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Biomedical subjects

S Rudikoff

Publications and source records attributed to S Rudikoff.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of a new subfamily of class I genes in the H-2 complex of the mouse.

A previously undescribed subfamily of mouse class I MHC genes, consisting of two to three members, has been identified. The structure and organization of one of these, Mb1, has been determined. Mb1 consists of five exons with open reading frames and potentially encodes a class I-like transmembrane protein. In the genome, Mb1 is linked to the H-2 complex, mapping telomeric to Qa. However, this gene has low (ca. 60%) nucleotide identity with other class I sequences and is no more related to mouse class I genes than to class I genes from other species. Mb1 transcripts have not been found in a variety of adult tissues or cell lines, suggesting that, if Mb1 is expressed, its expression is highly regulated. From DNA sequence identity and intron-exon organization, Mb1 appears to be a primordial gene which antedates mouse speciation and which has evolved independently of the rest of the class I gene family. Examination of various species of wild mice demonstrates the presence of a discrete Mb1 subfamily over long evolutionary periods of time.

Amino Acid Sequence↗
PubMed1988

Evolution of the IgA heavy chain gene in the genus Mus.

To examine questions of immunoglobulin gene evolution, the IgA alpha heavy chain gene from Mus pahari, an evolutionarily distant relative to Mus musculus domesticus, was cloned and sequenced. The sequence, when compared to the IgA gene of BALB/c or human, demonstrated that the IgA gene is evolving in a mosaic fashion with the hinge region accumulating mutations most rapidly and the third domain at a considerably lower frequency. In spite of this pronounced accumulation of mutations, the hinge region appears to maintain the conformation of a random coil. A marked propensity to accumulate replacement over silent site changes in the coding regions was noted, as was a definite codon bias. The possibility that these two phenomena are interrelated is discussed.

Amino Acid Sequence↗
PubMed1988

Modern evolution of a single-copy gene: the immunoglobulin C kappa locus in wild mice.

The immunoglobulin kappa light-chain constant region gene (C kappa) has been cloned and sequenced from five wild mouse species. Analysis of these data has permitted an assessment of single-copy gene evolution during a limited time period as defined by the genus Mus. Sequence conservation was found to be as high (or higher) in the 5' and enhancer regions as in the coding region. The pattern of substitutions throughout these genes suggests that parallel evolution has occurred frequently and that substitutions at replacement sites have not decreased significantly, owing to saturation during this period of approximately 10 Myr. Phylogenetic relationships have been determined among these wild species as well as among members of the genus Rattus.

Animals↗
PubMed1987

Amino acids at the site of V kappa-J kappa recombination not encoded by germline sequences.

Murine V kappa-J kappa recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain VH-D-JH assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both kappa and heavy chain genes. We have examined the origin of an unusual amino acid, Ile, found at the site of V kappa-J kappa recombination in antigalactan antibodies, by sequence analysis of the corresponding rearranged and germline genes. Results indicate that the Ile codon can be generated by use of a single nucleotide 3' of the V kappa segment in combination with the second and third nucleotides of the first codon of J kappa 5 or J kappa 4. However, several antigalactan antibodies express Ile in combination with J kappa 2. An Ile codon cannot be generated by recombination in any reading frame between germline V kappa and J kappa 2 segments. These results suggest that the origin of the Ile codon in lines using J kappa 2 may represent a novel even in murine light chain assembly, possibly similar to the de novo addition of nucleotides observed in heavy chain gene recombination.

Amino Acid Sequence↗
PubMed1987

Purification and NH2-terminal sequence of a plasmacytoma growth factor derived from the murine macrophage cell line P388D1.

Plasmacytoma growth factor (PCT-GF), a putative macrophage-derived lymphokine essential for the in vitro viability and proliferation of early generation plasmacytomas, was purified from conditioned medium of the murine macrophage cell line P388D1. The purification of PCT-GF was accomplished by a batch concentration on trimethylsilyl-controlled pore glass beads, followed by: gel filtration chromatography; hydrophobic interaction HPLC; and reverse-phase HPLC. SDS-PAGE analysis of the purified PCT-GF revealed a single band of Mr 23,000. The amino terminal sequence of PCT-GF was established as NH2-Pro-Thr-Ser-Gln-Val-Arg-Arg-Gly-Asp-Phe-Thr-Glu-Asp-Thr-Thr-Pro-Asn- Arg-Pro-Val-Tyr-Thr. No significant homology was found between this sequence and proteins in the National Biomedical Research Foundation database, suggesting that PCT-GF is a new lymphokine unrelated to previously described growth and differentiation factors.

Amino Acid Sequence↗
PubMed1987

Identification of cysteine 656 as the amino acid of hepatoma tissue culture cell glucocorticoid receptors that is covalently labeled by dexamethasone 21-mesylate.

Recent results using proteases suggest that dexamethasone 21-mesylate (Dex-Mes) labeling of the rat hepatoma tissue culture (HTC) cell glucocorticoid receptor occurs at one or a few closely grouped cysteine residues (Simons, S.S., Jr. (1987) J. Biol. Chem. 262, 9669-9675). In this study, a more direct approach was used both to establish that only one cysteine is labeled by [3H]Dex-Mes and to identify the amino acid sequence containing this labeled cysteine. Various analytical procedures did not provide the purification of the extremely hydrophobic Staphylococcus aureus V8 protease digestion fragment that is required for unique amino acid sequencing data. Therefore, Edman degradation was performed on the limit protease digest mixtures which appeared to contain only one 3H-labeled peptide. These degradation experiments revealed the number of amino acid residues between the NH2 terminus of each peptide and the [3H]Dex-Mes-labeled cysteine. A comparison of these amino acid spacings with the published amino acid sequence of the HTC cell glucocorticoid receptor (Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikstom, A-C., Gustafsson, J-A., and Yamamoto, K. R. (1986) Cell 46, 389-399) indicated that the one cysteine labeled by [3H]Dex-Mes is Cys-656. Further analysis of the receptor sequence for the presence of the observed grouping of proteolytic cleavage sites, but without any preconditions as to which amino acid was labeled, gave Asp-122 and Cys-656 as the only two possibilities. Potential labeling of Asp-122 could be eliminated on the basis of immunological and genetic evidence. We, therefore, conclude that the single Dex-Mes-labeled site of the HTC cell glucocorticoid receptor has been identified as Cys-656. Since several lines of evidence indicate that [3H]Dex-Mes labeling of the receptor occurs in the steroid binding site, Cys-656 is the first amino acid which can be directly associated with a particular property of the glucocorticoid receptor.

Affinity Labels↗
PubMed1987

Tissue-specific expression and structure of a divergent member of a class I MHC gene family.

The class I gene family of the major histocompatibility complex (MHC) of the miniature swine (SLA) contains seven members. Six of these are highly homologous, whereas the seventh, PD6, is only distantly related. A genomic clone containing PD6 has been isolated and characterized. The PD6 SLA gene, although only 55% homologous to the other SLA class I sequences, is still a member of the family; its size and exon/intron organization are similar to other class I genes. When aligned with other SLA genes, exons 1 to 6 of PD6 have open reading frames; exon 7 contains a termination codon. PD6 is transcribed in transfected mouse L cells where its expression is enhanced by interferon. In vivo expression of PD6 is observed in a variety of tissues, with the highest levels in mature lymphoid tissues. Peripheral T cells contain high levels of PD6-specific RNA relative to B cells, whereas no PD6 transcripts are detectable in thymocytes.

Amino Acid Sequence↗
PubMed1987

Idiotypic self binding of a dominant germline idiotype (T15). Autobody activity is affected by antibody valency.

We have previously described (1-3) an IgM antibody that binds to PC, expresses the T15 idiotype, and binds also to itself or T15 if insolubilized. Because of the simultaneous presence of complementary idiotopes and paratopes this type of antibody has been termed autobody. The self binding involves the antigen-binding site because the F(ab')2 fragment of T15, PC, and no other haptens inhibit the self binding. DNA sequence analysis of 11E7-1 using primer extension cDNA sequencing showed that the variable sequences of H and L chains of 11E7-1 are identical to the germline sequence of the prototype T15 idiotype. Furthermore, monomeric and dimeric T15 IgA were shown to bind to insolubilized T15 and other T15+ antibodies including 11E7-1. Thus, the self-binding activity is an inherent property of the T15 germline sequence. The self binding is highly dependent on the polymeric state of the binding antibody since the IgM pentamer of 11E7-1 is about three fold more effective than the T15 dimer and 50 times more than the T15 monomer. These data suggest that the self-binding activity of a germline-encoded idiotype may play an important role in the biology of its expression, and more specifically, may be responsible for the establishment of its dominant expression.

Amino Acid Sequence↗
PubMed1987

Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector.

The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.

Amino Acid Sequence↗
PubMed1987

Structure and expression of class I MHC genes in the miniature swine.

