Isolation and molecular analysis of the gene for cytochrome c1 from Kluyveromyces lactis

By ethyl methanesulphonate mutagenesis of the yeast Kluyveromyces lactis we have isolated five nuclear mutants that were unable to grow on non-fermentable carbon sources. The mutations were found to belong to three complementation groups. After functional complementation of the mutation in one of these mutants we have cloned the structural gene for cytochrome c ₁, named KlCYT1. This gene has been assigned to chromosome VI and its nucleotide sequence exhibited 74.3% identity to the homologous gene of S. cerevisiae. Received: 6 February 1996 / 2 April 1996     

The influence of a pulsating electric field on selenium accumulation in Kluyveromyces marxianus cells

K. marxianus was cultivated under conditions of PEF (pulse electric field) action and a selenium source. The culture duration after which cells were treated with PEF, the field exposure time and the selenium concentration in the medium were all optimized. Optimization of culture duration caused a 33% increase in selenium accumulation in cells as compared to the control with no PEF treatment. The highest selenium accumulation--about 167 microg/g dry mass (DM)--was recorded after 3-minute PEF treatment of 16-hour culture. A roughly two-fold increase in selenium content was achieved after optimization of culture duration and PEF treatment time. Finding the optimum selenium concentration in the medium brought about a 13-fold increase of selenium accumulation in the cells of K. marxianus.     

Extrachromosomal genetics in the yeast Kluyveromyces lactis. Isolation and characterization of antimycin-resistant mutants

Summary Antimycin-resistant (A^R) mutants of the yeast Kluyveromyces lactis, obtained either spontaneously or after manganese treatment, were isolated and genetically characterized. Most of the mutants obtained after manganese mutagenesis and two spontaneous mutants, tolerated high antimycin concentrations (more than 10 /gmg/ml) and were extrachromosomal. One mutant which grew only in low antimycin (1 /gmg/ml) showed a Mendelian type of inheritance. The extrachromosomal mutants could be assigned to at least two genetic loci (A _I ^R and A _II ^R ). Mutants representative of these two groups showed increased resistance to the antibiotic when the respiration of whole cells or mitochondria was studied. Extrachromosomal mutants of Saccharomyces cerevisiae resistant to antimycin were also induced with manganese, isolated and characterized. Comparative studies of the antimycin-resistant mutants of K. lactis and S. cerevisiae permitted the following observations: a) K. lactis is more resistant to antimycin, funiculosin, mucidin and diuron than S. cerevisiae, as are the A^R mutants; b) K. lactis shows correlated sensitivity to funiculosin differing in this aspect from S. cerevisiae; c) the antimycin-resistant mutants of K. lactis belonging to group 11 (A _II ^R ) were also resistant to diuron, tolerating concentrations of more than 200 /gmg/ml; d) all extrachromosomal antimycin-resistant-mutants of S. cerevisiae and some of the A^R mutants of K. lactis were more sensitive to mucidin than the wild type.Abbreviations diuron or DCMU 3-(3,4-Dichlorophenyl)-1,1dimethylurea - HQNO 2-n Heptyl-4-hydroxyquinoline N-oxide     

Isolation and characterization of KlUBP2, a ubiquitin hydrolase gene of Kluyveromyces lactis that can suppress a ts-mutation in CBF2, a gene encoding a centromeric protein of Saccharomyces cerevisiae

 The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases. Received: 6 July 1999 / 29 March 2000     

High efficiency transformation of Kluyveromyces marxianus by a replicative plasmid

Kluyveromyces marxianus can be transformed with an efficiency of 10⁵ transformants/g of DNA by a replicative plasmid using electroporation. In order to obtain this efficiency, we isolated ura^- mutants cells which can be complemented by the URA3 gene from Saccharomyces cerevisiae. The URA3 gene and KARS2, a replicative origin from Kluyveromyces lactis which functions in K. marxianus, were ligated together in a plasmid which can be used as a vector to transform this strain.     

