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At least 1,279 records · Page 71Linked to original sources
PubMed1978

Heterogeneity in the 3'-terminal sequence of ribosomal 5S RNA synthesized by isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei synthesize ribosomal 5S RNA of very nearly correct sequence and size. The in vitro product was resolved according to size on formamide-containing polyacrylamide gels and the fractions were subsequently hybridized to recombinant DNA containing the 5S genes from Xenopus mulleri. It could be shown that the 5S RNA synthesized in vitro differed only very slightly in size from the mature species labeled in vivo and contained a few extra nucleotides in some of the molecules. Analysis of the 3'-terminal base of molecules synthesized independently with four different nucleotides showed that the chains were almost exclusively terminated with uridine. Digestion of the in vitro product with ribonuclease T1 and analysis of the oligonucleotides on DEAE-Sephadex A-25 in the presence of 7 M urea revealed that the molar yield of the internal fragments agreed well with the expected theoretical values. The 3'-terminal fragments, however, were found to be present in three different species with the sequences CUUOH, CUUUOH, CUUUUOH which occurred with a frequency of about 60%, 20%, and 20%, respectively. From these data we conclude that 5S RNA synthesis in isolated HeLa cell nuclei was correctly initiated but that termination occurred with a slight ambiguity, adding either one or two uridine residues to some of the chains.

Base Sequence↗
PubMed1978

Isolation, purification, and properties of mouse heavy-chain immunoglobulin mRNAs.

A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.

Animals↗
PubMed1978

Identification of the transcribing DNA strand for the deoxynucleotide kinase gene of bacteriophage T2.

A modified procedure was developed which allows RNA--DNA hybridization reactions to be performed without the loss in translational capacity of mRNA which accompanies hybridization at elevated temperatures or in the presence of the denaturing agent formamide. Separated l and r strands of bacteriophage T2 DNA were hybridized in the presence of 4 M sodium perchlorate at 37 degrees C with total RNA from infected cells. After passage of the hybridization mixture through a nitrocellulose column to remove single-strand DNA and DNA--RNA hybrids, the eluent was measured for its capacity to promote deoxynucleotide kinase (gene 1) synthesis in an in vitro protein-synthesizing system derived from uninfected Escherichia coli. With this procedure, which should be of general use for any message whose product can be measured either enzymatically, immunologically, or by location in an acrylamide gel, it was demonstrated that deoxynucleotide kinase mRNA is transcribed from the l strand of bacteriophage T2 DNA. By titrating with l strand DNA, the number of deoxynucleotide kinase transcripts present 9 min after T2 phage infection at 30 degrees C was estimated to be about 38 copies per cell.

Coliphages↗
PubMed1978

Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase.

Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide. Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract. The in vitro synthesized polypeptides were identified by tryptic peptide analysis. Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657). Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed. When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.

Glyceraldehyde-3-Phosphate Dehydrogenases↗
PubMed1977

Resolution of ribonucleic acids by Sepharose 4B column chromatography.

Ribonucleic acids were resolved by molecular sieve chromatography on columns of Sepharose 4B. The elution positions of messenger ribonucleic acids were determined by detection of polyadenosine tracts and by support of protein synthesis in a messenger-dependent cell-free system. The elution position of other ribonucleic acid species from the Sepharose 4B was determined by formamide-sucrose density gradient centrifugation. Resolution of ribonucleic acids by this column was not dependent on molecular weight but rather on other properties such as secondary structure or the presence of poly(adenylic acid). The elution profiles of ribonucleic acids on cross-linked Sepharose 4B differed markely from those on conventional Sepharose and appeared to depend on molecular size alone. There was diminished resolution of high molecular weight ribonucleic acids on such columns.

Animals↗
PubMed1976

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.

Animals↗
PubMed1976

The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.

Animals↗
PubMed1976

Determination of molar ratios of vesicular stomatitis virus induced RNA species in BHK21 cells.

A modified procedure for analysis of RNA in denaturing formamide-polyacrylamide slab gels containing 6 M urea is described. Using this technique, in conjucntion with fluorographic analysis, we determined molecular weights and molar ratios of the various vesicular stomatitis virus (VSV) induced RNAs in BHK21 cells. A comparison of the molar ratios of virus-specific mRHAs and their putative protein products in these cells suggests that there is little, if any, translational control of viral gene expression during acute VSV infection.

Cell Line↗
PubMed1976

Purification of myosin translational control RNA and its interaction with myosin messenger RNA.

