Minor Transcripts Research Articles

Gamma-tubulin in trypanosomes: molecular characterisation and localisation to multiple and diverse microtubule organising centres.

A genomic clone from Trypanosoma brucei, which contains a full length gamma-tubulin gene, was isolated using degenerate oligonucleotide primers. The sequence of this clone predicts a protein of 447 amino acids having a high degree of homology with gamma-tubulins from human and Xenopus laevis (67.2% amino acid identity) and only 57.7% identity with the Plasmodium falciparum gamma-tubulin. Northern blot analysis of poly(A)+ selected RNA from a procyclic culture detects a major transcript of approximately 2.2 kb plus a minor transcript of approximately 3.6 kb. A fusion protein comprising almost the full length gamma-tubulin gene product (amino acids 8-447) plus an amino-terminal histidine tag has been expressed and purified from Escherichia coli and used to raise a polyclonal antibody. Immunofluorescence, using this antibody, shows classical centrosomal localisation in mammalian cells. In T. brucei gamma-tubulin is present in the basal bodies which subtend the flagellum and also at the anterior tip of the cell body where many minus ends of microtubules are located. Furthermore the antibody reveals a small subset of the sub-pellicular microtubules and a discrete dot within the nucleus which alters form with progression through the mitotic cycle. Evidence is also presented for discrete punctate staining within the microtubules of the cell body which may represent the presence of gamma-tubulin on the ends of individual microtubules. Our results indicate that gamma-tubulin is associated with diverse microtubule organising centres and structures in trypanosomes.

Expression of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene in Benign Prostatic Hyperplasia and in Prostate Carcinoma in Humans

The findings that normal rat prostates express functional LH/hCG receptors led us to test the hypothesis that benign prostatic hyperplasia (BPH) and prostate carcinomas may also express this receptor gene. The data revealed the presence of LH/hCG receptor transcripts and receptor protein in normal and hyperplastic but not in atrophic glands present in BPH tissue. Smooth muscle and blood vessels in stroma of BPH tissue also contained receptors. Prostate carcinomas contain lower and more heterogeneous receptor levels than BPH tissue. Two human prostate cancer cell lines (LNCaP and DU 145) that were investigated showed the presence of a major 4.5-kilobase transcript and several minor transcripts and also the protein of LH/hCG receptors. However, androgen-sensitive LNCaP cells contained more receptors than androgen-insensitive DU 145 cells. In summary, we demonstrate for the first time that BPH and prostate cancer tissues and cell lines express LH/hCG receptor gene. These findings suggest that higher LH levels in aged men may play a role in BPH and/or prostate carcinomas.

Cloning of a rat alpha1,3-fucosyltransferase gene: a member of the fucosyltransferase IV family.

We report the cloning of a rat alpha1,3-fucosyltransferase gene (rFuc-T), isolated from a rat genomic library by a PCR-cross-hybridization based cloning approach using primers derived from the conserved region of human alpha1,3-Fuc-T sequences. Comparison of the rFuc-T predicted amino acid sequence with those of previously cloned human and murine fucosyltransferases showed highest degree of homology to murine Fuc-TIV (87% identity) and human Fuc-TIV (78% identity), with lower homology (41-49% identity) to Fuc-TIII, V, VI, and VII. COS-1 cells transfected with the rFuc-Tgene expressed a fucosyltransferase activity with type 2 (Gal beta1-->4GlcNAc)-containing oligosaccharides and the glycolipid acceptor neolactotetraosylceramide but only low activity with sialylated substrates; the SSEA-1/Le(x) antigen was detected in transfected cells by immunocytochemistry. Based on these results, we surmise that rFuc-T is a member of the fucosyltransferase IV family. Northern blot analysis with a rFuc-T specific probe indicated a major transcript of 4.2 kb most abundantly expressed in rat spleen; minor transcripts of different sizes were detected in several tissues, including rat brain.

Expression and distribution of two alternatively spliced transcripts from the chicken alpha 2 (VI) collagen gene.

Two types of mRNA molecules with different 3' ends are transcribed from the chicken alpha 2(VI) collagen gene. The major splice variant encodes a polypeptide with a von Willebrand factor A domain at its carboxyl terminus. In the minor splice variant, this A domain is replaced by a novel motif which reveals some similarity to a fibronectin type III repeat. In situ hybridization experiments demonstrate that the major transcript is ubiquitously expressed. Substantial amounts are found in skeletal and cardiac muscle, gizzard, skin, tendon, liver, the wall of blood vessels, and the connective tissue of peripheral nerves. In contrast, the minor transcript is expressed at a very low level and can hardly be detected in any tissue by in situ hybridization. Only the aortic wall contains a considerable amount of this splice variant. However, no difference is observed by Northern blotting and the polymerase chain reaction in the ratio of the two transcripts when aorta and the other tissues are compared. Thus, the minor splice variant is not expressed in a tissue specific manner and, consequently, it is unlikely that it plays a tissue specific role. It might rather serve a general function in the structure and assembly of type VI collagen microfibrils.

Expression and distribution of two alternatively spliced transcripts from the chicken α2(VI) collagen gene

Two types of mRNA molecules with different 3′ ends are transcribed from the chicken α2(VI) collagen gene. The major splice variant encodes a polypeptide with a von Willebrand factor A domain at its carboxyl terminus. In the minor splice variant, this A domain is replaced by a novel motif which reveals some similarity to a fibronectin type III repeat. In situ hybridization experiments demonstrate that the major transcript is ubiquitously expressed. Substantial amounts are found in skeletal and cardiac muscle, gizzard, skin, tendon, liver, the wall of blood vessels, and the connective tissue of peripheral nerves. In contrast, the minor transcript is expressed at a very low level and can hardly be detected in any tissue by in situ hybridization. Only the aortic wall contains a considerable amount of this splice variant. However, no difference is observed by Northern blotting and the polymerase chain reaction in the ratio of the two transcripts when aorta and the other tissues are compared. Thus, the minor splice variant is not expressed in a tissue specific manner and, consequently, it is unlikely that it plays a tissue specific role. It might rather serve a general function in the structure and assembly of type VI collagen microfibrils. © 1996 Wiley-Liss, Inc.

Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells.

In the present studies we examined the regulation of insulin-like growth factor I (IGF-I) expression in porcine granulosa cells in vitro. Using Northern analysis and ribonuclease protection assays with exon-specific probes, we identified the IGF-I messenger RNA (mRNA) transcripts present in these cells under basal and hormone-stimulated conditions. We also assessed changes in secreted IGF-I using Western blots and correlated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its transcription, two major transcripts of approximately 1 and 7.5 kilobases, seen in freshly isolated granulosa cells and follicle wall and in single passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forskolin treatments or in control cells after long exposure of the autoradiographs, were attributed to incompletely processed RNA precursors. Ribonuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the transcripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probes, respectively. GH increased steady state levels of IGF-I mRNA 3-fold, FSH increased it approximately 10-fold, and forskolin maximally increased it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA coordinately increased both class 1 and class 2 transcripts, with the increase in class 1 greater than that in class 2. Multiple forms of IGF-I protein were seen under basal conditions and after hormone treatment. These were identified based on mRNA analysis and biochemical methods as both glycosylated and nonglycosylated IGF-IA prohormone, incompletely processed forms of prohormone, and the mature peptide. Changes in the levels of total protein were similar to the changes in mRNA (GH, 3-fold; FSH and forskolin, 10- to 20-fold). All forms of the protein changed coordinately, suggesting that these hormones had no major effect on the intracellular processing mechanism. IGF-binding protein-3 was able to bind to all IGF-I forms. These data conclusively demonstrate FSH and GH induction of ovarian IGF-I. The porcine granulosa cell culture system used in these studies should be an excellent system for studying the hormonal regulation of IGF-I expression.

Aromatase cytochrome P450 gene expression in endometrial carcinoma.

We analysed aromatase gene expression and its regulation in seven cases of endometrioid endometrial carcinoma. Immunohistochemistry revealed the presence of strong aromatase immunoreactivity in the stromal cells of carcinoma in five out of seven cases. A polymerase chain reaction after reverse transcription (RT PCR) revealed varying levels of aromatase transcripts (0.1-27.0 amol ng(-1) total mRNA) in five cases. The alternative use of multiple exons 1 was also examined by identifying various human aromatase transcripts specific for exons 1 in RT-PCR products. Gonadal type or exon 1d was primarily used in three cases in which aromatase overexpression was not detected. The two cases in which fibroblasts type or exon 1b was used with other exons 1 as minor transcripts demonstrated aromatase overexpression in immunohistochemistry and RT PCR analysis. Further studies are required, but alternative splicing as well as use of multiple exons 1 transcripts may result in increased aromatase expression in stromal cells observed in endometrial carcinoma.

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

We have cloned and expressed two cDNAs encoding the human lysosomal alpha-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from alpha-mannosidosis fibroblasts, and p-nitrophenyl-alpha-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, Km, and Vmax characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No alpha-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb alpha-mannosidase cDNA immunoprecipitated lysosomal alpha-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including alpha-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal alpha-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239-245) and several differences were found relative to the functional lysosomal alpha-mannosidase encoded by the 3-kb spleen cDNA.

Molecular analysis of the Rhodobacter capsulatus chaperonin (groESL) operon: purification and characterization of Cpn60.

The heat-shock protein Cpn60 (chaperonin, GroEL homologue) from the phototrophic bacterium Rhodobacter capsulatus B10 was purified to homogeneity and biochemically characterized. Native Cpn60 from R. capsulatus was shown to be a tetradecamer of 840 kDa similar to that of homologous chaperones characterized so far. Cpn60 possesses ATPase activity and promotes refolding of chaotropically denatured citrate synthase. The groESL operon of R. capsulatus was cloned using a degenerate oligonucleotide and sequenced. Two open reading frames (285 and 1,635 bp) were found; they encode Cpn10 and Cpn60, with corresponding deduced molecular masses of 10.6 and 57.6 kDa. The deduced amino acid sequences coincided perfectly with those of the amino terminus and of three tryptic peptides of purified Cpn60 from R. capsulatus. Strong evidence that R. capsulatus encodes only one copy of the groESL operon was obtained. Primer-extension analysis revealed that the groESL operon is transcribed by a -35/-10-type promoter, and that transcription was initiated from the same positions before and after heat-shock under both chemotrophic and phototrophic conditions. The major initiation site is immediately followed by the inverted repeat structure CIRCE, which has been found upstream of many bacterial heat-shock operons. A second minor transcript starts just after the CIRCE element. Although heat-shock induction of a groEL-lacZ fusion failed because of thermal inactivation of the fusion protein, Western blot analysis revealed a two- to threefold induction of cellular Cpn60 levels 45-75 min after shifting from 28 degrees C to 39 degrees C. Deletion mapping of the groESL promoter identified upstream of the promoter a 19-bp element that enhances groESL transcription eightfold and contains the AT-rich sequence dAAATTTTT, which is found at similar positions in heat-shock operons of other gram-negative bacteria.

The Promoter Structure of TGF-β Type II Receptor Revealed by “Oligo-capping” Method and Deletion Analysis

The promoter of TGF-β type II receptor lacks TATA box or CAAT box. In order to define the exact transcriptional start site(s), we used “oligo-capping” method developed by ourselves. The major transcripts were started from GAA located between −35 and −33 relative to 5′ end of cDNA. Other minor transcripts started from −4, +11 and +17. Deletion analysis of this promoter region revealed that it contained two adjacently located promoters (P1 and P2) capable of acting by itself. The P1 is located between −22 and +55 which covers the minor transcriptional start sites described above. The P2 is located just upstream of the P1 between −137 and −22. The results showed all of the transcripts were started either from P1 or P2.

An Alternatively Spliced C. elegans ced-4 RNA Encodes a Novel Cell Death Inhibitor

The C. elegans gene ced-4 is essential for programmed cell death. We report that ced-4 encodes two transcripts and that whereas the major transcript can cause programmed cell death, the minor transcript can act oppositely and prevent programmed cell death, thus defining a novel class of cell death inhibitors. That ced-4 has both cell-killing and cell-protective functions is consistent with previous genetic studies. Our results suggest that the dual protective and killer functions of the C. elegans bcl-2-like gene ced-9 are mediated by inhibition of the killer and protective ced-4 functions, respectively. We propose that a balance between opposing ced-4 functions influences the decision of a cell to live or to die by programmed cell death and that both ced-9 and ced-4 protective functions are required to prevent programmed cell death.

Human skin contains luteinizing hormone/chorionic gonadotropin receptors.

Normal human skin contains a major 4.5 kb and several minor mRNA transcripts and a 66 kDa protein of luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptors which are capable of binding exogenous 125I-hCG. The distribution of receptor transcripts and receptor protein are the highest in epidermis followed by hair follicles, sebaceous and sweat glands. LH/hCG receptors are co-localized with androgen receptors in all the skin appendages. These data are the first demonstration of skin containing LH/hCG receptors and would suggest that LH and hCG may regulate skin functions.

Ovine Endometrial Expression of Transforming Growth Factor β Isoforms during the Peri-Implantation Period1

During the estrous cycle and early pregnancy, the uterus undergoes a variety o morphological changes. Because of their powerful effects on angiogenesis, on extracellular matrix modification, and on cellular proliferation and differentiation, the transforming growth factor beta (TGF beta) family of polypeptides may be involved in the regulation of pregnancy-related endometrial modification. In this study, endometrial stead-state mRNA expression of the three isoforms, TGF beta 1, beta 2, and beta 3, was quantified, and the proteins were localized during the later part of the estrous cycle (Day 13 and 16) and during early pregnancy (Days 13 through 30) in sheep. TGF beta 1 mRNA was expressed as a single transcript with steady-state mRNA expression levels 2-fold higher on Day 16 of the estrous cycle than on Day 13 of the estrous cycle (p < or = 0.002) or Days 13 and 16 (p < or = 0.004) and 0.008, respectively) of gestation. During pregnancy, levels increased progressively to peak on Day 27 (2.5-fold above Day 16 of pregnancy; p < or = 0.0001) and then leveled off at Day 30. Immunocytochemical localization of TGF beta 1 demonstrated protein in glandular protein in glandular and luminal epithelium at all days examined. TGF beta 2 mRNA was expressed as five distinct transcripts, and mRNA expression levels were lowest in Day 16 pregnant endometrium. Thereafter the expression level increased steadily through Day 30 (p < or = 0.0003). TGF beta 2 protein was localized in epithelium, diffusely within the endometrial stroma, and in leukocyte-like cells within the stroma. TGF beta 3 was expressed as one major transcripts and two minor transcripts. As with TGF beta 1, there was a dramatic difference in TGF beta 3 levels between Day 16 of pregnancy and Day 16 of the estrous cycle (3.8-fold, p < or = 0.0001). In pregnant ewes, endometrial TGF beta 3 levels increased 1.9-fold (p = 0.13) between Days 16 and 23 and remained relatively constant through Day 30. Immunohistochemistry localized TGF beta 3 protein most prominantly in the subepithelial stroma of the caruncule from Day 16 of the cycle in endometrial tissue. The observed changes in mRNA and protein expression patterns of TGF beta s within the ovine endometrium suggest that TGF beta s play a role in restructuring and modifying endometrium for a subsequent estrous cycle and/or pregnancy.

Existence and expression of the untranslated first exon of aromatase mRNA in the rat brain.

Tissue-specific expression of aromatase activity and mRNA occurs by alternative utilization of multiple untranslated first exons and promoters in the human. The major transcript in the human brain contains the brain-specific first exon, "exon I-f". However, few reports on the untranslated first exon of aromatase mRNA in the rat brain have been available so far. In the present study, we investigated the existence and expression of exon I-f in the rat brain to elucidate the mechanism of the tissue-specific expression of the brain aromatase. Total RNA extracted from amygdala (AMY) was subjected to a reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the RT-PCR product had 89.4% homology to the corresponding region of exon I-f of the human aromatase cDNA. It was indicated that the major transcript in the rat AMY contained exon I-f by the use of a rapid amplification of cDNA ends (RACE). Furthermore, in order to determine the distribution of the aromatase mRNA with exon I-f, total RNAs from the hypothalamus-preoptic area (HPOA), AMY, testis and ovary were analysed by RT-PCR using the primers specific for the mouse exon I-f and the primers for the rat exon III-V. Significant levels of PCR products were found in all tissues with the highest level being in the ovary, using the primers for exon III-V. On the other hand, using the primers for exon I-f, the levels of signals from HPOA and AMY were higher than those from the testis and ovary. These results suggest that tissue-specific expression of aromatase mRNA occurs by an alternative utilization of multiple promoters in the rat, as in the human. It should be noted that minor transcripts containing exon I-f were observed in the testis and ovary.

Expression of tissue factor correlates with grade of malignancy in human glioma.

Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF belongs to the interferon receptor family and is one of the immediate early genes, suggesting that TF may participate in the regulation of cell growth. However, the correlation between the expression of TF and cell growth is still unclear. Expression of TF in 6 glioma cell lines and 44 glioma surgical specimens was studied by Northern blot analysis, Western blot analysis, immunohistochemistry, and in situ hybridization. Northern blot analysis showed that glioma cells expressed minor novel transcripts of 3.3 kb and 1.6 kb, in addition to the transcripts of 2.2 kb and 3.1 kb that were previously reported. Western blot analysis revealed that the level of TF protein did not correlate with that of TF transcripts. Although immunohistochemical analysis of surgical specimens showed that all gliomas were positive for TF, it was interesting that 1 of 10 benign gliomas (10%) was positive for TF (malignancy Grade I-II), 13 of 14 anaplastic astrocytomas (86%) (malignancy grade III) and 19 of 20 glioblastomas (95%) (malignancy grade IV) were moderately or strongly positive for TF. In situ hybridization showed the expression of TF mRNA in glioma cells. TF is expressed in glioma and the level of expression correlates with the histologic grade of malignancy.

Single nucleotide primer extension: quantitative range, variability, and multiplex analysis.

The quantitative measurement of transcription products from homologous alleles at a diploid locus has broad application for the study of mammalian gene expression. Single nucleotide primer extension (SNuPE) analysis is a simple and sensitive method for allelic transcript discrimination requiring only 1 bp difference between alleles. In this study the effective range, experimental variation, and the influences of poly(dT)-primed and gene-specific reverse transcriptions are characterized. The ability to analyze several genes from a single reverse transcription reaction is assessed as well. For the genes examined, the maximum range of detection is reached when the minor transcript represents 1/250 of the major allele. Relatively little error is seen within or between assays and linearity of response is maintained over an approximately thousandfold range.

Infection of macrophages by influenza A virus: characteristics of tumour necrosis factor-α (TNFα) gene expression

Human monocytes and murine macrophages were found to be susceptible to influenza A virus infection. We could show that virus was absorbed and de novo virus protein synthesis was initiated, but actual virus replication was extremely low; 24-36 h after infection, monocytes and macrophages died of apoptosis. Before cell death, an influenza A virus infection induced strong mRNA accumulation of cytokines: tumour necrosis factor-alpha (TNF alpha), interleukin-1 (IL1) and IL6. However, the translation into bioactive cytokine protein was rather low, and high cytokine production was only found when a secondary signal such as LPS was added. Influenza A virus infection of mononuclear phagocytes displayed a characteristic feature at the level of cytokine gene transcription which was not found with other viruses such as vesicular stomatitis virus: in addition to the regular 1.7-kb TNF alpha mRNA, a high molecular weight (hmw) TNF alpha mRNA of 2.4 kb was detected. This hmw TNF alpha mRNA did not contain intron or intergenic region elements, was polyadenylated and carried the regular 5' and 3' untranslated regions. The generation of hmw TNF alpha mRNA required exposure to fully infectious influenza A virus, since virus inactivation at 56 degrees C induced only regular and not hmw TNF alpha mRNA. Whether this unique hmw TNF alpha mRNA represents a virus-induced abnormality or only a superinduction of an otherwise minor TNF alpha transcript, and whether this mRNA species codes for a biologically active product, remain to be further studied.

Connexin 35: a gap-junctional protein expressed preferentially in the skate retina.

We have used low stringency hybridization to clone a novel connexin from a skate retinal cDNA library. A rat connexin 32 clone was used to isolate a single partial clone that was subsequently used to isolate seven more overlapping clones of the same cDNA. Two clones containing the entire open reading frame have a consensus sequence of 1456 bp and predict a protein of 302 amino acids length and molecular mass of 35,044 daltons, referred to as connexin 35 or Cx35. Southern blot analysis suggests that the cloned sequence lies in a single gene with one intron. Polymerase chain reaction amplification from genomic DNA and partial sequencing of this intron showed that it was approximately 950 bp in length, and located within the coding region 71 bp after the translation start site. Hydropathy analysis of the predicted protein and alignments with previously cloned connexins indicate that Cx35 has a long cytoplasmic loop and a relatively short carboxyl terminal tail. Multiple sequence alignments show that Cx35 has similarities to both alpha and beta groups of connexins and suggests that its origins may be near the divergence point for the two groups. Consensus sequences consistent with sites for phosphorylation by protein kinase C and by cAMP - or cGMP -dependent protein kinase were identified. Two transcripts were detected in Northern blot analysis: a 1.95-kb primary transcript and a 4.6-kb minor transcript. In RNA samples from 10 tissues, transcripts were detected only in the retina.

