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R C Huang

Publications and source records attributed to R C Huang.

At least 73 records · Page 4Linked to original sources
PubMed1981

Terminally redundant sequences in cellular intracisternal A-particle genes.

The sequences coding for intracisternal A-particle RNA form a family of related but not identical genetic elements which are present in 650 to 1,000 copies within the mouse genome. We showed that different intracisternal A-particle genes had a terminally redundant sequence of about 400 base pairs, one-half of which arose from the 3' end of the intracisternal A-particle RNA. A second portion of the redundant region did not contain 3-related sequences and was probably derived from the 5' end of intracisternal A-particle RNA. Thus, there were endogenous intracisternal A-particle genes in the cellular DNA-3'-5'--3'-5'-cellular DNA configuration identified for type B and C retroviruses. This indicated that the initial integration of intracisternal A-particle genes into the Mus musculus genome occurred by the same mechanism as the integration of other retroviruses. Two types of heterogeneity were identified among the 5' sequences of the two genes.

Animals↗
PubMed1980

Sequence organization of cloned intracisternal A particle genes.

Seven recombinant DNA clones containing mouse intracisternal A particle genes were isolated and analyzed by restriction enzyme digestion, Southern blot analysis and heteroduplex mapping. The sequence organization of the individual genes was found to differ, with one end of the gene region being most variable, while a central segment of 1.8 kb was missing from two of the clones. A third region, common to all the clones and containing the 3' end of the gene, is present in about 1800 copies per haploid genome, but the central portion is found in only 650 copies. The same reiteration frequency is found in both myeloma tumor and mouse liver DNA. The most abundant intracisternal A particle RNA in two different myeloma lines was found to be 3.5 kb, and RNA/DNA hybrids show that the RNA is homologous to all but a small internal segment of one of the clones.

Animals↗
PubMed1978

Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes.

The purine ribonucleoside triphosphate analogues adenosine 5'[gamma-S]triphosphate and guanosine 5'[gamma-T]triphosphate were used as affinity probes for studying RNA chain initiation in isolated nuclei from the mouse myeloma 66.2 cell line. Transcripts initiated with either nucleotide analogue were isolated by affinity chromatography on a mercury-agarose affinity column. The binding was specific and dependent upon the inclusion of the sulfur nucleotide analogues in the in vitro synthetic reaction. Several lines of evidence indicate that the affinity-labeled RNA is initiated in vitro. First, the sulfur nucleotide is recovered in high yield as a single nucleoside 5'[gamma-S]triphosphate 2',3'-monophosphate product following alkaline hydrolysis of RNA bound to the affinity column. Second, authentic ribosomal 5S RNA is known to initiate with GTP; in vitro 5S RNA is bound to mercury-agarose only if [gamma-S]GTP is used as the affinity label in the synthesis, and not if [gamma-S]ATP is used. Under the conditions studied, nuclei incorporated 1.2--2.4 pmole of UMP per 10(6) nuclei per min, and the rate of synthesis was unaffected by substitution of the nucleotide analogues for the normal nucleotides. The percentage of the total RNA synthesized that was incorporated into sequences initiated in vitro was 7.8 +/- 1.5% with [gamma-S]ATP and 9.6 +/- 6.4% with [gamma-S]GTP. The size of the total RNA synthesized, determined by sedimentation on sucrose density gradients containing dimethylsulfoxide, ranged from less than 5S to 45S, and the size of the affinity-labeled sequences ranged from less than 5S to 28S. Approximately half of the incorporation into RNA initiated in vitro was sensitive to a concentration of alpha-amanitin which selectively inhibits polymerase II activity. Most of the remaining incorporation into initiated sequences can be abolished by concentrations of alpha-amanitin that are inhibitory for polymerase III activity. Over 70% of the total incorporation into ribosomal 5S RNA transcripts was into sequences initiated in vitro. This initiation was catalyzed by polymerase III and was specific for GTP as the initiating nucleotide. The RNAase T1 fingerprint of the newly initiated 5S RNA indicates that this gene is accurately initiated and faithfully elongated in vitro. The use of these affinity label probes provides much greater sensitivity for studying the initiation of RNA in vitro.

