Sherie L. Morrison

Immunochemical characterization of binding sites of hybridoma antibodies specific for α(1 → 6) linked dextran☆☆☆★

The combining sites of seven BALB/c IgM, four BALB/c IgA and one C57BL/6 IgA hybridoma antibodies specific for α(1 → 6) linked dextran were probed by precipitin and precipitin inhibition assays. The 12 antibodies are able to bind to linear determinants in the interior of the dextran molecule; some have sites complementary to six α(1 → 6) linked glucose residues and others have sites complementary to seven α(1 → 6) linked glucose residues. From the analysis of the precipitins and precipitin inhibitions, it is concluded that no two hybridoma proteins have identical binding sites.

Association constants of hybridoma antibodies specific for α(1 → 6) linked dextran determined by affinity electrophoresis☆☆☆

Binding constants of monomers of seven BALB/c IgM, four BALB/c IgA, and one C57BL/6 IgA anti-α (1 → 6) dextran hybridoma antibodies with dextran B512 and with isomaltoheptaose were determined by affinity electrophoresis. Binding constants to dextran range from 1.52 × 105 to 4.43 × 105 ml/g for the five IgA monomers and from 1.70 × 103 to 6.10 × 104 ml/g for the seven IgM monomers. Antibody monomers containing both specific and nonspecific (derived from the myeloma cell that was used to generate the hybridomas) light chains are shown to have association constants with dextran 6 to 30-fold lower than monomers containing only specific light chain, suggesting that the association of specific heavy chain with nonspecific light chain does not result in an anti-dextran combining site. Binding constants with isomaltoheptaose range from 1.45 × 104 to 7.01 × 104/M for the IgA proteins and from 6.46 × 103 to 7.70 × 104/M for the IgM proteins. The binding constants with dextran and with isomaltoheptaose, and the electrophoretic, immunochemical and idiotypic characteristics of the hybridoma proteins are discussed.

Polypeptide products of nonsense mutations: I. Termination fragments from nonsense mutations in the Z gene of the lac operon of Escherichia coli☆

Abstract A sensitive, specific method of detecting the N-terminal region of the β-galactosidase molecule is described. This method is used to measure the quantities and molecular weights of nonsense termination fragments produced by many Z − nonsense mutants. The quantity of the fragment varies over a hundredfold range but is always less than the wild-type enzyme. There is no simple correlation between either map position or polarity and amount of fragment present. The molecular weights of the fragments increase continuously with map distance from the operator end of the Z gene.

Studies on mouse hybridomas secreting IgM or IgA antibodies to α(1→6)-linked dextran☆

Abstract Twelve mouse hybridomas secreting antibodies to dextran B512, identified by replica immunoadsorption screening of 100,000 immobilized hybridoma clones, were obtained. Among 11 hybridomas of BALB/c origin seven produce IgM and four produce IgA. One hybridoma of C57BL/6 origin synthesizes IgA. A κ light chain is synthesized by each of the 12 hybridomas in addition to the nonspecific κ light chain of the parent myeloma. The heavy chain is shown to associate preferentially with the specific (spleen cell derived) light chain. All hybridoma antibodies were purified from ascites by precipitation with dextran B512, followed by subsequent digestion of the dextran with dextranase. Although all the specific light chains migrate identically in SDS gels under reducing conditions, variations in migration are noticed among the heavy chains. Differences in migration among the IgA monomers and among the IgA polymers are seen on nondenaturing polyacrylamide gels. Densitometer scans of such gels show that more than 50% of the IgA hybridoma antibodies are in polymeric form. Implications of the preferential association of heavy chain with specific light chain, and of the size differences among the heavy chains for the generation of antibody specificity and diversity are discussed.

Genetically Engineered Antibody Molecules and Their Application

Immunoglobulin genes can be efficiently expressed following transfection into myeloma cells. Using protoplast fusion, transfection frequencies greater than 10(-3) can be achieved. Compatible plasmids containing two different selectible markers are used to simultaneously deliver heavy and light chain genes to the same cell. To produce molecules with differing specificities the rearranged and expressed variable regions can be cloned from the appropriate hybridoma. In some cases, variable regions from cDNAs can be inserted into the expression vectors. It is possible to manipulate the immunoglobulin genes and produce novel antibody molecules. Antibodies have been produced in which the variable regions from mouse antibodies have been joined to human constant regions. In addition, antibodies with altered constant regions have been produced. These genetically engineered antibodies provide a unique set of reagents to study structure-function relationships within the molecule. They also can potentially be used in the diagnosis and therapy of human disease.

