Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

Yeast flocculation: reconciliation of physiological and genetic viewpoints.

Yeast flocculation results from surface expression of specific proteins (lectins). Two flocculation phenotypes were suggested by physiological and biochemical tests, whereas genetic data suggested a larger number of mechanisms of flocculation. After reviewing the biochemistry, physiology and genetics of flocculation, a new hypothesis combining the data available from these different sources, is proposed. Flocculation results when lectins present on flocculent cell walls bind to sugar residues of neighbouring cell walls. These sugar receptors are intrinsic to the mannan comprising cell walls of Saccharomyces cerevisiae. Two lectin phenotypes were revealed by sugar inhibition studies. The gluco- and mannospecific NewFlo phenotype is not, as yet, found in genetically defined strains. Mannospecific flocculation (Flo1 phenotype) is found in strains containing the genes FLO1, FLO5 and FLO8. This phenotype is also found following mutation of the TUP1 or CYC8 loci, in previously non-flocculent strains. It is therefore proposed that the structural gene for mannospecific flocculation is common or possibly ubiquitous in non-flocculent strains and in consequence, FLO1, FLO5 and FLO8 are probably regulatory genes, exerting positive control over the structural gene. Flocculation expression requires lectin secretion to the cell surface. Many of the observed 'suppressions' of flocculation may be due to mutations of the secretory process, involved in transporting structural proteins to the cell wall. The possible involvement of killer L double-stranded RNA with flocculation is suggested, given the lectin properties of viral coat proteins and an association between L double-stranded RNA and the Flo1 phenotype.     

Mapping the putative RNA helicase genes by sequence overlapping.

An 'electronic' gene mapping procedure based on computer-aided search for overlapping gene sequences was used to identify adjacent genes and localize several putative RNA helicase genes to different chromosomes. PRP28 and AMD1 genes map to the right arm of chromosome IV next to sup2, which encodes a tyrosine tRNA. PRP16, previously mapped to chromosome XI, is tightly linked to MRP-L20. PRP22 is adjacent to PRE1, whose chromosomal location is currently unknown. The utility of this approach in yeast gene mapping is evaluated.     

Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: electrophoretic karyotypes.

Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S. paradoxus and S. bayanus were analysed using contour-clamped homogeneous electric field electrophoresis. Southern blot hybridization with eight cloned S. cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes in the three sibling species. A comparative study of Ty1, Ty2 and telomere-associated Y' sequences having multiple chromosomal location was also done. Chromosome length polymorphism was found in cultured strains of S. cerevisiae. Wild S. cerevisiae and S. paradoxus strains yielded chromosome banding patterns very similar to each other. The karyotype pattern of S. bayanus was readily distinguishable from that of S. cerevisiae and S. paradoxus. Southern blot analysis revealed a low degree of homology between the S. cerevisiae genes studied and the corresponding S. paradoxus and S. bayanus genes. The number of chromosomes appears to be 16 in all three species.     

Characterization of Kluyveromyces lactis subtelomeric sequences including a distal element with strong purine/pyrimidine strand bias

Telomeres are the specialized structures at the ends of eukaryotic chromosomes and are composed of short T/G-rich DNA repeats and the proteins that interact with them. Internal to telomeres are subtelomeric regions that are species-specific and often repetitive. The yeast Kluyveromyces lactis has telomeric tracts of 10-20 copies of a 25 bp repeat, but the subtelomeric regions have not previously been characterized in detail. Here we have cloned and characterized subtelomeric regions from 10 of the 12 chromosome ends. The amount of sequence examined was 0.7-10 kb for each subtelomeric region. We have identified a K. lactis subtelomeric element, the R element, which has a strong purine/pyrimidine strand bias and extends for about 2 kb. Internal to the R element, we found extensive similarity that is shared among half of the chromosome ends reported here. This similarity appears to include three putative gene families, two of which are also subtelomeric in Saccharomyces cerevisiae.     

NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis

An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast.     

The construction and cloning of synthetic genes coding for artificial proteins and expression studies to obtain fusion proteins

Synthetic genes coding for artificial proteins with predeflnedand nutritionally valuable amino acid compositions have beenconstructed and cloned In bacterial plasmid vector pKK233-2.The genes were constructed from three easily interchangeable‘cassettes’ encoding either essential, non-essentialor branched-chain amino acid residues. A potential hairpin loopstructure in the mRNA around the region of the ribosome bindingsite was probably the reason for blockage of translation fromthis vector. Two selected genes, AHB (containing one copy ofeach cassette) and A (consisting of six copies concatemerizedA₆cassette) were cloned into pUR300, a (ß-Gal fusionvector and expressed as fusion proteins (ß-Gal-AHBand (ß-Gal-A₆.     

Two proteins for the price of one: the design of maximally compressed coding sequences

The emerging field of synthetic biology moves beyond conventional genetic manipulation to construct novel life forms which do not originate in nature. We explore the problem of designing the provably shortest genomic sequence to encode a given set of genes by exploiting alternate reading frames. We present an algorithm for designing the shortest DNA sequence simultaneously encoding two given amino acid sequences. We show that the coding sequence of naturally occurring pairs of overlapping genes approach maximum compression. We also investigate the impact of alternate coding matrices on overlapping sequence design. Finally, we discuss an interesting application for overlapping gene design, namely the interleaving of an antibiotic resistance gene into a target gene inserted into a virus or plasmid for amplification.