Expression of rat thyrotropin-releasing hormone (TRH) gene in TRH-producing tissues of transgenic mice requires sequences located in exon 1.

TRH, an amidated tripeptide secreted by certain hypothalamic neurons, is a principal regulator of TSH secretion and thyroid hormone release. TRH is also produced by other neurons in the central nervous system, where it appears to function as a neuromodulator or neurotransmitter, and by certain endocrine cells, where it may act as an autocrine or paracrine factor. The genomic organization of the rat TRH (rTRH) gene is well understood; however, the domains of the rTRH gene that regulate expression are less well characterized. We observed that the region between -47 and +6 of the rTRH gene (relative to the transcription start site at +1) was active in CA-77 cells, a medullary thyroid carcinoma cell line model of TRH production, but was not active in transgenic mice. Inclusion of most of exon 1 (84 out of 103 bp; -47 to +84) increased promoter activity in CA-77 cells and was active in transgenic mice, principally in tissues that normally express the TRH gene. Further lengthening of the 5' end to -243, -547, or -776 retained this expression in TRH-producing tissues in transgenic mice, while further increasing activity in CA-77 cells. These results suggest that cis element(s) located within exon 1 are necessary for the expression of the rTRH gene in vivo.

The thyrotrophin-releasing hormone (TRH)-like peptides in rat prostate are not formed by expression of the TRH gene but are suppressed by thyroid hormone.

Thyrotrophin-releasing hormone (TRH)-immunoreactive peptides were extracted from rat prostate and divided into two groups by mini-column cation exchange chromatography. The amounts of the peptides in each group were determined by radioimmunoassay with a TRH antiserum. The unretained peptides which lacked a basic group and the retained peptides which possessed a basic group were further purified by high-performance liquid chromatography. The unretained fraction was found to contain a series of TRH-immunoreactive peptides, one of which corresponded chromatographically to synthetic pGlu-Glu-Pro amide and another to pGlu-Phe-Pro amide. None of the TRH-immunoreactive peptides in either fraction exhibited the chromatographic behaviour of TRH. Additional evidence for the absence of TRH gene expression in the prostate was obtained by Northern blot analysis and by application of polymerase chain reaction amplification, which failed to reveal TRH mRNA. Furthermore the preproTRH-derived peptide, preproTRH(53-74), could not be detected by radioimmunoassay. The influence of thyroid status was investigated on the levels of the TRH-like peptides in the prostate. Adult rats were treated chronically with thyroxine (T4) or propylthiouracil (PTU) and the concentrations of the TRH-immunoreactive peptides were determined by chromatography and radioimmunoassay. Treatment with T4 caused the levels of the neutral and acidic TRH-like peptides to fall to approximately one-third of the levels in the controls. No significant difference from the controls was seen in the concentrations of the peptides in the prostates of rats rendered hypothyroid by administration of PTU. The results demonstrate that rat prostate contains TRH-immunoreactive peptides which are not derived from the TRH gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin-releasing hormone gene expression in normal thyroid parafollicular cells.

The rat TRH gene encodes a 255-amino-acid precursor polypeptide, preproTRH, containing five copies of TRH and seven non-TRH peptides. Expression of this gene is well documented in the central nervous system, particularly in the hypothalamus. Thyroids also contain TRH immunoreactivity, but it is unknown whether this immunoreactivity results from expression of the TRH gene or from other genes encoding TRH-like products. Since the CA77 neoplastic parafollicular cell line expresses the TRH gene, we investigated whether TRH gene expression also occurs in normal thyroid parafollicular cells. Northern analysis of total thyroid RNA with a preproTRH-specific RNA probe identified a single hybridizing band the same size as authentic TRH mRNA found in hypothalamus and CA77 cells. Gel filtration analysis of thyroid extracts identified the same 7-kilodalton and 3-kilodalton species of immunoreactive preproTRH53-74 previously identified in hypothalamus and CA77 cells. Immunoreactive preproTRH115-151, not previously identified, was found in all three tissues. Part of this immunoreactivity comigrated with the synthetic preproTRH115-151 standard on gel filtration and reversed-phase HPLC. PreproTRH53-74 was localized to thyroid parafollicular cells by immunostaining. These findings demonstrate authentic TRH gene expression by normal rat thyroid parafollicular cells and establish the CA77 cell line as the only model system of a normal TRH-producing tissue. In addition to expanding the range of neuroendocrine peptides known to be produced by parafollicular cells, these results also suggest a potential paracrine regulatory role for TRH gene products within the thyroid.