Myosin messenger ribonucleoprotein-translational control ribonucleic acid (mRNP-tcNA) from myosin mRNPs found in embryonic chick muscle has been further purified by Dowex chromatography and, from a number of controls, it is suggested that this small RNA is not an artifact produced through the degradation of RNA during its isolation. This highly purified myosin mRNP-tcRNA is shown to have a molecular weight of 10 000 on formamide-acrylamide gels, and reacts stoichometrically (on a 1:1 mole ratio) with myosin mRNA. The stoichiometric interaction between myosin mRNA and myosin mRNP-tcRNP is demonstrated by ists ability to increase the nuclease resistance of the messenger, as well as inhibit its translation in a cell-free amino acid incorporating system.

Animals↗
PubMed1976

Isolation of L-cell messenger RNA which lacks poly(adenylate).

It has been found that centrifugation of (ethylenediamine)tetraacetic acid dissociated L-cell polyribosomes at 25 degrees C through preformed CS2SO4 density gradients containing 15% dimethyl sulfoxide resolves them into two distinct classes of particles that contain ribosomal and messenger RNA, respectively. Ribosomal RNA bands at a higher density than mRNA, presumably because it is more extensively stripped of protein by the action of CS2SO4 than mRNA. Undergraded RNA can be recovered from the gradients by trapping the particles on nitrocellulose filters and then eluting at 60 degrees C with a solution containing formamide and sodium dodecyl sulfate. When cells were labeled with [3H]adenosine, either in the presence or absence of a dose of actinomycin D which selectively inhibits rRNA synthesis, and their polyribosomes centrifuged through CS2SO4 density gradients, a large amount of the radioactivity recovered from the messenger-ribonucleoprotein region of the gradient had the size distribution of mRNA, but lacked poly(adenylate) (poly (A)). This material constituted 29-31% of the total mRNA. Histone mRNA was the most prominent component of poly(A)-lacking mRNA. However, 50-65% of the poly(A)-lacking mRNA was of larger size than histone mRNA.

Cell Fractionation↗
PubMed1976

Isolation and translation of calvaria procollagen messenger ribonucleic acids.

Procollagen messenger ribonucleic acid (mRNA) was isolated from the calvaria of 15-day-old chick embryos by chromatographing total RNA over oligo(dT)-cellulose two times, and then fractionating the twice-bound RNA on 85% Me2SO/0-20% sucrose gradients. When analyzed on 99% formamide gels, the 27-30S fraction had three sharp fluorescent bands, one corresponding to 27S ribosomal RNA (rRNA), the others having mobilities lower than 27S corresponding to molecular weights of 1 700 000 and 1 800 000. In wheat-germ, cell-free extracts, the 27-30S fraction directed the synthesis of two prominent collagenase sensitive polypeptides with mobilities corresponding to the calvaria pro-alpha chain markers. Twelve percent of this [3H]proline-labeled, wheat-germ product could be hydroxylated with prolyl hydroxylase.

Animals↗
PubMed1975

Endonuclease II of Escherichia coli: degradation of gammairradiated DNA.

Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage. The first type leads to single strand breaks in the DNA observed after exposure to alkali. This type of alkali-labile bond will be designated a spontaneous break. The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site. This site is not alkali-labile. After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites. There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2. The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2. The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation. The treatment can be before or after the preincubation. This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions. That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used. Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites. Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites. It has no effect when added at the end of the preincubation. Catalase added before the preincubation has no effect on either type of damage. It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates. The intermediates then undergo either depurination or depyrimidination. The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH. An alternative hypothesis would involve damage to the sugar rather than to bases. The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent. The chemical nature of either form of primary damage is not known.

Binding Sites↗
PubMed1975

Association of the polyadenylate segment of messenger RNA with other polynucleotide sequences in mouse sarcoma 180 polyribosomes.

Limited digestion of polysomal RNA with pancreatic ribonuclease releases a structure consisting of poly(A) associated with other polyribonucleotide sequences. This complex can be purified by oligo(dT)-cellulose chromatography. Heating for formamide treatment causes the dissociation of fragments free of poly(A) from the poly(A)-containing components. The two types of fragments tend to reassociate under annealing conditions, and this association is prevented by poly(U). Control experiments indicate that this structure is not an artifact generated during the manipulations. The same structure can be obtained by limited RNase digestion of polyribosomes, followed by deproteinization. The results suggest that the mRNA in polyribosomes may have a defined configuration caused by the interaction of the poly(A) sequence with another segment of the RNA.

Adenine↗
PubMed1975

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.

Base Sequence↗
PubMed2006

Annotating enzymes of unknown function: N-formimino-L-glutamate deiminase is a member of the amidohydrolase superfamily.