Formation of Hybridoma Clones in Soft Agarose: Effect of pH and of Medium

Optimal conditions for the formation of hybridoma clones in soft agarose are described. The hybridization frequency is shown to be highly dependent on the pH of the polyethylene glycol (PEG) solution used for fusion and on the cloning medium. Maximal numbers of clones are obtained when the PEG solution used for fusion is at pH 8.0–8.2.

Polypeptide products of nonsense mutations: II. Minor fragments produced by nonsense mutations in the z gene of the lactose operon of Escherichia coli

Abstract Untreated extracts of z -gene nonsense mutants of Escherichia coli were found to function as α-donors in a complementation reaction with XA21. In mutants which are operator-proximal to a certain point (mutant AP2177), the α-donating material was the nonsense fragment. In the region bounded by and including mutants AP2177 and NG521, extracts of mutants contained two species of polypeptides. One was the principle auto-α donor and corresponded to the nonsense fragment. A second fragment, α U1 , with a molecular weight of 46,000, functioned as the principle α-complementing material in untreated extracts. α U1 is a minor component (~10%) of the total z -gene protein. In extracts of nonsense mutants which are operator-distal to NG521, untreated extracts showed several peaks of α-complementing activity. One of these peaks was α U1 . In the two most operator-distal nonsense mutants studied, two peaks of auto-α donating activity were found. The map position of the end of α U1 coincides, as nearly as can be determined, with π(β).

ICR-191 and ethyl methanesulfonate induced mutagenesis at the immunoglobulin locus in the Y5606 cultured myeloma cell line.

The Y5606 mouse tumor synthesizing an IgG3, lambda immunoglobulin (Ig) was adapted to continuous growth in tissue culture. The spontaneous mutation rate at the Ig locus (approximately 3 X 10(-5)/cell/generation) in this cell line was found to be less than that in other cultured mouse myeloma lines. Treatment with either ICR-191 or ethyl methanesulfonate (EMS) increased the mutation rate approx. 100-fold. Spontaneous and ICR-191 induced mutants were synthetic variants that is they synthesized either heavy (H) or light (L) chains alone instead of the H and L chains synthesized by the parent. Following EMS treatment assembly variants which were synthesizing structurally altered H chains were isolated in addition to synthetic variants. The assembly variants appear to be a unique consequence of EMS mutagenesis.

An immunoglobulin heavy chain gene deletion at direct repeats: Nucleotide sequence and effect on mrna accumulation☆

The DNA from the mouse myeloma cell, I17, which produces aberrant gamma 2b heavy chain mRNAs, was cloned and sequenced. The I17 mutant, and its parent line 10.1, share a small deletion at the splice junction of the CH1 domain which results in the absence of CH1 sequences from the mRNA. In addition, the genomic DNA of I17 has a deletion of 253 nucleotides which fuses the CH2 and CH3 exons, causes a frameshift of the next 43 amino acids and results in a truncated protein. The deleted nucleotides are flanked by two direct repeats of the CAGCA pentamer in the normal gene. One copy of the repeat and the interposed DNA is removed in the mutant. The DNA deletion is colinear with the mRNA. Both I17 and 10.1 cells have decreased accumulation of the secretory-specific gamma 2b mRNA. The amounts of membrane-specific gamma 2b mRNA are also affected in the mutants.

The IgA myeloma W3129 contains a deletion in CH3 which prevents the formation of the membrane form of heavy chain mRNA

We report here a 54 base pair deletion in the CH3 exon of the alpha gene in the mouse myeloma W3129. This deletion results in a loss of 18 amino acids and a change from a glycine to a serine at position 464. The extent of the deletion was determined by sequencing a portion of CH3 cloned from a variant of W3129, and S1 nuclease protection showed the deletion pre-exists in the parental cell line. The deletion removes the donor splice site normally used in joining CH3 to the alpha membrane (MB) exon when forming MB-specific mRNA. Examination of cytoplasmic RNA by blot hybridization and S1 nuclease protection using MB-specific probes showed a complete lack of membrane mRNA in W3129 and its derivatives. An RNA transcript of unknown origin and function which includes sequences from the CH3-MB intron was seen in W3129 and in J558, an IgA, lambda myeloma with specificity for alpha (1----3) linked dextrans. We discuss the possible influence of the mutation on the W3129 protein. In contrast to the other myelomas studied in this laboratory, light chain loss variants are readily isolated from W3129 and are stable in their production of heavy chain [Dackowski and Morrison (1981) Proc. natn. Acad. Sci. U.S.A. 78, 7091-7095].