Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.     

Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts

Polyadenylated RNA isolated from senescent human diploid fibroblasts (HDF) inhibited DNA synthesis in proliferation-competent cells after microinjection, whereas polyadenylated RNA from young HDF had no inhibitory effect. Polyadenylated RNA from young cells made quiescent by removal of serum growth factors had a slight inhibitory effect on DNA synthesis. The abundance level of inhibitor messenger RNA (mRNA) from senescent cells was estimated at 0.8 and that of quiescent cells at 0.005 percent. These results demonstrate the existence of one or more antiproliferative mRNA's in nonproliferating normal human cells; these RNA's code for factors that either work antagonistically to initiators of DNA synthesis or regulate the expression of the initiators in some way. The abundance level of the inhibitory mRNA in senescent cells indicates the feasibility of developing a complementary DNA probe that will be useful in studying cell cycle control mechanisms.     

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.     

A single troponin T gene regulated by different programs in cardiac and skeletal muscle development

A cloned complementary DNA derived from a messenger RNA transiently present at low abundance levels in early chick embryonic skeletal muscle hybridizes to a messenger RNA present at high abundance levels in cardiac muscle. Genomic DNA hybridization and nucleotide sequence identity of complementary DNA's from both heart and skeletal muscle demonstrate that the messenger RNA's from both sources are encoded by the same gene. The encoded polypeptide is a troponin T sequence which is probably a cardiac isoform. This single copy troponin T isogene is governed by different regulatory programs in heart and skeletal muscle differentiation.     

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.     

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.     

Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand

The genome of the human immunodeficiency virus (HIV) is known to contain eight open reading frames (ORFs) on the minus strand of the double-stranded DNA replicative intermediate. Data presented here indicate that the DNA plus strand of HIV contains a previously unidentified ORF in a region complementary to the envelope gene sequence. This ORF could encode a protein of approximately 190 amino acid residues with a relative molecular mass of 20 kilodaltons if translation began from the first initiation codon. The predicted protein is highly hydrophobic and thus could be membrane associated. It is possible, therefore, that the HIV genome encodes a protein on antisense messenger RNA.     

Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.     

Norwalk virus genome cloning and characterization

Major epidemic outbreaks of acute gastroenteritis result from infections with Norwalk or Norwalk-like viruses. Virus purified from stool specimens of volunteers experimentally infected with Norwalk virus was used to construct recombinant complementary DNA (cDNA) and derive clones representing most of the viral genome. The specificity of the clones was shown by their hybridization with post- (but not pre-) infection stool samples from volunteers infected with Norwalk virus and with purified Norwalk virus. A correlation was observed between the appearance of hybridization signals in stool samples and clinical symptoms of acute gastroenteritis in volunteers. Hybridization assays between overlapping clones, restriction enzyme analyses, and partial nucleotide sequence information of the clones indicated that Norwalk virus contains a single-stranded RNA genome of positive sense, with a polyadenylated tail at the 3' end and a size of at least 7.5 kilobases. A consensus amino acid sequence motif typical of viral RNA-dependent RNA polymerases was identified in one of the Norwalk virus clones. The availability of Norwalk-specific cDNA and the new sequence information of the viral genome should permit the development of sensitive diagnostic assays and studies of the molecular biology of the virus.     

Genetic variation in the human insulin gene

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.     

Identification of a thyroid hormone receptor that is pituitary-specific

Three cellular homologs of the v-erbA oncogene were previously identified in the rat; two of them encode high affinity receptors for the thyroid hormone triiodothyronine (T3). A rat complementary DNA clone encoding a T3 receptor form of the ErbA protein, called r-ErbA beta-2, was isolated. The r-ErbA beta-2 protein differs at its amino terminus from the previously described rat protein encoded by c-erbA beta and referred to as r-ErbA beta-1. Unlike the other members of the c-erbA proto-oncogene family, which have a wide tissue distribution, r-erbA beta-2 appears to be expressed only in the anterior pituitary gland. In addition, thyroid hormone downregulates r-erbA beta-2 messenger RNA but not r-erbA beta-1 messenger RNA in a pituitary tumor-derived cell line. The presence of a pituitary-specific form of the thyroid hormone receptor that may be selectively regulated by thyroid hormone could be important for the differential regulation of gene expression by T3 in the pituitary gland.     

Molecular cloning of complementary DNA for the alpha subunit of the G protein that stimulates adenylate cyclase

A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.     

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.     

Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle

The alterations in morphology and function of the ovarian follicle as it matures, ovulates, and becomes a corpus luteum are dramatic. A variety of steroid and polypeptide hormones influence these processes, and the ovary in turn produces specific hormonal signals for endocrine regulation. One such signal is inhibin, a heterodimeric protein that suppresses the secretion of follicle-stimulating hormone from pituitary gonadotrophs. Rat inhibin complementary DNA probes have been used to examine the levels and distribution of inhibin alpha-and beta A-subunit messenger RNAs in the ovaries of cycling animals. Striking, dynamic changes have been found in inhibin messenger RNA accumulation during the developmental maturation of the ovarian follicle.     

Molecular cloning of a gene sequence regulated by nerve growth factor

Nerve growth factor (NGF) is essential for the development and differentiation of sympathetic or sensory neurons. A complementary DNA was cloned that corresponds to a gene sequence induced more than 50-fold in a cultured target cell line of pheochromocytoma cells (PC12 cells) 5 hours after the addition of NGF. The induced messenger RNA encodes a 90,000-dalton polypeptide that may represent one of the primary events in NGF-induced differentiation of neurons.     

Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors

Eleven complementary DNA (cDNA) clones were generated from messenger RNA isolated from abdominal light organs of the bioluminescent click beetle, Pyrophorus plagiophthalamus. When expressed in Escherichia coli, these clones can elicit bioluminescence that is readily visible. The clones code for luciferases of four types, distinguished by the colors of bioluminescence they catalyze: green (546 nanometers), yellow-green (560 nanometers), yellow (578 nanometers), and orange (593 nanometers). The amino acid sequences of the different luciferases are 95 to 99 percent identical with each other, but are only 48 percent identical with the sequence of firefly luciferase (Photinus pyralis). Because of the different colors, these clones may be useful in experiments in which multiple reporter genes are needed.