Mapping and nucleotide sequence of a newHLA class II light chain gene,DQB3

A genomic clone specifying a new HLA class II antigen β chain,DQB3, was isolated from a human genomic phage library using aDQB1 cDNA probe under low stringency conditions. Southern hybridization and nucleotide sequence analyses identified the β2 domain exon (exon 3) with several deleterious mutations and the CP-TM-CY exon [connecting peptide, transmembrane, and cytoplasmic regions, (exon 4)], but the first, second, and fifth exons encoding the 5′ UT-leader, the β1 domain, and the 3′ UT domain of normal β chains, respectively, were entirely missing. The nucleotide sequences of these two exons were distinct from those of other class II β chain genes, but slightly more related to theDQB1 andDQB2 genes than to other class II genes. TheDQB3 sequence mapped betweenDQA2 andDQB1, 15 kb upstream fromDQA2, by analysis of overlapping cosmid clones. This mapping was supported by the fact thatTaq I,Msp I, andBam HIDQB3 polymorphisms were perfectly correlated with theDQA2 polymorphism and not with any polymorphisms in theDR orDQ subregion, suggesting the presence of a hot spot for recombination betweenDQB3 andDQB1. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M26577.     

Genomic structure and chromosome location of the murine PDE1B phosphodiesterase gene

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby participating in regulation of the intracellular concentrations of these second messengers. The PDE1 family is defined by regulation of activity by calcium and calmodulin. We have cloned and characterized the mouse PDE1B gene, which encodes the 63-kDa calcium/calmodulin-dependent PDE (CaM-PDE), an isozyme that is expressed in the CNS in the olfactory tract, dentate gyrus, and striatum and may participate in learning, memory, and regulation of phosphorylation of DARPP-32 in dopaminergic neurons. We screened an I-129/SvJ mouse genomic library and identified exons 2–13 of the PDE1B gene that span 8.4 kb of genomic DNA. Exons range from 67 to 205 nucleotides and introns from 91 to 2250 nucleotides in length. Exon 1 was not present in the 3 kb of genomic DNA 5′ to exon 2 in our clones. The mouse PDE1B gene shares many similar or identical exon boundaries as well as considerable sequence identity with the rat PDE4B and PDE4D genes and the Drosophila dunce cAMP-specific PDE gene dnc, suggesting that these genes all arose from a common ancestor. Using fluorescence in situ hybridization, we localized the PDE1B gene to the distal tip of mouse Chromosome (Chr) 15. Received: 10 November 1997 / Accepted: 12 March 1998     

Active MHC class Ib genes in rat are pseudogenes in the mouse

The most telomeric class I region of the MHC in rat and mouse is the M region, which contains about 20 class I genes or gene fragments. The central part carries three class I genes—M4, M5, and M6—which are orthologous between the two species. M4 and M6 are pseudogenes in the mouse but transcribed, intact genes in the rat. To analyze the pseudogene status for the mouse genes in more detail, we have sequenced the respective exons in multiple representative haplotypes. The stop codons are conserved in all mouse strains analyzed, and, consistent with the pseudogene status, all strains show additional insertions and deletions, taking the genes further away from functionality. Thus, M4 and M6 indeed have a split status. They are silent in the mouse but intact in the closely related rodent, the rat.GenBank accession numbers: AF057065 to AF057072 (exon 3 of H2-M4 of reported mouse strains), AF057976 to AF057985 (exon 3 of RT1.M4 of reported rat strains), AF058923 and AF058924 (exon 2 of RT1.M4 of strains PVG and BN), AY286080 to AY286092 (exon 4 of H2-M6 of reported mouse stains), and AY303772 (full-length genomic sequence of RT1.M6-1^l)     

Differential transport requirements of HLA and H-2 class I glycoproteins

Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line × T-lymphoblastoid cell line (B-LCL × T-LCL) hybrid, 174 × CEM. T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or 137 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2D ^p or H-2K ^b class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.     

A novel expression vector for transfection of bovine MHC class I genes

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk⁻ cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3′-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5′-and 3′-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.     

Characterization of a class Ib gene of the rat major histocompatibility complex

The cDNA and a partial genomic sequence of a rat class I major histocompatibility (RT1) gene, 11/3R, is reported here. The sequence contains several unique amino acid residues at certain positions, mutations in exon 7 (which is not expressed), a mutation of the canonical exon 8 stop codon to a sense codon, and includes a long 3 unstranslated region (utr). The structure of exon 7 differs from that found in most rat class I genes and resembles exon 7 of most H-2K,D,L.Q genes. Parts of the 3 noncoding region are homologous to the RT1.A-4 and certain H-2 genes. Expression is detectable by northern blot analysis in mitogen-stimulated lymphocytes only, by polymerase chain reaction (PCR) in each tissue tested. After transfection into L cells 11/3R can be shown to be expressible at the cell surface. Probes derived from the 3 noncoding part crosshybridize with a number of restriction fragments which map to the RT1.C region, thus defining a subfamily of RT1.C region genes. Several members of this subfamily are deleted in the M1 RT1 mutant. The 11/3R gene presents typical features of a class Ib gene. Aspects of evolution and the potential of the gene are discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank molecule sequence data base and have been assigned the accession numbers X67503 ande X67504.     

Analysis of D2 d : a D-region class I gene

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2 ^d , D3 ^d , and D4 ^d , in addition to H-2D ^d and H-2L ^d . We have determined the DNA sequence of the D2 ^d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2 ^d gene is similar to other class I genes. It also contains similar 5 regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2^d molecule, we generated an exon-shuffled construct containing the promoter and exons 1–3, encoding the signal peptide, 1, and 2 external domains of the D2 ^d gene linked to exons 4–8, encoding the 3, transmembrane and cytoplasmic domains, of the H-2D ^d gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the 3 domain of H-2D^d, as well as by other class I-specific mAbs. Although D2^d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.     

Sequence of the mouse Q4 class I gene and characterization of the gene product

The Q4 class I gene has been shown to participate in gene conversion events within the mouse major histocompatibility complex. Its complete genomic nucleotide sequence has been determined. The 5 half of Q4 resembles H-2 genes more strongly than other Q genes. Its 3 end, in contrast, is Q-like and contains a translational stop signal in exon 5 which predicts a polypeptide with an incomplete membrane spanning segment. The presence of two inverted B1 repeats suggests that part of the Q4 gene may be mobile within the genome. Gene transfer experiments have shown that the Q4 gene encodes a ²-microglobulin associated polypeptide of M_r 41 000. A similar protein was found in activated mouse spleen cells. The Q4 polypeptide was found to be secreted both by spleen cells and by transfected fibroblasts and was not detectable on the cell surface. Antibody binding and two-dimensional gel electrophoresis indicate that the Q4 molecule is identical to a mouse class I polypeptide, Qb-1, which has been previously described.     

Complete nucleotide sequence of a functional class I HLA gene, HLA-A3: implications for the evolution of HLA genes

The complete nucleotide sequence of an active class I HLA gene, HLA-A3, has been determined. This sequence, together with that obtained for the HLA-CW3 gene, represents the first complete nucleotide sequence to be determined for functional class I HLA genes. The gene organisation of HLA-A3 closely resembles that of class I H-2 genes in mouse: it shows a signal exon, three exons encoding the three extracellular domains, one exon encoding the transmembrane region and three exons encoding the cytoplasmic domain. The complete nucleotide sequences of the active HLA genes, HLA-A3 and HLA-CW3, now permit a meaningful comparison of the nucleotide sequences of class I HLA genes by alignment with the sequence established for a HLA-B7-specific cDNA clone and the sequences of two HLA class I pseudogenes HLA 12.4 and LN- 11A . The comparisons show that there is a non-random pattern of nucleotide differences in both exonic and intronic regions featuring segmental homologies over short regions, which is indicative of a gene conversion mechanism. In addition, analysis of the frequency of nucleotide substitution at the three base positions within the codons of the functional genes HLA-A3, HLA-B7 and HLA-CW3 shows that the pattern of nucleotide substitution in the exon coding for the 3rd extracellular domain is consistent with strong selection pressure to conserve the sequence. The distribution of nucleotide variation in the other exons specifying the mature protein is nearly random with respect to the frequencies of substitution at the three nucleotide positions of their codons. The evolutionary implications of these findings are discussed.     

Gene conversion-like mechanisms may generate polymorphism in human class I genes.

The nucleotide sequences of the human class I major histocompatibility complex genes HLA-B27k and HLA-B27w have been determined. They differ by only four nucleotides over a stretch of 14 bp in exon 2, resulting in three amino acid exchanges at positions 77 (Asp to Asn), 80 (Thr to Ile) and 81 (Leu to Ala). The distribution of these nucleotide substitutions suggests a gene conversion-like event responsible for the generation of these HLA-B27 subtypes. The mechanisms underlying the generation of new polymorphic variants in man are therefore probably identical to the gene conversion-like events postulated in the generation of H-2Kbm class I mutants in the mouse.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Isolation,expression, and the primary structure of HLA-Cw1 and HLA-Cw2 genes: Evolutionary aspects