Cloning and characterization of KlCOX18, a gene required for activity of cytochrome oxidase in Kluyveromyces lactis

We describe the isolation and initial characterization of KlCOX18, a gene that is essential for the assembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis. Cells carrying a recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a ₃ . The KlCOX18 gene has been cloned by complementation of the respective nuclear mutation, sequenced, and disrupted. KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs. The corresponding protein exhibits 70.4% similarity to the Cox18p of Saccharomyces cerevisiae. It contains three possible membrane-spanning domains and a putative amino-terminal mitochondrial import sequence. The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c oxidase and respiratory activity. It is proposed that KlCox18p, like its S. cerevisiae counterpart, provides an important function at a later step of the cytochrome oxidase assembly pathway. Received: 25 March / 3 July 1997     

Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants

 We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2Δ and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Received: 13 October/27 November 1995     

Glutathione is an essential metabolite required for resistance to oxidative stress in the yeastSaccharomyces cerevisiae

  Glutathione (GSH) is an abundant cellular thiol which has been implicated in numerous cellular processes and in protection against stress caused by xenobiotics, carcinogens and radiation. Our experiments address the requirement for GSH in yeast, and its role in protection against oxidative stress. Mutants which are unable to synthesis GSH due to a gene disruption in GSH 1, encoding the enzyme for the first step in the biosynthesis of GSH, require exogenous GSH for growth under non-stress conditions. Growth can also be restored with reducing agents containing a sulphydryl group, including dithiothreitol, β-mercaptoethanol and cysteine, indicating that GSH is essential only as a reductant during normal cellular processes. In addition, the GSH 1-disruption strain is sensitive to oxidative stress caused by H₂O₂ and tert-butyl hydroperoxide. The requirement for GSH in protection against oxidative stress is analogous to that in higher eukaryotes, but unlike the situation in bacteria where it is dispensable for growth during both normal and oxidative stress conditions. Received: 20 September / 21 October 1995     

Amphimeric mitochondrial genomes of petite mutants of yeast. I. Flip-flop amphimers make up the mitochondrial genomes of ``palindromic' petite mutants of yeast

 The mitochondrial (mt) genomes of three spontaneous cytoplasmic ``palindromic' petite mutants of yeast were studied by restriction-enzyme analysis. These mt genomes were shown to be made up of an amplified ``master basic unit' consisting of two inverted segments (a and A) and of two different unique segments (d and t) separating them. The basic unit was called ``amphimeric', this term having been first proposed for certain lambda-phage mutants. We propose that in the mt genomes of the petite mutants studied, the four possible variants of the amphimeric basic unit form two – ``flip' and ``flop' – tetra-amphimeric repeat units datA-datA-DaTA-DaTA and DatA-DatA-daTA-daTA, respectively. These repeat units make two types of ``amphimeric' mt genomes which exist in equal proportions in the cell. In each mt genome, the duplicated segment regularly alternates in its direct and inverted orientation (a…A…a…A…), whereas the unique segments are arranged twice in tandem fashion and twice in inverted fashion (d…d…D…D…d…d… and t…t…T…T…t…t….). The only difference between flip and flop amphimeric mt petite genomes is the different relative orientation of the unique segments in the mono-amphimers. In the mono-amphimers of flip mt genomes, both unique segments are arranged in the same direction (d…t and D…T), whereas in the mono-amphimers of flop mt genomes, both unique segments are arranged in opposite directions (D…t and d…T). Control experiments on one spontaneous petite mutant (which was an ancestor of the mutants studied here) and on three independent, previously investigated, EtBr-induced mutants showed that all of them were, in fact, organized in the same way. Analysing our experimental data and the results published by others, we conclude that amphimeric organization is a general feature of mt petite genomes of yeast previously called ``palindromic' or ``rearranged'. Received: 2 November 1995     

The nuclear Kluyveromyces lactis MRF1 gene encodes a mitochondrial class I peptide chain release factor that is important for cell viability

