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PubMed1993

Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.

High-titer, site-specific antibodies have been produced against the rat kidney broad-spectrum, sodium-independent neutral and basic amino acid transporter (NBAA-Tr) whose cDNA we cloned earlier. These antibodies have allowed us to characterize the transporter protein in normal rat tissues and in various cellular and in vitro expression systems. Western analysis detected 84- to 87-kDa glycosylated species enriched in rat renal and jejunal epithelial cell brush border membranes. In vitro translation of NBAA-Tr complementary RNA in the rabbit reticulocyte lysate system yielded a 78-kDa protein, a molecular mass that was predicted by the amino acid sequence deduced from the cloned cDNA. Translation in the presence of rough microsomal membranes yielded a glycosylated 89-kDa species. Glycosylated 87- to 89-kDa species were also expressed in Xenopus oocytes microinjected with NBAA-Tr complementary RNA and in COS-7 cells transfected with NBAA-Tr cDNA. Localization of NBAA-Tr in renal and intestinal brush border membranes is consistent with its proposed role in transepithelial transport of amino acids.

Amino Acid Sequence↗
PubMed1980

Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins.

Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.

Animals↗
PubMed1989

Structure and coding properties of Bs1, a maize retrovirus-like transposon.

We have sequenced Bs1, an insertion element isolated from a null allele of the Adh1 locus encoding alcohol dehydrogenase in maize. The Bs1 element is 3203 base pairs (bp) in length, has 302-bp identical long terminal direct repeats (LTRs), and created a 5-bp flanking direct duplication of target Adh1 DNA upon insertion. The 5' LTR is followed by a canonical primer binding site with homology to the plant initiator methionyl-tRNA, and the 3' LTR is directly preceded by a polypurine stretch like that observed in retroviruses and retrotransposons. Bs1 encodes two overlapping open reading frames specifying peptides of 740 and 168 amino acids. The longer open reading frame specifies a peptide with amino acid homology to the protease and nucleic acid binding moiety of retroviruses and retrotransposons. The deduced amino acid sequence encoded by Bs1 lacks convincing homology to the polymerase (reverse transcriptase) encoded by retroposons, despite the fact that this polymerase-encoding domain is routinely the most conserved region of any such element. The sequence and relatively small size of Bs1 suggest that this element is a deleted retrotransposon that inserted into Adh1 with the aid of a reverse transcriptase function provided in trans. In vitro transcribed Bs1 complementary RNA was translated in vitro to produce both a protein of 81 kDa representing open reading frame 1 (ORF1) and one of the 95-kDa size predicted for the frame-shifted fusion of ORF1 and ORF2. As with many other retroposons, the efficiency of translational initiation at the AUG beginning ORF1 was not noticeably affected by the presence of one or more upstream, unproductive AUGs in the complementary RNA transcript.

Amino Acid Sequence↗
PubMed1996

Pharmacokinetic properties of several novel oligonucleotide analogs in mice.

Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2'-propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5'-octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5'-conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney.

Animals↗
PubMed1979

No homology detectable between Marek's disease virus (MDV) DNA and herpesvirus of the turkey (HVT) DNA.

The relation of four different strains of MDV and two strains of HVT was analyzed by gel electrophoresis of viral DNA digested by various restriction endonucleases and by filter hybridization of viral DNA with complementary RNA. The four MDV strains showed fragment patterns completely different from those of HVT upon digestion of the viral DNA with Bam H I, Eco R I, Hind III, Hpa, I, and Xho and separation of fragments on agarose gels. The cleavage patterns of the four MDV strains showed great similarities among each other as well as some differences between the individual strains. In the cleavage patterns of HVT a similar close relationship was observed between the two HVT strains with slight divergence between both. Filter hybridizations of viral DNA with labelled complementary RNA prepared from the DNA of the GA strain of MDV or from the DNA of the PH-THV1 strain of HVT revealed no cross-hybridization between the MDV and the HVT strains. cRNA prepared from the DNA of an MDV strain hybridized only to restriction enzyme fragments of the MDV strains transferred to nitrocellulose filters, but not to fragments of HVT DNA, and vice versa.

Animals↗
PubMed1997

Characterization and phylogenetic significance of rhinoceros luteinizing hormone beta (LHbeta) subunit messenger RNA structure, complementary DNA sequence and gene copy number.

