Commonly used rat strains.

It is critical that genetically defined animals be used in immunological studies so that data can be adequately compared both within and between strains to examine the genetic effects on the phenomena being studied. This appendix lists some of the most commonly used rat strains and their immunogenetic properties.

Cytogenetic localization of the growth and reproduction complex (Grc) in the rat and in the mouse and its position in relation to RT1.EC and other loci in the rat MHC.

The segment of rat chromosome 20 (RNO20p12) that contains the classical loci of the major histocompatibility complex (MHC; RT1.A-RT1.E) also contains genes affecting growth, reproduction and susceptibility to chemical carcinogens (the Grc) and multiple genes encoding class I MHC antigens (the EC region). The relative positions of the MHC, Grc, and EC region have not been demonstrated explicitly, although they have been postulated from genetic mapping studies. The present study was undertaken to map these regions cytogenetically by several different approaches using cosmids specific for the Rps 18, Hspa1 and Bat1 genes. The order was shown to be: centromere-Rps 18-Hspa1-Bat1-EC-Grc.

Biological effects of genes in the Grc and EC region of the rat major histocompatibility complex.

PROBLEM: To study the mechanism of action of major histocompatibility complex (MHC)-linked genes affecting reproduction, growth, and susceptibility to chemical carcinogens. METHOD OF STUDY: Tumors derived from rat embryonic fibroblasts were transfected with cosmids from the Grc and its linked regions, the unrelated A region, and a nonMHC region, or with genes from the Grc, Grc-linked, and nonMHC regions, to determine whether they could suppress tumor growth as determined by in vitro (soft agar) and in vivo assays. RESULTS: Tumor fibroblasts transfected with cosmids from the Grc or from the EC region decreased tumor growth in both the in vitro and in vivo assays. Transfection with individual genes from the Grc had no effect on tumor growth in either assay. CONCLUSIONS: The effects of the Grc on reproduction, growth, and tumorigenesis are mediated by extended genetic effects, i.e., by the conformation of the DNA in this region. Similar effects were seen following transfection with cosmids from the Grc-linked EC region, and this finding strengthens the hypothesis that the conformation of the DNA in this general region is critical for its function. A similar effect has been described for the locus control region (LCR) in the beta-globin gene family in the human.

Immunogenetic and oncogenic properties of rat trophoblast cell lines.

PROBLEM: The nature of major histocompatibility complex (MHC) antigen expression on rat placentas, trophoblast cell lines, and tumors derived from trophoblast cells was explored. METHOD OF STUDY: Cytohistochemical and flow cytometric analysis and molecular techniques. RESULTS: MHC antigen expression and genomic imprinting on the placenta and on trophoblast cells varies with the time of gestation and with the type of MHC antigen. CONCLUSIONS: There is no correlation in trophoblast cells between class I expression and cell ploidy, on the one hand, and malignant potential, on the other hand. Genomic imprinting of class I antigens in the rat placenta is a quantitative phenomenon.

Recent studies on the structure of the grc region in the rat.

PROBLEM: To explore the nature of the genes in the grc. METHOD: Chromosome walking of the grc and sequencing new genes. RESULTS: The RT1.O,N genes have been aligned, and two new types of genes, RT1.S1,2 and Rprl,2,3, have been discovered and mapped. An extensive physical map of the grc region is presented. CONCLUSION: The disease associations in both the rat and the human appear to cluster in two regions that map in approximately the same place in both species, given the translocation that occurred in the major histocompatibility complex (MHC) of the rat relative to the MHC of the human.

Physical mapping and structural analysis of new gene families RT1.S and Rps2r in the grc region of the rat major histocompatibility complex.

