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A V Taranin

Publications and source records attributed to A V Taranin.

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Expression of immunoglobulin kappa and lambda chains in mink.

The ratio of kappa and lambda chains of immunoglobulins varies significantly from one species to another. It has previously been thought that lambda was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent lambda and kappa types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK-1 containing kappa chain-encoding sequence. The cDNA insert of pIGK-1 included most of the V segment, as well as the J, C and 3' untranslated sequences. Mink V kappa sequence shown the highest homology with the human V kappa II subgroup genes (76-79%). Mink C kappa sequence was 53-63% homologous to C kappa of other species. The striking feature of mink C kappa chain is the presence of glutamine in the C-terminal position. Southern blot analysis suggested that mink haploid genome has one C kappa gene and multiple V kappa genes. The kappa:lambda chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the kappa- and lambda-producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the lambda chains, thereby confirming that lambda chains in this species are additionally subdivided into several subtypes.

Amino Acid Sequence

cDNA clones encoding mink immunoglobulin lambda chains.

Screening of a mink cDNA library with an antibody probe resulted in the isolation of clone pIGL-2 containing an Ig lambda chain coding sequence. The sequence comprised almost the entire V segment as well as J, C, and 3'-untranslated sequences. A second clone, pIGL-10, was isolated by rescreening the cDNA library with the use of pIGL-2 as a probe. pIGL-10 was found to contain a frameshift deletion of a single nucleotide in the C region. pIGL-2 and pIGL-10 were 81% homologous to each other in the FR3 of the V segment, and 95% of homology was found in their C regions. The J segments of the two clones differed in only one nucleotide position. Comparison of cloned lambda chain sequences with those of other mammals revealed that mink V lambda and C lambda genes have the highest homology with their human counterparts. The V lambda sequence of clone pIGL-2 appears to be a homologue of human subgroup III V lambda genes. Southern blot hybridization of mink DNA with the C lambda and V lambda probes derived from pIGL-2 revealed five or six hybridizing C lambda fragments and at least 11 hybridizing V lambda fragments. This suggested that the lambda genes in carnivores, like those in primates, have duplicated extensively during evolution.

Amino Acid Sequence

Monoclonal antibodies against heavy and light chains of domestic mink IgG.

A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).

Animals

Genetic polymorphism of IgG in the mink. IX. High proportion of allotype-producing lymphocytes in individuals with minor level of allotypes H3 and H4 in serum.

The levels of mink C gamma-allotypes (H3, H4, H6 and H8) were determined in sera, and the proportion of the corresponding allotype-synthesizing B cells was estimated in peripheral blood, spleen and mesenteric lymph nodes. Individual differences in H6 levels and, possibly, those in H8 were entirely dependent on the proliferation degree of the corresponding clone of B cells and also determined by the dosage of the structural gene. There was no correspondence between the great numbers of H3+, H4+ cells and low levels of H3 and H4 allotypes in the sera of the majority of minks with their minor expression. A possible cause of this discrepancy may be a blockade of the secretion of IgG by H3+, H4+ cells. There exists most likely a gene (or genes) controlling the blockade of IgG secretion. The regulation of C gamma-allotype expression is presumably effected in a manner specific to each of the allotypes.

Animals

Genetic polymorphism of IgG in the mink. VIII. A quantitative study of the expression of C gamma-allotypes (H3, H4, H6, H8) in sera.

The results of a quantitative study of the expression of mink C gamma-allotypes (H3, H4, H6, and H8) in sera are presented. H6 and H8 were found to be stably expressed, and the individual concentrations of the allotypes varied within one order of magnitude. Gene dosage effects were observed for H6 and H8: average sera allotype concentrations in homozygotes were twice those in heterozygotes. In contrast, the serum concentrations of H3 and H4 varied by three orders of magnitude, ranging from minor (2-200 micrograms/ml) to high (1-10 mg/ml). No gene dosage effects were observed for the expression of H3 and H4. Histograms for the population of H3 concentrations showed three peaks, sharply differing from those of H4, H6, and H8. There was no association between the minor expression of H3 and H4. The data obtained indicate that the expression of mink C gamma-allotypes is regulated by different allotype-specific mechanisms.