 We report the isolation and characterization of the Kluyveromyces lactis MRF1 gene encoding mitochondrial peptide chain release factor mRF-1. Over-expression of the KlMRF1 gene has a strong antisuppressive effect in a Saccharomyces cerevisiae mitochondrial nonsense suppressor strain. Inactivation of KlMRF1 results in a dual phenotype: most cells die after about 10–13 generations, while a small number of cells exceed this limit. We propose that the lethality is related to a loss of mitochondrial genome integrity. Surviving Klmrf1 cells are able to grow slowly on the non-fermentable substrate glycerol, indicating the existence of a second mitochondrial release factor activity. Our previous comparative analysis of class I release factors is refined by the incorporation of KlmRF-1 and ten recently identified prokaryotic release factor sequences. Received: 10 July/4 December 1995     

Isolation and characterization of sulfite mutants of Saccharomyces cerevisiae

Sulfite-resistant and sulfite-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. Genetic analysis indicated that one and four genes were responsible for the resistant and sensitive responses, respectively, and suggested that defects in methionine and cysteine metabolism were not involved. Some resistant alleles, all of which were dominant, conferred greater resistance than others. Mutations conferring sensitivity were recessive and one co-segregated with impaired respiration. Two of the sensitive mutants exhibited cross-sensitivity to other metabolic inhibitors: sulfometuron methyl, cycloheximide, oligomycin, and antimycin A. A 50% glutathione deficiency in one sensitive mutant was not sufficient in itself to account for its sensitivity. Screening of other relevant mutants revealed that relative to wild-type, met8 and a thioredoxin null mutant are sensitive, and met3 and met14 mutants are not. Reduced production of extracellular acetaldehyde, a compound that detoxifies sulfite, was observed in three of the four sensitive mutants. However, acetaldehyde was also underproduced in the resistant mutant. Because sulfite is a reducing agent, cells were tested for coincident sensitivity or resistance to ascorbate, selenite, dithiothreitol, nitrite, thiosulfate, reduced glutathione, and cysteine. No consistent pattern of responses to these agents emerged, suggesting that the response to sulfite is not a simple function of redox potential.Technical Paper Number 9984 of the Oregon Agricultural Experiment Station     

Isolation and characterization of S. cerevisiae mutants deficient in amino acid-inducible peptide transport

Summary The transport of small peptides into the yeast Saccharomyces cerevisiae is subject to complex regulatory control. In an effort to determine the number, and to address the function, of the components involved in peptide transport and its regulation, spontaneous mutants resistant to toxic di- and tripeptides were isolated under inducing conditions. Twenty-four mutant strains were characterized in detail and fell into two phenotypic groups; one group deficient in amino acid-inducible peptide uptake, the other with a pleiotropic phenotype including a loss of peptide transport. Complementation analysis of recessive mutations in 12 of these strains defned three groups; ptr1 (nine strains), ptr2 (two strains), and ptr3 (one strain). Isolation and screening of 31 additional N-methyl-N-nitro-N-Nitrosoguanidine (MNNG)-induced, peptide transport-deficient mutants produced one ptr3 and 30 ptr2 strains: no additional complementation groups were detected. Uptake of radiolabeled dileucine was negligible in ptr1 and ptr2 strains and was reduced by 65% and 90% in the two ptr3 mutants, indicating that all strains were defective at the transport step. We conclude that the S. cerevisiae amino acid-inducible peptide transport system recognizes a broad spectrum of peptide substrates and involves at least three components. One gene, PTR3, may play an indirect or regulatory role since mutations in this gene cause a pleiotropic phenotype.     

Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5′-decarboxylase gene,pyrG, as a unique transformation marker

  A 8.6-kb disruption cassette, referred to here as a pyrG-blaster and consisting of the Aspergillus niger pyrG gene flanked by a direct repeat that encodes the neomycin phosphotransferase of transposon Tn5 was constructed. Following transformation of a uridine/uracil auxotrophic pyrG strain of A. fumigatus, genomic insertions of the pyrG-blaster were obtained either by targeted gene replacement at the rodA locus, resulting in the formation of hydrophilic spores, or by ectopic integration. In both cases, recombination between the two elements of the direct repeat could be selected in the presence of 5-fluoro-orotic acid and resulted in the excision of the A. niger pyrG gene, producing A. fumigatus uridine/uracil auxotrophs that retained their additional mutant phenotype because of the persistence of one of the two elements of the direct repeat at the site of insertion of the pyrG-blaster. Selection for uracil/uridine prototrophy can therefore be used again to disrupt another gene. Received: 21 November 1995 / Accepted: 8 February 1996     

Isolation and characterization of yeast mutants with thermoconditional sensitivity to the bifunctional alkylating agent nitrogen mustard

Summary Selection of mutants of Saccharomyces cerevisiae sensitive to the DNA cross-linking agent nitrogen mustard (HN2) at two temperatures (23 °C and 36 °C) yielded two isolates with thermoconditionally enhanced (ts) sensitivity to the mutagen. Both were due to single recessive nuclear genes. Mutant allele snm1–2 ^ts showed mainly ts-sensitivity to HN2, whereas mutant allele snm2-1 ^ts conferred ts-sensitivity to HN2, half mustard (HN1) and UV. In temperature-shift experiments it was determined that the functions of SNM1 and SNM2 are needed for recovery within 6 to 7 h. after mutagen exposure during incubation at 23 °C on YEPD when HN2 and UV are applied. After HN1 treatment the SNM2 coded function is required for recovery for about 14 hrs. This possibly indicates a handling of UV- and HN2-induced lesions different from that of HN1-induced lesions.     

Grip force dynamics in the approach to a collision

This experiment investigated the prediction of load force (LF) in impulsive collisions inferred from anticipatory adjustments of grip force (GF) used to stabilise a hand-held object. Subjects used a precision grip to hold the object between thumb and index finger of their right hand and used the arm either: (1) to move the object to produce a collision by hitting the lower end of a pendulum, causing it to swing to one of three target angles, or (2) to hold the object still while receiving a collision produced by the experimenter releasing the pendulum from one of three angles. Visual feedback of the pendulum’s trajectory was available in the production task only. In all conditions, subjects increased GF in advance of the collision. In receiving the collision without advance information, subjects set GF levels to the mid-range of the experienced forces. When subjects possessed knowledge about the maximum angle of pendulum swing – either because they were going to produce it or because they were verbally informed – magnitude of the anticipatory-GF magnitude response was scaled to the predicted LF magnitude. Furthermore, GF was scaled to LF with a higher gain when producing compared to receiving the collision. This suggests that updating forward models through a semantic route is not as powerful as when the updating is achieved through the more direct route of dynamic exploration. Received: 31 July 1998 / Accepted: 16 March 1999     

Determination of amino acid pairs in human haemoglobin α chain sensitive to variants by means of a random approach

 In this data-based theoretical analysis, we use a random approach to analyse amino acid pairs in human haemoglobin a chain in order to determine which amino acid pairs are more sensitive to 133 positional variants in human haemoglobin a chain. The rationale of this study is based on our hypothesis and previous findings that variance is more likely to occur at randomly unpredictable amino acid pair positions than at randomly predictable positions. This is reasonable to argue as randomly predictable amino acid pairs are less likely to have deliberately evolved, whereas randomly unpredictable amino acid pairs have probably deliberately evolved in connection with protein function. A total of 93.99% of 133 variants occurred at randomly unpredictable amino acid pairs, which accounted for 68.57% of 140 amino acid pairs in human haemoglobin α chain. Thus randomly unpredictable amino acid pairs are more sensitive to variance in human haemoglobin α chain. The results also suggest that the human haemoglobin α chain has a natural tendency towards variants. Received: 16 May 2002 / Accepted: 18 July 2002