The luteinizing hormone (LH) beta subunit gene is expressed in the pituitary glands of all mammals, whereas the closely related chorionic gonadotropin (CG) beta subunit genes have been identified only in primates and equids, and are expressed in placenta. In the case of horses, there is a single-copy equine (e) luteinizing hormone/chorionic gonadotropin hormone beta subunit gene (eLH/CGbeta) that (1) is expressed in both pituitary gland and placenta, (2) encodes a characteristic carboxyl terminal peptide (CTP) extension, and (3) transcribes an atypically elongated 5'-untranslated region (UTR) in both pituitary and placenta. However, it is not known whether similar expression patterns and gene locus characteristics may be exhibited by other members of the order Perissodactyla (equid, rhinoceros and tapir species). To begin to investigate these possibilities, we undertook analysis of the rhinoceros (rn or rhino) LH/(CG?)beta gene locus and the rnLHbeta cDNA. Total RNA isolated from the pituitary gland of a female white rhino was used as template for amplifying rnLHbeta cDNA by reverse transcription-polymerase chain reaction. Following cloning of the amplified cDNA, nucleotide (nt) and deduced amino acid sequences were determined. The first in-frame stop codon occurred at codon position +122, suggesting that the rnLHbeta subunit does not contain a CTP. To assess gene copy number, Southern blot analysis of Indian rhino genomic DNA was performed. The resulting simple hybridization pattern indicated that, as in the horse and donkey, there is a single-copy gene at the rnLH/(CG?)beta gene locus. Primer extension mapping of the pituitary transcriptional start site of the rnLHbeta subunit gene revealed an 8 nt 5'-UTR which is similar to that reported for the majority of mammalian LHbeta transcripts. Northern analysis was consistent with the transcriptional start site findings. We postulate from these data that rhinos diverged from equids prior to the occurrence of the mutations causing CTP expression and adoption of a non-consensus 5'-UTR/proximal promoter region. However, these findings do not rule out the possibility of expression of a placental CGbeta subunit lacking a CTP in rhinos.

Amino Acid Sequence↗
PubMed1997

Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas.

OBJECTIVE: A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. METHODS: We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. RESULTS: C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. CONCLUSION: ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.

Animals↗
PubMed1993

Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression.

We have used a previously described 17-mer phosphorothioate (Monia, B.P., Johnston, J.F., Ecker, D. J., Zounes, M.A., Lima, W.F., and Freier, S.M. (1992) J. Biol. Chem. 267, 19954-19962) for structure-function analysis of 2'-sugar modifications including 2'-O-methyl, 2'-O-propyl, 2'-O-pentyl, and 2'-fluoro. These modifications were analyzed for hybridization affinity to complementary RNA and for antisense activity against the Ha-ras oncogene in cells using a highly sensitive transactivation reporter gene system. Hybridization analysis demonstrated that all of the 2'-modified oligonucleotides hybridized with greater affinity to RNA than an unmodified 2'-deoxy oligonucleotide with the rank order of affinity being 2'-fluoro > 2'-O-methyl > 2'-O-propyl > 2'-O-pentyl > 2'-deoxy. Evaluation of antisense activities of uniformly 2'-modified oligonucleotides revealed that these compounds were completely ineffective in inhibiting Ha-ras gene expression. Activity was restored if the compound contained a stretch of at least five 2'-deoxy residues. This minimum deoxy length correlated perfectly with the minimum length required for efficient RNase H activation in vitro using partially purified mammalian RNase H enzyme. These chimeric 2'-modified/deoxy phosphorothioates displayed greater antisense potencies in inhibiting Ha-ras gene expression, compared with the unmodified uniform deoxy phosphorothioate. Furthermore, antisense potency correlated directly with affinity of a given 2' modification for it's complementary RNA. These results demonstrate the importance of target affinity in the action of antisense oligonucleotides and of RNase H as a mechanism by which these compounds exert their effects.

Base Composition↗
PubMed1996

NIRCA: a rapid robust method for screening for unknown point mutations.

We describe a method for screening for dispersed point mutations, based on the observation that RNase frequently cleaves both strands of base pair mismatches in duplex RNA targets. The mismatched substrates are generated by in vitro transcription of wild-type and mutant templates amplified by the PCR or reverse transcription (RT)-PCR; bacteriophage promoters are incorporated into the PCR primers to permit both strands of the products to be transcribed into RNA. Complementary wild-type and mutant transcripts are hybridized and treated with RNase, and the cleavage products are separated on agarose gels and detected by visualization of the ethidium-stained sample under UV light. The method is thus non-isotopic, and since the cleavage products remain double-stranded during analysis, the labor-intensive RNase inactivation steps required in the original procedure can be eliminated. Also, nonspecific background cleavage is reduced so that longer target regions (1 kb) can be screened in a single step. The Non-Isotopic RNase Cleavage Assay (NIRCA) achieved a detection rate of 88%-90% in blind studies in a Factor IX model system, and it was also used to detect unknown p53 mutations in breast tumor samples. NIRCA provides a rapid method for sensitive, non-isotopic, high-throughput genetic screening.

Base Sequence↗
PubMed1998

Expression of alpha3, beta3 and gamma1 GABA(A) receptor subunit messenger RNAs in visual cortex and lateral geniculate nucleus of normal and monocularly deprived monkeys.

