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Objectives In the bovine placenta, intimate fetomaternal contact is restricted to placentomes. Within the placentomes fetal chorionic villi interdigitate with corresponding maternal caruncular crypts. The trophoblast epithelium covering the chorionic villi consists of 80% uninucleate trophoblast cells (UTCs) and 20% trophoblast giant cells (TGCs). TGCs migrate toward the endometrium and fuse with endometrial cells to form short-lived fetomaternal hybrid cells. Thereby the TGCs transport molecules of fetal origin across the placental barrier into the maternal compartment. The UTC/TGC ratio is constant during pregnancy because UTCs can differentiate into new TGCs to replace spent TGCs. However, our understanding of this differentiation process was sparse. Therefore, we collected the data to study the gene expression profiles in UTCs and TGCs and to identify differently expressed genes between the two trophoblast cell populations. Using Gene Ontology analysis, we wanted to identify biological processes and pathways that play an important role in the differentiation of UTCs into TGCs. Data description Bovine placentas were from days 118 to 130 of gestation. We obtained virtually pure UTCs and TGCs using a fluorescence-activated cell sorting (FACS) method. Total RNA was extracted from the UTC and TGC isolates, labeled and hybridized to Affymetrix Bovine Gene 1.0 ST Arrays.

In addition to many other functions, the placenta is a source of a vast number of autocrine, paracrine and endocrine factors. However, the spectrum of placental regulatory factors, their concentrations, gestational profiles and roles may differ considerably even between phylogenetically closely related species. Depending on the species, placental regulatory factors of a broad range of molecule classes have been found including (glyco-)proteins, peptides, steroids and prostaglandins. Local placental regulatory factors are especially important for the dialogue between the fetal and the maternal compartment immediately at the feto-maternal borderline and for the control of growth, differentiation and functions of the placenta itself. Moreover, placental hormones in a proper sense may also have effects in more remote targets within the maternal compartment, serving functions such as pregnancy-specific adaptations of maternal circulation, provision of hemotrophe to the fetus or the development and function of the mammary gland. Functions of placental hormones in the fetus proper are less clear but may be especially important before the establishment of a functional fetal endocrine system and near term within the highly species-specific networks of signals preparing and initiating parturition. This review takes a comparative view on the situation in different domestic animals focusing on ruminants and on placental hormones occurring at significant concentrations in the maternal circulation.

CYP17A1 encodes the key enzyme of androgen biosynthesis, P450c17. The gene is expressed in a number of steroidogenic tissues among them testis, ovary, placenta and adrenal gland. The proper analysis of CYP17A1 expression and of epigenetic parameters however, is hampered by the presence of more than one copy of the gene within the bovine genome. Therefore, as a prerequisite for future studies we characterized these copies and analyzed their promoter methylation and expression profiles in different tissues. DNA methylation levels were determined by bisulfite modification, amplification, cloning and sequencing. Transcription was analyzed by RT-PCR. From bovine genomic DNA three different CYP17A1 promoter sequences could be amplified with a sequence similarity of 94.8%, 95.6% and 98.7%. Based on these sequences we could reconstruct, by in silico analysis, the promoter regions and eight potentially coding exons of two loci, CYP17A1a and CYP17A1b, and the promoter region and truncated first exon of a third locus, CYP17A1x. By using locus-specific primers, only transcripts of CYP17A1a, but not of CYP17A1b could be detected in testis, epididymis, theca, corpus luteum, placental cotyledons, adrenal gland and preoptic brain area. Methylation analysis revealed that only the CYP17A1a promoter was hypo-methylated in the tested P450c17 active tissues, whereas both other copies showed higher levels of methylation. From these data we conclude that the bovine genome contains three paralogous copies of the CYP17A1 gene, of which two (CYP17A1b and CYP17A1x) might be silenced by epigenetic modification (promoter methylation).

