Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.     

Expression of human papillomavirus proteins in yeast Saccharomyces cerevisiae.

The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes.     

UV-inducible proteins in Saccharomyces cerevisiae

Summary Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae. The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons. This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins. The larger (78 kD) protein is induced in various rad strains and in a ° cir° strain. Attempts are being made to isolate the genes coding for these inducible proteins.     

Chromosomal proteins in Saccharomyces cerevisiae

Summary Proteins were isolated from purified yeast chromatin and subjected to two-dimensional electrophoresis. The cellular and the chromosomal content of the major nonhistone proteins was measured. Two polypeptides of molecular weights 55,000 and 53,000, identified as and tubulin, and a polypeptide of molecular weight 63,000, associated with the nuclear DNA to a very high degree, account for nearly 50% of the nonhistone proteins present in chromatin. Only one tenth of the RNA polymerase subunit with the molecular weight of 23,000 was associated with nuclear DNA following chromatin purification in metrizamide gradients.     

Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is characterized by asymmetric cell division and the asymmetric inheritance of spindle components during normal vegetative growth and during certain specialized cell divisions. There has been a longstanding interest in the possibility that yeast chromosomes segregate non-randomly during mitosis and that some of the differences between mother and daughter cells could be explained by selective chromatid segregation. This review traces the history of the experiments to determine if there is biased chromatid segregation in yeast. The special aspects of spindle morphogenesis and behavior in yeast that could accommodate a mechanism for biased segregation are discussed. Finally, a recent experiment demonstrated that yeast chromatids segregate randomly without mother–daughter bias in a common laboratory strain grown under routine laboratory conditions.     

Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose.     

Chromium resistant mutants of the yeast Saccharomyces cerevisiae

Summary Many strains of Saccharomyces cerevisiae do not grow on YPD agar containing 750 g/ml CrO₃. Mutants able to grow in the presence of 850 g/ml CrO₃ were obtained from such strains after UV mutagenesis. All of the mutants grew even in the presence of 1,000 /ml CrO₃. Chromium resistance was dominant or partial dominant over normal response, therefore it was impossible to determine the number of genetic loci by complementation analysis. However, the segregation of representative mutants strongly indicated that resistance was determined by single mutations. In addition, a limited analysis of recombination suggested that the chromium resistant mutations were located on a certain region of the yeast genome. Although it was determined that the mutants had slightly reduced rates of Cr⁶⁺ uptake, the exact mechanism of resistance was not discovered. According to the studies of interactions between resistant mutations and sensitive mutations, however, we have proposed a preliminary pathway of Cr⁶⁺ detoxification.     

Palindrome content of the yeast Saccharomyces cerevisiae genome

Palindromic sequences are important DNA motifs involved in the regulation of different cellular processes, but are also a potential source of genetic instability. In order to initiate a systematic study of palindromes at the whole genome level, we developed a computer program that can identify, locate and count palindromes in a given sequence in a strictly defined way. All palindromes, defined as identical inverted repeats without spacer DNA, can be analyzed and sorted according to their size, frequency, GC content or alphabetically. This program was then used to prepare a catalog of all palindromes present in the chromosomal DNA of the yeast Saccharomyces cerevisiae. For each palindrome size, the observed palindrome counts were significantly different from those in the randomly generated equivalents of the yeast genome. However, while the short palindromes (2–12 bp) were under-represented, the palindromes longer than 12 bp were over-represented, AT-rich and preferentially located in the intergenic regions. The 44-bp palindrome found between the genes CDC53 and LYS21 on chromosome IV was the longest palindrome identified and contained only two C-G base pairs. Avoidance of coding regions was also observed for palindromes of 4–12 bp, but was less pronounced. Dinucleotide analysis indicated a strong bias against palindromic dinucleotides that could explain the observed short palindrome avoidance. We discuss some possible mechanisms that may influence the evolutionary dynamics of palindromic sequences in the yeast genome.     

Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae

Summary Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glyccrol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

Investigation of Batten disease with the yeast Saccharomyces cerevisiae

The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.     