Comparison between parr and smolt Atlantic salmon (Salmo salar) α subunit gene expression of Na+/K+ ATPase in gill tissue

Increases in branchial Na(+)/K(+) ATPase activity during seawater adaptation of euryhaline fish species, have been well documented. During the parr-smolt transformation of salmonids this activity increases two to five fold and is used as an indicator of the transformation. In order to improve the understanding of differences in enzyme activity found between Atlantic salmonSalmo salar parr and smolt fish, we investigated the gene expression of the Na(+)/K(+) ATPase α-subunit(s) in gill tissue. Gill mRNAs were analyzed and quantified at distinct time points using Northern and Dot blot techniques. We amplified by PCR, a conserved region of the cDNA encoding the Na(+)/K(+) ATPase α-subunit of the rainbow troutOncorhynchus mykiss. The PCR products (670 bp) were cloned and all independent clones showed a sequence corresponding to the α subunit of the Na(+)/K(+) ATPase. The fragments obtained appeared as a heterogenous population of three sequences showing, when compared between each other, 86 to 93% identity. This suggests that different allelic forms of the α-subunit are expressed in gill tissue. Hybridization studies performed with these PCR probes revealed two mRNA species, a major 3.7 kb transcript and a minor transcript of 1.8 kb. Enhanced 3.7 kb transcript levels are concurrent with elevated enzyme activity in smolts during the March and April parrsmolt transformation of Atlantic salmon. Interestingly, our study disclosed that smolt fish only displayed a two-fold increase in transcript levels when compared to parr whereas enzyme activity showed a 4 to 5 fold increase. This suggests that the increase in the 3.7 kb mRNA content of gill tissue is probably not the only mediator leading to the rise in enzyme activity during parr-smolt transformation.

Localization and regulation of corticotropin receptor expression in the midgestation human fetal adrenal cortex: implications for in utero homeostasis.

Developmental changes in the responsiveness of the fetal adrenals to corticotropin (ACTH) play an important role in the regulation of the fetal hypothalamic-pituitary-adrenal axis. Responsiveness of adrenal cortical cells to ACTH is dependent on the extent of ACTH receptor expression. Therefore, we examined the localization and regulation of ACTH receptor expression in the midgestation (16-24 weeks) human fetal adrenal cortex. In situ hybridization analysis was used to localize messenger RNA (mRNA) encoding the ACTH receptor in sections of human fetal adrenal glands. Messenger RNA encoding the ACTH receptor was localized in cells from all cortical zones; abundance was higher in definitive zone than in fetal zone cells and was least abundant in the more central portions of the cortex. Regulation of ACTH receptor expression was studied using Northern blot analysis of total RNA extracted from primary cultures of fetal and definitive zone cells. Two major (1.5 and 3.5 kilobases) and, upon stimulation with ACTH, 3 minor (4.0, 6.0 and 10.0 kb) ACTH receptor mRNA transcripts were detected in RNA from fetal and definitive zone cells. In both cell types, ACTH-(1-24) increased the abundance of mRNA encoding the ACTH receptor 10- to 20-fold compared with untreated cells. The effects of ACTH-(1-24) on ACTH receptor expression in fetal zone cells were time- and dose-dependent. The ED50 for the stimulation of ACTH receptor expression by ACTH-(1-24) was 1-10 pM, and maximal response to 0.1 nm ACTH-(1-24) was detected after 12-16 h. Eight-bromoadenosine cAMP and forskolin also stimulated ACTH receptor expression in fetal zone cells and closely mimicked the effects of ACTH-(1-24). In contrast, stimulation of protein kinase C with 12-O-tetradecanoyl phorbol 13-acetate had no effect on ACTH receptor expression. Changes in ACTH receptor expression in response to ACTH-(1-24), cAMP and forskolin were paralleled by changes in expression of the P450 cholesterol side chain cleavage (P450scc) enzyme. These data demonstrate that expression of the ACTH receptor by the human fetal adrenal cortex is up-regulated by its own ligand and that this effect is mediated by a cAMP-dependent mechanism. In addition, the coordinate stimulation of ACTH receptor and P450scc expression by ACTH indicates that the gene for the ACTH receptor is one of a specific cohort of genes regulated by ACTH that are required to facilitate fetal adrenal cortical response to ACTH. ACTH regulation of its own receptor may represent a mechanism by which fetal adrenal responsiveness to ACTH is maintained and possibly enhanced during fetal development.