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S Rudikoff

Publications and source records attributed to S Rudikoff.

At least 91 records · Page 5Linked to original sources
PubMed1983

Two-dimensional gel analysis of swine histocompatibility antigens.

Miniature swine MHC antigens from three inbred herds were examined by two-dimensional gel electrophoresis. These antigens were found to constitute a series of complex glycoproteins displaying haplotype-specific patterns that allowed the distinction of both class I and class II molecules among the three haplotypes. Selected outbred pig antisera reacted with a subset of class I antigens, suggesting the presence of at least two distinct molecular species among these antigens. Similarly, alloantisera reacting with mouse Ia antigens and a monoclonal anti-human DR were shown to immunoprecipitate a subset of class II molecules. Examination of the cells from two recombinant haplotypes demonstrated that both independent recombinational events took place between the class I and class II genes.

Animals↗
PubMed1983

Murine thymocyte and splenocyte Ia antigens are indistinguishable by two-dimensional gel electrophoresis.

Ia antigens have been found on the surface of B lymphocytes, macrophages, epidermal Langerhans cells and on certain transformed cells. Ia antigens have also been detected on the surface of thymocytes but the biosynthesis of these antigens by thymocytes has been difficult to demonstrate. We describe the labeling of murine thymocytes with 35S-methionine and the subsequent analysis of Ia antigens by two-dimensional polyacrylamide gel electrophoresis. Cell elimination experiments demonstrated that the Ia antigens detected were not of B cell origin and were synthesized by a Thy-1-positive thymocyte. Ia antigens from thymocytes were found to be indistinguishable from spleen Ia preparations. Since T cell I region determinants have been postulated to be involved in cellular recognition phenomena, models addressing this recognition must allow for the observation that T and B cell I region molecules detected by antisera such as A. TH anti-A. TL are indistinguishable by two-dimensional gel analysis and are thus unlikely to be involved in the generation of specificity in recognition.

Animals↗
PubMed1982

Classification of mouse VK groups based on the partial amino acid sequence to the first invariant tryptophan: impact of 14 new sequences from IgG myeloma proteins.

Fourteen new VK sequences derived from BALB/c IgG myeloma proteins were determined to the first invariant tryptophan (Trp 35). These partial sequences were compared with 65 other published VK sequences using a computer program. The 79 sequences were organized according to the length of the sequence from the amino terminus to the first invariant tryptophan (Trp 35), into seven groups (33, 34, 35, 36, 39, 40 and 41aa). A distance matrix of all 79 sequences was then computed, i.e. the number of amino acid substitutions necessary to convert one sequence to another was determined. From these data a dendrogram was constructed. Most of the VK sequences fell into clusters or closely related groups. The definition of a sequence group is arbitrary but facilitates the classification of VK proteins. We used 12 substitutions as the basis for defining a sequence group based on the known number of substitutions that are found in the VK21 proteins. By this criterion there were 18 groups in the Trp 35 dendrogram. Twelve of the 14 new sequences fell into one of these sequence groups; two formed new sequence groups. Collective amino acid sequencing is still encountering new VK structures indicating more sequences will be required to attain an accurate estimate of the total number of VK groups. Updated dendrograms can be quickly generated to include newly generated sequences.

Amino Acid Sequence↗
PubMed1982

Polymorphism in immunoglobulin heavy chains suggesting gene conversion.

Complete heavy (H) chain variable region (V region; amino acids 1-118) sequences have been determined for three phosphocholine (PCho)-binding monoclonal antibodies of CBA mouse strain origin. Two of these were found to differ from the sequence of the BALB/c T15 germline VH segment (segment of the V region that includes amino acids 1-95) at four positions but were identical to the allelic form of T15 (C3) found in C57BL. The third VH segment, HP101.6G6 (6G6), was clearly the product of a second, related VH gene, probably the allele of the BALB/c V11 gene, a second member of the P-Cho VH gene family. Thus, more than one VH gene is capable of encoding heavy chains of PCho-binding antibodies. The 6G6 VH segment differs from VII at seven positions; four of these distinguishing amino acids are encoded in other membranes of the PCho VH gene family. We postulate that the origin of the 6G6 VH sequence can most easily be explained by a process of gene conversion occurring between the least three members of the PCho VH family.

