Synthesis of part of a mouse immunoglobulin light chain in a bacterial clone

Amster, O.; Salomon, Daniela; Zemel, O.; Zamir, Ada; Zeelon, Elisha; Kantor, Frida; Schechter, Israël

doi:10.1093/nar/8.9.2055

Cited by 6 publications

(5 citation statements)

References 28 publications

(11 reference statements)

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“…Secondary structure analysis, as described (13), revealed no hairpin loop sequestering the SD or initiation codon into double-stranded regions of the mRNA of either pCT54 19-1 or the S1 derivatives. Such base pairing interactions have been shown drastically to reduce translational efficiency of a number of genes, notably those for phageX cro (17), fibroblast Following electrophoresis, gels were stained with Coomassie blue (lanes [1][2][3][4][5][6][7] or subjected to analysis by Western blotting (lanes 8-10) using rabbit anti-X serum and iodinated protein A (24Ci/ml). Lane 1, purified MOPC104E myeloma protein standard indicating the position of authentic X protein; lane 2, E103S containing pNP3 after growth to stationary phase in L-broth; lanes 3-5, samples from E103S containing pNP3 taken at increasing absorbance during induction; lanes 6 and 7, respectively, soluble and insoluble fractions from pNP3 containing E103S; lane 8, unfractionated extract; lanes 9 and 10, respectively, soluble and insoluble fractions.…”

Section: Construction Of Plasmids For Expression Of X Light Chainmentioning

confidence: 99%

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Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised inE. coli

et al. 1984

Nucl Acids Res

154

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Genes for a murine mu heavy chain and a lambda light chain immunoglobulin have been inserted into bacterial expression plasmids containing the Escherichia coli trp promoter and ribosome binding site. Induction of transcription from the trp promoter results in accumulation of both light and heavy chain polypeptides in appropriate host strains. Both proteins were found as insoluble products. Following extraction and purification of the immunoglobulin containing fractions, antigen binding activity was recovered. The activity demonstrates essentially the same properties as the antibody from the hybridoma from which the genes were cloned.

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Section: Construction Of Plasmids For Expression Of X Light Chainmentioning

confidence: 99%

“…Thus, it is surprising that with the many studies on expression of eukaryotic genes in E. coli (1), little has been done on immunoglobulin genes. So far immunoglobulin genes have been expressed in modified forms at low levels in E. coli, usually as incomplete amino-terminal fusion proteins (2,3,4).…”

Section: Introductionmentioning

confidence: 99%

Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised inE. coli

et al. 1984

Nucl Acids Res

154

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“…The enhanced transposition is due to the particular combination of the cloned DNA and its position in the cloning vector. Recombinant plasmids containing longer stretches or the entire length of L-321 cDNA cloned by dc:dG "tailing" into the Pst I site of pBR322 did not acquire IS insertions (17,38), Likewise, scoring of a large number of individual transformant clones with pBR313 did not reveal any loss of the TcR marker as would result from IS insertions into the Tc region. It thus appears that the junctions of VBRI with the flanking plasmid sequences (pBR313 or pBR322), or another type of coupling between the cloned DNA and the cloning vector sequences are effective in the enhancement of transposition frequency.…”

Section: Discussionmentioning

confidence: 93%

“…Plasmid construction. Double stranded cDNA for L-321 was synthesized from purified mRNA (16) as described (17). Equimolar amounts of L-321 double stranded cDNA and pBR313 (18) were digested with Bam HI, ligated with T4-DNA ligase, and used to transform E. coli HB101.…”

Section: Methodsmentioning

confidence: 99%

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids

1982

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Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.

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“…Periplasmic proteins were extracted from lysozyme treated bacteria as described by Amster et a1. 14 ) Labeling of C2-casein by 125-iodine was performed by the Chloramine-T method according to the maker's instructions (Amersham). The radioimmunoassay was performed essentially as described by Enami and Nandi 15 ) except that the immunocomplex was pelleted by centrifugation at 2,500 rpm for 10 min, washed with 2.5 ml of 10 mM sodium phosphate, 0.14M NaCI, pH 7.4, and counted with a y-counter (Nuclear Chicago 1185 series).…”

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confidence: 99%