Five new genes were identified in the growth and reproduction complex (grc) region: RT1.S1, RT1.S2, Rps2r1, Rps2r2, and Rps2r3. The class Ib RT1.S1 and RT1.S2 genes have five distinct exons (1, 4, 5, 6, 7) similar to other class I major histocompatibility complex genes but the conventional exons 2 and 3 are absent. The genes are 97% similar, have CAAT and TATA boxes much upstream of the conventional position, obey the GT/AG rule in their exon-intron boundaries, and are transcribed at a low level in thymus and testis but not in the liver and spleen. The region between exon 1 and exon 4 was analyzed by obtaining transcripts by reverse transcription-polymerase chain reaction (RT-PCR) amplification which revealed the presence of four alternatively spliced mRNA transcripts of RT1.S1: 1) S1-1 (clones 14 and 16) has no exon between exons 1 and 4; 2) S1-2 (clones 7 and 8) has an exon of 45 nucleotides that can translate into 15 amino acids; 3) S1-3 (clone 5) has an exon of 42 nucleotides with a stop codon; and 4) S1-4 (clone 10) has two exons of 42 and 38 nucleotides, respectively, with stop codons. Only one RT1.S2 mRNA transcript was obtained, and it has an exon of 45 nucleotides between exon 1 and exon 4 which can form a peptide identical to the S1-2 isoform for that region. The 45 nucleotide exon between exon 1 and exon 4 was unique for RT1.S1 and RT1.S2 and only matched a sequence in the RT1.O intron region (nucleotides 2905 - 2949). The three ribosomal-protein-S2-related (Rps2r) genes are 94% - 98% similar; they are related to the genes encoding ribosomal protein S2 of the black rat and the LLRep3 genes of the mouse and the human and to the genes encoding Saccharomyces cerevisiae S4, Escherichia coli S5, and other members of prokaryote S5 family. The Rps2r1 gene is located just outside of the grc region. The Rps2r2 and Rps2r3 genes are in the grc and have multiple stop codons in their genomic sequences. The Rps2r1 mRNA transcript was identified by RT-PCR in the thymus and testis but not in the liver and spleen.

Malignant potential, major histocompatibility complex antigen expression, and genomic imprinting of rat trophoblast cell lines.

Invasive growth, variation in major histocompatibility complex antigen expression, and genomic imprinting are important properties of both trophoblast cells and malignant tumors. This study, undertaken to address these three issues, used cultured trophoblast cell lines derived from Day 11/12 rat placentas of all mating combinations of the DA and WF inbred strains. In addition, genomic imprinting was also examined in intact rat placentas from Days 11-19. There was no correlation in trophoblast cells between class I antigen expression, DNA content, and cell ploidy on the one hand and oncogenic potential on the other hand. The constitutive suppression of class II antigens in the trophoblast cells could not be abrogated by treatment with interferon-gamma, whereas such treatment always maximally induced class I antigen expression regardless of the initial resting levels. The trophoblast cells at Day 11/12 expressed both maternal and paternal class I antigens, and studies in whole placental tissues showed that the imprinting of the maternal class I antigens was manifested by a decreased level of expression rather than an absence of expression. Thus, genomic imprinting in the rat placenta is a quantitative, rather than an all-or-none, phenomenon.

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 the E/C region, or a large part thereof, and the other (110 kb) contained the grc 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 the E/C region using Eu-specific and LW2 (RT1.C)-specific primers showed that each cosmid contained both Eu-like and C-like genes. They are clearly different but closely associated, and they show some variation from the prototypic E (Eu) and C (LW2) genes, respectively. Comparison of DNA from grc+ and grc- strains of rats showed that the deletion in the grc- strains was approximately 50 kb, and that it was located on two of the three cosmids in the grc-region contig. The use of specific class I probes showed that the grc region contained tandemly duplicated RT1.O-RT1.N genes and that the RT.BM1 loci lay outside of the grc region. Neither contig reacted with probes specific for class II, TNFA, Hsp70, or RT1.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) and MHC-linked region of the rat is: A-E/C-grc-M.

Expression of the rat MHC class I gene in the human B lymphoblastoid cell line Hmy 2 CIR.

Rat MHC class I cDNA (e.g. RT1.A1) can be expressed optimally as a heterodimer consisting of the rat heavy chain and the human beta2-microglobulin in the human B lymphoblastoid cell line Hmy 2 CIR, which offers several advantages. Gene expression was detected by flow cytometry using anti-human beta2-microglobulin and anti-rat heavy chain antibodies.

Seminal fluid and the expression of MHC class I antigens in the placenta of the rat.

PROBLEM: To determine whether seminal fluid influences the expression of MHC class I antigens on the surface of basal trophoblast cells in the placenta of the rat. METHODS: Transfer of DA x DA embryos into a WF (allogeneic) or DA (syngeneic) recipient made pseudopregnant by hormonal treatment followed by mating with a vasectomized male (seminal fluid) or by mechanical stimulation (no seminal fluid). Antigen expression was determined by electron microscopic immunocytochemistry using the appropriate gold-labeled monoclonal antibodies. RESULTS: Seminal fluid did not affect the expression of MHC class I antigens on the surface of the basal trophoblast in either allogeneic or syngeneic matings. CONCLUSIONS: The suppression of the expression of paternal class I antigens on the surface of the basal trophoblast cells in allogeneic pregnancies most likely occurs at the genome level shortly after fertilization.