Amino Acid Sequence↗
PubMed1982

Somatic mutation in a cultured mouse myeloma cell affects antigen binding.

The S107 mouse myeloma cell line synthesizes an IgA antibody that binds the hapten phosphocholine and is similar if not identical in its heavy and light chain variable region sequence to the predominant antibody produced by BALB/c mice in response to immunization with phosphocholine. This cell line frequently and spontaneously generates somatic variants producing immunoglobulins with decreased ability to bind antigen. One such variant, S 107.U1, is described here. This variant has a decreased ability to bind phosphocholine when it is attached to a carrier, although its affinity for free hapten is the same as that of the parent. This decrease in antigen binding is associated with a single amino acid substitution at the fifth residue in the JH segment.

Amino Acid Sequence↗
PubMed1982

Characterization of a porcine genomic clone encoding a major histocompatibility antigen: expression in mouse L cells.

A porcine genomic clone encoding a major histocompatibility, (MHC) antigen was isolated by direct screening of a swine genomic library with a heterologous human MHC cDNA probe. Mouse L cells transformed with DNA from the clone stably express swine MHC antigen. Pig alloantisera specifically lyse transformant but not control cell lines in a complement-mediated cytotoxicity assay. Direct immunoprecipitation of radiolabeled cellular protein from transformed lines by pig alloantiserum results in the coprecipitation of swine MHC heavy chain and mouse beta 2-microglobulin, demonstrating the association of heterologous subunits of MHC antigens.

Animals↗
PubMed1982

Single amino acid substitution altering antigen-binding specificity.

S107, a phosphocholine-binding myeloma protein, has been cloned in soft agar, and an antigen-binding variant has been isolated and characterized. The variant does not bind phosphocholine attached to carrier or as free hapten in solution but does retain antigenic determinants (idiotypes) of the parent. Chain recombination experiments suggest that the defect in binding is entirely in the heavy chain. Amino acid sequence analysis showed a single substitution--glutamic acid to alanine at position 35--in the first hypervariable or complementarity-determining region. In terms of the three-dimensional model of the phosphocholine-binding site, glutamic acid-35 provides a hydrogen bond to tyrosine-94 of the light chain that appears to be critical for stability of this portion of the binding site. The removal of this bond and the presence of the smaller alanine side chain is thus consistent with the loss in binding activity. These results suggest that small numbers of substitutions in antibodies, such as those presumably introduced by somatic mutation, may in some situations be effective in altering antigen-binding specificity.

Amino Acid Sequence↗
PubMed1982

Transplantation in miniature swine. XII. N-terminal sequences of class I histocompatibility antigens (SLA) and beta 2-microglobulin.

Purified class I histocompatibility antigens (SLA) from three haplotypes were prepared by papain treatment of lymphoid cell membranes obtained from spleens and lymph nodes of miniature swine homozygous at their major histocompatibility complex. Antigens were purified by ion-exchange chromatography followed by gel filtration. Purity was analyzed by SDS-PAGE, and antigenic specificity by inhibition of complement-dependent, alloantiserum-mediated cytotoxicity. The SLA antigens were reduced and alkylated, and the component heavy and light chains were isolated by gel filtration under dissociating conditions. N-terminal amino acid sequences were obtained for SLAaa, SLAcc, and SLAdd heavy chains, as well as for the light chain, beta 2-microglobulin. The swine antigens showed high levels of homology with class I antigens from other animal species. Heterogeneity was observed among the swine haplotypes, and several of the positions at which substitutions were found are apparently invariant in other animal species. In contrast, only minimal sequence heterogeneity was detected within haplotypes, the basis of which may be of relevance to understanding the evolutionary development of these molecules.

Amino Acid Sequence↗
PubMed1982

kappa-Chain restriction in anti-galactan antibodies.

Complete kappa-chain variable region sequences were determined for 10 light chains from anti-beta-(1,6)-D-galactan-binding monoclonal antibodies. Eight of these light chains were from IgM and two from IgG proteins. Seven of the eight IgM light chains were identical from position 1 to 95, the region encoded by the light chain variable region gene. The eighth differed at a single framework position. The two IgG light chains were identical in sequence and differed from the IgM light chains at two framework positions. All 10 light chains have an lle at position 96, as has been previously reported in 5 of 6 anti-galactan myeloma proteins. This residue is the first amino acid normally encoded by the light chain J gene. None of the germ line J genes encodes an lle at position 96, and furthermore the lle codon cannot be generated by alterations in the frame of recombination between the codon for amino acid 95 and J gene codons. These light chains are thus the product of an unusual and reproducible recombination event that appears to employ an extra codon at the 3' end of the variable region gene. Three different J segments are potentially used in these light chains J1 or J2 was found in three chains and J5 in seven.

