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Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.

A full-length cDNA clone encoding the human gastric H,K-ATPase (EC 3.6.1.36)beta-subunit was isolated from a human gastric mucosal lambda gt10 library using oligonucleotide probes which were based on the cDNA sequence from rat and rabbit H,K-ATPase beta-subunits. The insert was 1407 bp in length and encoded a polypeptide of 291 amino acids with a MW = 33,367 Da. It exhibited 84.2%, 85.6% and 81.3% identity to the H,K-ATPase beta-subunits of rabbit, pig and rat, respectively.

Cruciferin (12S globulin) is the major seed protein in Brassica napus (oil seed rape). It is synthesized during seed development and consists of six subunit pairs. Each of these pairs is synthesized as a precursor containing one alpha and one beta chain. At least three different precursors exist (P1-3), giving rise to four different mature subunits (cru1-4). Several cruciferin clones were isolated from a seed mRNA cDNA library. Comparison of the deduced amino acid sequences of these clones to amino acid sequences of purified cruciferin chains and peptides identified them as coding for cru2/3 and cru4 subunits. From the amino acid sequences deduced from two overlapping cDNA clones, the precursor of the cru4 subunit was shown to consist of 465 amino acid residues. Comparison of cruciferin and cruciferin-related sequences from B. napus and Arabidopsis thaliana, respectively, suggested that early during evolution the Brassicaceae family only possessed two types of 11-12S globulin genes, like the present-day Fabaceae.

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.

Sequences of different sizes are generated when DNA from homozygous HLA-Dw/DR typing cells are digested with restriction endonuclease and analyzed by hybridization with a HLA-D region class II antigen beta-chain cDNA probe. The patterns of hybridization were highly polymorphic but one endonuclease, BamHI, defined sequences unique to all HLA-Dw/DR specificities 1-8 except HLA-Dw/DR 2 and 6; however, these two specificities were resolved with the enzyme EcoRI. Digestion with other endonucleases such as Pst I results in patterns of restriction fragments that differ between homozygous typing cells of the same HLA-Dw/DR specificity. HLA-D region beta-chain probes permit HLA-D region genotyping at the DNA level and may allow detection of genes controlling the association of HLA specificities with a wide variety of diseases.

Membrane-bound mRNA was isolated from Raji cells and enriched for message coding for the HLA-DR transplantation antigen alpha-chain by sucrose gradient centrifugation. Double-stranded cDNA was constructed from this mRNA fraction, ligated to plasmid pBR322, and cloned into Escherichia coli. By hybrid selection, a plasmid, pDR-alpha-1, able to hybridize with mRNA coding for the HLA-DR alpha-chain was identified. From the nucleotide sequence of one end of the insert an amino acid sequence was predicted which is identical to part of the amino-terminal sequence of an HLA-DR alpha-chain preparation isolated from Raji cells. This clearly shows that pDR-alpha-1 carries almost the complete message for an HLD-DR alpha-chain. From the nucleotide sequence of this plasmid it will be possible to predict the primary structure of an HLA-DR alpha-chain.

The murine major histocompatibility complex is known to express two class II molecules, A and E. They are composed of one alpha- and one beta-polypeptide chain. Recently, we reported the finding of an additional beta-chain second domain exon tentatively designated A beta 2. We describe here the nucleotide sequence of the A beta 2 gene and an A beta 2 cDNA clone. A beta 2 displays the same exon organization as the known expressed beta-chain genes, and the predicted A beta 2 polypeptide shows all the characteristic features of the expressed beta-chains. The predicted A beta 2 polypeptide shows 49-56% amino acid sequence identity to A beta and E beta chains and to the human DP, DQ, and DR beta-chains. These are 63-79% homologous to each other, indicating that A beta 2 is the most divergent member of the beta-chain family. The A beta 2 gene seems to be transcribed in the same types of cells as other class II genes. Detection of incompletely spliced A beta 2 mRNA and the finding of a cDNA clone containing an intron segment suggest that A beta 2 transcripts are processed slowly. Hybridizations with A beta 2 probes to restriction enzyme-digested genomic DNA indicate that the A beta 2 gene displays lower allelic polymorphism than the A beta gene.

cDNA for a beta-chain of HLA-DR antigens was cloned and the partial nucleotide sequence was determined. The data suggest that the beta-chain consists of approximately 230 amino acids, of which about 200 are exposed on the cell surface. The beta-chain appears to be composed of two exposed disulphide-containing domains. The arrangement of the disulphide loops suggests that the beta-chain is similar in structure to the HLA-A, B, C antigen subunits and the immunoglobulin chains. For the beta-chain domain closest to the membrane this similarity was verified at the level of primary structure. The partial amino acid sequence of the NH2-terminal domain did not display any apparent homology to HLA-A, B, C antigens and immunoglobulins. However, the similarity established here between the two types of major histocompatibility antigen subunits and the immunoglobulin chains suggests a common ancestral origin for at least some regions of these molecules.

The invariant gamma chain is transitorily associated with class II histocompatibility antigens during intracellular transport. We have isolated and sequenced a cDNA clone corresponding to the human gamma chain. mRNA hybridizing to the cDNA clone translated into a 33,000-dalton chain that associated specifically with class II antigen alpha and beta chains. The gamma chain consists of 216 amino acids. The two N-linked carbohydrates are attached to asparagines 114 and 120. A continuous stretch of hydrophobic and neutral amino acids occurs in positions 31-56 from the NH2 terminus. This region seems to constitute the transmembrane portion of the polypeptide chain. The positions of the carbohydrate moieties and the putative transmembrane segment indicate that the NH2 terminus of the gamma chain resides on the cytoplasmic side of the membrane. Cell-free translations in conjunction with radiochemical amino acid sequence analyses suggest that the gamma chain lacks an NH2-terminal signal sequence.

Hybridizations of HLA-DR and DC transplantation antigen beta chain cDNA probes to restriction enzyme digested genomic DNA were examined in regard to their potential in tissue typing. DNA from cells, typed by cellular techniques to be homozygous for the specificities Dw 1 to 8, gives rise to unique fragment patterns for each specificity. Hybridizations to DNA from unrelated individuals serologically typed to be DR identical occasionally reveal genetic differences in the class II antigen region, notably with the DC beta cDNA probe. In contrast, hybridizations to DNA from monozygotic twins displayed completely identical patterns with both types of probes. From these data it seems reasonable to conclude that genomic hybridizations with class II antigen probes will be useful in the definition of new class II antigen loci and alleles, in determination of paternity, in studies of class II antigen linked diseases and as a complementary method in conventional tissue typing.

Class II genes of the major histocompatibility complex (MHC) in the chicken have been investigated by Southern blot analysis using human cDNA probes for DQ alpha, DQ beta, DR alpha, and DR beta. Both beta probes but not the alpha probes cross-hybridized well with chicken DNA. The results indicated that the beta probes hybridized with at least two beta genes in the chicken MHC and there was no clear indication of a DQ-DR subdivision of chicken class II beta genes. The possibility of using human beta probes for MHC typing in the chicken was tested by using two homozygous individuals for each of 20 different, serologically defined, MHC (B) haplotypes originating from the domestic breeds of White Leghorn and Rhode Island Red, or from Red Jungle Fowl (the wild ancestral form). Genomic DNA samples from these individuals were digested with any one of the Eco RI and Pvu II restriction enzymes and hybridized with the DR beta probe. Restriction fragment length polymorphism (RFLP) was obtained with Pvu II only, which resolved seven different RFLP types. There was an excellent correlation between these RFLP types and the serological B typing since the RFLP type was identical within each pair of homozygotes. In addition to this broad survey of many haplotypes, a more detailed comparison was carried out on B21-like haplotypes originating from different breeds. No differences in restriction fragment patterns among these haplotypes could be resolved using any of the restriction enzymes Bg1 II, Eco RI, Hind III, Pst I, Pvu II, and Taq I.

A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic-acid-binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5' untranslated portion, the whole translated and the complete 3' untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse-human, hamster-human and rat-human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol-binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.

Class II antigens of the major histocompatibility complex (MHC) consist of a family of closely related cell surface-expressed glycoproteins. These antigens, which are genetically polymorphic, control important aspects of the immune response. At least three types of human class II antigens, namely, DR, DC and SB (refs 2-4), have been identified. All class II antigens are heterodimers composed of one alpha- and one beta-chain. The genes for both types of subunits are encompassed within the MHC. The general features of the DC and DR antigens have recently been elucidated. Much less is known, however, about the SB molecules. Here we describe the isolation of a cDNA clone as well as a genomic clone encoding a beta-chain whose amino acid sequence is compatible with the partial amino-terminal sequence of SB beta-chains.