The pre-ovulatory luteinizing hormone (LH) surge induces an extensive molecular, physiological, and morphological reorganization of the bovine follicle. This study was designed to elucidate if chromatin modulation is involved in the LH-induced gene regulation. Granulosa and theca of well-characterized large bovine follicles were isolated before and after the LH surge. CYP19A1, HSD3B1, and CYP17A1 transcripts, which encode key enzymes of steroid hormone biosynthesis, were quantified by real-time PCR (qPCR) and the degree of chromatin condensation was determined by DNase I protection assays. After LH, granulosa-specific CYP19A1 and theca-specific CYP17A1 transcripts were almost completely down-regulated. Also, the abundance of HSD3B1 transcripts was reduced. The promoter chromatin of HSD3B1 and particularly of CYP19A1 was significantly less accessible to DNAse I in both cell types after LH, whereas the chromatin accessibility of the CYP17A1 promoter changed only in the theca. Correlation analysis revealed partly, highly significant negative correlations between transcript abundance and protection from DNase I digestion of the corresponding chromatin. The data strongly suggest that LH induces cell type- and gene-specific chromatin condensation in the pre-ovulatory bovine follicle. This epigenetic mechanism might be involved in the pre-ovulatory down-regulation of genes.

The luteinizing hormone-induced morphological and physiological reorganization of the bovine follicle is preceded by a profound and well-orchestrated modulation of gene expression. In the present study, the cell type-specific methylation profiles of CYP11A1, HSD3B1, and CYP19A1, genes that encode key enzymes of steroid hormone biosynthesis, were analyzed to elucidate whether epigenetic parameters such as DNA methylation might be involved in gene regulation during luteinization. Transcript abundance and DNA methylation levels were determined in granulosa and theca of large dominant and late preovulatory follicles and in large granulosa lutein cells isolated from corpora lutea cyclica and graviditatis. Levels of the steroid hormones progesterone and estradiol-17beta were monitored to assess the physiological status of individual follicles. From our results, we conclude that (1) individual, even closely neighboring, CpG dinucleotides can show very different methylation levels; (2) proximal (

The transformation of the dominant follicle into a functional corpus luteum is accompanied by a profound molecular and morphological reorganization of somatic cell layers. Several studies have focused on gene expression during early processes of follicular differentiation as it relates to recruitment and selection of dominant follicles. However, little information exists on changes of gene expression profiles in late preovulatory follicles. This lack of information is addressed here to elucidate molecular mechanisms behind the LH-induced transition from the large dominant estrogen-active to the preovulatory follicle, an intermediate stage toward full luteinization. Transcripts encoding key molecules for the biosynthesis of steroid hormones and prostaglandins, as well as receptors for gonadotropic and growth hormones (Star, Cyp11a1, Hsd3b, Cyp17, Cyp19, Ptgs2, Fshr, Lhr, and Ghr), were quantified by real-time polymerase chain reaction (PCR) in the granulosa and theca of large dominant and late preovulatory follicles. The steroid hormones progesterone (P4) and estradiol-17beta (E2) were monitored to distinguish estrogen-active and estrogen-inactive follicles. We found that (1) independent of the follicular stage, the gene expression profile was very different in granulosa and theca; (2) the abundance of several key transcripts was lower in estrogen-inactive, compared with estrogen-active, dominant follicles; (3) in the granulosa of late preovulatory follicles, transcripts encoding steroidogenic enzymes and hormone receptors were largely down-regulated, whereas (4) progesterone and E2 were found at high concentrations in the follicular fluid. Collectively, our data show that late preovulatory follicles have a transient and unique gene expression profile and are clearly different from both the preceding and subsequent (follicular and luteal, respectively) stages.

