Expression of prohibitin in rat seminiferous epithelium

Subtractive hybridization was used to isolate cDNAs highly expressed in stages IX-XI of the cycle of the seminiferous epithelium in the rat. One of the cloned cDNAs was sequenced and shown to be homologous to a previously described cDNA encoding rat prohibitin. Northern blot analyses showed that 1.9- and 1.2-kb transcripts were present in Sertoli cells whereas 1.5-, 1.2-, and 0.7-kb transcripts were expressed in germ cells. Western blot analyses with anti-peptide antibody to prohibitin revealed only a single 30-kDa protein in testis. Immunocytochemistry demonstrated that prohibitin protein was expressed constitutively in adult Leydig cells and Sertoli cells at all stages. Immunoreactivity of prohibition was very low in preleptotene spermatocytes, very high in leptotene spermatocytes, and very low in zygotene spermatocytes. In pachytene spermatocytes, immunoreactivity was very high in stages VII-XI and was minimal during stages XII and XIV. No protein was detected in spermatogonia and spermatocytes undergoing mitotic and meiotic divisions, respectively. These studies show that the prohibitin gene is expressed differentially in testis. The expression pattern of the prohibitin gene in rat testis appears to correlate with a proposed antiproliferative role of prohibitin

Pure chromosome-specific PCR libraries from single sorted chromosomes.

Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases