Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs.

Parnes, Jane R.; Velan, Baruch; Felsenfeld, Adam; Ramanathan, Lata; Ferrini, Umberto; Appella, Ettore; Seidman, Jonathan G.

doi:10.1073/pnas.78.4.2253

Cited by 291 publications

(130 citation statements)

References 34 publications

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“…Binding of the plasmid DNA, prepared by the method of Holmes and Quigley [41] and purified by cesium chloride centrifugation [39] to a nitrocellulose filter was performed as described by Parnes et al [42].…”

Section: In Vitro Transcription/ Transpor T Assaymentioning

confidence: 99%

Energy requirement and kineties of transport of poly(A)‐free histone mRNA compared to poly(A)‐rich mRNA from isolated L‐cell nuclei

Schröder

Friese

Bachmann

et al. 1989

European Journal of Biochemistry

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ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)-rich mRNAs (actin and P-tubulin) from isolated L-cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)-free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone-H4-mRNA efflux was in the same range as that found for ATP-induced release of RNA from demembranated nuclei (about 15-20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)-rich RNA, but not the poly(A)-free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNAbinding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear-pore-complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)-rich mRNA.

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Section: In Vitro Transcription/ Transpor T Assaymentioning

confidence: 99%

Energy requirement and kineties of transport of poly(A)‐free histone mRNA compared to poly(A)‐rich mRNA from isolated L‐cell nuclei

Schröder

Friese

Bachmann

et al. 1989

European Journal of Biochemistry

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“…Transformants were probed with 32p-labelled cDNA prepared either by reverse transcription of poly(A) ÷ RNA extracted from actinomycin Dtreated infected cells or by reverse transcription of viral genomic RNA using calf thymus random primers (Taylor et al, 1976). Recombinant cDNA clones were identified by in vitro translation of hybrid-selected mRNA using a rabbit reticulocyte lysate system (Parnes et al, 1981). Putative RS virus clones were confirmed by radioimmunoprecipitation of the in vitro translated proteins using specific RS virus monoclonal antibodies (Ward et al, 1983).…”

mentioning

confidence: 99%

Nucleotide Sequence of the Respiratory Syncytial Virus Phosphoprotein Gene

Lambden

1985

Journal of General Virology

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SUMMARYA cDNA library was prepared from poly(A) + RNA extracted from respiratory syncytial (RS) virus-infected HEp-2 cells. A recombinant plasmid, pRSP68, encoding the RS virus phosphoprotein gene was identified by translation in vitro of hybridselected mRNA. The cloned cDNA insert of 1 kb was sequenced and a polypeptide of 241 amino acids with a molecular weight of 27166 was deduced from the sequence. The protein was relatively rich in polar amino acids and devoid of both cysteine and tryptophan. A second short open reading frame with a coding potential of 65 amino acids was identified and overlapped the 3' terminus of the phosphoprotein gene by 34 bases.Respiratory syncytial (RS) virus is the major pathogen causing severe lower respiratory tract infection in infants (Parrott et al., 1973). The virus is a pleomorphic, unsegmented, negativestranded RNA virus similar to the paramyxoviruses but placed in a separate genus, Pneumovirus, on the basis of its morphology and the lack of detectable neuraminidase and haemagglutinin activities. More recently, study of the genetic organization of RS virus has highlighted the differences between it and the typical paramyxoviruses . The RS viral genome is a single negative strand of RNA with a molecular weight of around 5 × 106, and is transcribed into 10 discrete polyadenylated mRNAs coding for 10 unique viral proteins . The gene order has been determined by transcriptional mapping using u.v. inactivation kinetics (Dickens et al., 1984). These studies provided evidence of a single promoter site for RS virus transcription and the gene order:

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“…These data suggest that the DR antigen heavy chain (the constant chain of the complex) may be encoded only once in the genome. The gene for (32-microglobulin, also a constant chain, is also thought to be a single-copy gene (23). The cDNAs made from mRNA and pDRH-2 do not appear to be sufficiently related to other DR heavy chains (32)(33)(34) to cross-hybridize with them.…”

Section: Resultsmentioning

confidence: 99%

cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.

Korman¹,

Knudsen²,

Kaufman³

et al. 1982

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

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A monoclonal antibody (HC 2. 1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papaintreated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning ofcDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.

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Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs.

Cited by 291 publications

References 34 publications

Energy requirement and kineties of transport of poly(A)‐free histone mRNA compared to poly(A)‐rich mRNA from isolated L‐cell nuclei

Energy requirement and kineties of transport of poly(A)‐free histone mRNA compared to poly(A)‐rich mRNA from isolated L‐cell nuclei

Nucleotide Sequence of the Respiratory Syncytial Virus Phosphoprotein Gene

cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.

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