CH Region Research Articles

Structural Evidence for Recombination at the Igh (H Chain) Complex Locus in BAB 14 Mice

Abstract Anti-PC antibodies from the recombinant mouse BAB 14 were evaluated by analytical isoelectric focusing in an attempt to gain information on the crucial question of whether the recombinational event in this strain occurred in V region structural genes or in allotype-linked regulatory genes. Immune sera from BAB 14, C57BL/6 and its congenic partner CB20 (both Ighb), BALB/c and its congenic partner BC8 (both Igha) were isofocused. Antibodies bearing two distinct idiotypes, T15 Id and M511 Id, were detected by an in situ localization procedure with radiolabeled anti-T15 and anti-M511 idiotypic antibodies. The IEF spectrotypic pattern of IgG antibodies of each strain revealed separate populations of T15 Id-positive (IEF-T15) and M511 Id-positive (IEF-M511) antibodies. The IEF-T15 and IEF-M511 spectrotypes in BAB 14 most closely resembled those of C57BL/6 due to the dominant charge effect of the CH region. However, the entire set of IEF-T15 bands and a portion of the IEF-M511 bands in BAB 14 show a slight but clearly evident anodal shift in its pI relative to C57BL/6. This finding of a shift, regardless of IgG subclass, demonstrates that the difference between BAB 14 and C57BL/6 resides in the VH region and not the CH region. Thus, there is direct evidence from structural data that H chains of BAB 14 are composed of a CH segment (allotype marker) commonly expressed by C57BL/Ka (Ighb) mice and VH segment commonly expressed by BALB/c (Igha) mice. These findings are discussed in terms of recombination in structural versus regulatory genes.

Complete inhibition of the expression of an idiotype by a mechanism of B-cell dominance.

Mice of the C.AL-20 strain, which express genes controlling CH regions of the AL/N strain on a BALB/c background, normally synthesize antibodies to the p-azophenylarsonate group (anti-Ar antibodies) with an idiotype characteristic of the A strain. The synthesis of the idiotype, as quantitated by a sensitive assay, can be completely inhibited by the transfer of leukocytes from BALB/c mice producing anti-Ar antibodies, which lack the idiotype. A number of control experiments show that the inhibition is not attributable to suppressor T cells and that the synergistic action of such cells is not required. The results indicate that B-cell dominance, mediated by secondary cells, can completely prevent the expression of unprimed cells with receptors of the same specificity. It is uncertain whether this effect is due entirely to selective capture of antigen by the secondary cells, or whether some type of active suppression by B cells is involved.

Allotype Suppression at the CHn Locus of IgM in Heterozygous Rabbits with Concomitant Suppression at the VHa Locus of All Ig Classes

Abstract Doubly heterozygous offspring, ♂n83a1/♀n82a2 or ♂n81a1/♀n82a2, of doubly homozygous dams, n82a2/n82a2, were injected neonatally with anti-n83 or anti-n81 antibody directed to the paternal n locus allotype. In each injected rabbit, the paternal n83 or n81 allotypes were suppressed and increased amounts of the maternal n82 allotype accounted for almost all of the serum IgM. This is the first report of allotype suppression in the rabbit mediated by antibody directed toward the CH region of any Ig class. A concomitant decrease in the paternal a1 Ig allotype and a compensatory increase in the maternal a2 allotype were also found. This finding supports the concept that IgM-bearing cells are the precursors of IgG-secreting cells.

Identification and Genetic Control of the n83 and n84 Allotypes of Rabbit IgM

Two additional allotypes of rabbit IgM, n83 and n84, have been identified and characterized with antisera obtained by cross immunization of rabbits with IgM. These allotypic specificities were not detected on IgG or IgA by double diffusion in agar gel and by quantitative radioprecipitin analyses. This finding implies that the specificities reside in the CH region of the mu-chain. Most of the IgM (88%) from an n83 homozygote reacted with anti-n83 Ab and most of the IgM (79%) from the net n84 homozygote reacted with anti-n84 Ab. The IgG or IgA from n83 or n84 rabbits did not precipitate to a significant extent (less than 6%) with the anti-n83 or anti-n84 Ab, respectively. That the n83 and n84 specificities are controlled at the n locus was verified by genetic analysis. Thus, four alleles are now known at the n locus, n81, n82, n83 and n84. The n locus is closely linked to the other six defined loci in the heavy chain chromosomal region and the allelic alternatives for each of the seven loci are coinherited by gene combinations called allogroups. Ten such allogroups are now defined with respect to the allele at the n locus.

]Kinetics and mechanisms of intermolecular ligand exchange : II. Diphenyltin- and dimethyltinacetylacetonate with acetylacetonate

The kinetics and mechanisms of intermolecular ligand exchange of acetylacetonate (acac) groups between “bulk” free acetylacetone (enol form) and R 2Sn-(acac) 2 (R  CH 3 or Ph) complexes have been re-investigated in chloroform- d (R  CH3) and chlorobenzene (R Ph) solutions by proton magnetic resonance spectroscopy using the total lineshape analysis in the temperature range: 12.7–37.0°C (R  CH 3;acac CHregion),−1.4 to 19.6°C (R  CH 3; acac CH 3 region), and 25.9–48.0°C (R Ph; acac CHregion). The activation energies and entropies of activation are: for R  CH 3, 7.2 ± 0.7 kcal/mol and −30 ± 3 eu (acac CH 3), and 6.3 ± 0.6 kcal/mol and −32 ± 2 eu (acac CH); for R  Ph 8.8 ± 2.7 kcal/mol and −31 ± 9 eu (acac CH). Concentration dependence studies for both the (CH 3) 2Sn(acac) 2Hacac and Ph 2Sn(acac) 2Hacac systems indicate that the rate of acetylacetonate exchange is first-order in the concentration of R 2Sn(acac) 2 and zero order in the concentration of “bulk” free ligand. The intermolecular ligand exchange phenomenon is believed to involve coordination number lowering; viz. a Sn-O bond rupture as the rate-determining step to yield a five-coordinate intermediate species with a dangling unidentate acetylacetonate ligand.

