Mouse H2 congenic intervals: analysis and use for mapping.

In this study we exploit the unique genetic resource of inbred mouse major histocompatibility complex (H2) congenic and recombinant strains to construct a high-resolution map of microsatellite loci in and around the H2 region, as well as an independent genetic map of other loci on mouse Chromosome (Chr) 17. Microsatellite loci were analyzed in 11 C57BL/10 (B10) strains to determine the size of the congenic interval in each. The length of the congenic interval found in each strain varied widely. Interestingly, the intervals were generally smaller than statistical expectations. However, the observed congenic intervals were still sufficiently long that these strains and probably wild-derived H2 congenics are an important source of genetic variability. The staggered ends of the various congenic intervals and the recombinants were used to construct the map. This map will be useful for physical cloning and to help localize novel genes. As evidence of the mapping application of congenic strains, locational information was derived about Trp53-ps and Stl.

A highly polymorphic microsatellite in the class II Eb gene allows tracing of major histocompatibility complex evolution in mouse.

A hallmark of major histocompatibility complex (MHC) genes is their extraordinarily high level of polymorphism. Polymorphic residues on MHC molecules determine which peptide ligands they bind and present to effector T lymphocytes. Although the genetic mechanisms responsible for MHC polymorphism have been delineated, the timetable and the pathway of their diversification remain unclear. To trace MHC evolution, we have characterized a highly polymorphic microsatellite containing tandem repeats (TRs) of two tetranucleotide units, TGGA and GGCA, located at the 3' end of the second intron in the class II Eb gene of mouse. On the basis of length as well as sequence variations, 11 TR alleles were defined in 55 inbred mouse strains, which included MHC recombinant haplotypes and haplotypes derived from different subspecies of mouse. In this extensive sampling, a striking concordance was observed between the serologically identified class II proteins and the associated TR alleles. Examination of several strains carrying the same MHC haplotypes as well as strains carrying recombinant MHC haplotypes indicates that TR alleles are extremely stable. These observations suggest that TR polymorphism predates the separation of various subspecies of mouse. On the basis of sequence divergence, a genealogical tree has been constructed to depict evolution of the different TR alleles. Finally, evidence is presented that suggests this microsatellite polymorphism is generated by slipped-strand mispairing during DNA replication.

Restriction fragment length polymorphism of the murine C4 and Slp genes: two C4 groups.

We have examined the related H-2 genes coding for the fourth component of complement (C4) and the sex-limited protein (Slp) from 30 inbred mouse strains by Southern blot analysis. With four restriction enzymes, 11 RFLP patterns distributed among 26 different H-2 haplotypes have been identified. Strains of the same serologic H-2 haplotype were found to have identical RFLP patterns. It was confirmed that the number of C4-related genes in most haplotypes is two, Slp and C4; but H-2SWI6 (SWI6) and SWI9, which have the same RFLP pattern, have four and Sw7 has five. Although C4 and Slp have many similarities, they also were found to contain distinctive features: relative to Slp, each C4 allele examined has two insertions totaling 1.1 kb located in introns 14 and 15; and each Slp allele examined, excluding hybrids, has a provirus insertion upstream. No other large deletions or insertions were detected. The RFLP patterns are also due to 10 polymorphic restriction sites, which have been placed on standard maps; two are associated with Slp and eight are associated with C4.Sk strains, the only strains that express low serum levels of C4, have the same RFLP phenotype as Sw14, Sw18, and Swx; Sk may have arisen from a recent common ancestor of these strains. Homologous recombination has been important in the formation of existing C4 alleles. However, based on complete linkage disequilibrium between three RFLP internal to C4, the haplotypes have been divided into two groups that may have functional significance.

Molecular mapping of murine I region recombinants. III. Crossing over at two discrete sites within the beta 1-beta 2 intron of the E beta gene.

The murine MHC provides a unique genetic system for studying meiotic recombination. A large number of murine H-2 recombinants cross over within a stretch of the E beta gene referred to as the E beta hot spot. The crossing over of eight such recombinants, derived from the s and k haplotypes, was studied at the nucleotide level. A 3-kb stretch of DNA, 3' to the beta 1 exon of the E beta gene, was sequenced after amplification of the genomic DNA from B10.S (one of the parental strains) by polymerase chain reaction. A number of sequence variations were identified with respect to B10.A (the other parental strain). Examination of these sequence variations by RFLP, simple sequence length polymorphism, as well as direct sequencing after polymerase chain reaction-amplification of genomic DNA from the recombinants led to unambiguous identification of the cross-over sites. Although all eight recombinants crossed over within the beta 1-beta 2 intron, two discrete nonoverlapping sites were involved. Five of the recombinants B10.BASR1, B10.ASR1, B10.ASR12, B10.HTT, and B10.S(9R) crossed over within a maximum of 395 bp of DNA 3' to the beta 1 exon. The remaining three recombinants B10.ASR7, B10.ASR11, and B10.S(8R) crossed over within 950 bp of DNA, adjacent to the cross-over site noted above. Each of these stretches of DNA was completely identical in the two parental haplotypes precluding further dissection of the cross-over sites. These cross-over sites are within those reported for the b and k recombination.

