Polymerase Chain Reaction Southern Blot Research Articles

Expression of sex hormone-binding globulin mRNA in human ovarian cancers

To know the role of sex hormone-binding globulin (SHBG) in the intracellular steroidal actions in human ovarian cancers, the expression of SHBG mRNA as a substitute for intracellular SHBG expression was investigated in normal ovarian tissues and ovarian tumors. In the present study, we used competitive reverse transcription-polymerase chain reaction-Southern blot analysis to evaluate SHBG mRNA levels. The expression of SHBG mRNA was detected in all normal ovaries and benign and malignant ovarian tumors analyzed. There were no significant differences in the mean SHBG mRNA levels among the three types of tissue. The expression in normal ovaries was significantly higher (p < 0.01) in premenopause, suggesting the predominance of a sex steroid hormone effect on ovarian SHBG synthesis. Relative overexpression of SHBG mRNA was observed in six out of 22 cases (27%) of ovarian cancer (three cases of endometrioid adenocarcinoma, two cases of serous cystadenocarcinoma and one case of mucinous cystadenocarcinoma) in comparison with normal ovaries and benign ovarian tumors. There was no difference in expression among the clinical stages of ovarian cancers. These data suggest that normal human ovaries and ovarian tumors might synthesize SHBG intracellularly, ovarian cancers might conserve an estrogen-associated property via SHBG and the regulation of intracellular SHBG expression might be changed in some cancers.

Canine distemper virus transcripts detected in the bone cells of dogs with metaphyseal osteopathy

Using the technique of in situ hybridisation, we have recently extended our observations that canine distemper virus (CDV) is present in the bone cells of patients with Paget's disease, and have shown that CDV is also detectable in the bone cells of dogs that are naturally infected with the virus. Since hybridisation was localised to bone cells within the metaphyses of the affected dogs, we investigated the possibility that CDV might be involved in the canine metaphyseal bone disorder, meitaphyseal osteopathy. Bone samples from three cases of metaphyseal osteopathy were examined for the presence of the CDV nucleocapsid (CDV-N) gene and the measles virus nucleocapsid (MV-N) gene, using 35S-labelled sense and antisense riboprobes. As with our previous findings in Paget's disease of bone, only the antisense probe was found to hybridise to the osteoblasts and osteoclasts within the affected metaphyses. No hybridisation was seen with the CDV-N sense and MV-N probes in any of the samples tested. Bone samples were also taken from one of the cases to check for the presence of the CDV-N gene using the polymerase chain reaction (PCR). Our findings with in situ hybridisation were confirmed by PCR and subsequent Southern blotting and probing with a 32P-labelled cDNA probe. The detection of CDV RNA within the bone cells of dogs with metaphyseal osteopathy suggests that this virus may be a cause of the disease and provides further, indirect evidence that CDV might be responsible for the bony abnormalities seen in Paget's disease of bone.

Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene.

The common translocation found in human follicular lymphoma, t(14;18)(q32;q21), results in deregulation of the BCL2 protoonocogene. The isolation of the intact gene would provide an essential substrate to analyze the molecular basis of this malignancy. Pulsed-field gel electrophoresis suggested that this three-exon gene was several hundred kilobases (kb) long. Therefore, a library of yeast artificial chromosome (YAC) clones was screened to isolate the intact BCL2 gene. Two clones, yA85B6 (200 kb) and yB206A6 (700 kb), were isolated by using polymerase chain reaction (PCR) assays specific for exon I/II and exon III, respectively. However, neither YAC contained the entire BCL2 locus. Since the two YACs were found to overlap by 60 kb, we sought to take advantage of the high recombination frequency in yeast and induce physical recombination between the two clones. Cells containing each YAC were mated and induced to undergo meiotic division and sporulation. Analysis of the resulting tetrads revealed a spore containing a single recombinant YAC of 800 kb. PCR assays and Southern blotting demonstrated that this recombined YAC contained the entire approximately 230-kb BCL2 gene. Furthermore, probe order was conserved and there was no evidence of overt rearrangements or deletions. These results indicate the feasibility of reconstructing large genomic segments with overlapping YAC clones to study genes spanning hundreds of kilobases.

Detection and Typing of Papillomavirus DNA in Formaldehyde-Fixed Paraffin-Embedded Tissue

Clinical specimens from nine patients with papillomatosis of the vocal cords and three patients with vocal cord polyps were evaluated for the presence of human papillomavirus (HPV) DNA using two complementary molecular hybridization techniques. In one method, involving polymerase chain reaction (PCR) amplification, HPV DNA sequences were replicated in vitro from tissue DNA extracted from paraffin sections prior to hybridization. Polymerase chain reaction amplification was compared with the standard method of Southern blot hybridization. Results of the two techniques for all nine laryngeal papillomas agreed completely: five patients harbored HPV type 6 and four HPV type 11. Both PCR amplification and Southern blot hybridization found two of the three polyps to be free of HPV infection, while PCR detected HPV type 18 in one polyp specimen that was reported negative by Southern blot hybridization, suggesting a greater sensitivity of PCR. Our results demonstrate that PCR amplification is as reliable and at least as sensitive as Southern blot hybridization. Moreover the PCR technique opens the way to the undertaking of a whole variety of retrospective studies using formaldehyde-fixed paraffin-embedded tissues.

Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique.

We have developed a rapid procedure for the detection of serum hepatitis B virus (HBV) DNA using the polymerase chain reaction (PCR) technique. HBV DNA is released from virions by incubating serum with 0.1 M NaOH for 60 min at 37 degrees C. The mixture is brought to neutral pH with HCl, and the HBV DNA sequences are detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with two successive sets of primer pairs. The detection limit of this method (i.e., 10(-5) pg of HBV DNA) is equivalent to that previously determined by one round of PCR amplification and Southern blot hybridization analysis. The advantages are that the assay can be completed in 1 day, is very sensitive, and does not require the use of radiolabeled reagents.