Sequence studies on the constant region of the Fd sections of rabbit immunoglobulin G of different allotype.

The amino acid sequence has been completed for the constant region of the Fd fragments of heavy chains from rabbit IgG (immunoglobulin G) of allotype Aa1 and Aa3. The amino acid sequence given by Fruchter et al. [(1970) Biochem. J. 116, 249-259] for the constant region of the Fd fragment from Aa1 IgG was extended and in in part corrected to give a continuous sequence of 140 residues. No allotype-related sequence variation was found in the constant section of the Fd fragment. This evidence confirms the view that the differences in sequence between the variable regions of Aa1 and Aa3 IgG [Mole et al., (1971) Biochem. J. 124, 301-318] are responsible for the allotypic specificities.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Preparation and characterization of chemically defined oligomers of rabbit immunoglobulin G molecules for the complement binding studies.

Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at -70 degrees C.     

HLA-DQ molecules form alpha-beta heterodimers of mixed allotype

Retroviral vectors with an internal cytomegalovirus major immediate-early gene enhancer/promoter regulating HLA class II gene expression were used to transfer HLA cDNA into human EBV-transformed B-lymphoblastoid cell lines. HLA-DQ2 beta and DQ3.2 beta cDNA were transferred into DQ3.2 and DQ2 homozygous lymphoblastoid cell lines, respectively. Serologic analysis of the infected cell lines with allospecific mAb demonstrated surface expression of these exogenous DQ molecules implying that DQ alpha-chains from DR3, DQ2-positive cells can pair with DQ3.2 beta-chains and, similarly, DQ alpha-chains from DR4, DQ3.2-positive cells can pair with DQ2 beta-chains. Immunoprecipitation of the introduced DQ3.2 beta molecule resulted in co-purification of the allotype-mismatched endogenous DQ2 alpha polypeptide. We also show that vectors with a cytomegalovirus major immediate-early gene enhancer/promoter result in higher levels of expression of the transduced gene compared to previously described HLA vectors with either the SV-40 early enhancer/promoter or the lymphotropic papovavirus-enhanced SV-40 promoter. Although deletion of HLA cDNA did sometimes occur in the process of generating virus-producing clones, the HLA cDNA is stably maintained in virus-producing clones, once it is generated. This retroviral expression system is a highly efficient way to study HLA gene function.     

Genetic variation at the immunoglobulin allotype loci in Creoles of Trinidad

The sera of a sample of 204 Creoles from Trinidad were tested for the presence of polymorphic gene complexes occurring on immunoglobulin light- and heavy-chain molecules including the allotypic markers IGKC 1, IGHA2 1 and 2, IGHG1 A, X, F, and Z, and IGHG3 G, G5, B0, B1, B3, B4, B5, C3, C5, S, and T. Nine IGHG (GM) haplotypes occur in polymorphic frequencies (greater than .01) in this population, including known African, Asian, Caucasian, and Amerindian marker haplotypes. Significant differences (P less than .01) were found in the frequency distributions of three IGHG (GM) haplotypes and the frequency of IGKC*1 in these data and data from Creole populations of Belize and St. Vincent. The Creoles of Trinidad and St. Vincent are more similar in IGHG (GM) haplotype distributions than are Trinidad and Belize populations. Previous testing has revealed no significant differences between St. Vincent and Belize Creoles at the Ig allotypic loci. Analysis of migration patterns in the Caribbean suggests that different rates of Asian migration have maintained regional diversity at these loci, while continuous gene flow from the eastern Caribbean to Trinidad has had a relative homogenizing effect on the gene pools of this area.     

Sequence studies on the heavy chain of rabbit immunoglobulin A of different alpha-locus allotypes.

The amino acid sequence was determined of part of the variable region of heavy chain from rabbit immunoglobulin A of allotypes a1 and a3. Two corrections of the primary sequence of Aa1 gamma-chains are reported; most of the structural correlates of the alpha-locus allotypes are confirmed. The amino acid sequence of the N-terminal 20 residues of alpha-negative molecules was also determined and found to be homologous to the human VhIII subgroup. These molecules are present in a much higher proportion in the alpha-chain pool than in the gamma-chain.     

Characterization and DNA sequence of the b6w2 allotype of the rabbit immunoglobulin kappa 1 light chain (b locus)

The b6w2 allotype of the constant region of the rabbit immunoglobulin kappa 1 (k1) light chain (b locus) was discovered in wild populations from northern Spain. At the serological level, the b6w2 allotype is characterized by the presentation of all b6-specific epitopes, while an allotypic determinant which is shared between the nominal b5 and b6 allotypes is lacking. The DNA fragment encoding the b6w2 allotype was amplified by means of the polymerase chain reaction, and sequenced directly by dideoxy-DNA-sequencing. When compared with the sequence of the nominal b6 allele, the b6w2 sequence differs at eleven nucleotide positions (96.5% similarity). This variation corresponds to amino acid replacements at 1) the three positions C-terminal to the peptidyl junction with the variable region (amino acid positions 109–111);2) the four positions N-terminal to the interdomain disulfide bond (167–170); and 3) two positions in the vicinity of the interchain disulfide bond (190 and 210). The nature and distribution of the observed nucleotide substitutions strongly suggest a possible role of the extra interdomain disulfide bond in the unusual evolutionary dynamics of the rabbit K1 light chain.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z48308     

Identification of rabbit genomic Ig-VH pseudogenes that could serve as donor sequences for latent allotype expression

Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.     