The HLA-Cw1 and -Cw2 genes were identified in a genomic library and their products characterized by biochemical methods. The HLA-Cw and -Cw2 genes, upon transfection in mouse L cells, give rise to class I antigen heavy chains that associate with neither mouse nor human beta-2 microglobulin. They are indistinguishable in isoelectric point from polypeptides identified as HLA-Cw1 and -Cw2 in human cells. The nucleotide sequence of HLA-Cw1 and -Cw2 and their comparison with HLA-Cw3, the only other known HLA-C sequence, reveal a characteristic pattern of locus-specific amino acids. A comparison of 13 different human class I primary structures leads us to speculate that the most variable region in HLA class I antigens, positions 61–83, could assume an alpha helical structure of critical importance for class I antigen function. The locus specificity and the higher degree of intralocus conservation in the COON-terminal region, especially in the transmembrane and cytoplasmic domains, must reflect evolutionary ancestry rather than positive selection. In view of the pattern and types of substitutions observed for HLA-C locus products, their function as immune response gene products is questioned.     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997     

Gene segments encoding membrane domains of the human immunoglobulin gamma 3 and alpha chains

The carboxyterminal region of the heavy chains, according to its hydrophilic or hydrophobic properties, determines whether the immunoglobulin will be secreted or membrane-bound. We have determined the nucleotide sequences of the human IGHG3, IGHA1, and IGHA2 membrane exons isolated from genomic DNA libraries. The IGHG3 M1 and M2 exons are separated by a long intron of 2.1 kilobases (kb) containing an highly repeated motif of 34 base pairs (bp). The IGHA1 and IGHA2 genes, like the mouse Igh-A gene, have a single exon encoding the extracellular, transmembrane, and cytoplasmic regions. For each class of immunoglobulins, the sequences of membrane exons are highly conserved between human and mouse, but no alignment is possible for the flanking regions. In contrast, for a same species, the sequences of the heavy chain membrane exons differ from one class to another. While the hydrophobic profile of the membrane core is well conserved, the cytoplasmic region differs in length and in composition. None of the intracellular domains presents the sequence implied in signal transduction, implying that membrane immunoglobulins need other proteins, which probably interact with the constant or membrane domain, to transmit signals leading to B-cell activation.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers M35288-91. Address correspondence and offprint requests to: M.-P. Lefranc.     

Transfection of murine LMTK− cells with purified HLA class I genes

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2K ^d, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the 137 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.Abbreviations used in this paper BSA bovine serum albumin - FCS fetal calf serum - mAb monoclonal antibody - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline     

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse

 A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2 ^bc ). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2 ^k , H2 ^b , and H2 ^bc , and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6-Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species. Received: 27 April 1998 / Revised: 4 June 1998     

Three extra copies of a C4-related gene in H-2 w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2 _w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5 end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5 flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3 side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2 ^w7 mouse.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers D90167-71.     

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F₁ haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2^k and H-2^b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K^k, H-2D^b (class I) and A^b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D^b exhibited partial X inactivation and only a proportion ( 30%) of the cells were targets for or stimulated H-2D^b-restricted H-Y-specific T cells. In contrast, H-Y eiptopes restricted by H-2^k (H-Y/K^k, H-Y/D^k) and A^b (H-Y/A^b) exhibited no X inactivation. Furthermore, no inactivation of H-Y/D^b, H-Y/A^b, or H-Y^k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2^k and H-2D^b class I molecules and A^b class II molecules may be the product of more than one gene.     

Comparative analysis of the structure and chromosomal assignment ofCD1: an evidence for different evolutionary histories between classic CD1 and CD1D class genes

The non-MHC-encoded CD1 family has recently emerged as a novel antigen-presenting system that is distinct from MHC class I and class II molecules. In the present study, we determined the genomic structure of that rat CD1, and compared with those of other previously reported CD1 genes. Rat CD1 was extremely similar to mouse CD1 genes, especially to CD1D1. It is of interest that a tyrosine-based motif for endosomal localization, identified in the human CD1b cytoplasmic tail, was conserved in all CD1 molecules except for CD1a, that was encoded by a single short exon. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the introns was also characteristic to each of the two classes of CD1 genes; classic (CD1A, CD1B, CD1C and CD1E), and CD1D, which have been categorized by comparison of coding regions. These findings support a hypothesis that the two classes have different evolutionary histories. In contrast to the absence of the classic CD1 genes in rats and mice, the entire region of nonpolymorphic CD1D gene has been conserved through mammalian evolution. Furthermore, we determined chromosomal localization of rat CD1 gene using the fluorescence in situ hybridization method with several probes derived from genomic rat CD1 clones. Similar to human and mouse CD1, rat CD1 mapped outside the MHC loci despite the structural and functional resemblance to MHC. Conserved syntheny of chromosomal segments of RNO2 and MMU3 is implied.