Locally produced estrogens are important paracrine regulators of placental growth and differentiation in cattle and sheep. The key enzyme of estrogen biosynthesis, aromatase cytochrome P450, is encoded by CYP19. Transcription of this gene in fetal cotyledons is the first regulatory step of placental estrogen production. The aim of the present study was to comparatively investigate if epigenetic mechanisms as tissue- and differentiation-specific DNA methylation might be involved in the regulation of placental CYP19 expression. Earlier investigations demonstrated that cattle and sheep use different promoters, P1.1 and P1.5 for placental CYP19 expression, respectively. During the present investigation, methylation of individual CpG dinucleotides within these promoter regions was analyzed with bisulfite direct sequencing in placental and luteal tissue. Transcript abundance in sheep was determined with qPCR. Both promoters contain only few CpGs and can therefore be classified as "low CpG densitiy regions". The average methylation levels of both promoter regions were significantly reduced in cotyledons compared to caruncles and corpus luteum of both species, which inversely coincides with high and low expression levels, respectively. It was evident however that even neighbouring CpGs can show very different, individual methylation levels. From the data we conclude that (1) CYP19 promoters are differentially methylated in ovine and bovine placental tissues of foetal and maternal origin; (2) DNA methylation is suggestively involved in the regulation of CYP19 expression; (3) the DNA methylation status on its own is not sufficient for the selection of tissue-and species-specific CYP19 promoters.

A quantitative trait locus (QTL) affecting milk fat percentage has been mapped to the centromeric end of the bovine chromosome 14 (BTA14). This genomic area includes the DGAT1 gene, which encodes acyl-CoA:diacylglycerol acyltransferase 1, the key enzyme of triglyceride biosynthesis. Genetic and biochemical studies led to the identification of the nonconservative DGAT1-K232A polymorphism as a causal mutation for the QTL. In addition to this, another polymorphism in the 5′-regulatory region of this gene, the DGAT1 variable number of tandem repeat (VNTR), also showed a strong association with milk fat percentage. This promoter VNTR polymorphism affects the number of potential Sp1 binding sites and therefore might have an impact on DGAT1 expression and also milk fat content. Hence, the DGAT1 VNTR polymorphism might be another causal mutation for the BTA14 QTL. However, evidence for Sp1 binding to this polymorphic site and for the capability of DGAT1 VNTR alleles to stimulate gene expression was lacking. In the current work Sp1-VNTR interactions were analyzed by EMSA. In addition, effects of DGAT1 VNTR alleles on gene expression were measured with reporter gene analyses. Conclusions from the results are that 1) the DGAT1 VNTR sequence is indeed a target for Sp1 binding; 2) DGAT1 VNTR alleles can stimulate gene expression in vitro and probably in vivo as well; and 3) although the stimulating effects of the different DGAT1 VNTR alleles did not show significant differences in vitro, their effects on transcription might be different in the chromatin context existing in vivo.

Gestagens and oestrogens are important regulators of pregnancy and parturition. The aim of the present study was the comparative quantification of steroidogenic transcripts in placenta and corpus luteum of cattle and sheep during pregnancy and post partum. Cyp19 transcript variants, derived from different promoters, as well as transcripts of Hsd3b, Cyp11A1, and Cyp17, encoding the steroidogenic enzymes P450arom, 3beta-HSD, P450SCC, and P450C17, respectively, were quantified by newly developed real-time PCR assays. All steroidogenic transcripts were detected in ovine and bovine corpus luteum and placenta during pregnancy, however at a very different concentration. In both species Cyp11A1 and especially Hsd3b transcripts predominated in corpus luteum, outnumbering transcripts of Cyp17 and Cyp19 by more than two and three orders of magnitude, respectively. Cyp19 transcript were found at high concentration in the placenta and at a very low concentration in corpus luteum. Cyp17 transcripts had a relatively low concentration in both, placenta and corpus luteum, however showed a peak of expression in the ovine and bovine term placenta. Tissue- and species-specific Cyp19 transcripts derived from different promoters were detected. In order to map all promoters, the bovine Cyp19 locus was reconstructed by in silico analysis. In the placenta, transcripts were primarily derived from the proximal promoter P1.5 in sheep, but from the distally located P1.1 in cattle. Corpora lutea of both species predominantly expressed P1.1 derived transcripts. Contrary to the bovine, the sheep corpus luteum also showed considerable P1.5 derived expression. This demonstrates that cattle and sheep use different promoters to direct Cyp19 expression during pregnancy.

