Regulation of clustered gene expression by cofactor of BRCA1 (COBRA1) in breast cancer cells.

Eucaryotic genes that are coordinately expressed tend to be clustered. Furthermore, gene clusters across chromosomal regions are often upregulated in various tumors. However, relatively little is known about how gene clusters are coordinately expressed in physiological or pathological conditions. Cofactor of BRCA1 (COBRA1), a subunit of the human negative elongation factor, has been shown to repress estrogen-stimulated transcription of trefoil factor 1 (TFF1 or pS2) by stalling RNA polymerase II. Here, we carried out a genome-wide study to identify additional physiological target genes of COBRA1 in breast cancer cells. The study identified a total of 134 genes that were either activated or repressed upon small hairpin RNA-mediated reduction of COBRA1. Interestingly, many COBRA1-regulated genes reside as clusters on the chromosomes and have been previously implicated in cancer development. Detailed examination of two such clusters on chromosome 21 (21q22) and chromosome X (Xp11) reveals that COBRA1 is physically associated with a subset of its regulated genes in each cluster. In addition, COBRA1 was shown to regulate both estrogen-dependent and -independent transcription of the gene cluster at 21q22, which encompasses the previously identified COBRA1-regulated TFF1 (pS2) locus. Thus, COBRA1 plays a critical role in the regulation of clustered gene expression at preferred chromosomal domains in breast cancer cells.

ABO genotyping of complete hydatidiform moles.

It has been suggested that the ABO blood group of a patient and her partner influence the clinical outcome for patients having a pregnancy with a complete hydatidiform mole (CHM). Since CHM lack red blood cells, it has not previously been possible to type CHM serologically and investigate the relationship between the blood group of the CHM and that of the patient. In the present study we have demonstrated the feasibility of using molecular genotyping to determine the ABO genotype of CHM, the ABO genotype being consistent with the androgenetic origin of CHM in all cases. In the series of 48 cases of CHM, the requirement for chemotherapy was not significantly different in those patients with a CHM of like blood group compared with those with a CHM of unlike blood group.

Ahaptoglobinaemia in Melanesia: DNA and malarial antibody studies.

To assess the relative contributions of genetic and acquired factors, particularly malaria, to the high frequencies of ahaptoglobinaemia found in Melanesia we have performed DNA and malarial antibody studies in a population from Vanuatu. No gene deletion or rearrangement was found on gene mapping in any ahaptoglobinaemic individual and the frequencies of the Hp1 and Hp2 alleles in the ahaptoglobinaemic group were similar to controls. However, antibodies to Plasmodium falciparum were significantly elevated in the ahaptoglobinaemics. These data suggest that malaria rather than genetic factors is the major cause of ahaptoglobinaemia in Melanesia.

Variation in alpha-1-antitrypsin phenotypes associated with penicillamine therapy.

During a detailed study of alpha-1-antitrypsin (AAT) by isoelectric focusing, of 130 individuals of the PiZ phenotype and their families, an unusual alpha-1-antitrypsin protein pattern was identified which related directly to the effect of penicillamine therapy. The effect, believed to be mediated by reaction with the free cysteine of the alpha-1-antitrypsin molecule, was demonstrable by in vitro experiments. It was not apparently associated with loss of the anti-proteinase properties of alpha-1-antitrypsin and was not specific to the Z gene products. It is however, a source of possible confusion in the accurate assessment of Pi phenotypes.

Identification of alcohol dehydrogenase and aldehyde dehydrogenase isoenzymes in human liver biopsy specimens.

A sensitive method for detecting isoenzymes of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in human liver biopsy specimens by simultaneous starch gel electrophoresis is described. All the currently recognised hepatic isoenzymes of ADH and ALDH which are concerned with ethanol metabolism can be identified. The method is suitable for amounts of liver biopsy material weighing as little as 2.5 mg and also allows spectrophotometric assay of ADH activity (at pH 8.8 and 11.0) and of ALDH.

Inosine triphosphate pyrophosphohydrolase deficiency in a kindred with adenosine deaminase deficiency.

A complete deficiency of inosine triphosphate pyrophosphohydrolase (ITPase) has been identified, together with high concentrations (mean 157 mumol/l) of the unusual nucleotide ITP, in the erythrocytes of 3 members of a consanguineous United Kingdom kindred. The defect has been noted previously in North America and Sweden, but even in presumed homozygotes some residual ITPase activity was reported. Homozygosity for the defect has not been associated previously with any clinical abnormality. In this kindred it was co-existent with adenosine deaminase (ADA) deficient severe combined immunodeficiency. Since the genes for both ITPase and ADA are localised on the same chromosome, segregation analysis of ITPase and ADA activity was undertaken in available kindred members. The results confirmed an autosomal recessive mode of inheritance for ITPase deficiency, but suggested that the co-existence with ADA deficiency was coincidental.

