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

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.

Electrophoretic molecular karyotype of the dermatophyte Trichophyton rubrum

The electrophoretic karyotype of the dermatophyte Trichophyton rubrum was established using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Five chromosomal bands of approximately 3.0 to 5.8 megabase pairs (Mbp) each were observed and together indicated that 22.05 Mbp of the total genome are organized as chromosomal macromolecules. In addition to establishing the number and size of T. rubrum chromosomes, these results open perspectives for the construction of chromosome-specific libraries and for the physical mapping of genes of interest, thus permitting future gene linkage studies in this pathogen. A detailed understanding of the karyotype and genomic organization of T. rubrum should contribute to further genetic, taxonomic and epidemiological studies of this dermatophyte.

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium alteredduring cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling.

18S-rDNA Sequencing, enzyme patterns and morphological characterization of trichophyton isolates

Dermatophytes, capable to use keratin of the host for nutrition, belong to one of the major groups of pathogenic fungi. Since dermatophytes are a closely related group they share various common features, and the morphology of isolates of a given species can be atypical, making species identification and differentiation even more difficult. Many methods have been explored in attempts to distinguish dermatophytes, but the combined use of different approaches for the investigation of the intraspecific and interspecific variability of Trichophyton continues to be scarce. Some studies have shown that amplified fragments of the small ribosomal DNA subunit 18S contains variable regions which can be used to discriminate between medically relevant yeast species, indicating that these regions could also be used for differentiation between dermatophytes. In our study, sequence analysis of the 18S-rDNA gene was combined with morphological and biochemical criteria in order to detect genetic differences between seven Trichophyton isolates and estimate their phylogenetic relationships. The results show that the isolates investigated belong to the Trichophyton group, which potentially contains the Trichophyton rubrum cluster.

The nucleation of microtubules in Aspergillus nidulans germlings

Microtúbulos säo filamentos compostos por dímeros das tubulinas alfa e beta e têm uma variedade de funçöes nas células vivas. Em fungos, os corpúsculos polares dos fusos säo geralmente considerados os centros organizadores dos microtúbulos. Com o objetivo de contribuir para uma melhor compreensäo dos processos de nucleaçäo dos microtúbulos no fungo filamentoso A. nidulans, nós utilizamos a droga antimicrotúbulo Benomil em experimentos de bloqueio e liberaçäo para depolimerizar e repolimerizar os microtúbulos. Após 20 segundos de reincubaçäo em meio sem Benomil, pequenos microtúbulos foram formados a partir de pontos distribuídos pela célula, sugerindo que os pontos de nucleaçäo de microtúbulos säo aleatoriamente distribuídos pelas hifas de A. nidulans. Como em A. nidulans o movimento nuclear é dependente de microtúbulos foi analisado se mutantes defectivos na distribuiçäo de núcleos ao longo das hifas (mutantes nud) possuíam algum defeito evidente nos microtúbulos. Os microtúbulos citoplasmáticos, dos fusos e astrais estäo presentes e aparentam-se normais em todos os mutantes nud, mas foi observada uma pequena distorçäo na proporçäo de fusos mitóticos longos e curtos nestes mutantes, comparados com o controle. Isto sugere que alguns núcleos de mutantes nud näo alcançam a fase tardia da divisäo celular, em temperatura näo restritiva.