Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the b-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.

Mutation accumulation in Tetrahymena

Background - The rate and fitness effects of mutations are key in understanding the evolution of every species. Traditionally, these parameters are estimated in mutation accumulation experiments where replicate lines are propagated in conditions that allow mutations to randomly accumulate without the purging effect of natural selection. These experiments have been performed with many model organisms but we still lack empirical estimates of the rate and effects of mutation in the protists. Results - We performed a mutation accumulation (MA) experiment in Tetrahymena thermophila, a species that can reproduce sexually and asexually in nature, and measured both the mean decline and variance increase in fitness of 20 lines. The results obtained with T. thermophila were compared with T. pyriformis that is an obligate asexual species. We show that MA lines of T. thermophila go to extinction at a rate of 1.25 clonal extinctions per bottleneck. In contrast, populations of T. pyriformis show a much higher resistance to extinction. Variation in gene copy number is likely to be a key factor in explaining these results, and indeed we show that T. pyriformis has a higher mean copy number per cell than T. thermophila. From fitness measurements during the MA experiment, we infer a rate of mutation to copy number variation of 0.0333 per haploid MAC genome of T. thermophila and a mean effect against copy number variation of 0.16. A strong effect of population size in the rate of fitness decline was also found, consistent with the increased power of natural selection. Conclusions - The rate of clonal extinction measured for T. thermophila is characteristic of a mutational degradation and suggests that this species must undergo sexual reproduction to avoid the deleterious effects detected in the laboratory experiments. We also suggest that an increase in chromosomal copy number associated with the phenotypic assortment of amitotic divisions can provide an alternative mechanism to escape the deleterious effect of random chromosomal copy number variation in species like T. pyriformis that lack the resetting mechanism of sexual reproduction. Our results are relevant to the understanding of cell line longevity and senescence in ciliates.

Relationship of PON1 192 and 55 gene polymorphisms to calcific valvular aortic stenosis

Introduction and Objectives - Paraoxonases may exert anti-atherogenic action by reducing lipid peroxidation. Previous studies examined associations between polymorphisms in the paraoxonase 1 (PON1) gene and development of coronary artery disease (CAD), with inconsistent results. Given the similarities in clinical and pathophysiological risk factors of CAD and calcific aortic valve stenosis (CAVS), we postulated a link between PON1 alleles and CAVS progression. Methods - We investigated the association between PON1 55 and 192 single nucleotide polymorphisms (SNPs), their enzyme activity, and CAVS progression assessed by aortic valve area and transvalvular peak velocity in 67 consecutive patients with moderate CAVS and 251 healthy controls. Results - PON1 paraoxonase activity was higher in CAVS patients (P<0.001). The PON1 genotype Q192R SNP (P=0.03) and variant allele (R192) (P=0.01) frequencies differed between CAVS patients and controls. Significant association existed between PON1 enzyme activity, phenotypic effects of PON1 192 genotype polymorphisms, and CAVS progression, but not between PON1 55 and high-density lipoprotein (P=0.44) or low-density lipoprotein cholesterol (P=0.12), between 192 genotype and high-density lipoprotein (P=0.24) or low-density lipoprotein cholesterol (P=0.52). Conclusion - The PON1 genotype Q192R SNP has an important effect on CAVS disease progression. This study helps outline a genotype-phenotype relationship for PON1 in this unique population.

Hélices e Espirais em Micro e Nanofibras Celulósicas

A celulose é o polímero renovável mais abundante do mundo. É conhecido pela sua excelente biocompatibilidade, propriedades térmicas e mecânicas. A celulose assim como os polipéptideos e o ADN, pertence a uma família de moléculas orgânicas que dão origem à formação de fases líquidas cristalinas (LCs) colestéricas. A Passiflora Edulis, tal como outras plantas trepadeiras, possui longas e flexíveis gavinhas que permitem à planta encontrar um suporte para se fixar. As gavinhas podem assumir a forma de espirais ou de hélices consoante sejam sustentadas por apenas uma ou por ambas as extremidades. As hélices apresentam muitas vezes duas porções helicoidais, uma esquerda e outra direita, separadas por um segmento recto denominado perversão. Este comportamento é consequência da curvatura intrínseca das gavinhas produzidas pela planta trepadeira. O mesmo comportamento pode ser observado em micro e nanofibras celulósicas fabricadas a partir de soluções líquido-cristalinas, numa escala três a quatro ordens de grandeza inferior à das gavinhas. Este facto sugere que o modelo físico utilizado tenha invariância de escala. Neste trabalho é feito o estudo de fibras e jactos que imitam as estruturas helicoidais apresentadas pelas gavinhas das plantas trepadeiras. As fibras e jactos são produzidos a partir de soluções líquidas cristalinas celulósicas. De modo a determinar as características morfológicas e estruturais, que contribuem para a curvatura das fibras, foram utilizadas técnicas de imagem por ressonância magnética (MRI), microscopia óptica com luz polarisada (MOP), microscopia electrónica de varrimento (SEM) e microscopia de força atómica (AFM) . A variação da forma das estruturas helicoidais com a temperatura parece ser relevante para o fabrico de membranas não tecidas para aplicação em sensores termo-mecânicos.