Gel electrophoretic fractionation of RNAs by partial denaturation with methylmercuric hydroxide

Chandler, Peter M.; Rimkus, Dan; Davidson, Norman

doi:10.1016/0003-2697(79)90063-0

Cited by 40 publications

(6 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The individual bands were identified from their mobilities relative to 4S RNA as reported (9). Methyl mercury/10% polyacrylamide slab gel electrophoresis was carried out by the system of Chandler et al (22). The rat liver RNA contained small nuclear (snRNA) species of defined length as follows: U2, U1, 5S, and 4.5S RNA representing 196, 171, 120, and 96 nucleotides per molecule (23,24).…”

Section: Methodsmentioning

confidence: 99%

Identification of the immunogenically active components of the Sm and RNP antigens.

White¹,

Gardner²,

Hoch³

1981

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The spectrum of cellular targets in the autoimmune diseases is both large and varied and includes among the nuclear components the so-called Sm and RNP antigens associated with systemic lupus erythematosus. The use of immunoaffinity chromatography with dual specificity for the Sm and RNP antigens has allowed for their substantial purification from rabbit thymus in parallel and in quantity. In lieu of a functional assay, the use of a counterimmunoelectrophoresis assay provided a sensitive and rapid means ofmonitoring the distribution ofthe two antigens during purification and ensured the isolation of complexes containing the components required for antigenicity. The resulting purified complex consisted of nine polypeptides, having molecular weights of approximately 9000 to 44,000 and two small RNAs of similar size. However, .limited proteolysis of the isolated complex suggested that most of these polypeptides were not actually required forantigenic activity.,Unlike Sm. in crude thymus extracts, purified Sm was RNase sensitive. Thus, one ofthe major diagnostic criteria used to distinguish Sm and RNP antigens in crude extracts was shown to be invalid for purified material, suggesting that both antigens from rabbit thymus are actually ribonucleoprotein complexes.Autoimmune diseases are characterized by the presence of circulating antibodies that react against components ofthe affected individual's own tissues (1-3). Isolation of the antigens associated with specific autoimmune diseases would facilitate the diagnosis of these diseases and, perhaps, would help in understanding the etiology of these diseases. Among the highly conserved cell components that can be targets of the autoimmune reaction are the so-called Sm and RNP (ribonucleoprotein) antigens. The presence ofantibodies to Sm is diagnostic of the disease systemic lupus erythematosus; anti-RNP antibodies are found in patients suffering from lupus and, more specifically, mixed connective-tissue disease (4, 5). Sm and RNP are both located in the nucleus, giving similar speckled immunofluorescent staining patterns (5). They have been reported to have similar molecular weights (6, 7), to copurify during ion-exchange chromatography (7,8), and to show partial antigenic identity (6,9). In practice, they have been distinguished by differential sensitivity to RNase (4, 10).There have been several reports in recent years on attempts to purify Sm and RNP (8,(11)(12)(13)(14). Most recently Lerner and associates (9, 15), using the technique of immunoprecipitation, identified both Sm and RNP as ribonucleoprotein complexes. We report here a large-scale purification ofthe Sm and RNP antigens in quantities sufficient for physical and chemical characterization. Moreover, the isolation protocol allowed for continuous monitoring of antigenicity, thus ensuring that we were working with antigenically active material. The isolated complexes contained both protein and RNA moieties; and each moiety was composed of a subset of polypeptides and oligoribonucleotides. By using t...

show abstract

Section: Methodsmentioning

confidence: 99%

Identification of the immunogenically active components of the Sm and RNP antigens.

White¹,

Gardner²,

Hoch³

1981

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…RNA was extracted from CHO cells using the method of Chirgwin et al (17) and electrophoresed in 1% agarose gels containing 10 mM methylmercuric hydroxide as described (18). RNA was visualized by staining the gel with 10 ug/ml ethidium bromide in 0.5 M ammonium acetate and then capillary-blotted onto Hybond-N membranes (Amersham).…”

Section: Rna Blot Analysismentioning

confidence: 99%

Characterization of cDNA sequences corresponding to three distinct HMG-1 mRNA species in line CHO Chinese hamster cells and cell cycle expression of the HMG-1 gene

Lee¹,

Pentecost²,

D'Anna

et al. 1987

Nucl Acids Res

View full text Add to dashboard Cite

We have isolated cDNA clones encoding the high mobility group (HMG) protein HMG-1 in line CHO Chinese hamster cells. The cDNA clones correspond to the three HMG-1 mRNA species detected on Northern blots. Three different polyadenylation sites are found to be used. The three mRNA species of sizes 1.05, 1.45 and 2.45 kb are generated by differential polyadenylation at sites 115 nucleotides, 513 nucleotides and 1515 nucleotides downstream from the stop codon. A perfectly conserved putative poly(A) signal AAUAAA is present upstream of only one of the three poly(A) sites. Two homologous but imperfect sequences exist upstream from the other two poly(A) sites. All three HMG-1 mRNA species maintain significant levels throughout the M, G1 and S phases of the cell cycle and the rate of large HMG protein (HMG-1 and HMG-2) synthesis increases approximately two-fold from G1 to S phase.

show abstract

“…Northern analyses RNA samples were isolated from anaerobically induced seedlings (1). 40 pg of total RNA or 3 ug poly(A ) RNA were run in each lane of a methyl mercury hydroxide agarose (1.5%) gel (9), transferred to DPT paper (10), hybridized to pZML793 probe and washed (8).…”

Section: Gel Electrophoresismentioning

confidence: 99%

Molecular analysis of the alcohol dehydrogenase 2 (Adh2) gene of maize

Dennis¹,

Sachs

Gerlach³

et al. 1985

Nucl Acids Res

314

View full text Add to dashboard Cite

The Adh2 gene of maize has a nucleotide sequence closely related to that of the maize Adh1 gene indicating that the two genes arose from a progenitor gene by a duplication event. The coding regions are 82% conserved at the nucleotide level and 87% conserved at the amino acid level. Each gene has nine introns in identical positions but their nucleotide sequences and lengths differ. Adh2 encodes a single mRNA and has a possible polyadenylation signal, AATAAT, fifteen bases upstream from the polyadenylation site. The Adh2 gene together with the Adh1 gene is induced by anaerobic conditions and under these conditions produces an increased level of mRNA. The 5' untranslated regions of the transcripts of Adh1 (100 bp) and Adh2 (126 bp) have diverged in sequence except for a conserved region which may be important for their anaerobic-specific translation. The 3' and 5' flanking sequences of the two genes are also divergent except for 11 bp of precise homology around the TATA boxes and three other 8 bp sequences further upstream. One of the common 8 bp sequences (CACCTCCC) is in a similar position (-150 bp to -200 bp) in the two genes and may have a regulatory function in gene expression.

show abstract

Gel electrophoretic fractionation of RNAs by partial denaturation with methylmercuric hydroxide

Cited by 40 publications

References 15 publications

Identification of the immunogenically active components of the Sm and RNP antigens.

Identification of the immunogenically active components of the Sm and RNP antigens.

Characterization of cDNA sequences corresponding to three distinct HMG-1 mRNA species in line CHO Chinese hamster cells and cell cycle expression of the HMG-1 gene

Molecular analysis of the alcohol dehydrogenase 2 (Adh2) gene of maize

Contact Info

Product

Resources

About