Analysis of the Transcripts of Bovine Papillomavirus Type 4

The transcripts of bovine papillomavirus type 4 (BPV4) were analysed through cDNA cloning and sequencing. Two cDNA's of particular interest were analysed. cDNA 7E11 was found to represent the 1 kb major transcript of BPV4. Primer extension and S1 nuclease protection mapping showed that this transcript had a multiple initiation site within the E6 orf region, with the major start site at nt870. cDNA and RNA sequencing showed that the transcript was spliced from nt1016 to nt3376. The polyadenylated 3' terminus was at nt4034, defining the early polyadenylation site of BPV4. Sequencing of cDNA's and RNA revealed alterations to the published genomic sequence (Patel et al., 1987) which changed the genomic organisation of BPV4. cDNA 7E11 was shown to encode a fusion orf in which the E4 orf was linked to an upstream ATG codon. Thus, as in other papillomaviruses, the major transcript of BPV4 encodes one of the E4 proteins (Doorbar et al., 1986). Orf homology analysis revealed that the BPV4 E4 orf is most like those of the carcinogenic HPV's 16, 18, 33 and 8. Since the E4 proteins are thought to be involved in the subversion of normal keratinocyte differentiation, this may be a significant finding. In addition, rare transcripts represented by cDNA Q were mapped by RNA sequencing and S1 nuclease protection. A 1kb transcript, which may be spliced, extends 5' to nt2834 and minor 5' termini at nt3063, nt3093 and nt3151 may have represented transcripts encoding the putative E2 repressor of BPV4

Application of <it>in situ </it>reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays

<p>Abstract</p> <p>Detection of disease-associated gene transcripts in primary disease tissues is frequently confounded by the presence of non-involved cell types. Alternative methods of detecting gene expression directly within tissues involve either the generation of antibodies, which can be a lengthy process and may suffer from lack of specificity, or amplification of reverse-transcribed cDNA in tissue sections (<it>in situ </it>RT-PCR). The latter method is highly specific and enables detection of transcripts in the cells originally responsible for their synthesis, but is highly destructive of tissue structures and can be carried out on only one or a few sections per experiment, resulting in low reproducibility. In this study, <it>in situ </it>RT-PCR was applied for the first time to commercially available tissue section microarrays enabling the examination of up to 70 different samples simultaneously. Modifications to the technique are detailed that preserved visible tissue and cellular structures and improved transcript detection whilst preventing significant generation of artefacts.</p

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs