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%.     

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.     

Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase

A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme. Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography. This preparation was used as template for synthesis of cDNA. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli DH1. Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation. The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis. One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA. The amount of the mRNA was greatly increased in the liver of rats given high protein diet.     

Molecular cloning of part of the mitochondrial DNA of Drosophila melanogaster

We report the construction of recombinant plasmids containing part of the mitochondrial DNA of $\text{Drosophila}\text{melanogaster}$. Of the four fragments of this DNA generated by the restriction endonuclease HindIII, two were successfully cloned into the HindIII site of the plasmid pCM2. Unexpectedly the other two fragments could not be isolated by cloning into the HindIII site of either pCM2 or pBR322. Part of a third fragment, containing the gene for the large ribosomal RNA, was incorporated into the PstI site of pBR322. We show that this recombinant plasmid contains sequences complementary to an abundant RNA species which is present in $\text{Drosophila}$ embryos and which binds to oligo-dT-cellulose.     

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA

P450_scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450_scc indicates P450_scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the $\text{PstI}$ site of pBR322 by dC·dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450_scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Synthesis and cloning of DNA complementary to mRNA of the bovine mammary gland

Poly(A+)mRNA from bovine mammary glands was used to synthesize double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst1 site by means of oligo(dG)-oligo(dC) tailing. After transfection of Escherichia coli JC5183, recombinant plasmid library containing 5400 clones was screened by serial rounds of colony hybridization in situ to total [23P] poly(A+)mRNA and electrophoretically homogenious [32P]16SmRNA of mammary glands. Then hybrid selection of mRNA and subsequent in vitro translation of selected mRNAs were performed. In this manner, recombinant clones coding for alpha S1- beta-, kappa-casein were identified. cDNA clones range in size from 35% for beta-casein, 65% for alpha S1-casein to about 95% for kappa-casein, in comparison with their respective mRNAs.     

Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.     

Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

We report here the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue (AE003774) was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product.     

Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.     

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.