Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family

Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences.      

Properties of natural double-strand-break sites at a recombination hotspot in Saccharomyces cerevisiae

This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3''-to-5'' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.     

Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.     

Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated with an Insertion of Telomeric Sequences Upstream of the His4 Locus in Yeast

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3'-hydroxyl groups.     

Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.     

Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae.

We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G + C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.     

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae.

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.     

The predicted presence of large helical structural variation in yeast HIS4 upstream region is correlated with general amino acid control on the CYC1 gene

A series of CYC1 constructions in which the upstream promoter portion has been replaced by a variety of HIS4 synthetic fragments has demonstrated that the 5' TGACTC 3' repeat is crucial for conferring amino acid general control. Efficient regulation, however, is obtained only with fragments containing both the repeat and flanking sequences. Analysis of the flanks shows the presence of a 16 nucleotide long sequence composed of alterations of two purines and two pyrimidines between the upstream and downstream repeats. Such a sequence has very large twist angle variations. Homologous sequence are observed in HIS1, HIS3, and in TRP5 upstream regions between copies of the repeat. Sequences which confer special structural characteristics may aid in protein recognition of the promoter region.     

Pichia pastoris as a host system for transformations.

We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P. pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P. pastoris cells.     

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.