Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites. This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMPI. Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMPI. In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene. Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMPI MIuI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element. The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S. cerevisiae.

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We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.

Site-directed mutagenesis has been used to produce a T-A change at nucleotide 70 of 4X174 genome. This generates an am codon, TAG, in the gene K reading frame without affecting the amino acid, leucine, encoded by the overlapping gene A. The gene K mutant produces small plaques on su-hosts. It has an identical latent period, but a more reduced burst size than that of the wild-type 4X174. The reduced burst size in the gene K mutant suggests that the gene K protein, although not essential, has a role in increasing infectivity by increasing the burst size threeto sixfold.