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PubMed1994

Antipeptide polyclonal antibodies that recognize a substance P-binding site in mammalian tissues: a biochemical and immunocytochemical study.

We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand-binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using 125I-substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.

Amino Acid Sequence↗

PubMedDate not supplied

Transfer and expression of DNA constructs into cells by using retrovirus vectors: a new tool for pharmacological research.

Recombinant retroviruses are useful vectors for introducing DNA sequences into eukaryotic chromosomes and obtain their expression. The principles and techniques for producing these vectors and their applications are discussed in view of their use in pharmacological research. Particularly, retroviral vectors seem to be attractive for expressing into mammalian cells antisense RNA, that is RNA complementary to target mRNA and capable of preventing the synthesis of the gene product.

Animals↗

PubMed1992

Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing.

Within the nucleus, pre-mRNA molecules are complexed with a set of proteins to form heterogeneous nuclear ribonucleoprotein complexes. A1, an abundant RNA binding protein present in these complexes, has been shown to bind selectively to single-stranded RNAs and destabilize base-pairing interactions. In this study.A1 is shown to promote the rate of annealing of complementary RNA strands greater than 300-fold under a wide range of salt concentration and temperature. Maximal annealing is observed under saturating or near saturating concentrations of protein, but annealing decreases sharply at both higher and lower concentrations of A1. Kinetic analysis shows that the rate of annealing is not strictly first or second order with respect to RNA at a ratio of protein/RNA that gives optimal rates of annealing. This result suggests that A1 protein may affect more than one step in the annealing reaction. Two polypeptides representing different domains of A1 were also examined for annealing activity. UP1, a proteolytic fragment that represents the N-terminal two-thirds of A1, displays very limited annealing activity. In contrast, a peptide consisting of 48 amino acid residues from the glycine-rich C-terminal region promotes annealing at a rate almost one-quarter that observed with intact A1. The RNA.RNA annealing activity of A1 may play a role in pre-mRNA splicing and other aspects of nuclear mRNA metabolism.

Heterogeneous Nuclear Ribonucleoprotein A1↗

PubMed1987

The sequence of the M RNA of an isolate of La Crosse virus.

The middle-size (M) genomic RNA of a New York State, U.S.A. isolate of La Crosse (LAC) virus has been cloned by a random priming procedure and its nucleotide sequence determined by the dideoxy method. The RNA was found to be 4526 nucleotides in length and to have a base composition of 34.2% U, 27.8% A, 20.6% C and 17.4% G. There is a single, long open reading frame in the viral complementary RNA that contains sufficient information to code for a protein of 1441 amino acids. In these respects, as in many others, the LAC virus M RNA and its encoded protein were very similar, if not identical, to those previously reported by other investigators for the closely related snowshoe hare virus. The M RNAs of the two viruses show 79% nucleotide sequence homology and 89% homology in the amino acid sequence of their encoded proteins. Several algorithms for predicting surface residues, as well as the Chou-Fasman rules for predicting secondary structure, were used to compare the LAC virus and snowshoe hare virus M gene proteins. These analyses identified 39 sites on the proteins as those most likely to be linear antigenic determinants that might contribute to the differences between the two viruses.

Amino Acid Sequence↗

PubMed1994

Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing.

Temporal and spatial distribution of a gene encoding murine bone morphogenetic protein 4 (mBMP-4) during fracture repair were investigated in mice by RT-PCR and in situ hybridization. For in situ hybridization, fractured ribs and surrounding tissues were decalcified and hybridized with a mBMP-4-specific complementary RNA probe labeled with digoxigenin-11 UTP. mBMP-4 messenger RNA (mRNA) was not detected in ribs without fracture, whereas it was detected only in the early phase of fracture from 12 to 72 h after the onset of fracture before new cartilage or bone formation. The mBMP-4 mRNAs were present in cells distributed in three distinct regions, namely, the proliferating periosteum, the medullary cavity, and the muscles near the fracture site. These BMP-4-positive cells did not express bone gla protein mRNA, which is a marker of the mature osteogenic cell. RT-PCR also showed a transient increase in the level of BMP-4 mRNA in the early phase of fracture repair. The findings provide us with some new information. (1) The BMP-4 gene is produced by less differentiated osteoprogenitor cells, not by differentiated osteoblasts. (2) The BMP-4 gene is enhanced by the impact of fracture and localized in callus-forming tissue before callus formation. Together with the activities of BMP-4, as was previously described, our results suggest that newly produced BMP-4 gene product is one of the local contributing factors in callus formation in the early phase of fracture healing.