The genome of the miniature swine, unlike other species, contains a relatively small class I MHC gene family, consisting of only seven members. This provides an excellent system in which to identify and characterize the regulatory mechanisms which operate to both coordinately and differentially regulate the expression of a multi-gene family. The structure of class I SLA genes, like other class I genes, consists of eight exons encoding a leader sequence, three extracytoplasmic domains, a transmembrane domain and intracytoplasmic domains. Despite the common structure, two sub-families of class I genes can be distinguished within the SLA family. One, containing the closely related PD1 and PD14 genes, encodes the classical transplantation antigens. Another contains the highly divergent PD6; the functions of the products of this subfamily, if any, are not known. The class I SLA genes share some common regulatory mechanisms, as evidenced by the fact that all three genes analyzed are transcribed in mouse L cells. Furthermore, interferon treatment of transfected mouse L cells enhances expression of all three genes. Both PD1 and PD6 are transcribed in vivo, where the highest levels of expression are observed in lymphoid tissues. Superimposed on the common patterns of class I gene expression are distinct ones, as evidenced by the findings that PD1 is preferentially expressed in B cells, whereas PD6 is preferentially expressed in T cells. These differences may reflect the extensive divergence of the 5' flanking sequences of these genes. Future studies will be aimed at elucidating the precise molecular interactions and mechanisms which give rise to the observed differential expression.

Animals↗
PubMed1986

Gene correction in the evolution of the T cell receptor beta chain.

Mutational mechanisms operating at the T cell receptor beta chain locus have been examined by comparison of the CT beta 1 and CT beta 2 gene sequences from Mus pahari, believed to be the oldest living species in the genus Mus, with those of inbred mice. Results indicate that a gene correction event independent of that suggested to have occurred in inbred mice has homogenized the M. pahari CT beta exon 1 sequences, minimizing diversity in this region of the molecule. These observations suggest that correction events such as gene conversion may occur frequently, even in pauci-gene families with as few as two members, and therefore play a significant role in gene diversification or homogenization of small as well as large gene families.

Amino Acid Sequence↗
PubMed1986

Genetic basis for expression of the idiotypic network. One unique Ig VH germline gene accounts for the major family of Ab1 and Ab3 (Ab1') antibodies of the GAT system.

Ig germline genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA either in pBR328 plasmid or in EMBL 3 phage. Three clones that gave a very strong positive hybridization signal with a VH anti-GAT-specific probe were completely characterized and sequenced. All three were greater than 95% homologous, with the exception of the 5' noncoding region, which was only 85% homologous but contained characteristic regulatory signals. One of these genes, H10, had a sequence that was completely identical to that of a cDNA derived from a GAT-specific BALB/c hybridoma. Southern blot analysis using Eco RI-digested DNA from rearranged GAT-specific hybridomas revealed that the same gene was used for other GAT-specific VH regions, including one differing from the H10 sequence by 12 nucleotides, which must have been generated by a somatic mechanism. The same H10 germline gene was also used, in most cases without any nucleotide substitution, in hybridomas of the Ab1' set of the GAT idiotypic cascade, suggesting that immunization with Ab2 (antiidiotypic) antibodies preferentially stimulates the direct expression of VH germline genes. Finally, the previous hypothesis that NPa and GAT VH genes were derived from the same germline gene was definitively confirmed, both from sequence data and Southern blot analysis.

Animals↗
PubMed1986

The galactan-binding immunoglobulin Fab J539: an X-ray diffraction study at 2.6-A resolution.

The crystal structure of the Fab of the galactan-binding immunoglobulin J539 (a mouse IgA,kappa) has been determined at a resolution of approximately 2.6 A by X-ray diffraction. The starting model was that obtained from the real space search described previously (Navia, M.A., Segal, D.M., Padlan, E.A., Davies, D.R., Rao, D.N., Rudikoff, S. and Potter, M. "Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5 A resolution." Proc. Nat. Acad. Sci. USA, 76:4071-4074, 1979). This Fab structure has now been refined by restrained least-squares procedures to an R-value of 19% for the 11,690 unique reflections between 8.0 A and 2.6 A. The rms deviation from ideal bond lengths is 0.025 A. The overall structure differs from McPC603 Fab, another mouse IgA,kappa antibody, in that the elbow bend, relating the variable and constant parts of the molecule, is 145 degrees vs. 133 degrees for McPC603. The region of the molecule expected to be the antigen binding site contains a large cavity with two clefts leading away from it. This has been fitted with a model of an oligo-galactan.

Animals↗