Showing papers by "Marcela Savoldi published in 2004"

Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis.

Abstract: Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37°C and 26°C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (α-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.

The Aspergillus nidulans npkA Gene Encodes a Cdc2-Related Kinase That Genetically Interacts With the UvsBATR Kinase

Abstract: The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity. We have used Aspergillus nidulans as a model system to characterize the DNA damage response caused by the antitopoisomerase I drug, camptothecin. We report the molecular characterization of a p34Cdc2-related gene, npkA , from A. nidulans . The npkA gene is transcriptionally induced by camptothecin and other DNA-damaging agents, and its induction in the presence of camptothecin is dependent on the uvsB ATR gene. There were no growth defects, changes in developmental patterns, increased sensitivity to DNA-damaging agents, or effects on septation or growth rate in the A. nidulans npkA deletion strain. However, the Δ npkA mutation can partially suppress HU sensitivity caused by the Δ uvsB ATR and uvsD153 ATRIP checkpoint mutations. We demonstrated that the A. nidulans uvsB ATR gene is involved in DNA replication and the intra-S-phase checkpoints and that the Δ npkA mutation can suppress its intra-S-phase checkpoint deficiency. There is a defect in both the intra-S-phase and DNA replication checkpoints due to the npkA inactivation when DNA replication is slowed at 6 mm HU. Our results suggest that the npkA gene plays a role in cell cycle progression during S-phase as well as in a DNA damage signal transduction pathway in A. nidulans .

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

Abstract: The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.