Conservation and diversity in the class I genes of the major histocompatibility complex : Sequence analysis of a Tiab gene and comparison with a Tlac gene ( multigene family / thymus leukemia antigen / DNA sequence )

The thymus leukemia (TL) antigens, encoded by class I genes in the Tla subregion of the major histocompatibility complex (MHC), are cell surface molecules expressed on thymocytes of certain strains of mice and on certain T-cell leukemias. In order to study the fine structure and interrelationships of genes of the TM subregion, a Tla-specific probe was isolated from the TL-encoding T13C gene ofBALB/c mice (Tb" haplotype). The probe hybridized with two Tla genes in the Thf haplotype (T13C and T3C) and with only one in the Tlab haplotype (T3b). Examination of this subset of Tla genes (T3b, T3Y, and T13c) by restriction enzyme analysis and oligonucleotide hybridization studies confirmed that T3b is the allele of T3C and that T3Y and T13c may have arisen by duplication. The T3b gene, while not transcribed in the tissues of the TLstrain C57BL6, was shown to be transcriptionally active in the TL-expressing leukemic cell line ERLD derived from that strain. The T3b gene was cloned and its complete DNA sequence was determined. These data permit complete comparison of two Tla-region genes, T3b and its homologue T13c, and allow us to conclude that these genes show extraordinarily high sequence conservation, in contrast to alleles of the H-2Kand H-2D-region genes. Comparison of T3b with other class I sequences in the H-2 and Qa subregions suggests that the T3-subset genes are the most divergent from other class I genes. The major histocompatibility complex (MHC) class I genes are a multigene group containing the H-2 genes (K, D, and L), the Qa genes, and the Tla genes. The number ofgenes in each subregion varies according to the haplotype, from 2 to 4 for the H-2 subregion, 8 to 10 for the Qa subregion, and 13 to 18 for the Tla subregion (1, 2). These genes have a number of features that distinguish them. The H-2 gene system, in particular the K and D genes and to a smaller extent the L gene, is marked by an extreme polymorphism (>50 alleles per locus; ref. 3) and diversity (15-20o differences in alleles) possibly associated with the role of the products in guiding T lymphocytes to react to cells bearing foreign antigens. On the other hand, those products of the Tla and Qa subregions that have been studied are significantly less polymorphic [e.g., only 6 alleles of Tla have been described (Tlaa-; ref. 4)]. In addition, unlike the H-2 products which are expressed on all nucleated cells, the TL and Qa products have a more restricted tissue distribution. The functions ofthe products of the Tla and Qa genes are unknown. To study the features of a specific gene (and its product) in the Tla subregion, we have cloned and determined the nucleotide sequence of the T3b gene,¶ which encodes a putative TL protein from the Tlab haplotype. The availability of this sequence allows the comparison of this gene to Tla genes of the Tlac haplotype, as well as to other class I genes, and refines our understanding of the interrelationships of the class I gene group. MATERIALS AND METHODS Mice and Cell Lines. Mice were obtained from The Jackson Laboratory (C57BL, A, BALB/c, 129); from L. Flaherty (Albany, NY) (A.CA, B6.AC2-Tlad, B6.AC1) or from Memorial Sloan-Kettering Cancer Center (B6.K1, B6.K2, C57BR-Gix(+), A-Tlab). The H-2, Qa, and Tla haplotypes of each strain are shown in Fig. 1. The TL-expressing ERLD leukemia cell line, which was induced in the C57BL strain, was kindly provided by E. Stockert (New York, NY). DNA and RNA Preparation and Analyses. DNA was prepared from spleen cells as described (5). Restriction enzymes were used under the conditions specified by the supplier (Bethesda Research Laboratories). Digested DNA was electrophoresed in agarose gels and transferred to GeneScreen (New England Nuclear) (6). Whole-cell RNA was prepared from fresh tissues as described by Auffray and Rougeon (7). RNA was size-fractionated in agarose/formaldehyde gels and transferred to nitrocellulose. The filters were hybridized, washed, and subjected to autoradiography as described (5). Isolation and 32P-Labeling of Cloned TL Probe. A 0.1kilobase (kb) Pst I-Sac I fragment was subcloned from X-17.3 (8) (containing the T13C gene) into pBR322. The recombinant plasmid was labeled by nick-translation and separated from unincorporated [a-32P]dGTP as described by Maniatis et al. (9). Preparation of TL-Specific Oligonucleotides. Oligonucleotides were chemically synthesized by either the phosphotriester (Bachem Fine Chemicals, City of Hope, CA) or the phosphoramidite (Applied Biosystems, Foster City, CA) solid-phase method. Sequencing of T3b Gene. Fragments of the H10 cosmid (2) were subcloned in both orientations, using M13 mplO, mpll, mp18, and mpl9 vectors (Pharmacia). DNA sequencing was performed according to Sanger et al. (10), using either the specific M13 primers (Pharmacia) or T3-specific oligonucleotides. Abbreviation: kb, kilobase(s). $We use terminology (to be described elsewhere) in which the Tla genes are designated by the letter T, followed by a number referring to gene position along the chromosome, with a superscript referring to the Tla haplotype. Thus the previously described gene 17.3A is called T13C (see Fig. 2). 1782 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 1783 RESULTS AND DISCUSSION Identification and Preparation of the TL-Specific Probe. To learn more about the interrelationships among Tla genes, we initiated studies to isolate and sequence a gene coding for a TL product in the Tlab haplotype. Since peptide map analysis (11) had shown an extraordinary similarity between TL products of the Tlab and Tiac haplotypes, and a DNA fragment that expressed a Tiac (T13) product was available (12), we subcloned from that DNA a 0.1-kb Pst I-Sac I fragment from the 3' end of exon III (designated pTL). This probe was specific for the Tla subregion and hybridized to a subset of Tla genes when genomic DNA from a panel of inbred and recombinant mouse strains (Fig. 1) was examined. The pTL probe was shown to be a single-copy probe for the Tlab haplotype, as it hybridized to a single Bgl II restriction enzyme fragment in C57BL DNA (Fig. 1). Similar results were obtained using BamHI, EcoRI, HindIIl, Sma I, andXba I restriction endonucleases (data not shown). The TL Probe Detects the T3b Gene. A panel of cosmids containing 26 class I genes spanning the H-2, Qa, and Tla subregions of C57BL mice (2) was screened with pTL (data not shown). Only the T3b gene from the H10 cosmid clone hybridizes with pTL, consistent with the finding that pTL hybridizes to a single gene in C57BL genomic DNA (Fig. 1). Interrelationships of the Subset of Genes Resembling T3. The pattern of hybridization of the pTL probe with genomic DNA (Fig. 1) revealed different numbers of genes for different Tla haplotypes. For example, Tlab had one band, both Tlac and Tlkhad two bands, and both Tlaa and Tiad had three bands. The finding of different numbers of genes resembling T3 in the various haplotypes is consistent with previous results (8) in which Tla subregions of the Tiac and Tlab haplotypes were aligned on the basis of restriction endonuclease maps (Fig. 2A). In that comparison, T13C was found to have no allele in Tlab because it resided on a cluster of genes, TJc'-TJ7c, that was not present in the C57BL mouse. Since T3b and T3C share all Kpn I and EcoRI sites, and T13C lacks one EcoRI site and has additional Xba I and Sma I sites (Fig. 2B), our data indicate that T3b is the allele of T3C and not the allele of T13c, thus confirming the alignment shown in Fig. 2A. Further support for the close relationship of T3b and T3C as compared to T13C comes from hybridization analyses with