Animals

A tissue-specific puff (Balbiani ring a) in Chironomus thummi may contain a gene encoding a 67-kDa protein which exhibits non-tissue-specific expression.

A 2.3-kb genomic clone has been isolated from the region where the tissue-specific puff, Balbiani ring a (BRa), is found on chromosome IV of the special lobe of Chironomus thummi salivary gland cells. The clone was characterized by nucleotide sequence analysis. Two clusters of direct tandem repeats were identified, as well as large and small open reading frames (ORFs). The large ORF was fused to an Escherichia coli lacZ gene. Antibodies against the beta-galactosidase/ORF fusion protein reacted selectively on Western blots with a 67-kDa protein. Western-blot analysis and immunoelectron microscopy showed that this protein was distributed in the cells of all larval tissues examined. We concluded that BRa, a tissue-specific puff, whose activity correlates with the synthesis of 160-kDa secretory protein [Kolesnikov et al., Chromosoma 83 (1981) 661-677], may also contain a gene which is not expressed in a tissue-specific manner.

Amino Acid Sequence

[Cloning and sequencing of immunoglobin lambda-chain cDNA in american mink (Mustela vison].

cDNA library in the lambda gt11 phage was constructed using poly (A+)-mRNA from mink spleen as a template. Immunoscreening of the library allowed the identification of 2 lambda-related clones containing 370 and 803 bp insertion (lambda IGL-1 and lambda IGL-2). Analysis of the primary structure of lambda IGL-2 demonstrated that it contains a large portion of V lambda-segment, J lambda-segment, C lambda-gene and its 3'-untranslated part. The nucleotide sequences known for the immunoglobulin genes were compared to the sequence of the lambda IGL-2 clone. The highest degree of homology was established for the rabbit lambda-genes, this being 63, 94 and 72% for the RF3 region of V lambda-segment, J lambda-segment and C lambda-region, respectively.

Animals

Genetic polymorphism of IgG in the mink. V. Two new genetic markers of the lambda light chains of mink immunoglobulins, L4 and L5.

Two new allotypes of the light (L) chains IgG, L4 and L5, were identified in the mink with dispecific antiserum produced by immunization with allogenic IgG. By means of hybrid IgG molecules and proteolytic fragments, L4 and L5 were localized on the C region of the L chain. L4 and L5 occurred frequently in the three mink populations studied and L4 and L5 are inherited independently of the known mink C gamma allotypes. L4 and L5 are encoded by closely linked genes. The antigenic specificities of L4 and L5 were not identified in the closely related Mustelidae and in the other mammalian representatives. Consequently, L4 and L5 are species specific to mink. Determination of the phenotype combinations of the five allotypes on the L chains (including the new L4 and L5) demonstrated the existence of seven combinations only with a predominance of L1,2,3; L4,5, and L1,2,3,4,5 phenotypes. Based on the results obtained, it is concluded that the mink C lambda locus has a complex organization. A model for the mink C lambda locus with at least three or possibly five linked genes is suggested.

Animals

[Analysis of expression of the gene from the BRa puff of Chironomus thummi encoding the low molecular weight secretory protein].

The plasmid containing F6.2 gene from the BRa of Chironomus thummi within the pUR 292 vector was constructed. Chimeric protein containing beta-galactosidase-F6.2 polypeptide was produced in Escherichia coli BMH71-18. The protein obtained has immunological similarity with secretion protein of 67,000 D from Ch. thummi salivary gland. The gene for sp67 is active in main and in special lobe of the gland. Based on the data obtained and on the previous results of the authors, conclusion is made that BRa is a complex locus containing several types of genes.

Animals

Mink-mouse hybridomas that secrete mink immunoglobulin G.

Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.

Animals

Genetic polymorphism of IgG in the mink. III. Instability of expression and the problem of the genetic control of C gamma-allotypes.

Quantitative expression of C gamma-allotype H4 of mink immunoglobulins was studied by enzyme-linked immunosorbent assay. The results presented suggest that production of H4 is under specific regulation. the concentration of H4 varies three orders of magnitude (10-10,000 micrograms/ml) from one mink to another. Fifteen percent of the sera of normal minks have the low H4 concentration, undetectable by the standard procedure of double immunodiffusion routinely used to test mink IgG allotypes. However, expression of these 'minor' allotypes may be significantly enhanced by hyperimmunization. Instability of this kind seems to be the main cause of earlier described deviations from Mendelian inheritance of C gamma-allotypes H2, H3 and H4.