Amanitins↗
PubMed1977

Incorporation of purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro.

Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5'-[gamma-S]triphosphates as one of the nucleotide substrates. Substitution of the thiol analogues for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with either adenosine 5'-[gamma-S]triphosphate or guanosine 5'-[gamma-S]triphosphate was isolated with high efficiency on mercury-agarose columns prepared by activation with low concentrations of cyanogen bromide. Sulfur was shown to be incorporated at the 5' end of RNA by identification of the tetraphosphate HSpppA32p liberated after alkaline hydrolysis of HS(A-32pU)n (alternating copolymer synthesized by the action of E. coli RNA polymerase on d(A-T)n-d(A-T)n with adenosine 5'-[gamma-S]triphosphate and uridine 5'-[alpha-32P]triphosphate as substrates). Transcripts elongated but not initiated with these thiol analogues did not bind to the affinity column. This technique provides an extremely sensitive assay for RNA synthesis initiation in vitro, since initiated transcripts containing radiolabel throught the entire transcript can be isolated.

Chromatography, Affinity↗
PubMed1977

Comparative study of hen yolk phosvitin and plasma vitellogenin.

Vitellogenin, the only phosphoprotein detectable in the plasma of laying hens, is present at an approximate concentration of 1 mg/mL and can be isolated by chromatography on diethylaminoethylcellulose. Vitellogenin has a molecular weight of 235 000--240 000 and contains approximately 3% phosphorus by weight. Evidence that this protein is the precursor of phosvitins includes its ability to act as an acceptor for phosphate with a phosvitin specific kinase, the generation of a peptide similar to phosvitin by trypsinization, and the presence of distinctive peptides of multiple clustered phosphoserine upon partial acid hydrolysis. This partial sequence similarity between phosvitins and vitellogenin has not been previously reported. The phosphorus content and amino acid composition of vitellogenin are consistent with a model which contains two phosvitins and one lipovitellin. The total molecular weights of these proteins (28 000 + 34 000 + 170 000 = 232 000) are close to that of vitellogenin.

Amino Acids↗
PubMed1977

Initiation and translation in vitro of mRNA for MOPC 315 immunoglobulin heavy chain and characterization of translation product.

An initiation study of mineral oil-induced plasmacytoma (MOPC) 315 heavy chain immunoglobulin (H315) in vitro has been conducted using formyl-[35S]methionyl-tRNAfMet and a highly purified 18 S message from MOPC 315 solid tumor in a crude rabbit reticulocyte lysate system. The product was specifically precipitated by antibodies directed against MOPC 315 immunoglobulin and H315. The in vitro H315 products terminally labeled with formyl-[35S]methionine or internally labeled with [3H]leucine were electrophoretically identical with in vivo H315 on sodium dodecyl sulfate-polyacrylamide gels. All of the [35S]-methionine was incorporated at the NH2 terminus, not internally, since there is a near complete recovery of [35S]methionine following one cycle of Edman degradation. The NH2-terminal cyanogen bromide peptide, CN2, of in vivo and in vitro H315 co-migrated exactly on gel electrophoresis under conditions which completely resolved two proteins differing in size by only 14 amino acids. These data strongly suggest that there is no NH2-terminal precursor of H315 in this system. Cyanogen bromide peptide profiles of in vivo and in vitro H315 were chromatographically indistinguishable. Three peptides, CN1, CN2, and CN4, which represent approximately 85% of the total amino acids of H315 were isolated and further characterized by electrophoresis and paper chromatography. All were very similar to the corresponding peptides of authentic H315. We conclude that the fidelity of H315 translation is preserved in vitro.

Cell Line↗
PubMed1976

Transcription in vitro of immunoglobulin kappa light chain genes in isolated mouse myeloma nuclei and chromatin.

