RNA-based mutation screening in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a cancer syndrome inherited in an autosomal dominant fashion. Four susceptibility genes are known, which code for DNA mismatch repair enzymes. The purpose of this study was to identify the HNPCC gene defects in a cohort of Australian HNPCC families and to evaluate the use of RNA-based screening methods. Six mutations were identified, four in the hMLH1 gene and two in hMSH2, by using a combination of DNA-based and RNA-based methods. One of the hMLH1 defects was a missense mutation, and the other five mutations would be expected to result in a shortened protein. These included a rare type of mRNA splicing mutation in hMLH1 exon 17. By use of reverse-transcriptase (RT) PCR, defective transcripts were detectable for three of the hMLH1 mutations but not for the fourth one, which was predicted to cause skipping of exon 15. Furthermore, many more alternative transcripts for the hMLH1 gene were found than previously described, and these were more abundant in the RNA samples prepared from whole blood than from lymphoblastoid cell lines. This confounded RNA-based screening for HNPCC mutations, because it was difficult to determine which aberrant RT-PCR fragment was the real hereditary defect. One of the splice-site mutations reported here causes skipping of exons 9 and 10, which also occurs as an alternative transcript. When the protein-truncation test was used, the results were indistinguishable between the patients in this family and controls. Other aberrant transcripts were also observed that varied in size between individuals but were unrelated to the hereditary defects. This study has important implications for the design of reliable diagnostic tests for HNPCC gene defects.

Cell-free extract(s) of Pseudomonas putida catalyzes the conversion of cyanides, cyanates, thiocyanates, formamide, and cyanide-containing mine waters into ammonia.

Our isolate, Pseudomonas putida, is known to be capable of utilizing cyanides as the sole source of carbon (C) and nitrogen (N) both in the form of free cells and cells immobilized in calcium alginate. In the present study, the cell-free extract(s) were prepared from the cells of P. putida grown in the presence of sodium cyanide. The ability of enzyme(s) to convert cyanides, cyanates, thiocyanates, formamide and cyanide-containing mine waters into ammonia (NH3) was studied at pH 7.5 and pH 9.5. The kinetic analysis of cyanide and formamide conversion into NH3 at pH 7.5 and pH 9.5 by the cell-free extract(s) of P. putida was also studied. The Km and Vmax values for cyanide/formamide were found to be 4.3/8 mM and 142/227 mumol NH3 released mg protein-1 min-1 respectively at pH 7.5 and 5/16.67 mM and 181/434 mumol NH3 released mg protein-1 h-1 respectively at pH 9.5. The study thus concludes that the cell-free extract(s) of P. putida is able to metabolize not only cyanides, cyanates, thiocyanates, and formamide but also cyanide-containing mine waters to NH3.

Chromosomal mapping of the human Mu class glutathione S-transferases to 1p13.

The chromosomal localization of the human Mu class glutathione S-transferase (GST) genes has been complicated by two factors; the total number of genes is unknown and there is a polymorphism that results from the presence or absence of the GSTM1 gene. Three human Mu class glutathione S-transferase isoenzymes, GSTM1, GSTM2, and GSTM3, have been characterized previously, and we have recently cloned and characterized GSTM4, another member of this class. Here we report that a probe derived from GSTM4 cross-hybridizes with the other three known human Mu class GST genes. In situ hybridization with the GSTM4 probe localized a major region of hybridization on chromosome band 1p13. Although there is a region of very weak hybridization on chromosome 6, these data indicate that the human Mu class gene family is largely clustered and not dispersed on different chromosomes. The identical hybridization patterns in individuals with or without the GSTM1 gene suggest that this locus is a component of the Mu class GST gene cluster.

Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript.

Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library. Both clones are incomplete and appear to result from alternative splicing. One clone is missing the sequence encoding exon 4 and the other is missing exon 8. The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones. This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process. By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli. In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.

Molecular genetics of the human glutathione S-transferase.

Multiple human cytosolic glutathione transferases have been described. These enzymes are the products of multiple genes that can be classified into at least four evolutionary classes. The genes encoding each class appear to be clustered on distinct chromosomes. Over-expression of glutathione S-transferase (GST) isoenzymes has been implicated in drug resistance and, conversely, deficiency of GST isoenzymes has been implicated in susceptibility to carcinogens. Some GST genes are expressed at varying levels in different individuals, and there is a frequent deficiency of the Mu class GST1 isoenzyme in all the racial groups studied so far. This deficiency is due to a deletion of the GST 1 gene. The Alpha class genes are located on the short arm of chromosome 6 and are closely linked, with less than 2 kb separating some genes. There is evidence for the existence of several pseudogenes in this cluster. A complete Alpha class gene has 7 exons and extends over 13 kb. The 5' flanking region of the gene encoding the GST2 type 1 isoenzyme has been cloned and sequenced. This region contains a number of putative promoter and enhancer elements that are similar to those found in rat and mouse Alpha class genes.