The bovine trophoblast produces significant amounts of estrogens. In maternal and fetal blood, estrogens occur predominantly in sulfonated forms, which are unable to bind to estrogen receptors (ESRs). However, estrogens may act as local factors in ESR-positive trophoblast cells or in the adjacent caruncular epithelium, which in addition to ESR highly expresses steroid sulfatase. Estrogen sulfonation is catalyzed by the cytosolic enzyme SULT1E1. Previous studies clearly indicated the trophoblast as the primary site of estrogen sulfonation. However, investigations into the cellular localization of SULT1E1 yielded conflicting results. In situ hybridization studies detected SULT1E1 mRNA only in trophoblast giant cells (TGCs), whereas in immunohistochemical experiments the SULT1E1 protein was virtually restricted to uninucleated trophoblast cells (UTCs). The aim of this work was to resolve this conflict by analyzing SULT1E1 expression in isolated UTCs and TGCs. Highly enriched pools of UTCs and TGCs were obtained from four bovine placentas (Days 118-130 of gestation) using an optimized fluorescence-activated cell sorting procedure. UTC and TGC pools were analyzed by quantitative RT-PCR and Western blot experiments to measure the amounts of SULT1E1 transcript and protein, respectively. In contrast to previously published results, both SULT1E1 transcript and SULT1E1 protein were clearly present in the UTC and TGC pools. However, some evidence indicated a higher transcript concentration in TGCs and a higher amount of protein in UTCs. Thus, our results resolve the conflicting results on the localization of SULT1E1 from earlier studies and suggest that posttranscriptional mechanisms play an important role in the control of SULT1E1 expression during TGC differentiation.

During this report the tissue-specific expression and promoter usage of the aromatase cytochrome P450 encoding gene, Cyp19, are compared between cattle and sheep. In addition, data will be presented on the identification of cis-acting regulatory sequences located in the bovine placenta-specific promoter 1.1. In cattle and sheep Cyp19 is mainly expressed in the foetal placental layer and ovarian granulosa cells but also in other organs as brain or testis. Differently spliced transcripts of Cyp19 which include an invariable coding region but a variable 5′-untranslated region could be detected in tissues of both species. However, in contrast to ovary and brain which express homologous transcript variants, different transcripts are present in placentae suggesting that also different placenta-specific promoter regions are active in cattle and sheep. The analysis of the chromatin structure of the main placental promoter 1.1 in different bovine tissues revealed that hypomethylation and the occurrence of DNaseI hypersensitive sites (HS) within this region are associated with promoter activity. Active regulatory elements were identified in reporter gene studies in JEG-3 choriocarcinoma cells. The co-localisation of an E-box element within one of the placenta-specific HS suggests that this element is important for Cyp19 expression in the bovine placenta.

Here we report on the genomic structure of the bovine aromatase cytochrome P450-encoding gene (Cyp19) and its tissue-specific transcript variants. The gene comprises at least 14 exons (1.1, 1.2a, 1.2b, 1.3,1.4, and 2-10) spanning more than 56 kilobases of genomic DNA. The coding area is confined to exons 2-10. Transcriptional start sites of Cyp19 were examined in granulosa cells, placenta, testis, adrenal gland, and brain, employing 5'-RACE (rapid amplification of complementary DNA ends) and primer extension. The analysis of 5'-RACE clones revealed six Cyp19 transcript variants that were different within their 5'-untranslated regions (5'-UTR). Yet, the coding region was identical in all clones. Although two of these 5'-UTR (the first 152 nucleotides of exon 2 and exon 1.4) are conserved among different species, four others (exons 1.1, 1.2a, 1.2b, and 1.3) did not show sequence homology to any other species. Transcription from exons 1.1 and 2 starts at several adjacent sites. In granulosa cells and placenta, but not in brain, a fraction of transcripts starting with exon 1.2a contains an additional untranslated exon, 1.2b, due to alternative splicing. Transcript variants comprising exon 1.1, 1.2a, 1.2b, or 1.3 were mainly found in the placenta, those with the 5'-UTR of exon 2 were predominant in granulosa cells, and transcripts with exon 1.4 prevailed in the brain. Estimates of Cyp19 transcript concentrations in six different tissues revealed high levels in granulosa cells and placenta, intermediate levels in testis and brain, and low levels in adrenal gland and liver. Our experiments demonstrate that six transcript variants of the bovine Cyp19 gene, including 9-11 exons, are expressed with tissue-specific preferences. These transcripts are presumably generated using five different promoter regions and tissue-specific alternative splicing.