Animals↗

PubMed1989

Physical map of the genome of sonchus yellow net virus, a plant rhabdovirus with six genes and conserved gene junction sequences.

We provide evidence that a plant rhabdovirus, sonchus yellow net virus (SYNV), is similar to most animal rhabdoviruses in the order of structural genes and in the nucleotide sequences at the gene junctions but that it differs in the presence and location of a putative nonstructural gene. From the patterns of hybridization of a library of recombinant DNA clones, we have shown that the SYNV genome is transcribed into a short 3'-terminal "leader RNA" and six mRNAs. The proteins encoded by the SYNV mRNAs, in order of the appearance of their genes in the SYNV genome, are designated 3'-N-M2-sc4-M1-G-L-5' (N, nucleoprotein; M, matrix protein; sc, protein encoded by SYNV complementary RNA; G, glycoprotein; L, large protein). The intergenic and flanking gene sequences are conserved and consist of a central core of 14 nucleotides (3'-UUCUUUUUGGUUGU/A-5') whose sequence is similar to the sequence at the gene junctions of vesicular stomatitis and rabies viruses. The SYNV core consists of an 8-nucleotide (3'-UUCUUUUU-5') transcription termination signal at the 5' terminus of each gene, a dinucleotide (GG) spacer whose complement does not appear in mRNA, and a tetranucleotide (3'-UUGU/A-5') that is complementary to the first four nucleotides at the 5' terminus of the SYNV mRNAs. These results, when compared with structural information available on animal rhabdoviruses, suggest that organization of structural genes and maintenance of signals thought to play important roles in regulation of transcription have been conserved during evolution in plant, insect, and vertebrate hosts. However, differences in number and location of putative nonstructural genes reveal some flexibility in genome organization that may be important in deducing taxonomic and evolutionary relationships among viruses causing diseases in phylogenetically diverse hosts.

Base Sequence↗

PubMed1995

Induction of heparin-binding growth-associated molecule expression in reactive astrocytes following hippocampal neuronal injury.

Heparin-binding growth-associated molecule is a potent neurotrophic factor. To obtain a better understanding of its role in the central nervous system, we studied the changes of its expression in adult rat brain after two types of neuronal injury. In the control hippocampus, expression of heparin-binding growth-associated molecule messenger RNA was confined to CA1 pyramidal neurons and some hilar cells. Following transient forebrain ischaemia, the messenger RNA expression decreased within the first two days. On day 4, however, both the messenger RNA level and the number of expression-positive cells markedly increased in the CA1 subfield, where the selective neuronal losses were seen following ischaemia. Double-staining with a heparin-binding growth-associated molecule complementary RNA probe and an anti-glial fibrillary acidic protein antibody revealed that most of the expressing cells were reactive astrocytes. Moreover, the protein induction of heparin-binding growth-associated molecule after neuronal injury was demonstrated by immunohistochemistry using the affinity-purified antibodies. This molecule was also induced after intraventricular kainate injection, which is known to cause selective pyramidal cell necrosis in the CA3 region. Four days after the insult, the number of cells expressing the messenger RNA prominently increased in the CA3 subfield ipsilateral to the injection. As observed after the ischaemic insult, most of the expression-positive cells were identified as astrocytes. The data presented here suggest that heparin-binding growth-associated molecule, produced by the reactive astrocytes, may play important roles in the repair process after neuronal injury.

Animals↗

PubMed1986

Analysis of the dosage compensation of a specific transcript in Drosophila melanogaster.

