sequence reported here has been entered in the EMBL Data Library under the Accession Number X87252.

Structurally conserved and functionally divergent yeast Ssu72 phosphatases.

The eukaryotic Ssu72 factor is involved in several RNA biogenesis processes. It has phosphatase activity on the carboxy‐terminal domain (CTD) of the major subunit of RNA polymerase II. The Kluyveromyces lactis Ssu72 (KlSsu72) shows in vitro phosphatase activity for the pNPP substrate, and this activity is inhibited by ortho‐vanadate. The expression of KlSsu72 in Saccharomyces cerevisiae shows defective CTD serine5‐P phosphatase activity and reveals the importance of Ssu72 for the normal CTD serine5‐P levels at two growth states. The divergence is emphasised by the remarkable changes in RNA14 alternative 3′‐end RNA processing, which are independent of the CTD serine5‐P levels.