Nucleotide sequence and structural analysis of the rat RT1.Eu and RT1.Aw3l genes, and of genes related to RT1.O and RT1.C.

A cDNA library was constructed using mRNA isolated from the R21 strain of rats which have the major histocompatibility complex (MHC) haplotype RT1.AlBlDlEu and the growth and reproduction complex (grc) genotype grc+. The cDNA clones that hybridized with the class I probes pAG64c and pARI.5 and were 1.3-1.7 kilobases were selected. Full-length clones were identified by sequencing partially the 5' and 3' ends of each clone, by the presence of a start codon at the 5' end, and by a polyadenylation sequence at the 3' end. The full-length cDNA clones were examined for in vitro transcription by transfection into human CIR cells using electroporation, and expression was detected by flow cytometry using monoclonal antibodies specific to the heavy chains and polyclonal antibody to beta 2-microglobulin. The RT1.Eu gene was transcribed and expressed optimally, and its nucleotide and deduced amino acid sequences differed significantly from the RT1.Aa, RT1.A(l), RT.Au, LW2, and 11/3R genes but only slightly from the RT1.K gene. The high level of sequence similarity between RT1.Eu and RT1.K suggests that the two genes may have originated from a common ancestral gene. In addition, three new genes (RT1.Aw3l, RT1.C-type, and RT1.O-type) were identified. The RT1.Aw3l gene is almost identical to RT1.A(l) with the exception of an in frame deletion of 21 nucleotides in exon 2 leading to a 7 amino acid deletion in the alpha 1 domain of the deduced amino acid sequence and 11 nucleotide substitutions and insertions in the rest of the sequence. It transcribed optimally, but no significant expression was detected. The RT1.C-type gene 119 is very similar (97%) to the LW2 gene in the 3' untranslated region, which suggests that it is in the RT1.C region. It transcribed optimally, but no significant expression was detected. The RT1.O-type gene 149 has all the features of a class Ib gene, but a premature stop codon in the alpha 1 domain causes incomplete translation. Its in vitro transcription was very low, and no expression was detected. These studies, combined with previous work, indicate that in the MHC of the R21 strain three class Ia genes (Eu, A(l), Aw3l) and three class Ib genes (C-type, O-type, N) are transcribed but only two class Ia genes (Eu, A(l)) are expressed.

Role of methylation in placental major histocompatibility complex antigen expression and fetal loss.

The purpose of this study was to determine the role of gene methylation on major histocompatibility complex (MHC) antigen expression in the rat placenta, specifically: 1) the constitutive suppression of labyrinthine class I genes and of all class II genes, 2) genomic imprinting of class I genes, and 3) the amount of fetal loss in relationship to gene methylation. Placentas from all four mating combinations, or a subset thereof, of the inbred DA and WF strains of rats were studied simultaneously through 1) molecular assessment of their levels of methylation at various stages of pregnancy and changes in methylation after treatment with 5-azacytidine and 2) immunohistochemical determination of their MHC class I and class II antigen expression. Treatment with 5-azacytidine caused demethylation of both class I and class II genes, the level depending upon the method of administration of the drug. Treatment with 5-azacytidine did not, however, alter the expression of either the class I or class II antigens. Hence, demethylation is not involved in the constitutive suppression of labyrinthine class I genes or that of all placental class II genes, and it is not involved in the genomic imprinting of placental class I genes. The effect of 5-azacytidine on fetal loss is due to its direct cellular effects and not to an immunological mechanism.

Genomic structure and organization of a Q-like gene in the GRC-G/C region of the rat.

In the rat homologue of the mouse Q/TL region, grc-G/C, a TL-like gene (RT1.N) has been identified recently. This paper reports on a Q-like gene, designated RT1.0, that maps in the same region. It contains a 5' untranslated region (UT), signal peptide, alpha 3 domain, transmembrane region, cytoplasmic domain (three exons) and 3'UT region. Comparison with mouse class-I genes shows that the greatest similarity is to the H-2Q, K, D and L genes; it is very different from the TL genes of the mouse and rat. A sequence that includes many CT repeats occurs in the 3'UT region of RT1.0 and in three to five other class I-hybridizing fragments. Thus, the MHC-linked region of the rat contains both Q-like and TL-like class-I genes.