GABA differentially regulates the gene expression of proopiomelanocortin in rat intermediate and anterior pituitary

The GABAergic regulation of proopiomelanocortin messenger RNA (POMC mRNA) levels in rat pituitary was investigated using molecular hybridization of DNA complementary to POMC mRNA. Endogenous GABA levels increased, in vivo, by inhibiting the GABA catabolic enzyme GABA-transaminase (GAT) with ethalonamine-O-sulfate (EOS) or with vinyl-GABA (VG). Rats were treated with VG (100 mg/kg or 800 mg/kg) or EOS (100 mg/kg), administered each second day. GABA levels in the neurointermediate lobe (NIL) and anterior lobe (AL) of the hypophysis and in the hypothalamus were significantly increased following 4 days of VG treatment (800 mg/kg). All treatments resulted in a 40-60% decrease in POMC mRNA levels after 4 days in the NIL but not in the AL. A similar decrease of about 60% in POMC mRNA levels in the NIL was seen when EOS was given in the drinking water (5 mg/ml). In this set of experiments the time course of alteration of POMC mRNA in the NIL and the concentration of alpha-MSH, a POMC-derived peptide, were analysed. After one day of EOS treatment, when POMC levels had already decreased by 40%, alpha-MSH levels were significantly elevated (34% above controls), possibly reflecting an inhibition of alpha-MSH secretion. However, after 4 and 8 days, POMC mRNA levels and tissue alpha-MSH levels had significantly decreased. When tested in vitro, on primary cultures of IL cells, GABA (10 microM) reduced POMC mRNA levels by 40% after 3 days of treatment. These results show that GABA exerts a direct inhibitory effect on POMC gene expression in the intermediate lobe.     

The mink proopiomelanocortin gene: characterization of cDNA and chromosomal localization

A cDNA library from the mink pituitary was screened using as probe a synthetic oligodeoxyribonucleotide, 5'-TTCATGACCTCCGA-3', corresponding to the endorphin region of bovine proopiomelanocortin (POMC) cDNA. As a result, several clones containing inserts complementary to POMC mRNA were identified. The sequence of one of the fragments (585 bp, 65% of the total length of mRNA) was determined. A high degree of homology (over 80%) among the primary structures of sequences from mink, man, and bovine cDNA POMC was established. With the cloned mink cDNA fragment as probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of mink POMC sequences and mink chromosomes in the mink-Chinese hamster panel allowed us to assign the POMC gene to mink chromosome 11.     

In vitro study on proopiomelanocortin messenger RNA levels in cultured rat anterior pituitary cells

The fundamental examination on the measurement of proopiomelanocortin (POMC) mRNA levels in cultured rat anterior pituitary (AP) cells was studied. In addition, the detailed study on time- and dose-related effects of corticotropin-releasing factor (CRF) and dexamethasone on the level of POMC mRNA in AP cells in vitro was examined. Basal levels of POMC mRNA in AP cells cultured with serum initially declined after 1-day culture, gradually increased and reached a peak after 3-day culture, and then slightly decreased after 4- and 5-day culture. These mRNA levels after 3-day culture did not change through subsequent 15-hr incubation without serum. CRF treatment caused a time- and dose-dependent increase in POMC mRNA levels. The minimum effective dose of CRF was 0.1 nM for 15-hr incubation. The significant increase in POMC mRNA levels was observed after 3 hrs of 1 nM CRF treatment with a 2-fold elevation seen after 15 hrs of exposure. Dexamethasone treatment caused a dose-dependent decrease in POMC mRNA levels in AP cells. The minimum effective dose was 0.1 microgram/ml and such mRNA levels did not decrease until 15 hrs of exposure.     

Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury

Overlapping cDNA clones for rat hepatocyte growth factor (rHGF) were isolated by cross-hybridization with the cloned cDNA for human hepatocyte growth factor (hHGF) and the nucleotide sequence of the cDNA was determined. The entire primary structure of rHGF was deduced from the sequence. Comparison of the amino acid sequences between rat and human HGFs revealed that the two sequences are highly conserved throughout the protein structures, suggesting that rat and human HGFs may be functionally similar. Responses of the rHGF mRNA during liver regeneration in rats were examined by Northern blot hybridization analysis with the aid of the cDNA probe for rHGF. The mRNA levels increased in the liver and spleen but not in the kidney after administration of carbon tetrachloride. At the maximum level of induction, the rHGF mRNA increased in the liver about 4.5-fold over its normal level. The mRNA levels also increased in the liver and spleen after administration of D-galactosamine. On the other hand, no obvious increase of the mRNA was observed in the liver and spleen after partial hepatectomy. These observations suggest that HGF may function as a regulator of liver regeneration following hepatic injury caused by hepatotoxins.     

5' sequence of porcine and rat pro-opiomelanocortin mRNA. One porcine and two rat forms

We have determined the nucleotide sequences of the 5' noncoding regions and the regions encoding the first 56 amino acids of rat and porcine pro-opiomelanocortin (POMC) mRNA. We accomplished this by sequencing cDNA produced by elongating specific DNA primers hybridized to neurointermediate pituitary mRNA. The nucleotide sequence of the 5' region of rat POMC mRNA fills a gap in our knowledge of the structure of this mRNA and the protein it codes for. While we have observed only a single porcine POMC mRNA, two different rat POMC mRNAs are detected. The rat POMC mRNAs differ by a 30-base insertion/deletion in their 5' noncoding regions. Its position and sequence suggest that it is the result of alternate modes of intron removal during RNA splicing.     

Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the beta-subunit of rat follicle stimulating hormone

To provide a hybridization probe for analysis of the regulation of rat gonadotropin subunit mRNA levels, an effort was made to isolate a cloned cDNA for the beta-subunit of rat FSH (FSH beta). Using a cloned bovine FSH beta cDNA as a hybridization probe, a rat pituitary lambda gt10 cDNA library was screened and a single, strongly hybridizing clone identified. The 874 base pair cDNA insert from this clone contains the complete sequence of rat FSH beta including an amino-terminal precursor segment. Hybridization of this cloned cDNA to rat pituitary RNA demonstrated the presence of an approximately 2.0 kilobase RNA species containing FSH beta sequences. Cloned rat cDNA was also used to demonstrate that estrogen treatment of ovariectomized female rats results in decreases in mRNA concentrations for FSH beta and the beta-subunit of LH with somewhat smaller decreases in alpha-subunit mRNA concentrations. Little or no change was detected in the mRNA for the beta-subunit of TSH.     

Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts.

We have isolated and sequenced overlapping cDNA clones for rat KC*. The 0.93 kb cDNA has a single open reading frame of 288 nucleotides, and substantial sequence identity with the platelet-factor 4 family members mouse KC, hamster gro, and human gro. Using cloned cDNA as a probe, expression of KC mRNA in lavaged rat alveolar macrophages (AMs) increased after lipopolysaccharide (LPS) treatment. We also studied expression in vitro by a rat fetal lung fibroblast cell line, RFL-6. Expression of KC mRNA in RFL-6 cells increased after treatment with interleukin 1 or with conditioned medium from rat AMs treated with LPS.     

Transforming growth factor-alpha in the mammalian brain. Immunohistochemical detection in neurons and characterization of its mRNA

In this communication, we demonstrate that adult mammalian brain neurons express transforming growth factor-alpha (TGF-alpha). We used the anti-TGF-alpha monoclonal antibody, MF9, to immunohistochemically localize TGF-alpha in human and rat brain. We found specific immunoreactivity in neurons throughout the brain which was not a result of cross-reactivity of MF9 with the neuropeptide, synenkephalin. Northern blot analysis of bovine and rat brain RNA using human and rat TGF-alpha cDNA probes, respectively, revealed a single 4.8-kilobase pair mRNA with approximately equal abundance in the bovine brainstem, cerebellum, hypothalamus, and cerebral cortex. Fetal rat brain had about 2-fold more TGF-alpha mRNA than did adult rat. The brain TGF-alpha cDNA was cloned from a human neonatal brainstem library. Four identical clones were isolated after screening 10(6) recombinant lambda gt11 phage. The sequence of the 894-base pair cDNA was virtually identical with the cDNA isolated from a human renal cell carcinoma. A single alanine codon was deleted in the brain cDNA at an exon-exon junction. The alanine deletion is within the amino-terminal region of the TGF-alpha precursor that is thought to be removed by proteolytic processing of the precursor to the mature growth factor. These studies indicate that the normal mammalian brain neurons express TGF-alpha.     

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy.

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.     

Cloning of cDNA to rat mammary-gland fatty acid synthase mRNA. Evidence for the expression of two mRNA species during lactation.

A cDNA library was constructed in the expression vector lambda gt11, by synthesizing cDNA from size-selected poly(A) RNA from lactating rat mammary gland, using random hexanucleotide primers. Using this library we identified two recombinants which, on addition of a lac z inducer, produced proteins recognized by affinity-purified anti-fatty-acid synthase antibody, and which, therefore, contained fatty acid synthase coding sequences. The inserts were subcloned, were shown to be between 500 and 600 base pairs in size, and to cross-hybridize. The cloned DNA was then used in Northern hybridizations with mRNA isolated at various stages throughout lactation. Two mRNA species were identified of approx. 9.7 and 10.4 kilobases, which increased and decreased in parallel during lactation, reaching a peak at 12-13 days. Both mRNA species disappeared rapidly if the pups were removed prematurely. This study provides evidence that, during hormonal induction in lactation, regulation of the level of fatty acid synthase protein can be accounted for by variation in the level of mRNA.     

Glucocorticoid regulation of proopiomelanocortin messenger ribonucleic acid content of rat hypothalamus

We have verified the possibility that the POMC gene of the rat hypothalamus might be subject to regulation by glucocorticoids. Adrenalectomy increased the concentration of POMC mRNA in anterior pituitary and in hypothalamus, but not in the neurointermediate lobe of the pituitary gland. Dexamethasone and, to a slightly lesser extent, corticosterone treatments reversed the adrenalectomy-induced increase in POMC mRNA concentrations in both anterior pituitary and hypothalamus. Dexamethasone caused a slight decrease of POMC mRNA levels in the neurointermediate lobe of the pituitary gland, while corticosterone had no effect. These results indicate that the POMC gene of the rat brain hypothalamus is also under negative control by glucocorticoids.     

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species.     

Molecular cloning of DNA complementary to rat alpha 2-macroglobulin mRNA

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein synthesized in the liver. Using an in vitro translation system coupled with solid-phase radioimmunoassay, alpha 2M mRNA activity was found to rise to a maximum level in 16-24 h after turpentine injection. Poly(A)+ RNA from turpentine-injected rat liver was converted to cDNA by the method of Okayama-Berg, and about 50,000 transformants were obtained. From these transformants, clones containing alpha 2M cDNA were selected using the following criteria: 1) alpha 2M cDNA should hybridize with synthetic oligonucleotides encoding portions of the alpha 2M amino acid sequence, 2) alpha 2M cDNA should hybridize preferentially with RNA which increases during inflammation, 3) mRNA which hybridizes with alpha 2M cDNA should encode a polypeptide which specifically reacts with antibody against alpha 2M, and 4) the cDNA should contain the nucleotide sequences encoding the amino acid sequences of alpha 2M. We found clones which fulfilled these criteria. Using the cDNA clone as a probe, we demonstrated that the level of alpha 2M mRNA in the liver of inflamed animal markedly increased up to 1000-fold. The size of the alpha 2M mRNA was about 4800 nucleotides in length by Northern analysis.     

Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA

Three cDNA clones encoding rat basic fibroblast growth factor (FGF) were isolated from 10(6) independent clones prepared from a pregnant mare serum gonadotropin (PMSG)-stimulated rat ovarian cDNA library. One of the cDNA clones contained the entire coding sequence for basic FGF. The other two possessed the sequence coding the carboxy terminal 61 amino acids of rat basic FGF, the putative upstream intron sequence, and a 3'-noncoding region. The cDNAs encoding rat basic FGF predict a molecule consisting of 154 amino acid residues, which is one amino acid shorter than the human and bovine basic FGF. Otherwise, there are only 5 conservative amino acid substitutions between the rat and the human/bovine sequences. Poly A+ RNA from brain cortex and hypothalamus show a single 6.0 kb band that hybridizes to the cloned cDNA probe by Northern analyses. The observation that basic FGF mRNA is below the limits of detection in adrenal, spleen, heart, lung, kidney, liver, stomach, small intestine, large intestine, testis, and ovary support the notion that the that the high levels of the protein found in these tissues is due to storage of the mitogen in the extracellular matrix and not continuous gene expression. The significance of the abundance of mRNA in tissues which are not undergoing either active angiogenesis or cell proliferation (hypothalamus and brain cortex) is unclear but emphasizes the potential neuronotrophic function of basic FGF.     

Molecular cloning of DNA complementary to bovine growth hormone mRNA

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.     

Synthesis, cloning and primary structure of DNA complementary to mRNA for human pituitary pro-opiomelanocortin

cDNA coding for the human pro-opiomelanocortin (POMC) has been cloned and sequenced. It codes for full size amino acid sequence of POMC and furthermore, contains most part of the 3'-terminal noncoding mRNA region and 60 nucleotides coding for signal peptide.     

Proopiomelanocortin (POMC) mRNA and POMC-derived peptides immunolocalization in the skin of Protopterus annectens, an African lungfish

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.