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line.

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.

Isolation, characterization, and DNA sequence of the rat somatostatin gene.

The gene encoding rat somatostatin has been isolated from a lambda phage gene library. Phage harboring the gene were identified by plaque hybridization using a nick-translated fragment derived from the cDNA for rat somatostatin. The transcriptional unit includes exons of 238 and 367 base pairs (bp) separated by one intron of 621 bp. The intron is located between the codons for Gln (-57) and Glu (-56) of prosomatostatin. Analysis of the nucleotide sequence 5' to the start of transcription reveals a number of sequences which may be involved in the expression of somatostatin. A variant of the "TATA" box, TTTAAA, lies 26 bp upstream from the start of transcription, and a sequence homologous to the "CAAT" box (GGCTAAT) is 92 bp upstream from the transcription start. A long alternating purine-pyrimidine stretch, (GT)25, which is similar to Z DNA-forming sequences in other genes, lies 628 bp 5' to the transcription start and is flanked by small repeats. Hybridization analysis shows that this region is highly repeated in the genome and that homologous sequences are located approximately 2 kilobase pairs downstream from the poly(A) addition site. Southern hybridization of the lambda clone with probes derived from brain or liver poly(A+) RNA demonstrates that another transcribed sequence lies about 7 kilobase pairs downstream from the poly(A) addition site of the rat somatostatin gene. Analysis of rat DNA suggests that there may be restriction-site polymorphisms in or near the gene or that additional somatostatin-hybridizing sequences may exist in the genome.

Cloning and characterization of a mRNA-encoding rat preprosomatostatin.

An undecanucleotide extended hybridization probe has been used to screen a rat medullary thyroid carcinoma cDNA library for clones which contain preprosomatostatin sequences. The nucleotide sequence encoding rat preprosomatostatin is reported. The sequence of cDNA contains 67 nucleotides in the 3'-noncoding region, 84 nucleotides in the 5'-untranslated region, and 458 bases corresponding to the coding region. The mRNA codes for a somatostatin precursor 116 amino acids in length (Mr = 12,773). The preprosomatostatin has a sequence of hydrophobic amino acids at the NH2 terminus, followed by a peptide of approximately 78 residues, which precedes somatostatin-14. The amino acid sequences of rat and human preprosomatostatin (Shen, L. P., Pictet, R. L., and Rutter, W. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4575-4579) differ by only 4 amino acid residues. Translation of rat poly(A) RNA in a rabbit reticulocyte cell-free system followed by immunoprecipitation with antisera directed against somatostatin-14 demonstrated the synthesis of a single protein having a molecular weight of 15,000. Two proteins having molecular weights of 14,000 and 15,000 are immunoprecipitated from a wheat germ cell-free translation mixture. Northern analysis of the somatostatin mRNA indicated that it is approximately 850 nucleotides in length. Analysis of several medullary thyroid carcinomas demonstrated that one tumor, designated WF, had immunoreactive somatostatin-14 in concentrations of 350 ng of somatostatin-14/mg of protein and somatostatin mRNA that represented 10% of the cellular poly(A) RNA. Cell lines derived from this tumor may provide an attractive system to investigate the regulation of somatostatin gene expression.

Cloning and biosynthetic studies of rat somatostatin.