The amount of steady-state level RNA complementary to the X-linked salivary gland secretion polypeptide gene Sgs-4 was measured in male and female third-instar larvae carrying one or two doses of a wild-type allele of the gene. RNA levels were found to be compensated in normal one-dose males and two-dose females and to be dosage-dependent within each sex. The presence of mutant alleles of male-less (mle) was found to reduce the level of Sgs-4 transcripts in males. These results support the contentions that dosage compensation is mediated by regulating the level of X-linked gene transcripts and that a product of the mle+ gene is involved in this process.

Alleles↗

PubMed1998

Optimization of the binding properties of PNA-(5')-DNA chimerae.

The synthesis and evaluation of PNA-(5')-DNA chimerae containing either a 5'-amide (i.e. 1a), a 5'-phosphodiester (i.e. 1b) or 5'-phosphonate linkages (i.e. 1c,d) at the junction site are described. The 5'-linkages could be installed using either 5'-amino-5'-deoxythymidine phosphoramidite 2, O-[2-(2-aminoethyl)-(thymin-1-ylacetyl)amino]ethyl phosphoramidite 3, N-(2-aminoethyl)-N-(thymin-1-ylacetyl)aminomethyl phosphonate 4 or N-(2-aminoethyl)-N-(allyloxycarbonyl)aminomethyl phosphonate 5 as building blocks, respectively. It is shown that PNA-(5')-DNA of type 1a-c have a higher binding affinity with complementary RNA than native DNA, and that the antisense activity is mainly due to RNase H.

Chimera↗

PubMed1982

Adenovirus-related RNA sequences in human neurogenic tumours.

Thirty two human tumours, mainly neurogenic, have been investigated for the presence of adenovirus-related RNA sequences. 3H-labelled tumour virus DNA probes derived from human adenoviruses types 2 and 12, bovine adenovirus type 3, and avian adenovirus CELO were hybridized in-situ on tumour kryostat sections under conditions that detect complementary RNA. Tumour virus-related RNA was detected in 62% of all tumours tested, but was not detectable in normal human brain tissues. Expression of tumour virus-related RNA was found in 2/4 astrocytomas, 2/4 metastatic brain carcinomas, 2/2 glioblastomas, 1/1 melanoma, 5/7 meningiomas, 4/4 neurinomas, 1/2 oligodendrogliomas, and 1/1 rhabdomyosarcoma. The presence of adenovirus-related RNA in the majority of human neurogenic tumours may reflect a viral involvement in the pathogenesis of these tumours.

Adenoviridae↗

PubMed1977

Immunologic selection against simian virus-40 transformed cells: concomitnat loss of viral antigens and early viral gene sequences.

A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.

Antigens, Viral↗

PubMed1963

Replicating form of a single-stranded DNA virus: isolation and properties.

The replicating form of single-stranded DNA virus has been isolated in pure form by chromatography on columns of methylated albumin. Its buoyant density in CsCl and "melting temperature" are characteristic of a double stranded DNA structure containing 43 percent guanine-cytosine. The nearest neighbors to uridylate were compared in the RNA synthesized when replicating-form DNA and mature single-stranded DNA were employed as templates in an in vitro system. The mature DNA component of the replicating duplex does not serve as the sole source of complementary RNA. The results agree best with the assumption that both strands of the replicating form function as templates. It is important to note that this is contrary to the situation found in the intact cell where only one of the two strands appears to be transcribed into message.

Cytosine↗

PubMed1993

Ileal proglucagon gene expression in the rat: characterization in intestinal adaptation using in situ hybridization.

