Kestutis Sasnauskas

Cloning and sequence analysis of a Candida maltosa gene which confers resistance to cycloheximide

A CYHR gene from Candida maltosa, which confers resistance to cycloheximide, was cloned in Saccharomyces cerevisiae. A 2.3-kb DNA fragment carrying this gene was sequenced, and an open reading frame able to encode 553 amino acids (aa) was found in the sequence. Computer searches of the GenBank, EMBL, SWIS-PROT and Gen-Pept databases using the FASTA program failed to detect any proteins with extensive similarities to the deduced aa sequence for CYHR. The cloned gene transforms S. cerevisiae at a frequency similar to auxotrophic markers and can be used as a dominant selectable marker for introducing recombinant plasmids into wild-type strains of S. cerevisiae, as well as for gene disruption experiments.

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.

Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene ( EPG1 )

The yeast Kluyveromyces marxianus strain BKM Y‐719 produces an efficient pectin‐degrading endopolygalacturonase (EPG) that cleaves the internal α‐1,4‐D‐glycosidic linkages to yield oligomers of varying sizes. The EPG1 gene encoding this industrially important EPG was cloned by using the polymerase chain reaction (PCR) technique and degenerate primers to generate a 135 bp DNA fragment with which a genomic library was screened. The cloned fragment contained an open reading frame (ORF) of 1083 bp, encoding a 361 amino acid polypeptide. The predicted amino acid (aa) sequence of EPG showed similarity with polygalacturonases (PGs) of fungi. Analysis of the aa sequence indicated that the first 25 aa constitute a signal sequence and a motif (C218XGGHGXSIGSVG230) that is usually associated with a PG active site. Pulsed‐field gel electrophoresis resolved chromosomal bands for K. marxianus BKM Y‐719 and using chromoblotting it seems thatEPG1 is present as only a single copy in the genome. The nucleotide sequence of K. marxianus BKM Y‐719 EPG1was submitted to the EMBL under Accession No. AJ000076. Copyright

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5-noncoding region and 292-bp 3-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.

Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins.

We have isolated mutants responsible for a super‐secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens α‐amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five‐fold excess compared to the wild‐type strain, was used for the cloning of genes, restraining the super‐secreting phenotype. In screening for genes affecting super‐secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super‐secreting phenotype. The first among the genes characterized, named KlSEL1 (‘secretion lowering’) showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin‐regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super‐secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super‐secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1. No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over‐secreting phenotype) could be attractive for further analysis of gene functions, super‐secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion. The KlSEL1 gene has been assigned EMBL Accession No. AJ488285. Copyright