Background Placenta-derived oestrogens have an impact on the growth and differentiation of the trophoblast, and are involved in processes initiating and facilitating birth. The enzyme that converts androgens into oestrogens, aromatase cytochrome P450 (P450arom), is encoded by the Cyp19 gene. In the placenta of the cow, expression of Cyp19 relies on promoter 1.1 (P1.1). Our recent studies of P1.1 in vitro and in a human trophoblast cell line (Jeg3) revealed that interactions of placental nuclear protein(s) with the E-box element at position -340 are required for full promoter activity. The aim of this work was to identify and characterise the placental E-box (-340)-binding protein(s) (E-BP) as a step towards understanding how the expression of Cyp19 is regulated in the bovine placenta. Results The significance of the E-box was confirmed in cultured primary bovine trophoblasts. We enriched the E-BP from placental nuclear extracts using DNA-affinity Dynabeads and showed by Western blot analysis and supershift EMSA experiments that the E-BP is composed of the transcription factors upstream stimulating factor (USF) 1 and USF2. Depletion of the USFs by RNAi and expression of a dominant-negative USF mutant, were both associated with a significant decrease in P1.1-dependent reporter gene expression. Furthermore, scatter plot analysis of P1.1 activity vs. USF binding to the E-box revealed a strong positive correlation between the two parameters. Conclusion From these results we conclude that USF1 and USF2 are activators of the bovine placenta-specific promoter P1.1 and thus act in the opposite mode as in the case of the non-orthologous human placenta-specific promoter.

The luteinizing hormone-induced morphological and physiological reorganization of the bovine follicle is preceded by a profound and well-orchestrated modulation of gene expression. In the present study, the cell type-specific methylation profiles of CYP11A1, HSD3B1, and CYP19A1, genes that encode key enzymes of steroid hormone biosynthesis, were analyzed to elucidate whether epigenetic parameters such as DNA methylation might be involved in gene regulation during luteinization. Transcript abundance and DNA methylation levels were determined in granulosa and theca of large dominant and late preovulatory follicles and in large granulosa lutein cells isolated from corpora lutea cyclica and graviditatis. Levels of the steroid hormones progesterone and estradiol-17beta were monitored to assess the physiological status of individual follicles. From our results, we conclude that (1) individual, even closely neighboring, CpG dinucleotides can show very different methylation levels; (2) proximal (300 base pair [bp] from the respective transcription start sites) but not distal CpGs show cell type-specific methylation levels; (3) higher methylation levels suggestively preclude high levels of gene expression; (4) DNA methylation is not involved in the transient (HSD3B1 and CYP11A1) respectively permanent (CYP19A1) down-regulation of gene expression in late preovulatory follicles; and (5) DNA methylation may have a role in the permanent shutdown of promoter 2-directed CYP19A1 expression in large (granulosa derived) lutein cells.

