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PubMed1984

Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced. These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation. Potential glycosylation sites in the preprotoxin peptide are identified. Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor.

Amino Acid Sequence↗

PubMed1984

Gene expression during Drosophila melanogaster oogenesis: analysis by in situ hybridization to tissue sections.

The pattern of gene expression for specific genes during oogenesis has been examined by in situ hybridization to RNA in sectioned ovarian preparations. The accumulation (1) of RNA complementary to heat-shock genes, both before and after heat shock; (2) of RNA complementary to three lambda phage recombinants which contain sequences expressed during oogenesis; and (3) of RNA complementary to the rudimentary gene have been analyzed. Gene expression in the ovaries is found to be dependent on both the cell type and the developmental stage of the ovarian chamber. In the nurse cells, the onset of vitellogenesis (stages 8-11) appears to be accompanied by a substantial activation of many different genes and there is a pronounced increase in the level of accumulation of complementary RNAs. Moreover, much of the RNA synthesized in nurse cells during vitellogenesis is ultimately transported into the growing oocyte where it accumulates. It is likely that this accumulation of RNA provides a source of gene products, either as message or proteins, for embryogenesis.

Animals↗

PubMed1974

Effect of interferon upon the primary and secondary transcription of vesicular stomatitis and influenza viruses.

The synthesis of viral complementary RNA by vesicular stomatitis virus in permissive cells can be distinguished operationally into two phases, primary transcription, which can be observed in cycloheximide treated cells, and secondary transcription, which represents the further synthesis of viral complementary RNA obtained in untreated cells. Pretreatment of mouse L cells or chicken embryo fibroblasts with the homologous interferon and, for chicken embryo fibroblast cells, poly(rI):poly(rC), inhibits the production of infectious virus and reduces the synthesis of viral complementary RNA to levels comparable to that obtained during primary transcription. Treatment of mouse L cells with interferon plus cycloheximide also gives levels of vesicular stomatitis viral complementary RNA synthesis comparable to that observed with the cycloheximide treatment alone. Similar results have been obtained for interferontreated cells subsequently infected with influenza virus (strain WSN). The results are interpreted as indicating that inhibition of virus development by interferon does not act at the level of primary transcription but rather at an intermediate step between primary and secondary transcription, such as viral protein synthesis.

Animals↗

PubMed1997

Peptidylarginine deiminase of the hair follicle: characterization, localization, and function in keratinizing tissues.

The enzyme peptidylarginine deiminase (PAD; EC 3.5.3.15) is responsible for the formation of protein-bound citrulline, a major amino acid in the inner root sheath (IRS) and in the medulla of the hair follicle. From mainly biochemical evidence, it is known that the substrate for the enzyme is trichohyalin and that trichohyalin granules gradually disappear to form a matrix with intermediate-like filaments in the IRS cells. In the medulla, the granules aggregate into large masses without filaments. The proteins in both the IRS and medulla are finally cross-linked by transglutaminase. A corollary of the apparent central role of PAD acting on the trichohyalin protein in these processes is that it should be present in the IRS and medulla cells, coincident with trichohyalin. Hair-follicle PAD has not previously been isolated. In the current study, the enzyme was isolated from wool follicles of adult sheep and peptide sequences were used to design DNA primers for the synthesis of PCR products from follicle mRNA. Subsequently, a PAD-specific complementary RNA probe and a trichohyalin complementary RNA probe were prepared for localization studies by in situ hybridization in wool follicles and the epithelia of the rumen, embryonic hoof, and tongue papillae. The experiments have revealed a striking co-expression of PAD and trichohyalin in all of these tissues. The amino acid sequence of the wool-follicle PAD molecule has been deduced from sequencing of the cloned PCR products.

Amino Acid Sequence↗

PubMed1978

RNA sequences complementary to citrus exocortis viroid in nucleic acid preparations from infected Gynura aurantiaca.

Molecular hybridization with (125)I-labeled citrus exocortis viroid RNA has been used to survey nucleic acid preparations from Gynura aurantiaca for viroid complementary molecules. A differential hybridization effect was detected between nucleic acid extracts from healthy and infected tissue in which significant RNase-resistant (125)I-labeled citrus exocortis viroid resulted in hybridization studies with the infected tissue extracts. Subsequent characterization indicated that RNA from infected tissue was involved in the formation of a duplex molecule with citrus exocortis viroid RNA and had properties of an RNA.RNA hybrid. Subcellular fractionation of infected tissue indicates that the complementary RNA is present in nuclear and soluble RNA fractions. This RNA may represent an intermediate molecule in the replication of the viroid or a pathogenic expression and may have a regulatory role in the host cell.

Journal Article↗

PubMed1998

Partial cloning and differential expression of ryanodine receptor/calcium-release channel genes in human tissues including the hippocampus and cerebellum.

Cellular Ca2+ signalling is an important factor in the control of neuronal metabolism and electrical activity. Although the roles of Ca2+-release channels are well established for skeletal and cardiac muscle, less is known about their expression and roles in the central nervous system, especially in the human brain. We have isolated partial complementary DNAs derived from the human ryanodine receptor Ca2+-release channel genes (ryr1, ryr2 and ryr3), and examined their expression in the human hippocampus and cerebellum. For comparison, we have included in our analysis an inositol trisphosphate Ca2+-release channel type I complementary RNA probe. All four messenger RNAs show widespread distribution in the human hippocampus, where ryr2 is the most abundant isoform, and all four are expressed in the human cerebellum. However, striking differences were seen between ryr and inositol trisphosphate Ca2+-release channel type I complementary RNA expression in the cerebellum, with inositol trisphosphate Ca2+-release channel type I messenger RNA being largely restricted to, and very highly expressed, in Purkinje cells, whereas ryr1, ryr2 and ryr3 were all expressed predominantly in the granular layer. The widespread expression of ryr isoforms in the human hippocampus and cerebellum suggests that ryanodine receptor proteins may have a central role in Ca2+ signalling and Ca2+ homeostasis in the human central nervous system. These may include roles in fundamental processes like synaptic plasticity. Furthermore, these Ca2+-release channels may be involved in pathogenic processes such as excitotoxicity, where excessive rises in intracellular Ca2+ concentration mediate neuronal cell death.

Aged↗

PubMed1979

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.

Animals↗

PubMed1994

Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.

Acrodermatitis↗

PubMed1975

Hybridization of pigeon globin messenger RNA with complementary DNA synthesized in vitro by reverse transcription: influence of the homopolymeric regions.

The kinetics of hybridization of pigeon globin messenger RNA with complementary cDNA synthesized by means of AMV reverse transcriptase is complex. Addition of poly A or poly U in excess to the reaction mixture normalized the kinetics. It is concluded that association of the complementary homopolymeric regions of mRNA and cDNA accelerates the complex formation between heteropolymeric sequences in a fraction of the molecules.

Adenine Nucleotides↗

PubMed1995

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Adenosine Deaminase↗

PubMed1996

Estrogen enhances endometrial estrogen receptor gene expression by a posttranscriptional mechanism in the ovariectomized ewe.

Prior influence of estrogen is required for many physiological effects of steroid hormones. This study addresses positive autoregulation of estrogen receptor gene expression in endometrium. Groups of ovariectomized ewes (n = 6) were treated with a single i.m. injection of 50 micrograms 17 beta-estradiol for 6, 12, 24, or 48 h or of vehicle for 24 h (control) prior to collection of endometrium. Three ewes received a regimen of estradiol-progesterone-estradiol (EPE) designed to mimic the estrous cycle. Northern analysis of endometrial RNA using an ovine estrogen receptor complementary RNA probe indicated that estradiol increased (p < 0.0001) estrogen receptor messenger RNA abundance over time to be 5-fold greater at 24 h postinjection. This effect also occurred after a period of progesterone dominance in EPE ewes (p < 0.05). Ribonuclease protection assays with a complementary RNA probe for the ovine progesterone receptor demonstrated that estradiol treatment increased progesterone receptor messenger RNA abundance at 48 h (p < 0.005). Nuclear runoff analyses indicated that whereas estradiol enhanced the transcription rates of progesterone receptor (p < 0.1) and 28S ribosomal RNA genes (p < 0.002), activity of the estrogen receptor gene was unchanged (p > 0.25). These results suggest that a physiological dose of estradiol, similar to the preovulatory surge of estrogen, up-regulates endometrial estrogen receptor gene expression by a posttranscriptional mechanism.

Amino Acid Sequence↗

PubMed1989

Plasmid library for the transcription of RNA probes complementary to the entire genome of the human immunodeficiency virus type 1 (HIV-1).

A plasmid (pBH10R3) containing a 9-kb Sst I fragment of HIV-1 (clone BH-10) inserted in pSP64, an in vitro expression vector, has been used for the transcription of anti-sense HIV-1 RNA. With this system, the transcripts obtained in vitro were not usually full length (1 to 2 kb long) and they predominantly span the 3' end ORF and ENV regions of the viral genome. We have rearranged the HIV-1 genomic sequences with respect to the SP6 promoter in the pSP64 vector and have obtained a series of new constructs allowing the expression in vitro of RNA transcripts complementary to other regions in the HIV-1 genome, including the 5' end of the ENV region as well as the TAT, POL, and GAG regions. In fact, the combined use of these constructs as templates for in vitro transcription allows the production of RNA probes spanning the entire viral genome. Compared with the 1- to 2-kb probes mentioned above, the combined use of such probes results in a several-fold increase in the sensitivity of molecular hybridization for the detection of HIV-1 nucleic acid sequences. Also, these constructs enable the preparation of RNA probes that have the potential to detect restriction polymorphisms throughout the HIV-1 genome.

Gene Library↗

PubMed2005

Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: implication for modular evolution of RNA viruses.

The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- (Cucumber necrosis virus, CNV), carmovirus (Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and post-recombinational amplification of new recombinant viruses.

Base Sequence↗

PubMed1992

Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology.

Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer than 15 could inhibit human DNA polymerases and RNase H activities, in a linkage number-dependent manner. Longer oligomers were more potent inhibitors than shorter ones. Kinetic studies indicated that S-dC28 was a competitive inhibitor of DNA polymerase alpha and beta with respect to the DNA template, whereas it was a noncompetitive inhibitor of polymerases gamma and delta. S-dC28 was also a competitive inhibitor of RNase H1 and H2 with respect to RNA-DNA duplex. Susceptibility of these enzymes to inhibition by S-dC28 was in the order of delta approximately gamma greater than alpha greater than beta and RNase H1 greater than RNase H2. Structural-activity relationships were explored with a group of S-dC28 analogs that have phosphorothioate internucleotide linkages at various positions. The inhibitory effect depended on the total number of thioate linkages, rather than the position of the linkages within the oligomer or the chain length itself. No sequence specificity was found. In the presence of the complementary RNA, antisense phosphorothioates (S-oligos) exerted a biphasic effect on RNase H activity. At low concentrations S-oligos could enhance the cleavage of the RNA portion of S-oligo-RNA duplex, whereas at high concentrations (in excess of the complementary RNA) S-oligos could inhibit RNase H and protect the complementary RNA from degradation. Together, these results suggest that the non-sequence-specific inhibitory effect of S-oligos should be taken into consideration in designing antisense inhibitors. This inhibitory activity could be avoided by decreasing the number of phosphorothioate linkages at the backbone, and S-oligos of 15-20 residues are preferable in antisense molecule design.

Base Sequence↗

PubMed1998

Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.

Chemical Phenomena↗

PubMed2002

Structure and spatio temporal expression of the full length DNA complementary to RNA coding for alpha2 type I collagen of zebrafish.

