Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis.

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.

Relative contributions of MCM1 and STE12 to transcriptional activation of a- and alpha-specific genes from Saccharomyces cerevisiae.

We have examined the relative contributions of MCM1 and STE12 to the transcription of the a-specific STE2 gene by using a 367 bp fragment from the STE2 5'-noncoding region to drive expression of a reporter lacZ gene. Mutation of the MCM1 binding site destroyed MCM1.alpha 2-mediated repression in alpha cells and dramatically reduced expression in a cells. The residual expression was highly stimulated by exposure of cells to pheromone. Likewise, the loss of STE12 function reduced lacZ expression driven by the wild-type STE2 fragment. In the absence of both MCM1 and STE12 functions, no residual expression was observed. Thus, the STE2 fragment appears to contain two distinct upstream activation sequences (UASs), one that is responsible for the majority of expression in cells not stimulated by pheromone, and one that is responsible for increased expression upon pheromone stimulation. In further support of this idea, a chemically synthesized version of the STE2 MCM1 binding site had UAS activity, but the activity was neither stimulated by pheromone nor reduced in ste12 mutants. Although transcription of alpha-specific genes also requires both MCM1 and STE12, these genes differ from a-specific genes in that they have a single, MCM1-dependent UAS system. The activity of the minimal 26 bp UAS from the alpha-specific STE3 gene was both stimulated by pheromone and reduced in ste12 mutants. These data suggest that at alpha-specific genes STE12 and MCM1 exert their effects through a single UAS.

The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene.

Two proteins, alpha 1 and pheromone/receptor transcription factor (PRTF), bind cooperatively to the upstream activation sequences (UAS) of yeast alpha-specific genes and thereby activate their transcription. In these protein-DNA complexes, the PRTF moiety interacts with a degenerate dyad symmetric sequence, the P box. PRTF contributes also to the regulation of a second set of cell-type-specific genes, the a-specific genes. We used two in vitro assays to show that PRTF is encoded, at least in part, by the MCM1 gene. In one assay, truncated MCM1 proteins encoded by deletion derivatives of the MCM1 gene formed protein-DNA complexes of novel mobility, demonstrated that MCM1 can bind to the P-box-containing DNA. Second, antibodies raised to a synthetic MCM1 polypeptide retard the migration of PRTF-DNA complexes in gel mobility shift assays. This result indicates that PRTF, defined as an activity that binds cooperatively with alpha 1 to alpha-specific UAS elements, shares an epitope with MCM1. In addition, we show that MCM1 deletions that remove the carboxy-terminal 129 codons of 286 total codons encode truncated MCM1 molecules that are competent to activate transcription in vivo, indicating that the carboxy-terminal residues are not required for this process.

Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes.

STE3 mRNA is present only in Saccharomyces cerevisiae alpha cells, not in a or a/alpha cells, and the transcript level increases about fivefold when cells are treated with a-factor mating pheromone. Deletions in the 5' noncoding region of STE3 defined a 43-base-pair (bp) upstream activation sequence (UAS) that can impart both modes of regulation to a CYC1-lacZ fusion when substituted for the native CYC1 UAS. UAS activity required the alpha 1 product of MAT alpha, which is known to be required for transcription of alpha-specific genes. A chromosomal deletion that removed only 14 bp of the STE3 UAS reduced STE3 transcript levels 50- to 100-fold, indicating that the UAS is essential for expression. The STE3 UAS shares a 26-bp homology with the 5' noncoding sequences of the only other known alpha-specific genes, MF alpha 1 and MF alpha 2. We view the homology as having two components--a nearly palindromic 16-bp "P box" and an adjacent 10-bp "Q box." A synthetic STE3 P box was inactive as a UAS; a perfect palindrome P box was active in all three cell types. We propose that the P box is the binding site for a transcription activator, but that alpha 1 acting via the Q box is required for this activator to bind to the imperfect P boxes of alpha-specific genes. Versions of the P box are also found upstream of a-specific genes, within the binding sites of the repressor alpha 2 encoded by MAT alpha. Thus, the products of MAT alpha may render gene expression alpha or a-specific by controlling access of the same transcription activator to its binding site, the P box.