The increasing evidence of fetal developmental effects on postnatal life, the still unknown fetal growth mechanisms impairing offspring generated by somatic nuclear transfer techniques, and the impact on stillbirth and dystocia in conventional reproduction have generated increasing attention toward mammalian fetal growth. We identified a highly significant quantitative trait locus (QTL) affecting fetal growth on bovine chromosome 6 in a specific resource population, which was set up by consistent use of embryo transfer and foster mothers and, thus, enabled dissection of fetal-specific genetic components of fetal growth. Merging our data with results from other cattle populations differing in historical and geographical origin and with comparative data from human whole-genome association mapping suggests that a nonsynonymous polymorphism in the non-SMC condensin I complex, subunit G (NCAPG) gene, NCAPG c.1326T>G, is the potential cause of the identified QTL resulting in divergent bovine fetal growth. NCAPG gene expression data in fetal placentomes with different NCAPG c.1326T>G genotypes, which are in line with recent results about differential NCAPG expression in placentomes from studies on assisted reproduction techniques, indicate that the NCAPG locus may give valuable information on the specific mechanisms regulating fetal growth in mammals.

Placental oestrogens play an important role as local regulators of placental growth and differentiation during gestation, and toward term they are also involved in the preparation of parturition. They are synthesized within the fetal cotyledons of placentomes by aromatase cytochrome P450 (P450arom; EC 1.14.14.1), the product of the Cyp19 gene. The first step of regulation of P450arom expression, and hence enzyme activity and oestrogen production, takes place at the level of Cyp19 transcription, which is driven by a proximal promoter region, P1.5/2, in the sheep placenta. The aim of the present study was to find out if different Cyp19 expression levels, which previously had been observed in ovine placentome tissues, correlate with the tissue-specific chromatin structure of the promoter. To this end, we investigated the chromatin structure across the P1.5/2 region in caruncles and cotyledons from 100 and 125 days pregnant ewes, and in term placentae, respectively, by analyzing the DNA methylation and the accessibility to restriction digestion. Our data show that: (1) cotyledonal DNA was significantly lower methylated than caruncular DNA; (2) methylation of cotyledonal DNA was low at 100 and 125 days of pregnancy, and increased to a significant higher level in term placentae; and (3) concurrently, cotyledonal chromatin became inaccessible to restriction digestion at term of gestation. The results imply that DNA methylation and chromatin accessibility of the P1.5/2 promoter region correlate with expression levels of the Cyp19 gene. Mol. Reprod. Dev. 75: 1–7, 2008. © 2007 Wiley-Liss, Inc.

Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue-specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue-specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta-specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis-regulatory elements, an E-box in HS1 and a trophoblast-specific element-like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta-derived nuclear factors. In conclusion, observed tissue-specific differences of the chromatin structure which correlate with tissue-specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle.

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

The aim of the present study was the characterization of the ovine aromatase cytochrome P450 encoding gene (Cyp19) and the analysis of its tissue-specific expression. Two loci with considerable sequence identity were found (Cyp19 and Cyp19b). From Cyp19, tissue-specific transcript variants with different untranslated first exons but identical coding regions could be identified. Cyp19b transcripts were not detected. In the sheep brain and ovarian granulosa cells transcript variants, starting with the untranslated exons 1.4 and 2, respectively, were preferentially found. Exons 1.2 and 1.3 which had been described in bovines could not be detected in sheep and the major 5′ untranslated region of the bovine placental transcript, exon 1.1, was also not found to predominate in the sheep placenta. However this exon frequently was combined with a new untranslated exon (exon 1.1a) thus generating an alternative splice variant. The main placental transcripts in sheep had a different first exon (exon 1.5). Two alternatively spliced variants of this transcript were found with tissue-specific preference. From the present data it can be concluded: (a) that the ovine genome contains two copies of Cyp19 of which only one is transcribed and may encode a functional protein; and (b) that in spite of being closely related species, sheep and cattle have remarkable differences concerning tissue-specific transcript distribution and presumable promoter usage.

