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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.
Mol Cell Biol 1987 Jun
PMID:Poly-L-ornithine-mediated transformation of mammalian cells. 360 Jun 62

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.
Mol Pharmacol 1987 Feb
PMID:Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs. 380 92

The effect of phenobarbital on the level of rat liver apolipoprotein A-I (apo-A-I) mRNA was studied. Poly(A+)-RNA isolated from livers of control or phenobarbital-treated rats was translated in vitro in the rabbit reticulocyte lysate system and immunoprecipitated with rabbit antiserum against rat apo-A-I. The immunoprecipitate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of apo-A-I mRNA was estimated from the incorporation of [35S]methionine into the apo-A-I band. It was found to be elevated 4-fold by 16 hr after rats received a single injection of phenobarbital. To study the effect of phenobarbital on the level of rat liver apo-A-I mRNA, a recombinant plasmid which contained a cDNA insert corresponding to rat liver apo-A-I mRNA was isolated and used to hybridize total liver poly(A+)-RNA from control and phenobarbital-treated rats. There were 4.8- and 10-fold increases in the amount of hybridization to mRNAs from rats after they were treated with phenobarbital for 8 and 16 hr, respectively. Thus, phenobarbital increases the level of rat liver apo-A-I mRNA.
Mol Pharmacol 1985 Mar
PMID:Phenobarbital induces rat liver apolipoprotein A-I mRNA. 391 79

Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1985 Jan
PMID:Regiospecific gamma-conjugation of methotrexate to poly(L-lysine). Chemical and biological studies. 396 26

The purpose of these experiments was to obtain proinsulin mRNA from catfish pancreatic islets and synthesize its cDNA. Poly(A)-rich mRNA was electrophoresed on preparative agarose-urea gels. One RNA fraction was obtained which translated predominantly preproinsulin. This mRNA was estimated to be approx. 210 000 Mr (650 nucleotides) when electrophoresed under denaturing conditions. [3H]Proinsulin cDNA was hybridized to excess RNA to monitor purification of mRNA from total islet RNA. Greater than 94% of proinsulin messenger contained poly(A) sequences. [3H]Proinsulin cDNA hybridized to its template mRNA with a Rot 1/2 of 4.4 x 10(-3) mole x sec/l. The overall purification was 80-fold by this type of analysis. Thermal denaturation studies indicated a high degree of fidelity of hybrid formation between [3H]proinsulin cDNA and proinsulin mRNA. Proinsulin comprised 20% of total islet protein when synthesis was measured in vivo (Albert and Permutt, 1979) and 12-20% when total islet mRNA was translated in a cell-free system. Using the [3H]proinsulin cDNA probe it was estimated that proinsulin mRNA accounted for approx. 15% of total islet mRNA.
Mol Cell Endocrinol 1980 Aug
PMID:Isolation of catfish proinsulin messenger RNA and synthesis of its complementary DNA. 615 87

Poly(A+)--RNA from rat ventral prostate was isolated using oligo(dT)-cellulose chromatography. 45% of the total poly(A+)--RNA was a single peak at 10S as demonstrated by centrifugation in a 5-20% sucrose gradient containing 1% SDS. By using complementary DNA probes, it was shown that the 10S RNA contained the major abundance class of poly(A+)--RNA. Denaturing agarose-gel analysis revealed 2 major bands in the 10S poly(A+)--RNA preparation approx. 600 NT and 500 NT (NT = nucleotides) long, resp. Double-stranded 32 P-DNAs complementary to light side and heavy side of the 10S poly(A+)--RNA peak were synthesized and isolated using reverse transcriptase and hydroxyapatite (HAP) chromatography. Approx. 40% of the first strand of the cDNAs were converted to double-stranded structures with a Tm of 88 degrees C. HAP purified double-stranded material was 92% resistant to S1 nuclease. the DNA--DNA reannealing profile of double stranded 32 P-cDNA enriched for the 500 NT band gave a Cot 1/2 of approximately 7 X 10(-4) moles X sec X 1(-1) indicating a complexity for this enriched synthetic gene of 500-600 nucleotide pairs (NTP).
Mol Cell Endocrinol 1980 Sep
PMID:Purification of major abundance class of poly(A+)-RNA from rat ventral prostate. 615 88

