Murine myeloma immunoglobulin heavy-chain mRNA. Isolation, partial purification, and characterization of .gamma.1, .gamma.2a, .gamma.2b, .gamma.3, .mu., and .alpha. heavy-chain mRNAs

Faust, Charles; Heim, Isabel; Moore, J M

doi:10.1021/bi00573a027

Cited by 14 publications

(4 citation statements)

References 54 publications

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“…After an 18-hour incubation at 4° C with renin-specific antiserum (10 /JL\ antiserum per 25 /il translation mixture), immune complexes were isolated by adsorption with S. aureus (IgG Sorb, New England Enzyme Center, Boston, Massachusetts). 17 Nonspecific adsorption of radiolabeled translation products to the heat-killed S. aureus was eliminated by incubation of the bacteria with a 100,000 g supernatant of female gland homogenate (1 ml supernatant per 100 /x\ packed volume cells). 6 Pretreated 5. aureus (1 fi\ packed cells per fi\ antisera) were incubated with the antiserum/ translation product mixture for 15 minutes at 4° C with gentle agitation, and immune complexes were deposited by centrifugation for 10 seconds in a Brinkman Microfuge.…”

Section: Mrna-directed Cell-free Translationmentioning

confidence: 99%

Influence of androgen on translatable renin mRNA in the mouse submandibular gland.

Pratt¹,

Dzau²,

Ouellette³

1984

Hypertension

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SUMMARY In mature outbred Swiss male mice, submandibular gland renin enzyme activity is 4-and 10-fold higher than in glands of prepubescent males and mature females, respectively. Levels of translatable renin mRNA have been studied in mouse submandibular gland during postnatal development and following administration of testosterone. The [ 35 S]methionine-labeled cell-free translation products directed by male glandular mRNA contain a 47 ± 2 kd renin precursor that is not detected in products coded by prepubescent male or female gland mRNA. This cell-free synthesized precursor is detected immunochemically only in the translation products of gland mRNA from males of 33 days or older and from females receiving testosterone administration, a pattern consistent with the measurements of renin enzyme activity. This increase in biologically active renin mRNA is a selective one, since unfractionated male and female mRNAs have similar overall nucleotide sequence complexity corresponding to 1% of mouse single copy DNA. The cDNA transcribed from male gland mRNA reacts 5-and 10-fold faster with the template mRNA than with female or prepubescent male gland mRNA, respectively, which indicates that the male gland contains abundant nucleotide sequences that exist at low concentration in the female or prepubescent male. Selective hybrid arrested translation confirms that the levels of renin mRNA are lower in the glands of prepubescent males than in those of the mature males. These data indicate that the regulation of renin enzymatic activity by androgens is mediated by an increase in the levels of translatable renin mRNA both during postnatal development and after testosterone administration. (Hypertension 6: 605-613, 1984) KEY WORDS • postnatal development hybrid arrested translation cDNA hybridization • saturation hybridization T HE mouse submandibular gland (SMG) contains an enzyme that resembles renal renin (EC3.4.99.19) physically, chemically, and immunologically.' Studies employing DNA recombinants complementary to SMG renin mRNA have demonstrated that extensive structural homology exists between SMG and renal renins 2 and that at least two genetically distinct forms of renin exist in mouse SMG.3 The high levels of SMG renin and of its mRNA 2 " 6 relative to those of the kidney and the sexually dimorphic expression of SMG renin and several

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Section: Mrna-directed Cell-free Translationmentioning

confidence: 99%

Influence of androgen on translatable renin mRNA in the mouse submandibular gland.

Pratt¹,

Dzau²,

Ouellette³

1984

Hypertension

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“…The rat myeloma, IR162 (obtained from Dr. H. Metzger, NIH), and murine myelomas were propagated by serial subcutaneous transplantation (Mach et ai, 1975 tumors were excised and polysomes were prepared, RNA was extracted from the polysomes, and mRNA was purified on poly(dT)-cellulose columns (Faust et al, 1979).…”

Section: Propagation Of Myeloma Tumorsmentioning

confidence: 99%

A Cloned cDNA Probe for Rat Immunoglobulin Epsilon Heavy Chain: Construction, Identification, and DNA Sequence

Kindsvogel¹,

Reddy²,

Moore³

et al. 1982

DNA

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IgE has clinical importance because it is responsible for immediate hypersensitivity. Studies of IgE expression in rats appear to contradict current models for immunoglobulin gene expression. To study rat IgE expression at the RNA and DNA levels, we have constructed a cDNA for part of the rat epsilon (epsilon) heavy chain that is expressed by a rat myeloma, IR162. The rat epsilon-chain clone was initially identified by an efficient selection scheme. DNA sequencing of the 580-bp cDNA revealed that it encoded 176 amino acids that were 50% homologous to the human epsilon H chain. The sequence begins near the end of the CH2 domain and ends 31 amino acids into the CH4 domain. Cysteines important for the structure of the human IgE were conserved in the rat epsilon H-chain. The identity of the cloned epsilon cDNA was confirmed by comparison with a portion of the constant region gene for mouse epsilon H chain. The mouse and rat nucleotide sequences were 79% homologous.

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“…To the aqueous phase from the extraction, 3 vol of ethanol were added and transported back to the laboratory on dry ice. Isolated crude RNA was further purified, as described (17,18); poly(A)+ RNA was precipitated and stored at -700C.…”

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confidence: 99%

Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.

Casadei¹,

Powell²,

Kenten³

1990

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

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A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli. The gene was expressed as apoaequorin and, by using luciferin isolated from RenilUa reniformnis, its activity was found essentially identical to native aequorin. The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion ofapoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody. The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene. Transfection of the chimeric gene into a cell line expressing the complementary Al light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten. The Ca2+-dependent bioluminescent activity of aequorin was also recovered.

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Murine myeloma immunoglobulin heavy-chain mRNA. Isolation, partial purification, and characterization of .gamma.1, .gamma.2a, .gamma.2b, .gamma.3, .mu., and .alpha. heavy-chain mRNAs

Cited by 14 publications

References 54 publications

Influence of androgen on translatable renin mRNA in the mouse submandibular gland.

Influence of androgen on translatable renin mRNA in the mouse submandibular gland.

A Cloned cDNA Probe for Rat Immunoglobulin Epsilon Heavy Chain: Construction, Identification, and DNA Sequence

Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.

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