Assessment of infection with polyomaviruses BKV, JCV and SV40 in different groups of Cuban individuals.

We investigated the frequency of BKV, JCV and SV40 reactivation in three groups of Cuban patients by multiplex nested PCR assay of 40 paraffin-embedded colorectal neoplasm tissues, 113 urine samples, and 125 plasma samples from 27 transplant recipients, and cerebrospinal fluid (CSF) from 67 HIV-1-infected individuals with central nervous system (CNS) disorders. None of these polyomaviruses were detected in colorectal neoplasms. JCV DNA was detected in 2 of 67 patients (2.9%) with CNS disorders, but neither BKV nor SV40 was identified. BKV was found in urine from 38.5% and 28.6% of adult and pediatric transplant recipients, respectively. In adult renal transplant recipients, excretion of BKV in urine was significantly associated with episodes of acute rejection (p=0.012) and with excretion of HCMV in urine (p= 0.008). In Cuba, the polyomaviruses studied here could not be related to colorectal neoplasms, and JCV was rarely detected in CSFs of HIV-1-infected individuals, whilst BKV reactivation was found to occur frequently in organ transplant recipients.

Assessment of immunological competence and SV40 specific recall immunity in malignant pleural mesothelioma.

Diffuse malignant pleural mesothelioma (MPM) is the third most common lung malignancy showing rising incidence with 250,000 deaths expected from it in Western Europe over the next 35 year. The tumour is generally resistant to conventional treatment and there is urgent need for novel preventative and therapeutic measures to combat this growing public threat. Finding of SV40 DNA sequences in a high proportion (40-90%) of several series of MPM cases, and suggestion of its potential co-carcinogen role provide a rationale for the development of novel anti-MPM vaccines incorporating SV40 gene sequences or antigenic determinants. As a prelude to adopting this approach, general T cell function was examined in relatively early cases of MPM presenting for biopsy or debulking surgery. CD8+ T cell responses were studied using antigenic epitopes of common viral antigens covering a broad range of haplotypes. 74.1% (20/27) of MPM patients and 80% (8/10) of the control subjects showed T cell responsiveness to the viral peptides mix, whilst a small proportion showed SV40 specific recall immunity.

Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.

A statistical model for locating regulatory regions in genomic DNA.

In addition to genes, chromosomal DNA contains sequences that serve as signals for turning on and off gene expression. These signals are thought to be distributed as clusters in the regulatory regions of genes. We develop a Bayesian model that views locating regulatory regions in genomic DNA as a change-point problem, with the beginning of regulatory and non-regulatory regions corresponding to the change points. The model is based on a hidden Markov chain. The data consist of nucleotide positions of protein-binding elements in a genomic DNA sequence. These positions are identified using a reference catalogue containing elements that interact with transcription factors implicated in controlling the expression of protein-encoding genes. Among the protein-binding elements in a genomic DNA sequence, the statistical model automatically selects those that tend to predict regulatory regions. We test the model using viral sequences that include known regulatory regions and provide the results obtained for human genomic DNA corresponding to the beta globin locus on chromosome 11.

Assessment of liposomal transfection of ocular tissues in vivo.

A possible route to the treatment of inherited retinal degenerations such as retinitis pigmentosa (RP) is the application of somatic gene therapy by the transfer and expression of corrective functional genes in ocular tissue. Cationic liposomes are established vehicles for the delivery and expression of exogenous genes in mammalian cells both in vitro and in vivo1. We report here a preliminary assessment of liposome-mediated transfer of a plasmid carrying the reporter gene lacZ (encoding the enzyme beta-galactosidase) into tissues of the adult rabbit eye.

In vivo assessment of the Z-DNA-forming potential of d(CA.GT)n and d(CG.GC)n sequences cloned into SV40 minichromosomes.

Alternating repeated d(CA.GT)n and d(CG.GC)n sequences constitute a significant proportion of the simple repeating elements found in eukaryotic genomic DNA. These sequences are known to form left-handed Z-DNA in vitro. In this paper, we have addressed the question of the in vivo determination of the Z-DNA-forming potential of such sequences in eukaryotic chromatin. For this purpose, we have investigated the ability of a d(CA.GT)30 sequence and a d(CG.GC)5 sequence to form left-handed Z-DNA when cloned into simian virus 40 (SV40) minichromosomes at two different positions: the TaqI site, which occurs in the intron of the T-antigen gene, and the HpaII site, which is located in the late promoter region within the SV40 control region. Formation of Z-DNA at the inserted repeated sequences was analyzed through the change in DNA linkage associated with the B to Z transition. Our results indicate that regardless of: (1) the site of insertion (either TaqI or HpaII), (2) the precise moment of the viral lytic cycle (from 12 h to 48 h postinfection) and (3) the condition of incorporation of the SV40 recombinants to the host cells (either as minichromosomes or as naked DNA, relaxed or negatively supercoiled), neither the d(CA.GT)30 nor the d(CG.GC)5 sequence are stable in the left-handed Z-DNA conformation in the SV40 minichromosome. The biological relevance of these results is discussed.

On the distribution of the nucleotides in seven completely sequenced DNAs.

Several Markov chain models (up to fourth order) have been fitted to the sequences of the seven DNAs presented in Fuchs et al. (1980). Two methods for determining the order of Markov chain are applied to the data. The two methods lead to different conclusions and we discuss these discrepancies. When the distribution of the nucleotides in a DNA sequence is investigated, it is suggested that the study on the order of the Markov model should be supplemented with additional analysis.