Amino Acid Sequence↗
PubMed1981

Nucleic acid and protein sequences of phosphocholine-binding light chains.

An 18-kilobase DNA fragment containing the sequence coding for both the variable and constant regions of the S107 mouse immunoglobulin light chain was cloned from total cellular DNA. The complete nucleotide sequence of the kappa-chain variable-region gene is reported. Determination of the amino acid sequence encoded by the DNA is found to be identical to the protein sequence of the T15 light chain through residue 88. Direct sequence analysis confirmed that the J1 joining segment is used in the recombination event producing the active kappa light chain gene.

Amino Acid Sequence↗
PubMed1981

Immunoglobulin heavy chains from anti-inulin myeloma proteins: evidence for a new heavy chain joining segment.

Immunoglobulin heavy chains have been shown to be encoded by at least 3 widely separated genetic elements, designated variable (V), diversity (D), and joining (J), which undergo rearrangement during somatic differentiation to produce the active gene form. The D segment codes for a portion of the 3rd hypervariable region and thus potentially contributes significantly to structural diversity in this portion of the molecule. Heavy chains from anti-inulin proteins are unusual in that they essentially lack a 3rd hypervariable region. Thus, if a D segment exists in these proteins, it is extremely short, possibly 1 to 2 amino acids, and more likely serves a framework function rather than introduces structural diversity in the 3rd hypervariable region. We have completed the heavy chain variable region amino acid sequence from proteins AMPC1 and T957 bringing to 6 the number of complete sequences from this group. All of these proteins lack a 3rd hypervariable region. In addition, substitutions are found within the J segments of AMPC1 and T957, which are unlikely to be generated by the recombination event. The occurrence of Pro at position 105 in both of these J segments in contrast to the Gln found in all other heavy chains using this J segment suggests the possible existence of a previously unidentified J segment gene.

Amino Acid Sequence↗
PubMed1981

Transplantation in miniature swine. IX. Swine histocompatibility antigens: isolation and purification of papain-solubilized SLA antigens.

Histocompatibility antigens have been purified by papain treatment of crude cellular membranes obtained from spleen and mesenteric lymph nodes of individual miniature swine that are homozygous at the major histocompatibility complex. The purification protocol included ultracentrifugation, gel filtration, ion-exchange chromatography, and in some instances preparative isoelectric focusing. This procedure was applied to the preparation of sLA antigens from the 3 haplotypes available in our partially inbred miniature swine herd. The purity of the SLAdd molecules was evidenced by: 1) a single activity peak of approximately 50,000 daltons on gel-permeation chromatography; 2) the antigen elution as a symmetrical peak from ion-exchange resins and from isoelectric focusing columns; 3) the presence of 2 predominant polypeptide chains on SDS-PAGE at apparent m.w. 43,000 and 13,000 daltons; and 4) specific immunoprecipitations by alloantisera and by anti-beta 1 microglobulin antisera. The antigenic activity of SLA products was analyzed by inhibition of complement-mediated cytotoxicity and by removal on anti-beta 2-microglobulin affinity columns. Heavy and light chains were separated by gel-filtration on Sephacryl S-200 in 6 molar guanidine. These SLA products, which have thus been shown to be pure by a variety of criteria, are now readily available for structural analysis and for analysis of biologic activity.

Animals↗
PubMed1981

Allelic forms of anti-phosphorylcholine antibodies.

Anti-idiotype antisera were prepared to 2 phosphorylcholine (PC) binding myeloma proteins, T15 of BALB/c origin and C3 of C57BL origin. These antisera were shown to recognize the respective determinants on induced anti-PC antibodies in the 2 parental strains. Genetic analysis of these V region markers in backcross progeny of (BALB/c X C57BL/6) X BALB/c demonstrated that the 2 determinants segregated as simple Mendelian alleles linked to the allotype locus. The genetic data are consistent with previously reported structural studies suggesting the allelic nature of the T15 and C3 heavy chain variable regions. The expression of the T15 and C3 antigenic determinants in Ig congenic and recombinant inbred strains derived from BALB/c-C57BL/6 crosses further supports the concept of allelism although a single exception was observed. Strain distribution analysis has indicated the potential existence of other alleles of the T15 gene in addition to C3.