The functional assignment of enzymes that catalyze unknown chemical transformations is a difficult problem. The protein Pa5106 from Pseudomonas aeruginosa has been identified as a member of the amidohydrolase superfamily by a comprehensive amino acid sequence comparison with structurally authenticated members of this superfamily. The function of Pa5106 has been annotated as a probablechlorohydrolase or cytosine deaminase. A close examination of the genomic content of P. aeruginosa reveals that the gene for this protein is in close proximity to genes included in the histidine degradation pathway. The first three steps for the degradation of histidine include the action of HutH, HutU, and HutI to convert L-histidine to N-formimino-L-glutamate. The degradation of N-formimino-L-glutamate to L-glutamate can occur by three different pathways. Three proteins in P. aeruginosa have been identified that catalyze two of the three possible pathways for the degradation of N-formimino-L-glutamate. The protein Pa5106 was shown to catalyze the deimination of N-formimino-L-glutamate to ammonia and N-formyl-L-glutamate, while Pa5091 catalyzed the hydrolysis of N-formyl-L-glutamate to formate and L-glutamate. The protein Pa3175 is dislocated from the hut operon and was shown to catalyze the hydrolysis of N-formimino-L-glutamate to formamide and L-glutamate. The reason for the coexistence of two alternative pathways for the degradation of N-formimino-L-glutamate in P. aeruginosa is unknown.

Amidohydrolases↗
PubMed1996

Frayed wires: a thermally stable form of DNA with two distinct structural domains.

In aqueous solutions containing mono- and divalent cations, the oligodeoxyribonucleotide d(A15G15) readily self-assembles into high-molecular weight species that resolve as discrete bands on native and denaturing electrophoresis gels. The complexes consist of an integer number of strands of d(A15G15), the number of strands range from one (the monomer) to greater than nine. The complexes form within a few minutes even at low concentrations (2 microM strands) of d(A15G15). The relative concentration of species is determined by the solvent conditions. The complexes are resistant to standard denaturation conditions, 50% formamide heated to 95 degrees C for 2 min followed by electrophoresis in 7 M urea at 55 degrees C. In the proposed model for the oligomers and polymers of d(A15G15), several molecules of d(A15G15) interact via a stem of tetraplex structure formed by the guanine residues. The 15 guanine residues in the stem account for its high stability. The 5' end adenines form single-stranded arms that are displaced from the guanine-containing stem. The arms can participate in the formation of Watson-Crick base pairs with dT10 and other partially complementary oligodeoxyribonucleotides such as d(CT15). Engagement of the arms in interactions with other strands does not affect the distribution of the species between different conformations. On the other hand, the addition of the fully complementary oligodeoxyribonucleotide d(C15T15) to the polymer leads to the disappearance of the high-molecular weight complexes and results in the formation of a canonical Watson-Crick base-paired duplex. The type and concentration of the cation present in solution determine which conformation d(A15G15) will adopt. Divalent cations at millimolar concentrations lead to the formation of the polymer, while the presence of the monovalent cations stabilizes lower-molecular weight complexes consisting of two to six strands of d(A15G15).

Base Sequence↗
PubMed1997

The relative stabilities of base pair stacking interactions and single mismatches in long RNA measured by temperature gradient gel electrophoresis.

The thermal stability of RNA duplexes differing by a single base pair (bp) substitution or mismatch were investigated by temperature gradient gel electrophoresis (TGGE). All base pair substitutions and mismatches were examined at six sites, and limited changes were investigated at three other sites. DNA templates for in vitro transcription were generated by the polymerase chain reaction (PCR). Transcribed forward and reverse single stranded RNAs were annealed to form 345 bp dupex RNA. Solution melting curves of selected RNAs were in good agreement with the predicted three step transitions. Parallel TGGE was used to determine the relative stabilities of the RNAs, and perpendicular TGGE was employed to obtain mobility transitions and midpoint transition temperatures (Tmu) of the RNAs' first melting domain. The gel solvent included formamide and urea. The Tmu values of the first melting domain were influenced by the identity of the base pair substitution or mismatch as well as by the site's neighboring base pairs. The difference in the transition temperatures (deltaTmu) between pairs of RNA ranged from 0 to 5 degrees C. deltaTmu values were used to determine free energy differences (deltaDeltaG). For RNA pairs distinguished by a base pair substitution, the deltaDeltaG values were closely correlated with free energy differences calculated from stacking free energies determined from melting studies in 1 M Na+ [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261.] An algorithm was developed using the free energies of terminal mismatches [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261] that provided very good agreement with experimental free energies for the single internal mismatches.

Base Composition↗
PubMedDate not supplied

Directed covalent immobilization of aminated DNA probes on aminated plates.

A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).

Adsorption↗