The Saccharomyces cerevisiae RAD9 cell cycle checkpoint gene is required for optimal repair of UV-induced pyrimidine dimers in both G(1) and G(2)/M phases of the cell cycle.

Cells respond to DNA damage by activating both cellular growth arrest and DNA repair processes. In Saccharomyces cerevesiae the RAD9 gene controls DNA damage-mediated cell cycle arrest that is known to allow efficient repair. To ascertain whether RAD9 plays a role in DNA repair per se, the removal of UV-induced photolesions was assessed in synchronized isogenic normal and rad9 cells using the high resolution primer extension technique. The results show that RAD9 is indeed involved in the removal of photolesions from both the transcribed and the non-transcribed strands of the reporter GAL10 gene, in G(1)- as well as G(2)/M-arrested cells. Interestingly, these data also reveal that in both normal and rad9 mutant, the repair strand bias towards the transcribed stand is more pronounced in G(2)/M- than in G(1)-arrested cells. These data indicate that RAD9 coordinate the cellular response to DNA damage by activating both cell cycle checkpoint and excision repair.

A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila.

Deep orange and carnation are two of the classic eye color genes in Drosophila. Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments. A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion. Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones. Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures. Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells. These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.

Deficient 17beta-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17beta-estradiol.

Aberrant aromatase expression in stromal cells of endometriosis gives rise to conversion of circulating androstenedione to estrone in this tissue, whereas aromatase expression is absent in the eutopic endometrium. In this study, we initially demonstrated by Northern blotting transcripts of the reductive 17beta-hydroxysteroid dehydrogenase (17betaHSD) type 1, which catalyzes the conversion of estrone to 17beta-estradiol, in both eutopic endometrium and endometriosis. Thus, it follows that the product of the aromatase reaction, namely estrone, that is weakly estrogenic can be converted to the potent estrogen, 17beta-estradiol, in endometriotic tissues. It was previously demonstrated that progesterone stimulates the inactivation of 17beta-estradiol through conversion to estrone in eutopic endometrial epithelial cells. Subsequently, 17betaHSD type 2 was shown to catalyze this reaction, and its transcripts were detected in the epithelial cell component of the eutopic endometrium in secretory phase. Because 17beta-estradiol plays a critical role in the development and growth of endometriosis, we studied 17betaHSD-2 expression in endometriotic tissues and eutopic endometrium. We demonstrated, by Northern blotting, 17betaHSD-2 messenger ribonucleic acid (RNA) in all RNA samples of secretory eutopic endometrium (n=12) but not in secretory samples of endometriotic lesions (n=10), including paired samples of endometrium and endometriosis obtained simultaneously from eight patients. This messenger RNA was not detectable in any samples of proliferative eutopic endometrium or endometriosis (n=4) as expected. Next, we confirmed these findings by demonstration of immunoreactive 17betaHSD-2 in epithelial cells of secretory eutopic endometrium in 11 of 13 samples employing a monoclonal antibody against 17betaHSD-2, whereas 17betaHSD-2 was absent in paired secretory endometriotic tissues (n=4). Proliferative eutopic endometrial (n=8) and endometriotic (n=4) tissues were both negative for immunoreactive 17betaHSD-2, except for barely detectable levels in 1 eutopic endometrial sample. Finally, we sought to determine whether deficient 17betaHSD-2 expression in endometriotic tissues is due to impaired progesterone action in endometriosis. We determined by immunohistochemistry the expression of progesterone and estrogen receptors in these paired samples of secretory (n=4) and proliferative (n=4) eutopic endometrium and endometriosis, and no differences could be demonstrated. In conclusion, inactivation of 17beta-estradiol is impaired in endometriotic tissues due to deficient expression of 17betaHSD-2, which is normally expressed in eutopic endometrium in response to progesterone. The lack of 17betaHSD-2 expression in endometriosis is not due to alterations in the levels of immunoreactive progesterone or estrogen receptors in this tissue and may be related to an inhibitory aberration in the signaling pathway that regulates 17betaHSD-2 expression.

Deleterious missense mutations and silent polymorphism in the human 17beta-hydroxysteroid dehydrogenase 3 gene (HSD17B3).

