Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes.

BACKGROUND: The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily. RESULTS: Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 gamma-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily. CONCLUSIONS: This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.

Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine.

Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5'-nuclease activity of Taq DNA polymerase, were developed for screening viral variants in lamivudine-treated patients' sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients' sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine(552) aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients' sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5'-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5'-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.

Concomitant chemo/radiotherapy for advanced carcinoma of the head and neck.

Giving chemotherapy and radiotherapy simultaneously (concomitant therapy) is one approach to improving results in advanced head and neck cancer. To assess the feasibility of one such regimen, 25 patients with advanced squamous carcinoma of the head and neck were treated with a continuous intravenous infusion of 5-fluorouracil, 1 g/m2 per 24 h for Days 1-5 (105 h) and mitomycin-C 14 mg/m2 intravenously on Day 3 during the first week of radiotherapy. Twenty had Stage IV disease; four Stage III; and one Stage II. Ages ranged from 21 to 73 years (median 60 years). The tumours involved were as follows: oral cavity (6); nasopharynx (8); oropharynx (5); secondary node from unknown primary (3); hypopharynx (2); paranasal sinus (1). Radiotherapy was delivered as 10 Gy per week (total dose 60-70 Gy). Chemotherapy was well tolerated and all received the intended dose. Mild nausea occurred in five patients and three experienced transient vomiting. A generalized "early" mucositis affected 16 out of 25 (64%), caused interruption of radiotherapy in three patients, and is thought to be chemotherapy related. Twenty-two patients received the dose of radiotherapy intended, and two stopped prematurely at 53 and 56 Gy. Three episodes of neutropaenic infection occurred. Two recovered uneventfully, but one toxic death occurred in a patient with alcoholic cirrhosis. A complete response was seen in 21 (84%). For 17 patients with non-nasopharyngeal carcinoma the 2-year survival is 40%, 24% disease free. The concomitant use of 5-fluorouracil, mitomycin and radiotherapy is well tolerated in this group of patients.