Removal of Qa-2 antigen alters the Ped gene phenotype of preimplantation mouse embryos.

Embryo survival is influenced by both genetic and environmental factors. Previous research in our laboratory has identified one gene associated with embryonic survival, the Ped gene, a gene that is linked to the major histocompatibility complex (MHC) of the mouse. The Ped gene has been shown to influence the rate of preimplantation embryonic cleavage division, as well as litter size, birth weight, and weaning weight. Genetic mapping of the Ped gene has located it in the Q region of the MHC and has suggested that possible Q region genes encoding the Ped gene are Q3, Q5, Q6, Q7, Q8, and/or Q9. Whereas the protein products of the Q3 and Q5 genes are unknown, the protein product of the very similar Q6, Q7, Q8, and Q9 genes is the Qa-2 antigen. Two forms of membrane-bound Qa-2 antigen are known: glycosylphosphatidylinositol (GPI)-linked and transmembrane bound. Only the GPI-linked form is sensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC). The first purpose of the present study was to determine the nature of the linkage of the Qa-2 antigen to the cell surface of preimplantation mouse embryos. It was found that all detectable Qa-2 antigen on the embryonic cell surface is sensitive to cleavage by PI-PLC and is therefore bound to the cell membrane by a GPI linkage. Furthermore, removal of Qa-2 antigen from the embryonic cell surface slows down the rate of development of preimplantation mouse embryos. These results suggest the likelihood that the Qa-2 antigen is the Ped gene product.     

Identification of two major histocompatibility complex class Ib genes, Q7 and Q9, as the Ped gene in the mouse

The Ped (preimplantation embryonic development) gene influences the rate of preimplantation embryonic development and subsequent embryonic survival. The protein product of the Ped gene, the Qa-2 protein, is a major histocompatibility complex (MHC) class Ib protein. There are two alleles of the Ped gene, fast (Qa-2 [+]) and slow (Qa-2 [-]). Qa-2 is encoded by four very similar MHC class Ib genes: Q6, Q7, Q8, and Q9. Recent research in our laboratory has shown that the Ped phenotype is potentially encoded by the Q7 and/or Q9 gene because the Q7 and Q9 genes, but not the Q6 or Q8 gene, are expressed during preimplantation mouse embryonic development. In this study we utilized microinjection of transgenes to assess the functional roles of both the Q7 and Q9 genes in control of the rate of preimplantation development. The Q7 gene, the Q9 gene, and a combination of the Q7 and Q9 genes were microinjected into Ped slow zygotes, and the Ped phenotype and cell surface expression of Qa-2 protein were assayed after a 72-h or 96-h incubation period. We found that the microinjected individual Q7 and Q9 genes increased the rate of preimplantation development. Simultaneous injection of the Q7 and Q9 genes did not have a synergistic effect on the Ped phenotype. Microinjection of the Q7 and/or Q9 genes resulted in protein expression in 10-25% of the microinjected embryos. These results show that both the Q7 and Q9 genes encode the mouse Ped phenotype.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

Expression of a class I MHC transgene: effects of in vivo α/β-interferon treatment

Transgenic mice containing a swine class I major histocompatibility complex (MHC) gene,PD1, express swine MHC (SLA) antigen. The tissue distribution of PD1 RNA parallels that observed in the swine, indicating that the expression ofPD1 is regulated and thattrans-acting factors involved in this regulation have been conserved between the species. Although PD1 RNA levels were much greater in transgenic spleen than in thymus, no difference in the chromatin organization of thePD1 gene was detected. In both tissues, a single DNase I hypersensitive site mapped within the 5′ flanking region. In vivo treatment of the transgenics with mouse α, β-interferon increases PD1 expression in a number of tissues. In the spleen, this increase parallels that observed for the endogenous transplantation antigen, K^b, but differs markedly from the differentiation antigen, Qa-2. Increases in cell surface expression of both PD1 and K^b occurred equally in splenic T- and B-cell populations following α,β-interferon treatment. In contrast, Qa-2 expression in B cells was enhanced by α,β-interferon, whereas it was unaffected in T cells and thymocytes.     