Conversion of the C4d.2 serologic allotype of murine complement component C4 to the C4d.1 allotype by site-specific mutagenesis.

C4d.1 and C4d.2 are serologically defined allotypes of murine complement component C4. Previous studies in Shreffler's laboratory have shown that the structural difference between the two allotypes lies within a single tryptic peptide of the C4 alpha-chain and that the sequences of this fragment from the two allotypes (determined from nucleic acid sequences of genomic clones) differ only by the substitution of arginine in C4d.2 for glutamine in C4d.1. Hence this single amino acid change apparently is responsible for the rather striking serological difference between the two allotypes. To test this conclusion, we have used site-specific mutagenesis to alter the sequence of a full-length C4 cDNA that was derived from a mouse strain expressing the C4d.2 allotype. We substituted a glutamine codon for the arginine codon at the specified site and expressed both mutant and parent recombinant C4 proteins by transient transfection of COS cells. We found that an alloantiserum specific for C4d.1 reacts with the mutant protein but not the parent whereas an alloantiserum specific for C4d.2 reacts with the parent protein, as expected, but not the mutant. These results confirm that a single amino acid difference specifies the C4d.1 and C4d.2 allotypes.

Demonstration of an unusual allelic variation of mouse factor H by the complete cDNA sequence of the H.2 allotype.

Three allotypes of murine factor H have been identified serologically in the previous study (denoted H.1, H.2, and H.3). A cDNA clone coding for the entire length of murine factor H was isolated from a library constructed from the livers of STR/N mice which have H.2 allotype and was fully sequenced. The insert of this clone (STR309) contained 4184 nucleotides and consisted of a 47-bp 5' noncoding region, a 54-bp coding for leader peptide, a 3648 bp for the mature factor H protein, and a 435-bp 3' noncoding region. Compared with the previously reported sequence of the cDNA clone (MH8) isolated from B10.WR mice that have H.1 allotype, the size of the protein coding region was exactly the same, but 21 nucleotide substitutions resulting in 15 amino acid replacements were observed. The amino acid replacement/nucleotide substitution ratio (0.71) is far higher than those observed in the allotypic variations of other proteins. Four 15-base oligonucleotide probes specific for either STR309 or MH8 were synthesized and used in Northern blot analysis. The probes specific for STR309 hybridized with mRNA isolated from the livers of STR/N mice but not with mRNA from the livers of BALB/c mice that have H.1 allotype, whereas the reverse pattern was observed with the oligonucleotide probes specific for MH8. These results strongly suggest that the nucleotide sequence of STR309 represents H.2 allotype of factor H protein, providing an example of an unusual allotype with high ratio of amino acid replacements to nucleotide substitutions.

Structural basis for the C4d.1/C4d.2 serologic allotypes of murine complement component C4.

The C4d.1 antigenic specificity was first defined serologically in 1959 as an H-2-associated cellular alloantigen first designated "G," later H-2.7. It was subsequently shown to be an allotype of component C4 of the C system, with the antigenic determinant carried on the C4d proteolytic fragment of the alpha-chain, thus the designation C4d.1. Alloantisera defining an antithetical Ag, C4d.2, were also prepared. Previous studies in our laboratory showed that the structural difference between the two specificities resides in a single tryptic peptide of C4d. As an efficient approach to definition of the amino acid difference(s) involved, genomic clones covering the C4d regions from two H-2 haplotypes of the C4d.1 type have been prepared and sequenced, and compared with two sequences already available for C4d.2-type molecules. The results indicate that the rather striking serologic difference between C4d.1 and C4d.2 is attributable to the single amino acid substitution of arginine in C4d.2 for glutamine in C4d.1. The substituted residue is in a highly hydrophilic region of the C4 molecule, at a position homologous to one that contributes to the Chido/Rodgers serologic difference of human C4 molecules. This substitution also determines a new Pst I site in C4d.1 strains. A HindIII restriction fragment length polymorphism between C4d.1 and C4d.2 has also been observed.

Studies on recombination within the mouse H-2 complex: IV. Characterization of new recombinant haplotypes H-2t7, H-2t8, H-2as2, and H-2as3.

