Cloning of GAP Gene from K. fragilis.

The K. fragilis CBS 397 gene library was screened with a GAP probe, which was designed according to the homology with S. cerevisiae, K. lactis, K. marxianus GAP gene. One positive clone pG1 containing GAP1 gene was isolated and confirmed by Southern hybridization. The GAP1 gene was partially sequenced. By using a fragment of the clone as a probe, another positive clone pG2 was acquired and also confirmed by Southern hybridization. The GAP2 gene from pG2 was completely sequenced. The upstream sequences of both genes were shown to have promoter activity. The fragment of pG1 could hybridize with three chromosomes of K. fragilis.

The Mutation of mRNA Translation Initiation Region Influences Gene Translation.

In order to investigate the relationship between the structure of the mRNA translation initiation region (TIR) and gene expression, We mutated multiple sites of the 5' end of IFN-alpha8 and GM-CSF genes by site-directed mutagenesis without changing their amino acid sequences. SDS-PAGE showed that the protein products of mutated genes increased greatly in recombinant clones, as compared with their native genes. RNA dot blot revealed that the difference of their corresponding amount of mRNA transcribed between the native and the mutated genes was negligible. These results imply that the elevated expressions are attributed mainly to increased translation level. The prediction of mRNA secondary structure suggests that the delta G of TIR may have close relations to the expression level.

Cloning of the K. fragilis PDI Gene.

The K. fragilis CBS 397 gene library was screened by the PDI probe, which was designed according to the homology with the S. cerevisiae PDI gene. One positive clone was found and confirmed. In order to learn its physical map,Southern hybridization was done on this positive clone. The K. fragilis PDI gene was found to locate in chromosome V genome. Its sequence was also made partly known by DNA sequencing. From the method of the turbidimetric assay of insulin disulfide reduction, we have also demonstrated that the cloned PDI gene can exhibit PDI activity.

The Construction and Application of the Multi-copy Integration Vector in K. lactis.

The vector pIRK was constructed by using a 2.2 kb EcoRI fragment of rDNA from Kluyveromyces lactis for targeted homologous recombination, with the URA3 gene from Saccharomyces cerevisiae acting as a selection marker. By the examination of the copy number, stability and chromosomal location of the vector in K. lactis transformants the results demonstrated that: (1) of the different transformants, the average copy number of the plasmid pIRK was 120 per cell; (2) after 50 generations of growth in rich medium, the vector displayed high stability. (3) all integration events occurred in the chromosome IV where genomic rDNA located. Using this vector, the LAC4 gene cloned from K. fragilis was expressed. The yield of beta-galactosidase related directly to the vector's copy number. The highest activity of beta-galactosidase produced by transformants was 8.6 times higher than that produced by the wild type strain of K. fragilis under the same conditions.