Green fluorescent protein as a convenient and versatile marker for studies on functional genomics in Drosophila

Gene function can be deduced from lack or gain of activity. For the manipulation of gene doses or activity in Drosophila, a set of P-based vectors was constructed containing green fluorescent protein as marker. pBLUEi, pGREENi and pYELLi were designed for large insert transformation. Mosaicism was generated in vivo with pFlipG which is also ideal for targeted gene disruption. Tissue-specific gene silencing in vivo was performed with the vector set pHIBS and pUdsGFP. pHIBS allows easy cloning and shuttling of double-headed constructs. With pUdsGFP, double stranded RNA can be produced in defined patterns and the area of interference simultaneously visualized by green fluorescence. We demonstrate nearly complete silencing of a ubiquitously expressed gene in a tissue-specific manner.     

Epigenomics: genome-wide study of methylation phenomena

Epigenetics is one of the key areas of future research that can elucidate how genomes work. It combines genetics and the environment to address complex biological systems such as the plasticity of our genome. While all nucleated human cells carry the same genome, they express different genes at different times. Much of this is governed by epigenetic changes resulting in differential methylation of our genome--or different epigenomes. Individual studies over the past decades have already established the involvement of DNA methylation in imprinting, gene regulation, chromatin structure, genome stability and disease, especially cancer. Now, in the wake of the Human Genome Project (HGP), epigenetic phenomena can be studied genome-wide and are giving rise to a new field, epigenomics. Here, we review the current and future potential of this field and introduce the pilot study towards the Human Epigenome Project (HEP).     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

Tumour class prediction and discovery by microarray-based DNA methylation analysis

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.     

Dermatophyte infection and colonization in dogs from South Santiago, Chile

Our main aim was to determine the dermatophyte infection and colonization prevalence in canines from South Santiago, Chile. We studied 241 dogs, 121 of them presented cutaneous lesions suggestive of dermatophytosis and the other 120 were free from lesions and were considered clinically healthy. Dermatophytes were isolated from the lesions of 48.8% of animals with cutaneous diseases and from 5% of healthy dogs. Microsporum canis was the principal species isolated with a frequency of 98.3% from dermatophytosis and from all healthy carriers. The direct microscopic test showed a sensibility of 85% with a positive and negative predictive value of 74 and 79%, respectively. The highest prevalence of dermatophytosis were detected in animals up to one year old (p <= 0.05) and in dogs with high level of irritation, scaly skin, itching and alopecia (p < or = 0.05). The lesions were detected more frequently in head-neck and anterior members (p < or = 0.05). This study shows some clinical characteristics and a frequency of dermatophytes in canines that can be interesting for laboratory professionals and veterinarians.     

Availability and use of molecular hydrogen as an energy substrate for Helicobacter species

Molecular hydrogen is produced in the large intestine of animals due to the fermentation reactions of sugar catabolism. The gastric pathogen Helicobacter pylori and the liver pathogen Helicobacter hepaticus have the capacity to use molecular hydrogen as a respiratory substrate. The amount of the gas within tissues colonized by these pathogens is ample, and use of H2 significantly increases the stomach colonization ability of H. pylori.