Thomas J. GillIII

Mapping in the mouse of the region homologous to the rat growth and reproduction complex (grc)

One stock of rats homozygous for the RT11 haplotype have a small body size and their reproductive capacity is impaired, particularly in males, in comparison to the R T l l / h e t e r o z y g o t e s (Gill and Kunz 1979; Kunz et al. 1980). The genes responsible for the effects on fertility and body size are separable by recombination, being at a distance of 0.07 centimorgans (cM) from each other and at a distance of 0.45 cM from the R T 1 . A locus (Kunz et al. 1980). The two genes are referred to as f i ( fer t i l i ty) and d w 3 ( d w a r f 3 ) , while the region occupied by them has been designated g r c ( g r o w t h a n d r e p r o d u c t i o n c o m p l e x ) . The grc is located between the R T 1 . E and R T 1 . G genes, which are believed to delineate a region homologous to the mouse Q a / T l a region (Inomata et al. 1986). The order of loci in the R T 1 complex is A . . .

The RT1.G locus in the rat encodes a Qa/TL-like antigen.

A new antigenic system in the rat homologous to theQa/TL antigen system in the mouse has been characterized. It was detected by antibodies raised in donor-recipient combinations that were matched for theRT1. A, B, D, E loci in the major histocompatibility complex (MHC): (R11×BN)F1 anti-BN.1L(LEW), (R18×BN)F1 anti-BN.1L, and BN.1LV1(F344) anti-BN.1L. Absorption analyses using these antisera and a variety of inbred, congenic and recombinant strains identified three alleles,RT1.Ga,Gb,Gc, of whichGc is a null allele. The strain distribution of these alleles was determined, using 37 strains of rats representative of all of the prototypic haplotypes and a number of congenic and recombinant strains. The use of the congenic and recombinant strains showed that theRT1.G locus was linked to the MHC and that the most probable gene order wasA-E-G. Testcross analysis showed that the map distance betweenA andG was 1.4 cM(4/285 recombinants). The RT1.G antigen has a heavy chain ofMr 46 000 and is present on both T and B cells.

Physical mapping of the E/C and grc regions of the rat major histocompatibility complex

Alignment of class I-hybridizing cosmids from an R21 (AlBlDlEugrc+) genomic DNA library gave two contigs: one [150 kilobases (kb)] encompassed theE/C region, or a large part thereof, and the other (110 kb) contained thegrc region which has genes influencing resistance to chemical carcinogens (rcc), fertility (ft), and growth (dw-3). Amplification of gene sequences in the four cosmids in theE/C region usingEu-specific andLW2 (RT1.C)-specific primers showed that each cosmid contained bothEu-like andC-like genes. They are clearly different but closely associated, and they show some variation from the prototypicE (Eu) andC (LW2) genes, respectively. Comparison of DNA fromgrc+ andgrc− strains of rats showed that the deletion in thegrc− strains was approximately 50 kb, and that it was located on two of the three cosmids in thegrc-region contig. The use of specific class I probes showed that thegrc region contained tandemly duplicatedRT1.O-RT1.N genes and that theRT.BM1 loci lay outside of thegrc region. Neither contig reacted with probes specific for class II,TNFA, Hsp70, orRT1.M genes. The data presented here and the previous data in the literature (summarized in Gill et al. 1995) suggest that the gene order in the major histocompatibility complex (MHC) andMHC-linked region of the rat is:A-E/C-grc-M.