Animals

Genetic polymorphism of IgG in the mink. IV. Identification and genetic control of L3 allotype of the light chains.

A new allotype of the mink light chains, designated L3, was identified. This allotype is inherited as a Mendelian character at a frequency of 0.46 in the mink population. Data were obtained indicating that L3 is independent of the C gamma-heavy chain allotypes of mink immunoglobulins. The gene L3 is closely linked to a gene encoding L1, another light chain allotype. Alloantigens L1 and L3 are presumably markers of the light chains of two different subtypes. In contrast to L1, which occurs in many mammalian species, L3 is species-specific, i.e., it is a case of light chain polymorphism representative of the whole mink species.

Animals

[Immunoglobulin (IgG) allotypes of the American mink with Aleutian disease].

Mink Aleutian disease (AD) is characterized by intensive proliferation of B-lymphocytes and hypergammaglobulinemia. Populational distribution of five genetic immunoglobulin markers (light chain allotype L1 and C gamma-allotypes H2, H3, H6 and H8) in minks of different coat color (Sapphire, Standard and Topaz) was studied. The groups of infected minks differed significantly from healthy ones in the distribution of the H3 allotype: the frequencies of some phenotypes--H3, H6, H8 and L1, H3, H6, H8 (Sapphire, Standard). H2, H3, H6, H8 and L1, H2, H3, H6, H8 (Sapphire) were increased significantly. At the same time, the frequencies of H6, H8; L1, H6, H8 and H2, H6, H8; L1, H2, H6, H8 were decreased in the AD population. The preferential stimulation of proliferation of the H3 + B-lymphocyte clones is suggested.

Aleutian Mink Disease

Genetic polymorphism of IgG in mink. I. Identification of 8 allotypes.

By means of intraspecific immunization of domestic mink (Mustela vison Schr.), 8, in all probability, complex IgG allotypes were detected in their sera. Based on the results of analysis of the preparations of the IgG heavy (H) and light (L) chains, as well as proteolytic IgG fragments, we assigned the allotypes detected to three groups: (1) marker of the L chain, L1; (2) allotypes of the C region of gamma-chains (H2, H3, H4, H6, and H8) and conformational allotype H7; (3) conformational allotype 5 with unknown location on the chains.

Animals

Genetic polymorphism of IgG in mink. II. A genetic analysis of allotypes.

Population distribution and inheritance pattern were analyzed in mink IgG allotypes: L1 (L chains), H2, H3, H4, H6, H7, and H8 (the constant region of the H chains, i.e. C gamma-allotypes) and conformational allotype 5 with unknown chain localization. Contrary to expectation, neither allelism, nor close linkage were demonstrated for these allotypes. The major feature of the inheritance of H2, H3, and H4 C gamma-allotypes, as well as allotype 5, was significant excess of negative (without these allotypes) progeny in the F1 generation from monohybrid cross. The explanation offered for this departure of the C gamma-allotypes from normal Mendelian genetics suggests widespread latencies of their expression in mink.

Animals

Structure of mink immunoglobulin gamma chain cDNA.

Two cDNA clones, encoding mink Ig gamma chains were characterized. The pIGG47 clone contains a part of the leader segment, VDJ and C regions, and pIGG14 contains a part of the J and a complete C region. The clones differ by only four nucleotides in the C region, and they most probably represent allelic variants of the same gene. The V gene segment of pIGG47 was found to be highly similar to human VHIII subgroup sequences; there was 86-87% similarity for the whole V gene segment and 91% for the VHIII specific regions (codons 65-87). Southern blot analysis demonstrated that a high proportion of mink VH genes is VHIII related. The V gene segment used as a probe revealed 19-23 bands in mink DNA under stringent conditions. This is in agreement with our previous data showing that a high proportion of mink Ig contains an 'alternative' binding site for protein A, a feature common to VHIII-related molecules. According to Southern blot analysis there may be 5-7 C gamma genes at the mink IgH locus.

Amino Acid Sequence