Immune RNA (I-RNA) was extracted form lymphoid organs of BALB/c mice immunized with AKR lymphoid cells. Incubation of normal BALB/c spleen cells with this I-RNA (but not with normal RNA) resulted in leukocyte migration inhibition reactions (LMIR) against AKR extracts but not against purified protein derivative or BALB/c sarcoma extracts. This transfer was abolished by pretreating I-RNA with RNAse but not with pronase. The active fraction of I-RNA was retained by and could be eluted from Poly-U Sepharose columns. Normal cells pretreated with I-RNA also reacted in the presence of an anti-idiotypic anti-serum of anti-(BALB/c anti-AKR) specificity. Pretreatment of cells with anti-idiotypic serum plus complement did not inhibit the subsequent transfer of LMIR with I-RNA. Idiotypic receptors were expressed on I-RNA treated cells less than one hour after I-RNA treatment. Using an I-RNA of double specificity, the results suggested that I-RNA entered into and acted on the cells through a nonspecific mechanism. Finally, I-RNA could induce BALB/c anti-AKR idiotypic markers in C57Bl/6 cells, genetically committed for different idiotypes, while RNA extracted from C57Bl/6 immune cells could not induce in BALB/c cells their own genetically acquired idiotypes. This series of data would prove that I-RNA acting as a mRNA is able to induce in normal noncommitted cells the de novo synthesis of antigen receptors similar or identical to those present in the surface of in vivo immunized lymphoid cells of the same strain.
Mol Cell Biochem 1980 Dec 16
PMID:Mechanism of immune transfer by RNA extracts. Immune RNA induces the synthesis of idiotype-bearing antigen receptors in noncommitted cells. 616 92

Cytoplasmic processing events in the poly(A) region of mRNA from Physarum polycephalum are reviewed. Two classes of poly-containing RNA [poly(A)+ RNA] exist in the cytoplasm. One contains very short poly(A) sequences, averaging about 15 adenylate residues, while the other contains relatively long poly(A) sequences, averaging about 60 residues. Molecules with short poly(A) sequences are found exclusively in the polysomes while those with long poly(A) sequences are restricted to the free cytoplasmic mRNP. Since proteins are associated with only the long poly(A) sequences the poly(A) . protein complex is also restricted to the free mRNP. The long poly(A) sequences are relatively short-lived. They are degraded by two distinct processes, a shortening process in which 15-20 residues are gradually removed and a turnover process in which long poly(A) tracts are rapidly converted to the short sequences. This process, along with the dissociation of the poly(A) . protein complex, occurs when poly(A)+ RNA molecules located in free mRNP are transferred to the polysomes. Poly(A) . protein complex dissociation appears to precede poly(A) turnover during translational selection. The significance of these processing events in relation to mRNA maturation is discussed.
Mol Biol Rep 1981 May 22
PMID:Cytoplasmic processing events in the polyadenylate region of Physarum messenger RNA. 616 54

A goat immunized with poly(A).poly(dT) produced three distinct antibody populations. The major one was specific for RNA-DNA hybrids and was purified from precipitates made with poly(I).poly(dC). It also reacted with hybrids of mixed base composition made with E. coli RNA polymerase. The other populations were purified with poly(A).poly(U) or poly(dT). Three rabbits also produced mainly hybrid-specific antibody in response to poly(A) . poly(dT). A goat immunized with poly(I).poly(dC) formed antibodies reactive with poly(I) and others reactive with poly(dC) but none specific for hybrid structure. Three rabbits did not respond to poly(I).poly(dC). Measurements of reactions with anti-inosine sera, thermal denaturation and sensitivity to S1 nuclease indicated that poly(I).poly(dC) is a less stable helix than poly(A).poly(dT) or poly(I).poly(C). Poly(A).poly(dT) is the more suitable synthetic immunogen for the production of hybrid-specific antibodies.
Mol Immunol 1982 Mar
PMID:Comparison of poly(A).poly(dT) and poly(I).poly(dC) as immunogens for the induction of antibodies to RNA-DNA hybrids. 617 64

During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components.
Mol Biol Rep 1983 Aug
PMID:Discrete poly(A)- RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5'-termini. 619 19


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