Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzylozymethyl cellulose or nitrocellulose sheets

Bittner, Michael; Kupferer, Patricia; Morris, Charles F.

doi:10.1016/0003-2697(80)90182-7

Cited by 428 publications

(107 citation statements)

References 21 publications

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“…The DNA was then purified by hydroxylapatite column chromatography (22), which resulted in DNA that was significantly free from contaminating RNA and polysaccharides. A genomic library of EcoRI fragments was prepared by using the X Charon 3A vector (23) Fragments that were complementary to the chloroplastid 5S rRNA were characterized and identified for DNA sequence analysis by electroblothybridization techniques (24,25). The complementary DNA was prepared by digesting the hybrid plasmid with EcoRI restriction endonuclease and purifying the inserted DNA on a 0.8% agarose slab gel (24).…”

Section: Methodsmentioning

confidence: 99%

Structure and evolution of the 4.5–5S ribosomal RNA intergenic region fromGlycine max(Soya bean)

1987

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The nucleotide sequence for the 4.5-5S ribosomal DNA region from the chloroplastids of soya beans was determined as the basis of further comparative studies on the structure and evolution of this intergenic region. Comparisons with other plant sequences as well as equivalent sequences in eubacteria suggest that the longer internal transcribed spacer regions of plants have evolved, at least in part, by DNA sequence duplications and that the presence of the 4.5S rRNA in chloroplast may result from the accidental acquisition of a RNA maturation site during the evolution of longer internal transcribed spacer regions. Estimates of the secondary structures also indicate only a very limited retention of structural features and suggest that the primary role of the intergenic sequences may be to bring processed sites into close proximity. INTRODUCrIONWhile the ribosomal RNAs of chloroplasts more closely resemble those of eubacteria, the chloroplast ribosomes of flowering plants contain two low molecular weight RNA components (1-4), the commonly observed 5S RNA and a somewhat smaller 4.5S rRNA. Sequence studies indicate that all of the chloroplast rRNAs are transcribed as a common larger precursor molecule, the sequences being arranged in the order 5'-16S-23S-4.5S-5S-3' (5-7). As ribosomal RNA from many origins have been sequenced, comparisons have clearly shown (5-8) that the 4.5S rRNA sequence is not unique to plant chloroplasts; it is actually equivalent to the 3' end of the eubacterial 23S rRNA and is the result of an extra cleavage or processing step in the chlorplast rRNA precursor.Although most mature ribosomal RNAs are products from the processing of larger precursor molecules, in many instances relatively little is known about the structural features which underlie these cleavages or the enzymatic mechanisms by which these cleavages occur (e.g. ref.

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Section: Methodsmentioning

confidence: 99%

Structure and evolution of the 4.5–5S ribosomal RNA intergenic region fromGlycine max(Soya bean)

1987

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“…Earlier studies on Xenopus oocytes by Prives (1988, 1989) and Prives and Foukal (1991) showed that injection of single-stranded oligodeoxynucleotides (oligos) complementary to parts of Ul or U2 caused truncation (Ul) (Bittner et al, 1980) and was hybridized with 32P-labeled antisense probes against various snRNAs. Because of the large amount of snRNA in a GV, autoradiographic exposures of a few hours were adequate.…”

Section: Introductionmentioning

confidence: 99%

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

Tsvetkov

Jantsch

et al. 1992

MBoC

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The five small nuclear RNAs (snRNAs) involved in splicing occur on the loops of amphibian lampbrush chromosomes and in hundreds to thousands of extrachromosomal granules called B snurposomes. To assess the role of these snRNAs during transcription and to explore possible relationships between the loops and B snurposomes, we injected singlestranded antisense oligodeoxynucleotides (oligos) against Ul and U2 snRNA into toad and newt oocytes. As shown before, antisense Ul and U2 oligos caused truncation of Ul and complete destruction of U2 snRNAs, respectively. However, injection of any oligo, regardless of sequence, brought on dramatic cytological changes, including shortening of the chromosomes and retraction of the lateral loops, with concomitant shutdown of polymerase II transcription, as well as disappearance of some or all of the B snurposomes. When injected oocytes were incubated for 12 h or longer in physiological saline, these changes were reversible; that is, the chromosomes lengthened, transcription (detected by 3H-UTP incorporation) resumed on newly extended lateral loops, and B snurposomes reappeared. In situ hybridization showed that loops and B snurposomes had negligible amounts of U2 snRNA after recovery from injection of the anti-U2 oligo, whereas these structures had normal levels of U2 snRNA after recovery from a control oligo. Thus, the morphological integrity of B snurposomes and lampbrush chromosome loops is not dependent on the presence of U2 snRNA. Because transcription occurs in the absence of U2 snRNA, we conclude that splicing is not required for transcription on lampbrush chromosome loops.