Complementary RNA probes derived from complementary DNA specifically subcloned from monkey tissue were used to localize, by in situ hybridization histochemistry, the relatively rare alpha3, beta3 and gamma1 subunit transcripts of the GABA(A) receptor in visual cortex and lateral geniculate nucleus of normal monkeys and in monkeys that had been deprived of vision in one eye. Overall, levels of alpha3, beta3 and gamma1 subunit transcripts were very low. In the primary visual cortex (area 17) they were concentrated in layers II and VI and in a stratum of white matter subjacent to layer VI. The localization and density of the three messenger RNAs closely resembled those of other rare (alpha2, alpha5 and beta1) transcripts but their distribution also overlapped that of the predominant alpha1, beta2 and gamma2 subunit transcripts. In area 18, alpha3 and beta3 transcript distribution resembled that in area 17, with the addition of a third band of hybridization in layer IV for beta3. Gamma1 subunit transcript localization in area 18 differed significantly from that in area 17, with increased expression restricted to layer IV. In the dorsal lateral geniculate nucleus, beta3 and gamma1 transcripts were expressed at low levels across all layers while alpha3 transcripts were restricted to the magnocellular layers. Following 15 and 18 day periods of monocular deprivation, induced by intravitreal injections of tetrodotoxin, levels of alpha3 receptor subunit transcripts showed modest reductions in layer VI of area 17 and in deprived geniculate laminae of adult animals. Reductions in alpha3 transcript levels were much more pronounced in layer IVCbeta of a five-month-old monkey deprived for the same time. Levels of beta3 and gamma1 transcripts were unaffected by monocular deprivation in cortex and geniculate at any age. Taken together with studies of other GABA(A) receptor transcripts, these results demonstrate the heterogeneity of GABA(A) receptor messenger RNA expression in the monkey geniculo-striate pathway and the varied response to reduced neuronal activity.

Animals↗
PubMed1997

Inhibition of eFGF expression in Xenopus embryos by antisense mRNA.

We studied the effects in Xenopus embryos of overexpression of antisense RNA complementary to the messenger RNA of eFGF. We show that the expression of sense RNA can be severely depressed in the presence of an excess of antisense RNA. This occurs by both partial destruction of the message and by a depression of translation of the residual message. The diminution of inducing activity of eFGF, measured in animal cap assays either by activation of the Brachyury gene or by morphology, parallels the reduction of translation. Endogenous eFGF expression is reduced to a similar extent, again by a combination of mRNA destruction and inhibition of translation. This shows that the overexpression of antisense RNA is, contrary to general opinion, a potentially useful technique for studying gene function in Xenopus embryos. However, in the case of eFGF, there is little or no overall phenotypic effect on whole embryos. This is probably because of the presence of several other FGFs with overlapping expression domains in the early embryo.

Animals↗
PubMed1989

Kinetics of complementary RNA-RNA interaction involved in plasmid ColE1 copy number control.

Binding of a small antisense RNA (RNA I) to the primer transcript (RNA II) of plasmid ColE1 inhibits formation of primer for DNA polymerase I-mediated plasmid replication. It is thought that RNA I and RNA II transiently interact via their single-stranded loop regions to form an unstable complex that subsequently converts into a more stable complex by hybridization. Rom (or Rop) protein enhances the inhibitory effect of RNA I on replication by enhancing the binding of the two RNAs. In this paper, we develop a model for the kinetics of the RNA I-RNA II binding reaction, estimate the rate constants, and provide a quantitative description of the effects of Rom protein. We show that the reaction kinetics are consistent with a stepwise binding model in which Rom protein binds to RNA I and RNA II, while the RNAs are held together in a transient complex. Mutations that replace C.G pairs by T.A pairs in the RNA loop regions and thus display weaker hydrogen bonding between the loop regions should be associated with an increased rate of dissociation for the unstable complex. Our model predicts that such destabilization of the loop interactions leads to a greater enhancement in the binding rate by Rom protein. The available data support this prediction.

Bacterial Proteins↗
PubMed1990

Effect of actinomycin D on replication of satellite tobacco ringspot virus RNA in plant protoplasts.

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea (Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 micrograms/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.

Dactinomycin↗
PubMed1995

Dissecting RNA-protein interactions: RNA-RNA recognition by Rop.

The ColE1 plasmid of E. coli encodes a small RNA-binding protein, Rop, which is involved in the regulation of plasmid copy number. Rop, a 4-helix bundle protein, facilitates sense-antisense RNA pairing by binding to the transiently formed hairpin pairs of RNA I and the complementary RNA II. We have identified the residues of Rop that are involved in RNA recognition. The residues form a narrow stripe down one face of the bundle and are symmetrically arranged, with recognition centered about two phenylalanine residues. Our results suggest that these phenylalanine residues interact with the loop region of the hairpin pair, with additional interactions between eight polar residues and the phosphate backbone. By modifying the identity of residue 14, we have created a variant of Rop that displays altered RNA binding specificity. The results of our studies allow us to present a detailed picture of RNA-protein recognition in a novel model system.

Amino Acid Sequence↗