BACKGROUND: Proglucagon-derived peptides are potential mediators of the adaptive response of the terminal ileum to massive small bowel resection. Ileal proglucagon messenger RNA (mRNA) levels increase during ileal adaptation. The present study explored the cellular basis of this response. METHODS: Sections of control ileum, ileum 4 days after resection, and pancreas were analyzed by in situ hybridization with 35S-labeled complementary RNA (cRNA) probes. RESULTS: Both the proglucagon and the peptide YY cRNA probes hybridized to discrete cells in the ileal mucosa, the disposition of which corresponds to that reported for intestinal L cells. Four days after resection there was a marked increase in the intensity of the signal for both probes without an increase in cell number. Insulin and histone H3 probes were used as controls to confirm the specificity of the hybridization seen with the L-cell specific, proglucagon, and peptide YY probes. CONCLUSIONS: The increase in proglucagon mRNA levels after massive small bowel resection is caused by an increase in the cellular content. The parallel increase in PYY mRNA levels implies an L cell--rather than a proglucagon gene--specific response.

Adaptation, Physiological↗

PubMed1990

Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.

Aging↗

PubMedDate not supplied

Synthesis and properties of oligonucleotide chimeras containing 5'-amino-2'-deoxy-2'-fluoroarabinonucleosides.

Oligonucleotide analogues comprised of 2'-deoxy-2'-fluoro-beta-D-arabinose units joined via P3'-N5' phosphoramidate linkages (2'F-ANA(5'N)) were prepared for the first time. Among the compounds prepared were a series of 2'OMe-RNA-[GAP]-2'OMe-RNA 'chimeras', whereby the "GAP" consisted of DNA, DNA(5'N), 2'F-ANA or 2'F-ANA(5'N) segments. The chimeras with the 2'F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5'-amino derivatives, i.e., 2'F-ANA > DNA > 2'F-ANA(5'N) > DNA(5'N). Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E. coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2'F-ANA > 2'F-ANA(5'N) > DNA(5'N). The corresponding relative rates observed with the human enzyme were: gap DNA > 2'F-ANA(5'N) > 2'F-ANA > DNA(5'N).

Amines↗

PubMed1993

Nucleotide sequence and possible ambisense coding strategy of rice stripe virus RNA segment 2.

The complete nucleotide sequence (3514 nucleotides) of RNA segment 2 of rice stripe virus (RSV), the prototype member of tenuivirus group, was determined. In the virus-sense RNA an open reading frame (ORF) is present which encodes a 199 amino acid protein of M(r) 22,762. Another long ORF encoding an 834 amino acid protein with M(r) 94,047 (94K) exists in the virus-complementary RNA. Between these two ORFs, there is a long non-coding intergenic region of 299 nucleotides. The sequence suggests that RNA 2 has an ambisense coding strategy as found for RSV RNAs 3 and 4. The putative 94K protein carries stretches with an amino acid sequence showing weak similarity to parts of the membrane glycoproteins of Punta Toro and Uukuniemi phleboviruses of the family Bunyaviridae, suggesting a possible distinct evolutionary relationship between the animal phleboviruses and the plant tenuiviruses.

Amino Acid Sequence↗

PubMed1973

Partial transcription of murine type C viral genomes in BALB c cell lines.

The mouse cell line, BALB/c 3T3, and its derivatives transformed either spontaneously or by treatment with a variety of external agents, were analyzed for cytoplasmic RNA complementary to DNA products prepared from the Kirsten strain of murine sarcoma-leukemia virus, and from an endogenous type C virus of BALB/c 3T3. Although none of these cell lines spontaneously releases complete type C virions, they all contain RNA which is partially homologous to a portion of the 35S RNA isolated from these viruses. The parental cell line, BALB/c 3T3, contains a low level of viral-related RNA, and there is an increased amount of this RNA in some of the transformed cells. The RNA detected represents only a fraction of the viral RNA found in virus-producing cells. The formation of RNA:DNA hybrids was detected by equilibrium centrifugation in Cs(2)SO(4) density gradients and by analysis with a single-strand-specific nuclease from Aspergillus oryzae. Viral DNA products prepared either from an endogenous reaction with whole virus in the presence of actinomycin D or from purified 70S viral RNA as template using avian myeloblastosis virus DNA polymerase yield comparable data. In addition, all of the BALB/c lines examined produce detectable levels of murine type C virus group-specific antigen.

Animals↗

PubMed1987

Tobacco mosaic virus replicase and replicative structures.

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase. Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity. Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography. The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126,000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.

Chromatography, Agarose↗