Non-random distribution of the Ty1 elements within nuclear DNA of Saccharomyces cerevisiae

Ty1 homologous sequences appear to be non-randomly distributed among different density classes of nuclear yeast DNA. Characteristic patterns of Ty1 containing EcoRI fragments can be generated from the various DNA fractions. The sequences are particularly enriched in the A + T rich part of the main nuclear DNA fraction, while the frequency in the rDNA containing heavy satellite DNA is low. The transposon however, seems to be present in this dense fraction, at least for some strains.     

The genomic RNA in Ty1 virus-like particles is dimeric

The yeast retrotransposon Ty1 resembles retroviruses in a number of important respects but also shows several fundamental differences from them. We now report that, as in retroviruses, the genomic RNA in Ty1 virus-like particles is dimeric. The Ty1 dimers also resemble retroviral dimers in that they are stabilized during the proteolytic maturation of the particle. The stabilization of the dimer suggests that one of the cleavage products of TyA1 possesses nucleic acid chaperone activity.     

Phylogenetic classification of transporters and other membrane proteins from Saccharomyces cerevisiae

On the basis of functional and phylogenetic criteria, we have identified a total of 229 subfamilies and 111 singletons predicted to carry out transport or other membrane functions in Saccharomyces cerevisiae. We have extended the Transporter Classification (TC) and created a Membrane Classification (MC) for non-transporter membrane proteins. Using the preliminary phylogenetic digits X, Y, Z (for new families, subfamilies, and clusters, respectively), we allocated a five-digit number to 850 proteins predicted to contain more than two transmembrane domains. Compared with a previous TC of the yeast genome, we classified an additional set of 538 membrane proteins (transporters and non-transporters) and identified 111 novel phylogenetic subfamilies. Electronic Publication     

Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae.

The mode of replication of the L double-stranded RNA (dsRNA) present in virus-like particles in Saccharomyces cerevisiae was examined by density transfer experiments. After transfer to light medium, significant amounts of fully heavy dsRNA persisted over a number of cell doublings. In addition, very little material of hybrid density was ever formed, and the accumulation of fully light material began as early as 0.5 doubling after transfer to light medium. Our results are compatible with a conservative mode of replication or with a semiconservative mode of replication carried out by a small portion of the total dsRNA population. In additional experiments the synthesis of dsRNA relative to the cell cycle was studied. This was done by determining the ratio of short-term to long-term radioactive label in size-separated cell fractions of a prelabeled exponential culture. The ratio of short-term to long-term label remained constant for all fractions, implying that dsRNA is synthesized throughout the cell cycle, increasing through the cell cycle at an exponential rate.     

Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris

Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.     

The effect of a-factor on hybrid formation by protoplast fusion in Saccharomyces cerevisiae

Abstract a_˜-Factor, unlike α-factor, does not significantly enhance hybrid formation by protoplast fusion in the yeast Saccharomyces cerevisiae . When Mat α cells are treated with a-factor prior to being proto-plasted and fused, the frequency of hybrid formation is only slightly increased over unarrested controls.     

Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.     

Study of cell-mediated response in mice by HPV16 L1 virus-like particles expressed in Saccharomyces cerevisiae

The first vaccine against human papillomaviruses (HPV) formulated with HPV16 L1 virus-like particles (VLPs) produced in yeast was approved by the FDA in June 2006. Nevertheless, there have been few studies of the immunogenicity in mice of VLPs. In this study, we evaluated the cell-mediated immune response to VLPs produced in Saccharomyces cerevisiae. After immunization of mice with HPV16 L1 VLPs, we measured splenocytes proliferation and the levels of IFNgamma, IL2, IL4, and IL5. Splenocytes proliferation was significantly increased and a mixed Th1/Th2 response was indicated. IgG subtype immunoresponses were strongly induced and IgG1 titers were higher than those of IgG2a.     

The production and immunogenicity of human papillomavirus type 58 virus-like particles produced in Saccharomyces cerevisiae

Human papillomavirus (HPV) is the cause of most cases of cervical cancer. HPV type 58 (HPV58) is the second most frequent cause of cervical cancer and high-grade squamous intraepithelial lesions (HSIL) in Asia and South / Central America, respectively. However, there is no vaccine against HPV58, although there are commercially available vaccines against HPV16 and 18. In this study, we produced HPV58 L1 protein from Saccharomyces cerevisiae, and investigated its immunogenicity. We first determined the optimum period of culture for obtaining HPV58 L1. We found that a considerable portion of the HPV58 L1 resulting from 48 h culture cannot be recovered by purification, while the HPV58 L1 resulting from 144 h culture is recovered efficiently: the yield of HPV58 L1 finally recovered from 144 h culture was 2.3 times higher than that from 48 h culture, although the production level of L1 protein from 144 h culture was lower than that from 48 h culture. These results indicate that the proportion of functional L1 protein from 144 h-cultured cells is significantly higher than that of 48 h-cultured cells. The HPV58 L1 purified from the 144 h culture was correctly assembled into structures similar to naturally occurring HPV virions. Immunization with the HPV58 L1 efficiently elicited anti-HPV58 neutralizing antibodies and antigen-specific CD4+ and CD8+ T cell proliferations, without the need for adjuvant. Our findings provide a convenient method for obtaining substantial amounts of highly immunogenic HPV58 L1 from S. cerevisiae.