Immunoglobulin Genes in a Mouse Myeloma and in Mutant Clones

Milstein, C.; Adetugbo, Kayode; Brownlee, George G.; Cowan, Nicholas J.; Proudfoot, Nick J.; Rabbitts, T H; Secher, David S.

doi:10.1016/b978-0-12-651050-8.50011-9

Cited by 11 publications

(13 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Firstly, the shift in mobility of the mRNA affects both the V and Cregions at the same time. Secondly, the deletion, which starts at or very near the V-C integration point [9] does not affect the ability of the isolated mRNA to be translated in a cell-free system. …”

Section: Discussionmentioning

confidence: 99%

“…The large size of this mRNA molecule, and the likelihood of contamination of preparations with 18-S ribosomal RNA, prompted us to study a mutant cell line (IF2 [5]) which synthesises an H-chain of considerably reduced molecular weight. The reduced molecular weight is the result of a deletion of the CH3 homology region of the heavy chain, resembling the deletions observed in human H-chain disease protein [9]. Here we report the purification, characterisation and partial sequence analysis of the IF2 H-chain mRNA.…”

mentioning

confidence: 99%

“…Table 2. Oligonucleotides between obligatory G residues greater than 20 bases apart Nucleotide sequences are derived theoretically from the IF2 H-chain amino acid sequcnce [9] using the genetic code. Possible derivatives following digestion with pancreatic ribonuclease are shown below each theoretical sequence.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Purification and Sequence Analysis of the mRNA Coding for an Immunoglobulin Heavy Chain

Cowan

Secher

Milstein

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

A mutant cell line (IF2) derived from the mouse myeloma MOPC 21 has been used for the isolation and sequence analysis of H-chain mRNA. The IF2 cells synthesise an H-chain of reduced size in which the CH1 homology region is missing. Sizing of the IF2 H-chain mRNA and wild-type H-chain mRNA revealed that the deletion is expressed at the mRNA level. The mutant H-chain mRNA sedimented at 16-S, enabling effective resolution from 18-S ribosomal RNA. In experiments using IF2 cells labelled with [32P]phosphate, the 16-S mRNA was purified by oligo(T)-cellulose chromatography. Polyacrylamide gel analysis of the poly(A)-containing fraction showed the presence of a single radioactive band. Comparison of the mobility of this band relative to markers of known molecular weight revealed that the molecule contained about 1600 nucleotides. Digestion of the 32P-labelled mRNA with T, ribonuclease and two-dimensional fractionation of the resulting oligonucleotides yielded a 'fingerprint' suitable for a preliminary sequence analysis. By using the established amino acid sequence of the IF2 H-chain and a knowledge of the genetic code, 14 oligonucleotides were assigned within the constant region and four within the variable region of the IF2 H-chain. This sequence data accounts for 19.5 "/, of the coding region. Several other oligonucleotides, which could not be assigned within the coding region but which occurred in approximately molar yield, have also been partially characterised. These oligonucleotides are presumably derived from the untranslated regions of the mRNA.Although purification procedures have now been developed for a number of eucaryotic mRNAs, little is known of the structural properties of these molecules. A detailed knowledge of mRNA structure is required to define its coding properties and to establish the nature of the untranslated regions, which may be important in the control of transcription and translation and in the transport of ribonucleoproteins from nucleus to cytoplasm, The structure of immunoglobulin mRNA is of particular interest because of its relevance to basic immunological problems. Each heavy (H) and light (L) chain of the immunoglobulin molecule consists of a region of variable (V) and constant (C) amino acid sequence. The V-region confers the antibody specificity, whereas the C-region is invariant within each type and class of chain. E,vidence based on the segregation of genetic markers suggests that the H and L-chains are encoded by separate pools of V and C-genes, so that each polypeptide is the result of expression of one gene from each pool [ 11. Hybridisation experiments, performed to define the number of genes in cellular DNA, depend on a knowledge of the structure and purity of the

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Purification and Sequence Analysis of the mRNA Coding for an Immunoglobulin Heavy Chain

Cowan

Secher

Milstein

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The cysteine and proline residues at positions 231 and 232 is the COOH terminus of the hinge region and is highly conserved among mouse y1 (43) electron microscopy have the structure indicating the presence of a short intervening sequence at the middle of the large Rloop, suggesting that our conditions for formation of R-loops were not optimal for detecting a short intervening sequence (about 100 bases). The smaller whisker at the outer end of the smaller R-loop probably corresponds to the V sequence of MPC 11 mRNA, which is not encoded by XgtWES-IgH22.…”

mentioning

confidence: 99%

“…Among them the most common is the deletion of a whole domain from the C sequence, which comprises a tandem array of three or four domains (49)(50)(51)(52)(53). A mouse -yl chain mutant (IF2) was also reported to lack the whole CHI domain (43). Another set of variants seems to arise by recombination between different C genes at the junction of domains (54,55).…”

mentioning

confidence: 99%

Cloning immunoglobulin gamma 2b chain gene of mouse: characterization and partial sequence determination.

