Isolation of DNA probes specific for rat chromosomal regions 19p, 19q and 4q and their application for the analysis of diethylstilbestrol-induced aneuploidy in binucleated rat fibroblasts.

DNA probes specific for rat chromosomes 19p, 19q and 4q were isolated, characterized and used for the detection and analysis of diethylstilbestrol(DES)-induced aneuploidy. By denaturing and partially reassociating total genomic DNA a new rat repetitive DNA family was isolated, which was located on chromosome 19p21. Sequencing of a number of subclones from cos76-1 and other clones of this so-called 76-family revealed that the repeat units are interrupted with large areas of other (unique) DNA. Consequently, after fluorescence in situ hybridization (FISH) the signals in interphase nuclei are large and spread out. The other two probes, cos25 (chromosome 4q) and cos42-47 (chromosome 19q), were isolated by screening cosmid libraries with probes isolated previously in our laboratory. The repeat unit of cos25 is a 2174 bp long EcoRI unit that contains three Sau3A sites and is tandemly organized. Sequencing of subclones of cos42-47 revealed that this probe was in fact the 5S RNA gene, located on 19q12. In order to determine if these probes were suitable probes for aneuploidy detection, two series of dual colour FISH with the combinations cos25/cos76-1 (4q/19p) and cos42-47/cos76-1 (19q/19p) were carried out on slides from an in vitro micronucleus assay with DES. With all three probes used, an increase in binucleated cells with non-disjunction or chromosome loss was observed in the DES-treated cultures. Scoring of additional micronucleated cells on slides hybridized with the cos25/cos76-1 (4q/19p) probes revealed that the hybridization signal of probe cos25 (4q) was over-represented in the micronuclei of the control cultures. The simultaneous use of the 19q and 19p probes is a particularly valuable tool for the detection of aneuploidy, since it allows distinction between aneugenic and clastogenic events in binucleated cells. Results of this analysis showed that apart from aneuploidy, DES also induced structural chromosome aberrations, although to a lesser extent.

The rat N-ras gene; interference of pseudogenes with the detection of activating point mutations.

The PCR technique in combination with selective hybridization to mutation specific oligonucleotides, is a widely used methodology for the detection of activating point mutations in ras oncogenes. In the present paper we demonstrate for the N-ras gene of the rat that processed pseudogenes do interfere with this method. A first indication for this interference came from the sequence analysis of cloned PCR fragments of exon 1, amplified with primers derived from previously reported exon sequences of the mouse N-ras gene. Between different clones originating from one PCR reaction, a marked sequence heterogeneity is observed and this is shown to be the result of the presence of at least two different processed pseudogenes of the rat N-ras gene. These two pseudogenes, together with the wildtype N-ras gene and a small 3' part of the unr gene, were eventually cloned and their genomic organization and nucleotide sequences determined. Furthermore, representative examples of the confounding effects of these pseudogenes on the screening for activating point mutations are presented. Taken together, our results demonstrate that intron-specific amplification is a prerequisite for the unambiguous detection of activating point mutations in the N-ras gene of the rat.

Chromosomal localization of the rat N-ras protooncogene by fluorescence in situ hybridization.

The rat N-ras protooncogene has been assigned to chromosome 2q34 by fluorescence in situ hybridization on rat metaphase chromosomes. This was accomplished using two recently isolated genomic clones with a length of 8.2 and 4.1 kb.