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Gene products encoded by the major histocompatibility complex often exhibit a high degree of polymorphism. In humans the HLA-DR polymorphism is due to more than 50 alleles with varying exon 2 sequences. Each group of DRB alleles contains a certain form of the basic simple repeat motif (gt)n(ga)m in intron 2. Identical alleles can be differentiated on the basis of the hypervariable repeat. In this study focused on cattle (Bos taurus) we identified different Bota-DRB alleles in a limited survey by amplification via polymerase chain reaction and sequencing. In addition DRB exon 2 sequences were also obtained from eight additional hoofed animal species (seven horned artiodactyls and one pig) revealing artiodactyl-specific polymorphic and nonpolymorphic substitutions. In the genus Bos the intronic simple repeat variability was compared with exonic DRB polymorphism. As in humans all Bota-DRB exons were always associated with specifically organized basic simple repeat structures. Yet the extent of simple repeat variability was lower in cattle compared to humans. Selective breeding in the process of domestication might be responsible for the diminished intronic hypervariability. Nevertheless, the hypermutable simple repeat sequences have been preserved in the same position and with the same principal structure for at least 70 x 10(6) years of evolution. Unexpectedly, the rate of intronic simple repeat and exonic changes appear quite similar.

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A number of cell surface glycoproteins can be specifically and completely released from intact cells of Saccharomyces cerevisiae with 0.5% mercaptoethanol. Among these proteins is one with a molecular mass of 22 kDa, which is synthesized only in haploid a cells treated with the peptide mating pheromone a factor. This protein could be radiolabeled in vivo with [2-3H] Saccharomyces cerevisiae mating pheromone a factor causes a first-cycle growth arrest in G1 when added to haploid a cells (1). In addition, the shape of the cells changes ("shmoo" formation), as does their cell surface, thereby increasing agglutinability with a cells, their mating partners (2-4).So far, few biochemical changes correlating with a-factor treatment have been reported. Thus, the overall cell wall composition changes (5), and a significant increase in the amount of chitin synthesized (6, 7) and in chitin synthase activity (6) is observed.Stetler and Thorner (8) have presented evidence that transcription of a number of genes is turned off, or in some cases on, after a factor administration. However, corresponding proteins have not been described up to now.It will be shown here that a protein of22 kDa is synthesized in S. cerevisiae a cells only when the cells are treated with a factor. This protein is solely O-glycosylated; it can be quantitatively removed from intact cells under mild conditions. Since the protein inhibits agglutination ofa with a cells, it most likely is the agglutinin of a cells. Radiolabeling was continued for 90-120 min, incorporation of the radioactivity being continuous under these conditions, and then cells were harvested by centrifugation. MATERIALS AND METHODSExtraction of Cell Surface Glycoproteins. Cells from radiolabeled cultures were washed twice with 2 mM Tris HCl (pH 8.0) and transferred to 1.5-ml capped Microfuge tubes. The cells were resuspended in 2 mM Tris HCl (pH 8.0) containing 0.5% 2-mercaptoethanol, and the tubes were shaken vigorously and routinely for 2 hr at room temperature with an Eppendorf mixer. Then cells were sedimented by centrifugation, the -supernatant was collected, and the cells were washed once with 0.2 ml of Tris HCl (pH 8.0). The supernatants (mercaptoethanol extract) were pooled, NaDodSO4/ PAGE sample buffer was added, and the extract was concentrated in a Speed-Vac concentrator (Savant). After mercaptoethanol treatment, 0.25 ml of 20 mM Tris HCl (pH 7.4) containing 10 ,ug of zymolyase 60,000 (Miles) per ml was added to the intact cells remaining after extraction with mercaptoethanol, and the suspension was shaken for 7 min at room temperature before centrifugation for 2 min. Few, if any, yeast cells are lysed by this treatment. NaDodSO4/ PAGE sample buffer was added to the supernatant, and this zymolyase-released material was concentrated. The amount of radioactivity in the fractions was determined, and samples of the fractions were submitted to NaDodSO4/PAGE (10) on gels containing 10% or 12% acrylamide. Radiolabeled bands were detected by fluorography. To quantify the rel...

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYP1 gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.