The predicted amino acid sequence of rat preprosomatostatin has been obtained by cloning and subsequent DNA sequence analysis of a cDNA obtained from mRNA prepared from a rat medullary thyroid carcinoma (MTC). The predicted preprosomatostatin is 116 amino acid residues in length. Somatostatin-14 is located at the C-terminus of the preprohormone and the amino terminus contains a 'signal' peptide of 24 amino acids. A somatostatin amino terminal protein of 78 residues is found between the signal peptide and somatostatin-14. Both somatostatin-14 and somatostatin-28 are observed in the thyroid tumour C-cells. A comparison of the rat and human preprosomatostatin amino acid sequences shows only 4 substitutions observed in 116 amino acids. Patients with localized MTC often exhibit high serum calcitonin levels while patients showing cellular heterogeneity in the MTC appear to have lower calcitonin levels and a virulent neoplasia with a grave prognosis. Numerous rat MTCs have been examined by two dimensional gel electrophoresis. It is possible to distinguish characteristic differences in protein profiles of tumours producing high levels of calcitonin from those showing low calcitonin and high somatostatin levels. This analysis can be done with less than 1 mg of tissue and may represent a valuable prognostic tool in evaluating the clinical variability of MTC.

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels.

The structure of cloned DNA complementary to catfish pancreatic somatostatin-14 messenger RNA.

Pancreatic poly(A) RNA isolated from the channel catfish (Ictalurus punctatus) was enriched for sequences corresponding to somatostatin mRNA on isokinetic sucrose gradients. Double-stranded cDNA was synthesized and inserted into the Pst I site pBR322 via the poly(dG) . poly(dC) tailing method. Escherichia coli was transformed with this DNA, and colonies containing somatostatin cDNA sequences were identified by hybridization using a primer-extended somatostatin cDNA. The somatostatin cDNA was obtained by extending a 5'-labeled undecanucleotide primer complementary to somatostatin mRNA with reverse transcriptase using catfish poly(A) RNA as a template. The synthetic primer d(T-T-C-C-A-G-A-A-G-A-A) was deduced from the amino acid sequence Phe-Phe-Trp-Lys present in somatostatin-14. Twenty positive colonies were obtained upon screening 2000 transformants. The restriction maps of the plasmid DNA obtained from the positive colonies were examined. Nineteen of these plasmids contained sequences corresponding to somatostatin-14, while one contained a sequence corresponding to somatostatin-22. The nucleotide sequence of pancreatic somatostatin-14 is reported here. The cDNA contains 350 nucleotides in the 3' noncoding region, 345 nucleotides in the coding region, and 104 nucleotides in the 5'-untranslated region. The mRNA codes for a precursor to somatostatin which is 114 amino acids in length. The preprosomatostatin has a sequence of hydrophobic amino acids at the NH2 terminus, followed by a connecting peptide of approximately 75 amino acids. The sequence Arg-Lys precedes somatostatin-14. Analysis of genomic DNA from the channel catfish reveals that somatostatin-14 and somatostatin-22 are present on different restriction fragments.

Purification and characterization of an enkephalin aminopeptidase from rat brain.

Rat brain enkephalin aminopeptidase was purified to apparent electrophoretic homogeneity. Enzyme activity was monitored during the purification by using ([3,5-3H2]Tyr)-Met-enkephalin and Tyr-beta-naphthylamide as substrates. It was shown that the enzyme activities resulting in hydrolysis of the tyrosine residue of ([3,5-3H2]Tyr)Met-enkephalin and formation of beta-naphthylamine from Tyr-beta-naphthylamide copurified. The homogeneous enzyme had a specific activity of 10.5 mumol of beta-naphthylamide hydrolyzed min-1 mg-1. Hydrolysis of Met-enkephalin yielded the products L-tyrosine and the tetrapeptide Gly-Gly-Phe-Met. Subsequent removal of glycine from Gly-Gly-Phe-Met was not observed with the purified enzyme. The homogeneous aminopeptidase has an apparent molecular weight of 115000 on Sephadex G-200 and a molecular weight of 102000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enkephalin-degrading enzyme had a pH optimum of 6.5-7.0 and exhibited maximal activity at 40 degrees C. Enzyme activity was inhibited by metal chelators, and it was found that 1 mol of Zn2+ was associated with 1 mol of enzyme (102000 Mr). The enzyme hydrolyzes various neutral and basic amino acid beta-naphthylamides but will not utilize acidic, D-amino acid, or N-terminal-blocked amino acid beta-naphthylamides as substrates.