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“…Hybridizations to Gene Screen Plus required an increase in sodium dodecyl sulfate (SDS) to 1%, as recommended by the manufacturer. We found, independently of other investigators (26), that it was necessary to reduce the temperature of hybridization and stringency washing to visualize fragments of a size <150 bp on blots from acrylamide gels. Hybridization was performed at 55°C in 5x SSPE, and blots were washed at 55°C in 5x SSPE (1 x SSPE = 0.18M NaCI, 10 mM NaP04, pH 7.7, 1 mM EDTA).…”

Section: Methodsmentioning

confidence: 71%

“…Electroblotting (26,27) was performed in low-salt buffer from 5% acrylamide gels to Gene Screen Plus, according to directions supplied with Zeta-Probe blotting membrane (28) and the Bio-Rad Trans-Blot apparatus (Bio-Rad, Rockville Center, NY).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Rabbit Genes for Synovial Cell Collagenase

Fini

Gross

Brinckerhoff

1986

Arthritis & Rheumatism

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To provide tools for understanding collagenase gene expression in rheumatoid arthritis, we have isolated and characterized genomic clones for rabbit synovial cell collagenase. These clones represent 2 types of collagenase gene, at least 1 of which is transcribed in synovial fibroblasts. By examining the rabbit genome in situ, we provide evidence that there are only 2 different synovial cell collagenase genes found in a haploid genome. Amplification of these genes is not a mechanism far collagenase messenger RNA induction by phorbol esters.The metalloproteinase, collagenase, plays a primary role in the joint destruction seen in rheumatoid arthritis (1). Vast excesses of this enzyme are secreted by the fibroblasts of the rheumatoid pannus. The fact that collagen breakdown can be initiated only by collagenase makes this enzyme rate-limiting in collagenolysis. One approach to the successful control of collagenolysis in diseases such as rheumatoid arthritis may lie in inhibiting the production of collagenase. Thus, an understanding of mechanisms regulating Studies in our laboratory, designed to understand the control of collagenase secretion, have been facilitated by the development of a cell culture system of synovial fibroblasts from rabbits (2). These cultures secrete negligible levels of collagenase when untreated. However, they can be stimulated to secrete major amounts of collagenase by treatment with numerous agents, including the tumor promoter, phorbol myristate acetate (PMA) (3), and crystals of monosodium urate monohydrate (4). Furthermore, additional agents such as retinoic acid or dexamethasone can reverse or suppress induction (5). To date, experiments with human synovial cells have corroborated our findings with the rabbit cell studies, and have thus validated the rabbit system as a model (6,7).Earlier studies demonstrated that inducers act to increase the synthesis of new collagenase protein rather than stimulate the release of previously synthesized enzyme stored intracellularly (8). With the development of a complementary DNA (cDNA) clone for collagenase, we showed that on induction, an increase in immunoreactive collagenase secreted into the culture medium is directly correlated with increased collagenase messenger RNA (mRNA) levels (9). Several steps in RNA metabolism could contribute to the increase in collagenase message levels, including increased transcription from the collagenase gene(s), greater mRNA stability, or changes in processing of pre-mRNA to mRNA. To study the contribution of these processes to mRNA induction, as well as the mechanisms by which they operate, requires more than a cDNA clone, which is merely a copy of mRNA. The collagenase gene itself must be isolated

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Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzylozymethyl cellulose or nitrocellulose sheets

Cited by 428 publications

References 21 publications

Structure and evolution of the 4.5–5S ribosomal RNA intergenic region fromGlycine max(Soya bean)

Structure and evolution of the 4.5–5S ribosomal RNA intergenic region fromGlycine max(Soya bean)

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

Characterization of Rabbit Genes for Synovial Cell Collagenase

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