Alleles↗
PubMed1980

kappa Chain joining segments and structural diversity of antibody combining sites.

Immunoglobulin kappa light chains are coded for by at least three distinct gene segments designated variable, joining, and constant. The joining gene codes for the 13 amino acid segment linking the variable and constant regions. This peptide includes the last amino acid (96) in the third complementarity-determining region and thus could introduce structural diversity. We have determined the light chain variable region sequences from three myeloma proteins with beta(1,6)galactan-binding specificity, bringing to six the number of light chains sequenced from proteins demonstrating this specificity. Five of these have isoleucine at position 96 and the sixth tryptophan. This substitution appears to be accommodated with no significant change in association constant for a beta(1,6)galactan hapten. Additionally, as many as nine substitutions are found in both light and heavy chain complementarity-determining regions between members of this group although only minimal variations in hapten binding affinity are observed. The isoleucine found at position 96 in five of the kappa chains could not be coded for by any of the joining gene nucleotide sequences previously observed and would require a novel nucleotide sequence at the recombination site between variable and joining genes to produce the observed protein structure. Alternatively, there may exist joining gene segments not yet detected.

Amino Acid Sequence↗
PubMed1980

Allelic forms of the immunoglobulin heavy chain variable region.

The complete variable region sequence of the heavy chain from a phosphorylcholine-binding myeloma protein of C57/BL allotype has been determined. When this sequence was compared with the germ line-coded heavy chain variable region sequence of BALB/c phosphorylcholine-binding proteins, five differences were observed. Four of the substitutions were located in the framework portion of the variable region and the fifth in the "J" or joining segment. Two of the framework substitutions were found at positions 14 and 16. Previous studies have shown that heavy chains from all anti-phosphorylcholine antibodies induced in C57/BL mice have the same amino acids at positions 14 and 16 as the C57/BL myeloma protein described in this communication. It has therefore been concluded that these residues are encoded in the C57/BL germ line in contrast to two alternatives in the BALB/c genome. This finding, in addition to the 96% homology found between the C57/BL and BALB/c sequences, suggests that these structures represent allelic forms of an entire variable region.

Alleles↗
PubMed1979

Structural evidence for independent joining region gene in immunoglobulin heavy chains from anti-galactan myeloma proteins and its potential role in generating diversity in complementarity-determining regions.

We have determined the variable region sequences of four heavy chains from beta(1-6)D-galactan-binding myeloma proteins. Two of these proteins are identical to position 100 which is located in the third complementarity-determining region (CDR-3). The remaining two differ at a total of 8 positions over the first 100 amino acids, and all of the differences can be explained by single-base mutations at the DNA level. When an assessment is made of the protein segment following CDR-3, which has been termed "J segment" or "FR4," a completely different pattern of variation is observed. The J segments from the four proteins can be divided into two sets. Members of each set share a series of linked amino acids not found in members of the alternative set. The two proteins identical to position 100 have J segments from the two different sets, suggesting that recombination has occurred between V and J genes. An examination of the CDR-3 sequences from the four heavy chains reveals substitutions at positions 100 and 105. Gly is found at 100 in two of the proteins and His in the remaining two. In the two proteins with Gly-100, the following J sequence is limited to one of the two sets of J segments defined by linked amino acids. Similarly, the two heavy chains with His-100 have J segments from the second set. Thus, at the protein level an apparent association is seen between CDR-3 and J segment. If CDR-3 should be found linked to J segment at the DNA level, a new mechanism would be introduced for increasing antibody diversity by recombining various CDR-3 plus J genes with genes coding for the remainder of the variable region. Alternatively, if CDR-3 were coded for by the V gene, then the recombination of V with J may provide an opportunity to introduce mutations in CDR-3. In this case the linkage of amino acids in CDR-3 and the J segments would suggest that recognition signals are used such that certain V genes only pair with a given J gene.

Amino Acid Sequence↗