Sequence studies of the Fd section of the heavy chain of rabbit immunoglobulin G

A partial amino acid sequence was given by Cebra, Steiner & Porter (1968b) of the N-terminal half of the heavy chain of rabbit immunoglobulin G. This was extended and in part corrected to give a continuous sequence of 136 residues, which together with other work accounts for three-quarters of the total sequence. Evidence is given suggesting that there is a limited region of 10–15 residues that are exceptionally variable in the heavy chains from pooled rabbit immunoglobulin G.     

Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine s chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1^b (IgG_2a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1^a, Igh-1^d, Igh-1^e and Igh-1^j allotypes. The genetic studies of (BALB/c x C57BL/6)F₁ and backcross F₂ animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.Abbreviations used in this paper PCA passive cutaneous anaphylaxis - RID radioimmunodiffusion - i.p. intraperitoneally - EA egg albumin - Igh-C immunoglobulin heavy chain constant region locus - DNP 2,4-dinitrophenyl - PBS phosphate-buffered saline - NMS normal mouse serum - KLH keyhole limpet hemocyanin Visiting investigator supported by the Scientific and Humanistic Development Council from the Central University of Venezuela, currently at the following address: Consejo de Desarrollo Cientifico y Universidad Central de Venezuela, Av. Principal Urb. La Floresta Ota., Silenia Caracas, Venezuela.     

Tests of association of immunoglobulin allotype genes and viral oncogenesis in chickens

Chickens from Regional Poultry Research Laboratory (RPRL) inbred line 6₃ are resistant to virally-induced Marek's disease (MD) and lymphoid leukosis (LL) and are relatively strong regressors of virally-induced Rous sarcomas. In contrast, RPRL line 100 chickens are highly susceptible to MD and LL and are weaker regressors of Rous sarcomas than line 6₃. RPRL lines 100 and 6₃ differ for alleles at the IgG-1 (G-1) allotype locus, but have identical IgM-1 (M-1) allotype alleles. To test the possible association of the G-1 locus with variations in resistance to virally-induced tumors, homozygous and heterozygous genotypes among F₃ crosses were infected. F₃ chickens with different G-1 types were comparable in their resistance to MD tumors following inoculation with the JM strain of the MD virus, and for their ability to regress Rous sarcoma tumors induced by the Rous sarcoma virus (RSV) RAV-1. However, following RAV-1 virus infection a smaller proportion of G-1 ^a /G-1 ^a F₃ or F₄ birds developed LL tumors than G-1 ^a /G-1 ^e and G-1 ^e /G-1 ^e birds. Genes determining immunoglobulin heavy chains were therefore associated with a recessive resistance to B-cell lymphomagenesis in chickens.Deceased     

Idiotype-specific helper cells (BH) express immunoglobulin markers and employ T cell function-associated molecules

An Lyt-1+ population, distinct from T cell subsets, that helps expression of B cell responses to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten was characterized. This lymphoid population, called BH, is present in the spleens of normal and athymic mice and preferentially helps the expression of plaque-forming B cells that carry NPb idiotypic determinants. To define the mechanism by which this cell population functions, the roles of T and B lymphocyte function associated antigens were studied. The data indicate that BH cells express immunoglobulin receptor components, i.e., IgM, IgD heavy chain, and lambda light chain immunoglobulin markers as well as the J11d marker associated with immature B cells. BH cells may also express determinants identical to or cross-reactive with the T cell-associated antigens L3T4a, L3T4b, and LFA-1 as defined by treatment with monoclonal antibodies specific for these antigens. In addition, L3T4a- and LFA-1- but not Lyt-1-like antigens appear to be functionally involved in BH-dependent helper activity, since augmentation of NPb idiotypic PFC responses was blocked with anti-L3T4a or anti-LFA-1 monoclonal antibodies. Further analysis of BH-containing populations indicates that T cells are probably not involved in BH cell function and therefore are not responsible for the presence of Lyt-1, L3T4a, or L3T4b determinants in this T-independent system. The relationship of this helper cell subset to conventional T and B cell populations is discussed.     

Evolutionary studies on pancreatic colipase

In this evolutionary study the following criteria have been used to prove the existence of colipase: 1. It restores the activity of human and porcine pancreatic lipase inhibited by bile salt. 2. It cross-reacts with antisera to human and porcine colipases. 3. Its restoration of lipase activity, inhibited by bile salt, in the tributyrin assay system, is prevented by antiserum to colipase. 4. It is a heat-stable, low-molecular-weight protein (molecular weight about 10 000 by gel-filtration). The occurrence of colipase has been verified in the exocrine pancreatic cells from hagfish (Myxine glutinosa), ratfish (Chimaera monstrosa), rayfish (Raja radiata), Greenland shark (Somnius microcephalus) and dogfish (Squalus acanthius). No colipase activity could be found in the gastric juice of crayfish (Pacifastacus leniusculus). These results indicate that colipase envolved in the vertebrates before the organized exocrine pancreatic gland and occurred simultaneously with the bile salts/bile alcohols.