Non-isotopic in situ detection of mRNA for interleukin-4 in archival human tissue.

Non-isotopic in situ hybridisation (NISH) is a rapid and sensitive method currently used in molecular pathology to identify DNA in fresh and archival human biopsies. In this study we have extended NISH technology for mRNA detection to include the use of digoxigenin-labelled riboprobes to localise low abundance mRNA for interleukin-4 in paraffin embedded material (from active inflammatory bowel disease). As this methodological approach makes the retrospective study of cytokine gene expression in archival material possible for the first time, it could act as a prototype for studies of cytokine involvement in various human diseases.

Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots.

A dual-labeling technique was developed for direct quantification of specific mRNA using a flatbed liquid scintillation counter. This method simultaneously measures cpm of 32P- and 35S-labeled probes bound to RNA dot blots and subtracts counts due to nonspecific background radioactivity bound to the filter. Probes for T-cell receptor and beta-actin (as the internal standard) were hybridized both separately and simultaneously to RNA isolated from five different sources. There was concordance between the radioactivity measured from single- and dual-hybridizations for each combination of 35S- and 32P-labeled probes. This methodology directly quantifies specific mRNA sequences bound to membranes and has potential for measuring gene dosage, without the need for re-probing or densitometric analysis.

Papillomavirus screening in cervical cell samples using dual-label dot-blot analysis.

The presence of human papillomavirus (HPV) in cervical cells is closely related to the development of cervical carcinoma. Detection of virus may be by Southern blot, dot blot or the highly sensitive polymerase chain reaction. Whatever method is employed, there are problems of false negatives due to poor clinical samples in which the DNA may be degraded or is absent altogether. Here we describe a new method of dual labelling for dot blots using a 32P-labelled probe for HPV and a 35S-labelled probe for human actin genes. The samples were counted on a Beta-plate flat-bed scintillation counter and the data analysed to separate the activities of the two isotopes. The counts from the actin probe show whether human DNA is present or not and false negatives from this cause may thereby be eliminated. The counts due to HPV when compared with those for actin give a quantitative measure of HPV abundance for the particular sample and this may have clinical relevance.

Loss of heterozygosity in malignant melanoma at loci on chromosome 11 and 17 implicated in the pathogenesis of other cancers.

Forty-six cases of sporadic melanoma have been investigated for loss of heterozygosity at 4 loci: D11S29 (11q23), YNZ22 (17p13.3), TP53 (17p13.1); and NM23 (17q22). Each of the loci is thought to be important in the pathogenesis of other tumours. Mutations were found infrequently at the YNZ22, NM23, and TP53 loci. At D11S29, however, the frequency of mutation in the melanoma samples was high (67%) and mutations at this locus were associated with younger age at presentation. This region of chromosome 11 is also commonly mutated in breast cancers and haematological malignancies. Genetic aberrations at D11S29 may therefore represent nonspecific mutations found in several malignancies or part of a pathway common to the malignant phenotype.

Quantification of oncogene dosage in tumours by simultaneous dual-label hybridization.

Gene amplification and allele loss occur in a variety of human tumours and some have prognostic value. Therefore, techniques which facilitate detection and quantification of gene dosage could have wide applicability in cancer research. Using the INT-2 gene as a model system, a quantitative procedure has been developed for measuring gene copy number using dual-label hybridization to DNA dot blots. A probe specific for the INT-2 gene was labelled with [alpha-32P]dCTP and a probe to beta-actin, the control locus, was labelled with [alpha-35S]dATP. Flat-bed scintillation counting was used to detect and separate the emissions resulting from each bound probe, and gene dosage was calculated from the ratio of INT-2 to the beta-actin probe compared with the ratio derived from constitutional DNA. Calculated ratios of greater than 1.22 and less than 0.78 indicated gene amplification and allelic loss respectively, at the 99% confidence limit derived from the population of 35 constitutional DNAs. The results were validated by RFLP analysis. It is expected that this technique will permit precise gene dosage quantification in many areas.

A Xenopus borealis homeobox gene expressed preferentially in posterior ectoderm.

We have isolated a new homeobox-encoding gene (XhoxB.1) from Xenopus borealis which has a homeobox exon identical to that of the murine Hox1.7 gene, except for one amino acid. XhoxB.1 transcripts of 2.1 and 7.0 kb are first detected at the late gastrula stage, accumulate until the tailbud stage and are most abundant in the posterior third of the dorsal part of the embryo.

Episomal and integrated human papillomavirus in cervical neoplasia shown by non-isotopic in situ hybridisation.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.