Molecular biology of the HLA class I and class II genes.

The human major histocompatibility complex (the HLA complex) encodes two classes of cell surface heterodimeric glycoproteins which regulate the immune response to foreign antigens. Molecular analysis of the HLA class I and class II genes has yielded considerable information about their organization and structure. The class I genes consist of the polymorphic A, B and C genes and the non-polymorphic Qa, Tla-like genes. The HLA class II region contains multiple alpha and beta genes and pseudogenes which map into three subregions: DR, DQ and DP. The class II genes share similar gene structures, but vary in their degree of polymorphism.

Multiple mechanisms regulate the expression of murine immune response genes.

The immune response antigens (la antigens) are heterodimeric cell-surface glycoproteins that regulate lymphocyte and macrophage interactions leading to the production of antibody against foreign antigens. The class II genes in the I region of the murine major histocompatibility complex encode the alpha and beta chains of the two known la antigens, I-A and I-E. Molecular cloning of the I region led to the discovery of three DNA regions, A beta 2, psi A beta 3, and E beta 2, which share some nucleotide homology with the classical class II beta genes. In this report we present evidence that A beta 2 is transcribed in spleen and in a cultured B cell hybridoma. A beta 2 mRNA is also found on membrane-bound polysomes, suggesting that it is translated. In contrast to the classical I region genes, A beta 2 transcripts are not induced in macrophage cells by gamma-interferon (gamma-IFN). This difference in sensitivity to gamma-IFN suggests that B cells and macrophages regulate the expression of the class II genes by distinct mechanisms.

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.

Immune interferon (IFN-gamma) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-gamma induces parallel expression of two other class II major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-gamma at similar rates but from initial levels that are 50% of maximal. After removal of IFN-gamma, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells.

How damaged is the biologically active subpopulation of transfected DNA?

Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of double-strand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex.

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.

Isolation of cDNA clones for the p33 invariant chain associated with HLA-DR antigens.

HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.

Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes.

At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence.

Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.

cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.

Isolation of cDNA clones encoding HLA-DR alpha chains.

HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.

Defined oligomeric SV40 DNA: a sensitive probe of general recombination in somatic cells.

We have constructed well defined oligomeric molecules of simian virus 40 (SV40) DNA as probes for investigating mechanisms by which cultured somatic cells recombine DNA. Restriction enzyme fragments from different temperature-sensitive mutants were joined in a head-to-tail orientation to create partial dimers 1.84 genome lengths in size. These molecules are too large to fit into a viral capsid. Therefore an assay that depends on production of progeny virus after infection with oligomeric DNA is a selective measure of precise conversion of oligomers to monomers. By constructing oligomers from appropriate combinations of temperature-sensitive DNAs, we have been able to study the conversion process in several defined regions of the SV40 genome. Our results indicate that conversion of oligomers to monomers occurs uniformly throughout the genome and is not dependent on normal viral DNA replication. These data indicate that conversion occurs primarily by general, homology-dependent recombination. At least one secondary mechanism that generates a low level of wild-type progeny was also detected. Studies with heteroduplex molecules indicate that repair of mismatched bases may be the secondary mechanism.

Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA.

Classical approaches to analysis of mitotic recombination by use of simian virus 40 (SV40) are limited in usefulness because of low frequencies of recombination. To bypass the apparent rate-limiting step in normal SV40 recombination, oligomeric SV40 was constructed in vitro by ligation of mixtures of pairs of linear DNAs carrying genetically distinct temperature-sensitive mutations. Cultured monkey cells infected with the unfractionated ligation products yielded frequencies of nonparental recombinant progeny that were increased up to 500-fold relative to cells infected with a mixture of the untreated circular molecules. Pairwise crosses were performed with tsB4, tsB8, and tsBC11 DNAs, using unfractionated oligomers constructed from linear molecules cleaved by EcoRI or BamHI. In each cross the fraction of progeny with nonparental genotypes was roughly proportional to the physical distances between the mutant sites. These results suggest a random, rather than site-specific, conversion of oligomers to monomers. Somewhat surprisingly, nonligated mixtures of linear tsB4 and tsB8 DNAs, created by EcoRI digestion, produced a 40-t to 100-fold increase in the frequency of nonparental progeny. These results indicate that intermolecular associations must occur with fairly high efficiency between these linear molecules.