Synchronous Regulation of VH and CH Allotypes among Ig Molecules in Multi-Heterozygous Rabbits Suppressed with Anti-VH Allotype Antibody: Emergence of IgA from Suppression Prior to IgG and IgM

Heterozygous rabbits of genotype a1n81f73g74/a2n82f71g75 were suppressed at birth for the VH region a1 allotype. At 8 weeks of age, quantitative analysis of serum IgG, IgM, and IgA molecules showed that the VHa1 specificity was effectively suppressed in the three classes of Ig and that the suppression was extended to the CH region n81 specificity on mu-chains as well as to the CH region f73 and g74 specificities on alphaf and alphag chains. At 26 weeks of age, analysis of serum IgG and IgM molecules showed that a1 was still suppressed to approximately the same extent in both Ig classes and the suppression was still extended to the CH region n81 specificity. However, at 26 weeks, the percentage of molecules with a1 specificity had doubled among serum and colostral IgA molecules and this increase was extended to the CH region f73 and g74 specificities. Thus, the suppressed allotypes reappeared first among IgA molecules. Our data are consistent with a regulatory mechanism which controls and synchronizes the expression of the VHa and the CH allotypes expressed on the same heavy chain. The order of the re-expression of the suppressed allotypes with respect to Ig class may allow further definition of selective regulatory mechanisms for the synthesis of Ig classes.

Antibody differentiation: apparent sequence identity between variable regions shared by IgA and IgG immunoglobulins.

We have analyzed a pair of human myeloma immunoglobulins (biclonal proteins) of the IgG and IgA classes from a single patient, GR. The light chains are identical in amino-acid sequence over 40 residues at their NH2-terminus, hwereas the heavy chains are identical throughout 45 residues of their NH2-terminus. Additional chemical and serological studies suggest the light chains and variable regions of the heavy chains (VH) are very similar, if not identical. The implications of these and of other published studies are discussed with regard to (i) the association of one VH region with multiple constant regions of the heavy chain (CH regions), (ii) two alternative types of V-C joining mechanisms, (iii) the differentiation of antibody-producing cells, and (iv) three categories of biclonal immunoglobulins.

Molecular energy levels of azines; ab initio calculations and correlation with photoelectron spectroscopy

Non-empirical calculations using a standard gaussian atomic orbital basis (LCGO) have been carried out for known and unknown azines. The wavefunctions show pseudo D6h symmetry and have been correlated with those of benzene; this correlation has been used to interpret the combinations of ‘lone pair’ orbitals that occur. In the case of the tetrazines the splitting of the lone pair levels leads to extensive overlap with the CH region of energy so that mixing occurs and 1,2,4,5-tetrazine shows five orbitals which can be described as ‘lone pair orbitals’. The orbital energy ordering is effectively identical with that obtained from recent double ζ calculations on a few of the molecules; these energies correlate well with photoelectron spectra and obey the relationship IPobs= 0·790IPcalc. Correlations using CNDO/2, INDO, and the Extended Huckel Method (EHM) are less satisfactory both in grouping of the orbitals and absolute energies. The LCGO band assignments are generally in good agreement with those made by Lindholm et al. based upon the EHM. The thermodynamic stability of the azines with respect to fragmentation is discussed; the difference in energies between the classical alternating bond lengths and the observed ones for selected examples are in the region 6–10 kcal mol–1.

Correlations between amino acid sequence and conformation of immunoglobulin light chains. II. Sequence comparison and the pattern of nonpolar residues.

A detailed comparison of the primary structure of 15 individual immunoglobulin light chains including 6 human k chains, 2 mouse k chains, and 7 human. chains is given. Their chemical similarities and the possible conformational influence of the invariant residues are discussed. Some preliminary results on heavy chains are also considered. The most striking structural feature common to all L chains examined is the pattern of distribution of nonpolar, but not of polar or ionized residues along the sequences. Both variable (VL) and constant (CL) regions of L chains contain approximately 55 nonpolar residues. Out of these, 30 nonpolar residues are found in all VL, regions at structurally identical sequence sites and 33 in all CL regions. This frequency of consistently nonpolar residues is similar to that in globin chains and is probably due to thermodynamical and stereochemical requirements. The globular folding of L chains is essentially stabilized by hydrophobic interactions and most of the consistently nonpolar sequence positions are likely to represent intrachain hydrophobic contact sites. These, however, require well‐defined distances between the interacting groups. There is no significant similarity between the pattern of nonpolar residues of the VL, and Ch regions. However, the rule of isopolar substitution suggests a similar folding of all VL, (and possibly Vrd regions, on the one hand, and a similar folding of all CL (and possibly CH) regions, on the other. The low a‐helical content of L chains is explained, and some conclusions concerning future structural studies are drawn.