Genetic and immunological characterization of naturally occurring recombinant B3 rats

The B-stock population of rats was bred for homozygosity at the loci controlling coat color. In this process, theAg-B1 andAg-B3 haplotypes became fixed in Hardy-Weinberg equilibrium. Extensive immunization and absorption studies showed that the specificities in the B-stock rats homozygous for theAg-B1 haplotype were the same as those found in the inbred F344 strain (Ag-B1), and that the specificities in the rats homozygous for theAg-B3 haplotype were the same as those found in the inbred BN (Ag-B3) strain. A homozygous line derived from the rats carrying theAg-B3 haplotype (B3) has the mixed lymphocyte reactivity and antibody responsiveness to poly (Glu52Lys33Tyr15) characteristic of the inbred strains in theAg-B4 group. Thus, it represents a naturally occurring recombination between the loci controlling MLR and immune responsiveness, on the one hand, and those controlling the Ag-B antigens on the other. Antibody responsiveness segregated with theAg-B3 haplotype in crosses between the B3 homozygotes and the low responder BUF and M520 strains; hence, this recombination is a stable one. There was no linkage of antibody formation or haplotype to coat color. The finding of a strain with a naturally occurring recombination in the major histocompatibility complex between the loci controlling mixed lymphocyte reactivity and the Ag-B histocompatibility antigens provides evidence for the separateness of these loci. Since the portion of the genetically determined mechanism controlling antibody responsiveness which is linked to the MHC was that characteristic of the MLR type, it too must lie outside the region defined by the serological specificities of theAg-B haplotype.

CML characterization of a product of a second class I locus in the rat MHC.

In the rat, genes that control the expression of target antigens detected by cell-mediated lympholysis (CML) are present in the major histocompatibility complex (MHC). The relationship of these loci, CT and Ag-L, to each other and to other loci within the MHC is unknown. In this report, we demonstrate the existence of a CML target antigen in the (DA × BN)F1 anti-DA.11(BI) strain combination. The gene coding for this antigen is linked to the RT1 complex as indicated by the CML reactivity of targets from backcross and congenic animals. Inhibition studies demonstrated that this antigen has the widespread tissue distribution characteristic of class I antigens, and the gene coding for this CML antigen maps coincident with the RT1.E class I locus as indicated by the lysis of targets from the recombinant strains r10 and r11. The CML can be blocked by antisera directed against a product of the RT1.E locus. The locus controlling this CML reactivity, like CT and Ag-L, has been separated from RT1.A by recombination; unlike CT and Ag-L, the product of this CML locus appears to be identical with an RT1.E allelic product that has been serologically identified and biochemically characterized.

Linkage of the locus encoding the A chain of α-crystallin (Acry-1) to the major histocompatibility complex in the rat

Alpha crystallin is one of three types of structural protein found in mammalian eye lens. Two distantly related genes encode the c~-crystallins, c~ A 2 and c~ B 2 (Berns et al. 1972, 1973). Other forms of e-crystallin arise by deamidation of e A 2 and e B2 (Delcour and Bouchet 1978) or, in some species of rodents, via alternative splicing of eA chain precursor in RNA (King and Piatigorsky 1984). The amino acid sequences of c~A-crystallin from rats and mice are identical (King et al. 1982, de Jong et al. 1975), which is consistent with the highly conserved nature of this protein (Wilson et al. 1977, Kramps et al. 1977). Using restriction fragment length polymorphisms, Skow and Donner (1985) mapped the locus encoding mouse eA-crystallin (Acry-1) to chromosome 17, between glyoxalase (Glo-1) and H-2K of the major histocompatibility complex (MHC). The present study utilizes a similar experimental approach to demonstrate linkage of the rat ~A locus, designated Acry-1, to the MHC. The DNA was prepared from spleens of inbred and congenic strains of rats maintained by the Department of Pathology, University of Pittsburgh. The strains used in this study and their MHC haplotypes were: ACP (avl), DA (avl), BUF (b), PVG (c), KGH (9), BN.1G(KGH)(9), BI (/), WKA (k), OKA (k), LEW (f), F344 (~vl), NBR (fvl), BN (n), MR (o), WF (u), BN.1U(WF) (u), YO (u), and TAL (dvl). The DNA preparation and restriction enzyme analysis are described in detail elsewhere (Skow and Donner 1985). Briefly, DNA was digested to completion with various restriction endonucleases, fractionated by submarine electrophoresis in 0.75% agarose gels, denatured, and transferred to nylon membranes (Zetabind, AMF Cuno). The DNA fragments containing cA-coding regions were detected by autoradiography after hybridization to 32p-labeled eA cDNA prepared from mouse eA mRNA (King et al. 1982). Five endonucleases were used to search for restriction polymorphisms in or near the c~A gene of the rats: Bam HI, Eco RI, Hinc II, Hind III, and Pst I. Polymorphism