Isozymes of 17beta-hydroxysteroid dehydrogenase (17betaHSD) regulate levels of bioactive androgens and estrogens in a variety of tissues. For example, the 17betaHSD type 3 isozyme catalyzes the conversion of the inactive C19-steroid androstenedione to the biologically active androgen, testosterone, in the testis. Testosterone is essential for the correct development of male internal and external genitalia; hence, deleterious mutations in the HSD17B3 gene give rise to a rare form of male pseudohermaphroditism termed 17betaHSD deficiency. Here, 2 additional missense mutations in the HSD17B3 gene in subjects with 17betaHSD deficiency are described. One mutation (A56T) impairs enzyme function by affecting NADPH cofactor binding. A second mutation (N130S) led to complete loss of enzyme activity. Also, a single base pair polymorphism in exon 11 of the HSD17B3 gene is described. The polymorphic A allele encodes a protein with a serine rather than a glycine at position 289 (GGT --> AGT). The frequency of the G allele (Gly) was 0.94, and that of the A allele (Ser) was 0.06. No difference in the frequencies of the G and A alleles was detected in 32 apparently normal women and 46 women with polycystic ovary syndrome. Enzymes bearing either glycine or serine at this position have similar substrate specificities and kinetic constants. The current findings boost to 16 the number of mutations in the HSD17B3 gene that impair testosterone synthesis and cause male pseudohermaphroditism, and add 1 apparently silent polymorphism to this tally.

17beta-hydroxysteroid dehydrogenases: physiological roles in health and disease.

Androgens and estrogens play crucial roles in the growth and development of sex organs. Interconversion of these hormones between biologically active and inactive forms is catalyzed by 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isozymes. Aberrations in the regulation or expression of the various 17beta-HSD isoforms has been implicated in the genesis/progression of hormonally dependent cancers of various tissues, including ovary, breast and prostate; in the predisposition of women with upper body obesity to several types of diseases, such as non-insulin dependent diabetes mellitus; and in the abnormal development of sexually ambiguous individuals, as seen in 17beta-HSD-deficient male pseudohermaphrodites. Of the five known 17beta-HSD isozymes, deleterious mutations in the type 3 isoform were found to give rise to male pseudohermaphroditism. The 16 mutations characterized to date include 12 missense mutations, three splice junction mutations, and one small deletion that results in a downstream premature stop codon. 17beta-HSD has also been studied in other species. The most notable species difference observed is the placental expression in humans of the 17beta-HSD type 1 isoform.

Cell type-specific expression of 17 beta-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver.

The enzymatic actions of the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, 17 beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. 17 beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive 17-ketosteroid counterparts. The expression pattern of 17 beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the 17 beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of 17 beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.

Assignment of the human peroxisomal branched-chain acyl-CoA oxidase gene to chromosome 3p21.1-p14.2 by rodent/human somatic cell hybridization.

PCR and rodent/human somatic cell hybrids were used to localize the human peroxisomal branched-chain acyl-CoA oxidase gene. Oligonucleotide primers were chosen to specifically amplify human hBCox DNA. The amplified sequence contained two restriction enzyme sites which were used to verify the authenticity of the amplified DNA. Initially, the gene was localized to human chromosome 3 by screening genomic DNA from a hybrid mapping panel. Additional hybrids retaining well-characterized fragments of human chromosome 3 were screened to further localize the gene to 3p21.1.p14.2.

Physiology and molecular genetics of 17 beta-hydroxysteroid dehydrogenases.