In cattle, the CYP19 locus comprises the aromatase cytochrome P450-encoding gene (CYP19) and a homologous pseudogene (CYP19P1). It has been assigned to chromosome region 10q26. Cloning of genomic DNA revealed that the CYP19 gene covers more than 56 kb. Its precise extent is still unknown because the DNA spanning the untranslated first exon 1.1 and the coding region (exons 2 to 10) have not been isolated. Furthermore, the chromosome arrangement of closely linked CYP19 and CYP19P1 was also not clear. To establish a high resolution physical map of the entire CYP19 locus, fluorescence in situ hybridization to extended bovine genomic DNA fibers (fiber FISH) was performed. The results demonstrate (1) that the clone containing exon 1.1 is located about 19 kb upstream from the CYP19 coding region. (2) Within the chromosome region 10q26 CYP19 and CYP19P1 are arranged “tail-to-head”, being separated by a distance of about 24 kb between the labeled clones. (3) The physical size of the bovine CYP19 locus amounts to a minimum of 130 kb.

In Bacillus subtilis the abrB gene product negatively affects the transcription of some genes activated during the transition from vegetative to stationary phase of growth. Interaction of AbrB with the promoters of two such genes, spoVG, a sporulation gene, and tycA, an antibiotic biosynthesis gene, was studied by DNase I and hydroxyl radical footprinting. Two binding areas within the leader and promoter regions of tycA were identified. In spoVG the binding site is located at the A + T-rich region upstream of the promoter. Hydroxyl radical footprinting revealed that the AbrB-protected regions, in both the tycA and spoVG promoters, are short A + T-rich regions that are separated by one helical turn, indicating that AbrB binds to one face of the helix. To examine the role of spoOA in the expression of abrB-controlled genes, the levels of AbrB protein in Spo + and in spoOA cells were determined by Western blot analysis. In wild-type cells AbrB was detected only during vegetative growth, whereas in spoOA cells a high level of AbrB was detected during both the vegetative and stationary phases of growth. These findings support a model in which (i) spoOA negatively affects abrB expression, and (ii) the repression of the transition state-activated genes tycA and spoVG in spoOA cells is due to constitutive expression of AbrB, which acts as a repressor.

Cellular oncogenes are frequently activated or deregulated in human malignant tumors. We have analyzed the expression of cellular oncogenes in human leukemia by Northern blot experiments in a case of acute myeloid leukemia (AML). In this case the cellular oncogenes N-myc, c-myb, c-fes and c-met were expressed at high levels. This is in contrast to normal peripheral blood mononuclear cells (PBMC) or chronic lymphocytic leukemia (CLL) cells, where these genes could not be detected. c-src2-specific RNA was not seen in either AML or PBMC cells but readily appeared in CLL cells. This appears to represent further evidence that in leukemic cells multiple cellular oncogenes might be activated.

BACKGROUND: CYP19A1 encodes the aromatase which catalyzes the final reaction of estrogen biosynthesis. The bovine genome also contains a non-coding copy of CYP19A1, the transcribed pseudogene CYP19P1. Whereas CYP19A1 is transcribed in all estrogen-producing tissues, mainly in the placenta and gonads, the CYP19P1 transcript so far was detected in the placenta. Strikingly, one sequence segment of both transcripts exhibits an exceptional high identity of 98%, which implies selective pressure and suggests some kind of function. Only recently, indeed, coding-independent functions of several transcribed pseudogenes were reported. Therefore, we analyzed CYP19P1 and CYP19A1 transcripts with the aim to detect clues for gene–pseudogene interference. FINDINGS: The CYP19P1 transcript was first examined in silico for the presence of microRNA coding sequences and microRNA targets. Further, to identify tissues where CYP19P1 and CYP19A1 transcripts are co-expressed, as a pre-requisite for transcript interference, expression profiling was performed in a variety of bovine tissues. Our in silico analyses did neither reveal potential microRNA coding sequences, nor microRNA targets. Co-expression of the CYP19 loci was demonstrated in placental cotyledons and granulosa cells of dominant follicles. However, in granulosa cells of dominant follicles the concentration of CYP19P1 mRNA was very low compared to CYP19A1 mRNA. CONCLUSIONS: CYP19P1 and CYP19A1 transcripts might interfere in placental cotyledons. However, in granulosa cells of dominant follicles relevant interference between gene and pseudogene transcripts is unlikely to occur because of the very low CYP19P1/CYP19A1 transcript ratio.