Frank Diehl

Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray

A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.

Human Papillomavirus Type 16 E6 Promotes Retinoblastoma Protein Phosphorylation and Cell Cycle Progression

ABSTRACT We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G1/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16INK4a, CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16INK4a. The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21WAF1/CIP1 were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21WAF1/CIP1 gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21WAF1/CIP1 levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G1 arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16INK4a) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.

Expression profiling of cervical cancer biopsies using high-resolution dual-label imaging of 33 P and 3 H on microarrays

Expression profiling of cervical cancer biopsies using high-resolution dual-label imaging of 33 P and 3 H on microarrays

Improving DNA-Chip Technology

DNA-microarrays have become a synonym for the type of analyses that aim at the understanding of cellular functioning in a comprehensive manner. Although still in a relatively early phase, the methodology has already proven its worth. Nevertheless, improvements on various aspects are necessary for making DNA-chips a routine tool in research and diagnostics. In this manuscript, some chemical developments toward this end are being reviewed.