Messenger RNA sequences for immunoglobulin kappa light chain were synthesized in vitro in isolated mouse myeloma nuclei using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) and from isolated myeloma chromatin using exogenous Escherichia coli RNA polymerase. The in vitro RNA was transcribed using 5-mercuriuridine triphosphate and separated from in vivo RNA by chromatography on an agarose sulfhydryl affinity column. Template restriction is retained in vitro since synthesis of kappa chain messenger RNA, As determined by hybridization with complementary DNA, was much more efficient in nuclei and chromatin isolated from myeloma 66.2 tissue culture cells, a kappa-chain-producing cell line, than from MOPC 315 tissue culture cells, a lambda-chain-producing cell line. Transcription of kappa chain messenger RNA was 25 times more efficient in myeloma 66.2 nuclei than in myeloma 66.2 chromatin.

Cell Nucleus↗
PubMed1975

Low molecular weight RNA species from chromatin.

Several methods of preparing low molecular weight RNA from chick embryo chromatin have been examined. Traditional methods for dissociating chromatin utilizing high concentrations of salt (greater than 2 M) followed by high-speed centrifugation resulted in very low yields of RNA. Increased yields of RNA were obtained by treating chromatin at lower salt concentration (0.2-0.5 M). By using low salt extraction and sodium dodecyl sulfate-phenol deproteinization, six to eight low molecular weight homogeneous RNA species were isolated from chick embryo chromatin and mouse myeloma chromatin. In the myeloma system, all these RNAs are metabolically stable. Each component is homogeneous as examined by gel electrophoresis and hybridizes with mouse DNA at a rate consistent with a single species. There are multiple gene copies for these RNA species in the mouse genome, varying from 100 to 2000 copies for the different species. One of these RNAs is identical with 5S rRNA. In addition, the redundancy of genes for 18S, 28S, and 5S rRNA and tRNA was determined. Approximately 300 copies for 18 and 28S rTRNA and 500 copies for 5S rRNA were found. tRNAs were on an average 110-fold redundant with about 55 different species measured.

Animals↗
PubMed1975

The conformation of proteins in chromatin. A circular dichroism study below 250 nm.

This paper is an investigation of the circular dichroism (CD) spectra of DNA and protein in chromatin. The circular dichroism (CD) of chromatin below 250 nm is due to DNA and protein peptide chromophores. The spectrum in this region is resolved into contributions from salt-extractable proteins (histone and non-histone proteins extractable with sodium chloride), residual non-histone proteins (not extractable with 3 M sodium chloride), and DNA. Below 250 nm, DNA in chromatin has the same CD spectrum as DNA free in solution, in contrast to the CD of DNA above 250 nm (Hjelm, R. and Huang, R. C., (1974), Biochemistry 13, 5275). Histones and salt-extractable non-histone proteins in chromatin are seen to have an average CD like those observed for globular proteins. The average CD of the residual non-histone proteins is consistent with a population of proteins with more extended conformation. The CD of each of these components is found to be the same in chromatins isolated from tissues having different nuclear synthetic activities: chick embryo brain, pig cerebellum, myeloma K41, calf thymus, and chicken erythrocyte.

Animals↗
PubMed1975

The contribution of RNA and non-histone proteins to the circular dichroism spectrum of chromatin.

This paper is an investigation of the contribution of low salt extractable RNA and non-histone proteins to the circular dichroism of chromatin. Circular dichroism (CD) of chromatin above 250 nm is due mainly to DNA and is different from that of DNA free in solution. In addition, to a smaller extent, we find that low salt extractable RNA and/or non-histone protein side chain chromophores contribute significantly to the spectra in this region and account for the major differences observed among the CD spectra of chromatins isolated from the five tissues studied; pig cerebellum, myeloma, calf thymus, chick embryo brain, and chick erythrocytes.

Animals↗
PubMed1975

Chromatin directed transcription of 5S and tRNA genes.

Chromatin prepared by gentle methods from mouse myeloma cells retained its ability to synthesize RNA using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). The transcription resembles that observed in vivo in several respects. The low-molecular-weight RNA species 5S RNA and the 4.5S precursor to 4S RNA, are transcribed accurately and transcription is reinitiated continually in vitro. Their synthesis was not inhibited by alpha-amanitin (1 mug/ml) as was found previously for these species in isolated nuclei.

Animals↗