17 beta-Hydroxysteroid dehydrogenases (17 beta-HSDs) are enzymes involved in both the activation and inactivation of androgens and estrogens. 17 beta-HSD type 1 shows a high specificity for C18 steroids and is the major isozyme in the granulosa cells of the ovary. Its role is to convert the inactive C18 steroid estrone to the active estrogen estradiol, which in turn locally promotes maturation of the follicle. In contrast, attenuation of estradiol action in the glandular epithelium of the secretory endometrium is achieved by expression of the oxidative type 2 isozyme that inactivates estradiol to estrone. An interesting feature of 17 beta-HSD type 2 is that the enzyme also possesses 20 alpha-HSD activity, i.e., it catalyzes the 20 alpha-oxidation of the inactive C21 steroid 20 alpha-dihydroprogesterone to the active progestin progesterone. As the type 2 enzyme is also active on androgens, it may play a general role in the peripheral inactivation of androgens and estrogens, thus determining their steady-state levels in target tissues. The reductive 17 beta-HSD type 3 is predominantly expressed in the testis and converts the inactive C19 steroid androstenedione to the active androgen testosterone. The importance of the type 3 enzyme in male steroid hormone physiology is underscored by the genetic disease 17 beta-HSD deficiency. Mutations in the 17 beta-HSD3 gene impair the formation of testosterone in the fetal testis and give rise to genetic males with normal male Wolffian duct structures but female external genitalia. To date, 15 mutations have been identified in 18 subjects with the disease.

Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias.

Congenital familial non-haemolytic hyperbilirubinaemias are potentially lethal syndromes caused by genetic lesions that reduce or abolish hepatic bilirubin UDP-glucuronosyltransferase activity. Here we describe genetic defects that occur in the UGT1 gene complex that cause three non-haemolytic unconjugated hyperbilirubinaemia syndromes. The most severe syndrome, termed Crigler-Najjar syndrome type I, is mainly associated with mutations in exons 2 to 5 that affect all UGT1 enzymes and many of the mutations result in termination codons and frameshifts. Crigler-Najjar type II syndrome which is treatable with phenobarbital therapy is associated with less dramatic missense mutations or heterozygous expression of mutant and normal alleles. Gilbert's syndrome, the most prevalent (2-19% in population studies) and mildest of the three syndromes is principally caused by a TA insertion at the TATA promoter region upstream of the UGT1A1 exon. Current methods used for the diagnosis and treatment of these diseases are discussed.

Assignment of the human peroxisomal palmitoyl-CoA oxidase gene to chromosome 17q23-qter by PCR technique.

Human peroxisomal palmitoyl-CoA oxidase plays a pivotal role in the beta-oxidation of fatty acids. Its importance is reflected by the severity of the disease associated with its deficiency in man. The gene was previously mapped to chromosome 17q25 with a FISH technique and is now confirmed using a PCR technique.

Cosegregation of intragenic markers with a novel mutation that causes Crigler-Najjar syndrome type I: implication in carrier detection and prenatal diagnosis.

Crigler-Najjar syndrome type 1 (CN-1) is a familial disorder characterized by severe unconjugated hyperbilirubinemia and jaundice and leads to kernicterus, neurological damage, and eventual death unless treated with liver transplantation. Previous reports identified mutations in the UGT1 gene complex to be the cause of the disease. The total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate of a CN-1 patient was determined by enzymological and immunochemical analysis. A novel homozygous nonsense mutation (CGA-->TGA) was identified in the patient by the combined techniques of PCR and direct sequencing. This mutation was located in exon 3 of the constant region in the gene complex which is common to all phenol and bilirubin UGTs. The segregation of the mutation in the patient's family was analyzed and confirmed the recessive nature of the disease. Newly developed intragenic polymorphic probes (UGT1* 4 and UGT-Const) were used on Southern blots of MspI-digested genomic DNA of the patient and his family. The segregation of individual alleles within the family was observed from haplotypes generated. Comparison of the segregation of haplotypes with the mutation for the patient and his family revealed the allele identified by the A1-B1-C2 haplotype to be carrying the mutation. The risk of recombination occurring is negligible, because of the intragenic nature of the probes. This study demonstrates the potential usefulness of these probes in carrier detection and prenatal/presymptomatic diagnosis.

Chromosomal assignment of human phenol and bilirubin UDP-glucuronosyltransferase genes (UGT1A-subfamily).

DNA probes were prepared from the 5'-terminal portion of four cDNA clones encoding human phenol and bilirubin UDP-glucuronosyltransferases (UGTs). An additional sequence common to all four clones was isolated from the 3'-terminal portion of one of the clones (UGT1A1). The four specific and the one common DNA sequences were used as probes on a panel of 16 human--rodent somatic cell hybrid DNAs by Southern-blot analysis. The results obtained indicate that all four cDNA clones are encoded by gene(s) located on human chromosome 2.