Gene order in the major histocompatibility complex of the rat

The loci in the major histocompatibility complex (MHC) of the rat which code for class I and class II antigens—RT1.A and RT1.B, respectively — have previously been separated by laboratory-derived recombinants and by observations in inbred and wild rats. Closely linked to the MHC is the growth and reproduction complex (Grc) which contains genes influencing body size (dw-3) and fertility (ft). These phenotypic markers were used in this study to orient the A and B loci of the MHC. Two recombinants were used for mapping. The BIL(R1) animal is a recombinant between the MHC and Grc, and it carries the haplotype RT1.AlBlGrc+. The r10 animal is an intra-MHC recombinant, and it has the haplotype RT1.AnB1Grc. These recombinants were characterized serologically, by mixed lymphocyte reactivity, by immune responsiveness to poly (Glu52Lys33Tyr15) and by the presence of the dw-3 gene. The data demonstrate that the gene order of the loci is: dw-3-RT1.B-RT1.A.

Comparison of rat MHC class I antigens by peptide mapping

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.An, RT1.Au, and RT1.Eu were used for immunoprecipitation of antigens biosynthetically radiolabeled with14C- or3H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (An and Au) differed by 30%, whereas the products of two different loci in the same haplotype (Au and Eu) differed only by 1–3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in Au have been substituted by an arginine and probably by a tyrosine residue, respectively, in Eu. The high degree of homology between the products of theA andE loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.

THE GENETIC CONTROL OF THE ANTIBODY RESPONSE IN INBRED RATS

SummaryThe antibody response of genetically inbred rats to poly(Glu52Lys33Tyr15) is controlled by a complex polygenic system which includes at least two autosomal genes and a sex influence, which may also be genetically determined. The genetic control of the quantity, binding constants, and specificity of the antibody formed is linked to the major histocompatibility locus. Factors other than the major genetic ones and the sex influence also affect the quantity of antibody formed, since animals of the same genotype can make significantly different amounts of antibody, depending upon the crosses by which they acquire the major histocompatibility alleles.After immunization with poly(Glu52Lys33Tyr15) the low responders make fewer antibody-producing cells, are not capable of mounting a delayed hypersensitivity reaction to the polypeptide and appear to be deficient in their ability to produce the specific IgM antibody. Immunization of the low responders with antigen aggregated with methylated bovine serum albumin enhances the quantity of antibody formed, increases the binding constants and crossreactivity of the antibody and enhances the delayed hypersensitivity response. In contrast to the findings with the L-amino acid polypeptide, there does not appear to be any genetic control over the antibody response to the D-amino acid enantiomorph poly(dGlu52dLys33dTyr15), which is minimal in all strains.

Detection of an Ss-like protein in the sera of inbred and wild rats

A normal serum protein that crossreacts with rabbit anti-mouse Ss serum was isolated by alternating gel nitration and ion exchange chromatography from the inbred Long-Evans (LGE) rat strain. Rabbit antisera prepared against this protein detected it in the sera of all inbred and individual wild rats tested. The close physical and immunochemical similarity between this protein and the mouse C4 component of complement (Ss protein) indicates that this protein may represent the rat homolog of the mouse C4. Quantitative differences in the level of the Ss-like rat protein, comparable to those seen in Ss low mice, were not observed in 25 inbred strains or 22 individual wild rats. These quantitative results were supported by functional assays for total hemolytic complement and individual C2, C3, and C4 complement components. Sixteen inbred strains were examined and all had normal levels of activity for each of the assays.