Twenty distinct genetic types of collagen have been identified up to now. Their structure and function are not completely elucidated. We have chosen zebrafish as a model to bring information about the role of collagen during embryogenesis. In the present study, we isolated four overlapping DNA complementary to RNA clones covering the 4879 nucleotides of a zebrafish messenger RNA (mRNA) encoding a fibrillar procollagen chain. The comparison of its primary structure with known other vertebrate collagens allowed to conclude that it encodes collagen pro-alpha2(I) chain. The 5' untranslated region showed a typical stem-loop structure with three ATG codons which is found in mammals types I and III collagen chains (but not in type II), which are expressed in the same tissues. This suggests that the supposed regulatory role of the stem loop structure could be tissue specific. The comparison of the Gly-Gly doublets found along the helical domain of several species allowed to speculate that the Gly-Gly repeats could be a poikilotherm feature. Expression of pro-alpha2(I) was examined during zebrafish development by reverse transcriptase-polymerase chain reaction and in situ hybridization on whole embryo and tissue section. Col1a2 was expressed as early as stage10 h post fertilization (hpf) and two peaks of expression were observed at 20 and 48 hpf. alpha2 mRNAs, whose presence suggests a collagen synthesis, were detected principally in the superficial cell layers surrounding 20-72 hpf embryos which are characterized by an acellular collagen stratum. At 26-30 days, fibroblasts invade the dermis and take over from the epithelial cells to synthesize collagen. This suggests a fine regulation of collagen synthesis in these cells that remains to be elucidated. alpha2 mRNA were also detected in other tissues such as the tail fin primordium and the notochord primordium suggesting a participation of type I collagen in a pathway for notochord and tail formation.

5' Untranslated Regions↗

PubMed1988

Expression of naturally occurring RNA molecules complementary to the murine L27' ribosomal protein mRNA.

We report here the existence of two naturally occurring RNA molecules that are complementary to the murine L27' ribosomal protein (rp) mRNA. These transcripts are 1.8 and 1.0 kb in length, and are both found in poly(A)+ populations of cytoplasmic and polysomal RNA of a number of established cell lines and in all adult murine tissues examined with the exception of the testes, where only the 1.8-kb transcript was detected. The expression of the 1.8-kb transcript is also constant during mouse embryogenesis from days 11 through 18 of gestation, and during differentiation of P19 embryonal carcinoma cells, whereas that of the smaller transcript decreases at 14 days and was not detected in 16- and 18-day embryos or in differentiated P19 cells. At the structural level both countertranscripts share the same region of perfect or near perfect complementarity to the L27' rp mRNA, which spans more than 75% of the coding region of the latter. The 0.8-kb difference in length of the two countertranscripts lies mainly 3' of the divergence from complementarity to the rp sequence. Indirect evidence suggests that the countertranscripts do not originate from the active L27' rp gene copy. The possible biological significance of the co-existence of the countertranscripts with the housekeeping L27' rp mRNA within the same cell is discussed.

Animals↗

PubMed1978

A conserved and unique (AT)-rich segment in yeast mitochondrial DNA.

The mtDNA of the cytoplasmic petite mutant of yeast RD1A consists mainly of a perfect head-to-tail repetition of a known sequence of 66 consecutive AT and 2 GC base pairs. We have hybridized complementary RNA made on RD1A mtDNA with the mtDNAs of four different wild-type Saccharomyces strains that differ markedly in restriction fragmentation pattern. The tm's of the four heteroduplexes are identical to the tm of the homoduplex of RD1A mtDNA with complementary RNA of one repeat length. With all four wild-type mtDNAs this complementary RNA hybridizes mainly to a single restriction fragment of about 300 base pairs. This shows the conservation and individuality of at least one (AT)-rich segment in yeast mtDNA. The 300 base pair fragment has been mapped in the vicinity of the oxi-2 locus. The possible role of the (AT)-rich segment in the processing of the primary transcript of this region is discussed.

Adenine↗