Hepatic ADH and ALDH isoenzymes in different racial groups and in chronic alcoholism.

A method has been developed for simultaneous analysis of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzymes in small (2.5 mg) liver biopsy cores by starch gel electrophoresis. All the currently recognized hepatic isoenzymes coded by ADH1, ADH2, ADH3 and ADH4 can be detected as can the five ALDH isoenzymes. Using this technique we have investigated the isoenzyme composition of liver samples from English and Chinese subjects and a group of chronic alcoholics. Pronounced racial differences in frequency of ADH2 and ALDH phenotypes were found--only 2 (4%) of English controls had the "atypical" ADH2 variant whereas this was present in 42 (84%) of Chinese subjects, and whereas all the English subjects had the rapidly migrating mitochondrial isoenzyme of ALDH, this was absent in 27 (54%) of Chinese. No differences in ADH or ALDH phenotype were seen in the chronic alcoholics, all of whom were of English origin, compared with the English controls, but there was a reduction in overall ALDH activity and particularly in the mitochondrial isoenzyme in those with cirrhosis. The reduction in ALDH activity is probably acquired; by limiting acetaldehyde oxidation it could be responsible for the rapid deterioration in liver function in patients who continue drinking excessively.

Biochemical genetic analysis of human and rodent aldehyde dehydrogenase (ALDH).

ALDH isozymes have been characterized in terms of substrate and coenzyme specificity, heat stability, tissue distribution and electrophoretic properties. The activity of the isozymes has also been examined in rodent-human somatic cell hybrids in order to map the structural genes to specific chromosomes and to study the control of gene expression. One isozyme, designated ALDH3, which is very active against benzaldehyde, was found to show variable expression in hybrids made between rat hepatoma cells and human fibroblasts or fetal liver. Segregation analysis of these hybrids indicates that the structural locus for human ALDH3 may be on chromosome 17. The expression of rodent ALDH3 in these hybrids was extremely variable and not correlated with the appearance of the human enzyme. In hybrids expressing human and rodent ALDH3 no heteromeric isozymes were observed. The human "cytosolic" ALDH1 and "mitochondrial" ALDH2 isozymes did not appear to be expressed in any of the somatic cell hybrids examined.

Immunological detection of human phosphoglucomutase (PGM 1) subtypes.

Anti-phosphoglucomutase (PGM) antibodies have been produced by immunising a sheep with a purified preparation of rabbit skeletal muscle PGM and used to devise an immunological procedure for detecting PGM isozymes after isoelectric focusing. The anti-rabbit PGM antibodies cross react with human PGM and can be used to identify the PGM1 isozymes characteristic of this polymorphism. The patterns revealed by immunodetection are exactly comparable with those obtained by isozyme staining.

Immunological reactivity of native and modified alcohol dehydrogenase (ADH).

The extent to which treatment of horse liver alcohol dehydrogenase (ADH) by procedures known to disturb the Zn association induced conformation changes detectable by immunological techniques has been investigated. Treatment of ADH by sodium dodecyl sulphate or by total reduction and carboxymethylation leads to complete loss of reactivity with a rabbit immune serum against the native enzyme. After selective carboxymethylation the enzymatic activity was reduced but the preparation had the same immunological activity of the native enzyme. Similar results were obtained when ADH was treated with reagents known to react with the "functional" Zn atoms, such as sodium dietyldithiocarbamate, 1,10 phenanthroline, and 2,2' bipyridine. In contrast dialysis in 0.01M phosphate buffer containing 0.1mM EDTA removing the "structural" Zn atoms leads to a parallel decrease of enzymatic and immunological activity. Thus loss of "structural" Zn atoms affects the immune reactivity of ADH differently from the loss of "functional" Zn atoms.

Genetic polymorphism of human phosphoglycolate phosphatase (PGP).

1. A method has been devised for the detection after starch-gel electrophoresis of phosphoglycolate phosphatase (PGP) isozymes. 2. PGP isozymes can be detected in all human tissues including red cells, lymphocytes and cultured fibroblasts. The highest activities occur in skeletal muscle and cardiac muscle. 3. PGP is a relatively specific phosphatase which shows enhanced activity in the presence of mercaptoethanol at a neutral pH.4. Six different commonly occurring electrophoretic types of PGP have been identified. Family studies indicate that they are determined by three alleles at an autosomal locus (PGP). 5. The gene frequencies of PGP1, PGP2 and PGP3 in a random sample of Europeans were 0.826, 0.129 and 0.045 respectively. 6. The three-banded isozyme patterns seen in heterozygotes suggest that PGP is a dimeric enzyme.