Kataoka¹,

Yamawaki-Kataoka²,

Yamagishi³

et al. 1979

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

View full text Add to dashboard Cite

DNA from newborn mice was digested with restriction endonuclease EcoRI, and a 6.6-kilobase fragment encoding immunoglobulin 'y2b chain mRNA derived from MPC 11 myeloma was enriched about 100-fold by RPC-5 column chromatography and agarose gel electrophoresis. The 6.6-kilobase fragment was Immunoglobulin heavy chain genes consist of a family of variable region (V) genes and several constant region (C) genes. V and C genes are genetically linked and expressed as a cis combination (1-3). In a given lymphocyte, immunoglobulin genes on one allele are exclusively expressed (4-6). Recently, we have shown that specific C genes are deleted from one allele in mouse myelomas depending on which C genes are expressed (7,8). We have proposed an allelic deletion model as the mechanism of the V-C gene recombination that results in the expression of the specific immunoglobulin genes (7,8 two or more neighboring genes. For this purpose it is essential to clone two putative adjacent y chain genes. In this paper we will describe cloning of the -y2b chain C gene, which is proposed to be adjacent to the y1 chain gene, and its structure. EXPERIMENTAL PROCEDURES Partial Purification of DNA Fragment. High molecular weight DNA was extracted from whole newborn mice (killed within 24 hr after birth). DNA was digested with EcoRI, extracted with phenol, and precipitated with ethanol. Forty milligrams of EcoRI-digested DNA was chromatographed on an RPC-5 column (0.8 X 90 cm) as described (10,12). Fractions that hybridized with the y2b chain cDNA by in situ hybridization were pooled and concentrated by ethanol precipitation. The DNA obtained was applied to a 1% agarose gel (7 X 15 X 4 cm) and fractionated as described (13). Fractions were assayed by in situ hybridization with nick-translated y2b chain [32P]cDNA cloned in a plasmid (pG2b-4).Preparation and Screening of Recombinant Phage. Bacteriophage XgtWES-XB (9) was used as the EK2 vector and propagated in ED8656 (14). Cloning experiments were carried out in a P2 facility (15, 16). Bacterial strains NS428 [N205 (X Aamll b2 red 3 cI857 Sam7)I (17) and Xdg8O5 [W3350 (Xdgal8O5 cl857 Sam7)] were used for in vitro packaging.Partially purified 6.6-kilobase (kb) mouse DNA (2 jig) was ligated with 5.1 ,ug of XgtWES outer fragments in a 50-,ul reaction mixture with 0.6 unit of T4 ligase as described (18). The recombinant DNA was packed into coat proteins in vitro and the recombinant phages were plated by the method of Blattner et al. (19). Twenty sets of about 2000 plaques were directly transferred from LB broth agar plates to Millipore filters as described (20). The Millipore filters were hybridized to 32p-labeled Hha I fragment of pG2b-4 and autoradiographed. Recombinant phage DNA was prepared as described (18).In Situ Hybridization. DNA, fixed on a Millipore filter (21), was hybridized to an appropriate probe as described by Jeffrey and Flavell (22) except that 1 M NaCl/50 mM Tris-HCI, pH 7.4/10 mM EDTA replaced 0.9 M NaCl/0.09 M sodium citrate.The probes used were [32P]cDNA (specific ac...

show abstract

Monoclonal IgM cryoglobulinemia associated with gamma‐3 heavy chain disease: immunochemical and biochemical studies

et al. 1978

View full text Add to dashboard Cite

A patient (Mia) with a monoclonal IgM(kappa) cryoglobulin (cryo IgM) developed additional heavy chain disease proteins of the gamma3 subclass 8 years later. Biochemical studies of the cryo IgM indicated that the heavy chain was VHI, but the NH2-terminal amino acid sequence of the light chain did not permit a definite assignment of its Vkappa subgroup. Two major fragments of the gamma3 chain were distinguishable by electrophoresis in sodium dodecyl sulfate polyacrylamide gel. The smaller component (designated Mia F) had a molecular weight of approximately 30 000 and the larger component (designated Mia S) 35 000. Both fragments had G3m(21) and G3m(27) allotypic determinants. These data and the NH2-terminal amino acid sequence of the gamma chain fragments suggested that Mia S consists of the major part of the gamma3 hinge region plus the CH2 and CH3 domains of the gamma3 chain, whereas Mia F may be derived from the former as a result of postsynthetic cleavage. The partial amino acid sequence of the Mia S fragment is homologous to the hinge region amino acid sequence of human gamma3 chains reported in the literature, with only one amino acid difference out of the 11 residues compared. This difference may represent an allotypic difference within the gamma3 subclass. Alternatively, the production of Mia S may have resulted from the accidental derepression of a "silent" constant region gene not expressed in normal individuals.

show abstract

Immunoglobulin Genes in a Mouse Myeloma and in Mutant Clones

Cited by 11 publications

References 22 publications

Purification and Sequence Analysis of the mRNA Coding for an Immunoglobulin Heavy Chain

Purification and Sequence Analysis of the mRNA Coding for an Immunoglobulin Heavy Chain

Cloning immunoglobulin gamma 2b chain gene of mouse: characterization and partial sequence determination.

Monoclonal IgM cryoglobulinemia associated with gamma‐3 heavy chain disease: immunochemical and biochemical studies

Contact Info

Product

Resources

About