Four new intra-H-2 recombinants were characterized serologically and functionally. In two of these recombinants, B10.ASR1 and B10.ASR7, crossing over occurred between the A alpha and E alpha subregions, very probably in E beta since most intra-I region recombinants thus far investigated at the DNA level appear to involve recombination within the E beta gene. In the other two recombinants, B10.ASR2 and B10.ARS8, crossing over occurred between the S and D subregions. B10.ASR2 and B10.ASR8, crossing over occurred between the S and D subregions. B10.ASR7, which is serologically indistinguishable from B10.BASR1 and B10.S(8R), slightly stimulates and strongly responds to both of these strains in MLR. The probable location of the B10.S(8R) stimulatory product is E beta. The H-2 composition of B10.ASR1 is closest to that of B10.S(9R) and B10.HTT; therefore, precise definition of the cross-over point at the DNA level will be of particular interest.

Origin of the fourth component of complement related Chido and Rodgers blood group antigens.

We have reviewed the relationship between C4 and its related blood group and discussed the mechanisms whereby a fragment of C4 could become attached to erythrocytes (E). We hypothesize that there is chronic fluid-phase activation of C4 by either C1 to form C4b or spontaneous cleavage of the thioester to form iC4. These activated molecules bind to E. Proteolytic degradation of the bound C4b or iC4 would leave a covalently attached fragment of C4 on E and thereby give rise to the Ch and Rg blood group antigens. This system is of further immunopathologic interest since this 'normal' activation or turnover of C4 is closely regulated. In patients deficient in regulatory proteins, this spontaneous or normal turnover of C4 and C3 may initiate a pathologic condition.

Lymphokines regulate expression of cell surface antigens and production of suppressor factors by murine suppressor T cell hybridomas.

Suppressor T cell hybridomas specific for L-glutamic acid 60-L-alanine30-L-tyrosine10 (GAT) elaborate monoclonal suppressor factors (TsF) and bear surface markers such as H-2, I-J, and Thy-1 antigens. Over a period of years in culture, the production of TsF by such hybridomas has been constitutive and continuous; by contrast, the expression of surface markers has declined and some have been lost. The mixture of lymphokines in the supernatant fluid from concanavalin A-activated mixtures of allogeneic splenocytes (CAS) restored surface antigen expression, and the effective agents have been identified by using purified sources of lymphokines. Murine gamma interferon (IFN gamma) induced expression of H-2 class I antigens, but not I-J or Thy-1, in the hybridoma cells. Human interleukin 2 (IL-2) induced the increased expression of I-J and Thy-1 but not H-2 class I antigens. Interestingly, production of TsF was decreased by IFN gamma treatment but increased by IL-2, indicating some measure of regulation by lymphokines of the level of a protein that is constitutively produced.

Mice constitutive for sex-limited protein (SLP) expression contain multiple Slp gene sequences.

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins whose structural genes map to the S region of H-2. Serum C4 levels vary as much as 20-fold between C4 high (C4H) and C4 low (C4L) strains, and Slp expression can be null (SlpO), limited to male mice of a subset of C4H strains (Slp+), or "constitutive" (SlpC), in which female as well as male mice express Slp. In this study, we compare, by genomic Southern blot analysis, the C4 and Slp genes from eight congenic inbred mouse strains representative of three distinct phenotypes: C4H, Slp+ (two strains), C4H, SlpO (two strains), C4H, SlpC (three strains), and C4L, SlpO (one strain). By using cDNA probes that recognize both C4 and Slp genes, and are derived from the extreme 5' and 3' ends of the mRNA as well as internal coding sequences, we find no evidence to suggest that strain-specific variations in the expression of C4 and Slp are due to gross deletions of major portions of the structural genes. In most cases, two distinct C4/Slp genes are detected; hybridization with C4- and Slp-specific probes indicate that one of these is C4 and the other is Slp. The three SlpC strains are exceptional: they carry at least four C4/Slp genes; one of these hybridizes to the C4-specific probe whereas the remaining genes hybridize to the Slp-specific probe. Hence, multiple duplication of a gene containing Slp sequences has occurred in certain strains of mice, and this is accompanied by constitutive expression of the Slp protein.

The C4 and Slp genes of the complement region of the murine H-2 major histocompatibility complex.

Recent analyses, at the protein and DNA levels of structure, of the murine complement components C4 and the closely related sex-limited protein, Slp have led to new insights into the H-2/S region-linked C4 and Slp genes and their products. The primary products are 200 000 Da precursors which are cleaved, intracellularly and extracellularly, into the the mature alpha-beta-gamma-subunit molecules of plasma. Precursor order of subunits is beta-alpha-gamma; a complementary DNA clone spanning the alpha-gamma junction has been extensively analysed. The C-terminal of the alpha-chain is of particular interest because of post-secretion processing which differentiates 'secreted' and 'plasma' forms of C4, both apparently functional, and because allelic variants of C4 and the Slp protein, which differ substantially in molecular masses, owe their differences principally to different levels of glycosylation of the alpha-chain. Allelic variations in rate of C4 synthesis (C4-high compared with C4-low) have been analysed in cultures of hepatocytes and macrophages. Three distinct modes of genetic regulation of the expression of the Slp protein have been identified.