Trehalases from spores and vegetative cells of yeast Saccharomyces cerevisiae

Trehalase (THA) activity from S. cerevisiae spores and vegetative cells could be differentiated in cell-free extracts. THA from the vegetative cells has an optimal activity at neutral pH whereas biphase pH optimum in the spores was observed. The enzyme from the spores exhibited higher thermostability than that from the vegetative cells. The presence of magnesium ions was necessary mainly for THA activity from the vegetative cells. The effect of the other metal ions studied: Hg²⁺, Ag²⁺, Cu²⁺, Fe³⁺, Ni²⁺, Cd²⁺ etc. (Table II), on THA from both sources was almost the same, however, the spores THA was resistant to Pb²⁺ and especially to Zn²⁺. Moreover, the influence of inorganic polyphosphates and polyamines was also quite dissimilar. Polyphosphates inhibited THA from the vegetative cells and to a smaller extent from the spores. On the other hand, polyamines stimulated highly THA activity from vegetative yeast cells in contrast to spores one. The effect of these ions modulators would facilitate differentiating of THA activity in the cell-free extracts from both sources. These data could be interpreted as phenotypic reflections of trehalase genes expression in the S. cerevisiae cells.     

Localization of telomeres and telomere-associated proteins in telomerase-negative Saccharomyces cerevisiae

Cells lacking telomerase cannot maintain their telomeres and undergo a telomere erosion phase leading to senescence and crisis in which most cells become nonviable. On rare occasions survivors emerge from these cultures that maintain their telomeres in alternative ways. The movement of five marked telomeres in Saccharomyces cerevisiae was followed in wild-type cells and through erosion, senescence/crisis and eventual survival in telomerase-negative (est2::HYG) yeast cells. It was found that during erosion, movements of telomeres in est2::HYG cells were indistinguishable from wild-type telomere movements. At senescence/crisis, however, most cells were in G₂ arrest and the nucleus and telomeres traversed back and forth across the bud neck, presumably until cell death. Type I survivors, using subtelomeric Y′ amplification for telomere maintenance, continued to show this aberrant telomere movement. However, Type II survivors, maintaining telomeres by a sudden elongation of the telomere repeats, became indistinguishable from wild-type cells, consistent with growth properties of the two types of survivors. When telomere-associated proteins Sir2p, Sir3p and Rap1p were tagged, the same general trend was seen—Type I survivors retained the senescence/crisis state of protein localization, while Type II survivors were restored to wild type. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enzyme-substitution therapy with the yeast Saccharomyces cerevisiae in congenital sucrase-isomaltase deficiency

Sucrase-isomaltase deficiency is an inherited disaccharidase deficiency that leads to malabsorption of sucrose, with resulting diarrhea and abdominal distention and cramps. We investigated the sucrose-splitting effect of viable yeast cells in eight children with congenital sucrase-isomaltase deficiency, by means of the sucrose hydrogen breath test. This test is based on the fact that hydrogen is released from the malabsorbed sucrose by the colonic microflora. We found that 0.3 g of lyophilized Saccharomyces cerevisiae, given after loading with 2 g of sucrose per kilogram of body weight, reduced hydrogen excretion in all patients, on average by 70 percent, in parallel with a complete loss or evident reduction of clinical symptoms. In vitro, lyophilized and fresh S. cerevisiae (fresh baker's yeast) had appreciable sucrase activity, a low isomaltase and maltase activity, and virtually no lactase activity. The sucrase activity was more inhibited by undiluted than by diluted gastric juice. We conclude that patients with congenital sucrase-isomaltase deficiency who intentionally or unintentionally consume sucrose can ameliorate the malabsorption by subsequently ingesting a small amount of viable yeast cells, preferably on a full stomach.     

Electrophoretical and immunological comparison of the ribosomal proteins of the fungus Podospora anserina and the yeast Saccharomyces cerevisiae

Summary The ribosomal proteins of two ascomycetes Podospora anserina and Saccharomyces cerevisiae, were compared by two dimensional electrophoresis in two different gel systems and we found only five pairs of proteins which have kept homologous physico-chemical properties under these conditions. An immunological analysis was performed by radioimmunodetection of proteins blotted on nitrocellulose sheet after separation by electrophoresis, with four sera directed against the r-proteins of each subunit of these fungi. So, we pointed out many common antigenic sites present on proteins which do not co-migrate except for yeast L3 and L1 of P. anserina which have the same properties.     

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae

Summary For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step start. The threshold amount of cell mass (reflected as a critical size) necessary for start is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at start, termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for start, causing the smaller cells, produced in the previous cell cycle, to be delayed at start while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and start, nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.     

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae

Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.