Richard H. Goodman

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

SummaryIn situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity forin situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18–20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combinedin situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.

Cell-free translation of messenger RNA coding for a precursor of human calcitonin

Abstract Polyadenylated RNA, extracted from a human medullary thyroid carcinoma, was translated in cell-free systems prepared from wheat germ and reticulocyte lysates. The major product of the translations was a protein of 15,000 M R which was immunoprecipitated specifically with an antiserum to synthetic human calcitonin. Addition to the translation reactions of microsomal membranes, prepared from canine pancreas, resulted in the partial disappearance of the 15,000 M R polypeptide and the concomitant appearance of a smaller peptide (11,000 M R ), also immunoprecipitated specifically by antisera to calcitonin. These results indicate that human calcitonin is synthesized in the form of a precursor of 15,000 M R and suggest that the precursor contains a leader sequence that is cleaved from the polypeptide by enzymes associated with microsomal membranes.

Glucagon Precursors Identified by Immunoprecipitation of Products of Cell-free Translation of Messenger RNA

Polyadenylated RNA extracted from anglerfish islets was translated in a wheat germ cell-free system containing [35S]methionine in the presence and absence of microsomal membranes prepared from a canine pancreas. Labeled translation products were analyzed by immunoprecipitation with an antiserum to porcine glucagon, followed by electrophoresis of the translation products and immunoprecipitated proteins on SDS polyacrylamide gels. In the absence of microsomal membranes two proteins of Mr = 14,500 and Mr = 12,500 were specifically immunoprecipitated with antiglucagon serum. Addition of microsomal membranes to the translation reactions resulted in a diminution of the labeled protein of Mr = 14,500 and a marked increase in the immunoreactive protein of Mr = 12,500. The protein of Mr = 12,500 was resistant to degradation by proteolytic enzymes added to translation reactions, indicating that it was segregated within microsomal vesicles. These results are consistent with synthesis of anglerfish islet glucagon in the form of a pre-prohormonal precursor (Mr = 14,500) containing a leader sequence that is cotranslationally cleaved from the protein by enzymes associated with microsomal membranes to produce a smaller intermediate prohormonal precursor (Mr = 12,500) of pancreatic glucagon (Mr = 3500).

Somatostatin gene regulation: An overview

The somatostatinergic system has proven to be one of the best models of neuropeptide biology. Originally characterized as a hypothalamic regulator of growth hormone secretion, somatostatin also regulates the secretion of several other pituitary, pancreatic, and gastrointestinal (GI) hormones including thyrotropin-stimulating hormone, insulin, glucagon, and gastrin. Disorders in somatostatin metabolism have been proposed to contribute to the pathogenesis of Alzheimers disease, epilepsy, GI motility disorders, and diabetes. On a more basic level, studies of somatostatin action have integrated divergent concepts of intracellular signal transduction. Advances in the understanding of somatostatin biosynthesis have had an impact on areas outside the field of endocrinology by providing new concepts of eukaryotic gene regulation. This report focuses on the transcriptional regulation of somatostatin gene expression. Two aspects of somatostatin gene transcription will be considered--regulated expression by second messengers and tissue-specific basal expression.

Intestinal glucagon mRNA identified by hybridization to a cloned islet cDNA encoding a precursor

Poly(A) RNA was prepared from the intestine of anglerfish and was translated in a wheat germ cell-free system supplemented with 35S-methionine. SDS polyacrylamide gel electrophoresis of the labeled translation products revealed that the intestinal poly(A) RNA directs the synthesis of many proteins. Immunoprecipitations of the intestinal cell-free translation products with an antiserum to glucagon known to recognize anglerfish islet pre-proglucagon failed to identify an intestinal glucagon precursor. However, sensitive techniques of hybridization with a 32P-labelled cDNA containing the coding sequence for pancreatic glucagon identified a complementary RNA in the intestine. The mRNA of 620 bases is similar in size to the pre-proglucagon RNA in the islets (620–650 bases). These observations indicate that a gene encoding glucagon is expressed in the intestine, and that the mRNA encoding the intestinal glucagon precursor is of similar size to the pre-proglucagon mRNAs identified in the islets.

Characterization and Expression of the Gene‐Encoding Rat Thyrotropin‐Releasing Hormone (TRH)

The chemical characterization of TRH by Guillemin and Schally in 1969 provided the first direct evidence for the existence of hypophysiotropic hormones in the Although the structure of TRH (pGlu-His-Pro-NHZ) was determined nearly two decades ago, relatively little is known about the factors that control the synthesis of this regulatory peptide. Studies of TRH biosynthesis within the hypothalamus have been difficult to perform because of problems in detecting the low concentrations of the small peptide in hypothalamic tissues and the lack of TRH-producing cell lines. These problems have made attempts to study TRH biosynthesis by conventional methods, such as pulse-chase labeling with radioactive amino acids, difficult to perform and interpret. Molecular biological techniques can overcome many of the problems inherent in studying the biosynthesis of hypothalamic peptides, but require the isolation of appropriate cDNA or genomic probes and the identification of cell lines that express the appropriate gene products. In this manuscript, we report the cloning and characterization of the rat prepro-TRH gene3 and the identification of a TRH producing-cell line.4 With these tools we have begun to examine elements of the TRH gene necessary for expression.

Biosynthesis of rat preprosomatostatin.

The biologically active forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28) arise by post-translational cleavage of prosomatostatin. Prosomatostatin in turn is derived from a larger precursor, preprosomatostatin. We have previously reported the structure of a complementary DNA molecule encoding rat preprosomatostatin. The nucleotide sequence of this cDNA indicated that SS-14 and SS-28 are located at the carboxy-terminus of a 116 amino acid precursor. At the amino-terminus of the precursor is a hydrophobic region characteristic of a leader or pre-sequence. Sequential Edman degradations of cell-free translation products synthesized in the presence of microsomal membranes indicate that preprosomatostatin is cleaved within the endoplasmic reticulum to form prosomatostatin, a precursor of 92 amino acids. To begin to elucidate the factors which regulate the expression of the rat somatostatin gene, we have determined the sequence of the gene isolated from recombinant bacteriophage libraries. The gene spans 1.2 kilobases in length and is interrupted within the coding sequence of prosomatostatin by a single intron of 630 bases. A variant of the Goldberg-Hogness promotor, TTTAAA, is located 31 bases upstream from the transcriptional start point. A repetitive sequence was identified in the 5 region of the gene within 650 bases of the promoter. The nucleotide sequence of this region reveals an alternating GT sequence 42 bases in length characteristic of DNA with Z-forming potential. Such sequences are thought to influence the expression of other eukaryotic genes.

In Situ Hybridization of Somatostatin and Vasoactive Intestinal Peptide mRNA in the Rat Nervous System

Expression of neuronal genes encoding specific neurotransmitters is one of the major events that occurs in the developing nervous system. The signals that regulate the expression of neurotransmitter genes during development are poorly understood. Identification of the signals and mechanisms that regulate neuronal phenotypic expression during development will be facilitated by knowing when neurotransmitter genes are activated.