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G M Pfaffenbach

Publications and source records attributed to G M Pfaffenbach.

8 recordsLinked to original sources
PubMed1996

A rapid genotype assay for Pla haplotypes.

The method described here results in simple visual analysis of 1, 2, or 3 ethidium bromide-stained bands corresponding to the PlA1, PlA2 or PlA1/A2 phenotypes, respectively. The amplification primers, designed to yield a 711bp product which encompasses exon 2 and substantial intronic sequence both 5' and 3' of the exon enable exquisite resolution of the restriction enzyme digestion products which define the alleles. In addition to prediction of fetal risk of neonatal alloimmune thrombocytopenia using this method, an ethnically heterogeneous pool of platelet donors was screened for the platelet antigen PlA (HPA-1) and gave unequivocal results which should be useful for platelet matching.

Antigens, Human Platelet↗
PubMed1991

Analysis of the H-2Kbm8 mutant: correlation of structure with function.

The gene for H-2K class I major histocompatibility antigen on the bm8 variant was cloned and the DNA sequence compared with the parental gene. Sequence analysis demonstrated that seven nucleotides were changed with respect to the parental gene sequence spanning 24 nucleotides. These changes represent an alteration of four amino acids from the parent protein. As this mutation occurred in a single generation, a potential donor gene for such a complex mutation was suggested and identified. The Q4 gene class I-like molecule has a stretch of 95 nucleotides of identity in the region of the bm8 mutation. Genomic Southern analysis of the mutant and parental DNA with a gene-specific oligonucleotide demonstrated that the potential donor gene Q4 is a likely candidate sequence for such an event. The amino acid alterations for the H-2Kbm8 mutation are discussed in consideration of hte three-dimensional structure of the characterized human class I glycoprotein.

Animals↗
PubMed1990

Biochemical analysis of related, independently arising histocompatibility mutants: bm17 and KB-98 enlarge the "bg series" of H-2Kb mutants.

The "bg" series of MHC mutations is the most prevalent type of mutations of Kb in C57BL/6 mice screened by reciprocal tail skin grafting. The basis for identification of this series of mutations is the incompatibility of grafts between the parental B6 and the mutant. This series takes the longest to reciprocally reject the skin grafts. The series can be subdivided into "bg 1" and "bg 2" groups based on Kb-restricted recognition of virus-infected mutant target cells. The biochemical basis for these mutations are amino acid substitutions at residues 116 and 121 of the Kb transplantation antigen. These substitutions do not alter monoclonal antibody binding sites. The structural basis of MAb binding and the genetic basis of the mutation are discussed.

Amino Acid Sequence↗
PubMed1986

Murine major histocompatibility complex class-I mutants: molecular analysis and structure-function implications.

The class-I mutants have provided a model system for understanding the generation of diversity of the genes encoding the histocompatibility molecules K, D, and L, and the relationship of their structure to function. The complex nature of the alterations found in Kb molecules from mutant mice has been documented at the nucleic acid level for eight mutants. The clustered changes in the mutant genes are consistent with the hypothesis that genetic recombination between class-I genes generates the Kb mutants. Techniques using synthetic oligonucleotide probes to mutant DNA sequence demonstrated that other class-I genes were available as donors for interaction with the Kb gene to produce the mutations. Intriguingly, donor genes found in the K region (K1) and the D region (Db), as well as the Qa regions (Q4, Q10), were capable of the interactions. The amount of genetic transfer to Kb from other class-I donor genes may range from a potential minimum of 5 nucleotides to a potential maximum of 95 nucleotides. Genealogical analysis of several bm mutants has further indicated that at least some, if not all, of the gene interaction events generating Kb mutations occurred during mitotic amplification of the germ cells. Genetic recombination among class-I genes occurring in nature to the extent observed for the Kbm mutants could readily generate mosaic transplantation genes containing sequences derived from other class-I genes. Thus, it seems likely that genetic interaction plays a major role in the diversification and ongoing evolution of the MHC. The localization of altered amino acids in the in vivo mutant Kb molecules has directed our attention to recognition regions on the Kb product that play a major role in determining alloreactivity and H-2 associative recognition. The replacement of one or a few amino acids in either of the postulated recognition regions located in the alpha 1 domain (residues 70-90) or alpha 2 domain (residues 150-180) can have marked effects on biological function. While the majority of monoclonal antibodies recognize epitopes in one or the other recognition region, CTL recognize determinants dependent on the apparent interaction of amino acids located in both regions. These overall conclusions are supported to a large extent by studies on mutants derived from several sources, i.e. spontaneous mutants, mutagen-induced somatic variants, and products of hybrid H-2 genes. Studies of in vitro variants can provide a more refined approach for analysis of structure-function relationships through the introduction of minimal biochemical changes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2bm5 and B6-H-2bm16.

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tyrosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2Kb mutants analyzed.

Amino Acid Sequence↗
PubMed1983

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2bm6, B6.C-H-2bm7, and B6.C-H-2bm9.

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2bm5 (bm5) and B6-H-2bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2Kb molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2bm6 (bm6), B6.C-H-2bm7 (bm7), and B6.C-H-2bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from B6 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the Kb molecule.

Amino Acid Sequence↗
PubMed1983

Spontaneous H-2 mutants provide evidence that a copy mechanism analogous to gene conversion generates polymorphism in the major histocompatibility complex.

The analysis of H-2K products from spontaneously generated major histocompatibility complex (MHC) mutants and of the primary structure of other class I antigens suggests the genetic hypothesis that diversity in the MHC results from a copy mechanism analogous to gene conversion. The hypothesis was tested by making precise structural predictions about three partially characterized MHC mutants (bm1, bm3, and bm8). The predictions were based on consensus sequences among class I genes that differ from H-2Kb in the same region of the molecule as do the Kb mutants. In two cases (bm3 and bm8) we successfully predicted the correct amino acid substitution at positions known to be altered but for which the specific nature of the substitution had not been determined. In two additional cases (bm1 and bm8) we predicted and found both new mutation sites and the specific amino acid substitutions. The positions and identifications of the variant amino acids were determined by radiolabeled amino acid sequence analysis and DNA restriction endonuclease analysis. The interaction of MHC genes through a copy mechanism to generate diversity permits the introduction of multiple nucleotide base substitutions into class I sequences by a single genetic event. Such a mechanism may account in part for the large structural divergence among alleles of MHC loci and the high degree of MHC polymorphism among wild mice.

Amino Acid Sequence↗