Genetic polymorphisms ofQ region genes from wild-derived mice: Implications forQ region evolution

Both serological and DNA sequence analyses were performed to determine the extent of genetic polymorphism inQ region genes. A panel of Qa-2-specific monoclonal antibodies (mAbs) was tested on 35 wildderived and inbred mouse strains. Members of this reagent panel recognize multiple and distinct epitopes on the Qa-2-bearing molecule(s). Although quantitative variations in Qa-2 levels were observed, no structural polymorphisms were detected. All strains were either entirely positive or entirely negative with the complete set of reagents. Moreover, cell surface Qa-2 expression was not significantly affected by differences in age or sex of the mouse or cell cycle status. To confirm this apparent lack of genetic polymorphism, the polymerase chain reaction (PCR) technique was used to amplify a portion of the 3 end of theQ region genes,Q4 toQ9, from several independent wild-derived strains of mice. Sequence analysis of the amplified material revealed very little evidence of nucleotide divergence. All strains tested had aQ even DNA sequence identical to that ofQ6/Q8 in the B10 strain. Likewise, all tested strains had aQ odd DNA sequence identical toQ7/Q9 in the B10 strain. Two strains showed additionalQ even sequences, while all strains tested possessed additionalQ odd sequences. The observed lack of polymorphism suggests that theQ genes have evolved in a different manner fromH-2K andH-2D. Moreover, duplications of these genes appear to have arisen prior to nucleotide sequence divergence.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30896-30902.     

Molecular cloning and structural analysis of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) phosphoenolpyruvate carboxykinase (<Emphasis Type="Italic">CsPCK</Emphasis>) gene

Genomic sequence of the ATP-dependent phosphoeno/pyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing to ascertain the structure of theCsPCK gene. Analysis of a selected positive clone (λcscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns, spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5′-noncoding region of cucumberPCK cDNA, whereas Exon 2 comprises 12 nucleotides of the S′-noncoding region with an N-terminal PEPCK coding sequence. All the exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of theCsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for bothArabidopsis Chromosome 4 (Atg4)PCK and the rice PCX genes, which contain 13 and 12 exons, respectively. Two additionalArabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively) of the PEPCK peptide. TheCsPCK gene promoter has conserved plant-specific as-acting elements within 2 kb of the 5’ flanking region. Several common cis-acting elements of the isocitrate lyase (icl) and malate synthase(ms) gene promoters, identified in theCsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements are discussed here.     

The role of structurally conserved class I MHC in tumor rejection: contribution of the Q8 locus

The mouse multimember family of Qa-2 oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two "prototype" Qa-2 sequences, Q8 and Q9. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting alpha(1) and alpha(2) regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the "odd" and "even" Qa-2 loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.     

Preimplantation embryo development (<Emphasis Type="Italic">Ped</Emphasis>) gene copy number varies from 0 to 85 in a population of wild mice identified as <Emphasis Type="Italic">Mus musculus domesticus</Emphasis>

The preimplantation embryo development (Ped) gene regulates the rate of preimplantation embryonic cleavage division and subsequent embryo survival. In the mouse, the Ped gene product is Qa-2 protein, a nonclassical MHC class I molecule encoded by four tandem genes, Q6/Q7/Q8/Q9. Most inbred strains of mice have all four genes on each allelic chromosome, making a total of eight Qa-2 encoding genes, but there are a few strains that are missing all eight genes, defining a null allele. Mouse strains with the presence of the Qa-2 encoding genes express Qa-2 protein and produce embryos with a faster rate of preimplantation embryonic development and a greater chance of embryo survival compared to mouse strains with the null allele. There is extensive evidence that the human homolog of Qa-2 is HLA-G. HLA-G in humans, like Qa-2 in mice, is associated with enhanced reproductive success. The human population is an outbred population. Therefore, for a better comparison to the human population, we undertook an investigation of the presence of the genes encoding Qa-2 in an outbred population of mice. We used Real-Time Quantitative PCR to quantify the number of Qa-2 encoding genes in a population of 32 wild mice identified as Mus musculus domesticus both by morphologic assessment and by PCR analysis of their DNA. We found great variability in the number of Qa-2 encoding genes in the wild mice tested. The wild mouse with the highest number of Qa-2 encoding genes had 85 such genes, whereas we discovered one wild mouse without any Qa-2 encoding genes. Evolutionary implications of a range of Qa-2 encoding gene numbers in the wild mouse population are discussed, as well as the relevance of our findings to humans.     

Identification of a new CML target antigen controlled by a gene associated with theQa-2 locus

BALB/cBy anti-BALB/cJ spleen cells were tested in a secondary cellmediated lympholysis assay. The effector cells generated displayed a positive cytotoxic effect against Con A lymphoblasts from only those strains that were typed serologically as having theQa-2 ^a allele. Confirmation that the target antigen is controlled by a locus closely associated with or identical toQa-2 was obtained by the findings that target cells from B6.K2 (Qa-2 ^a,Qa-3 ^a) mice were lysed by the effector cells, while those from theQa-2, 3 congenic strain B6.K1 (Qa-2 ^b,Qa-3 ^b) were not. The fact that target cells from aQa-2-positive/Qa-3-negative strain (DBA/1,Qa-2 ^ai,Qa-3 ^b) were killed indicates that the target antigen is controlled, at least in part, by theQa-2 locus, not the Qa-3.There is no observedH-2 genetic restriction for this cytotoxic effect, since target cells which have theQa-2 ^a allele but differ from the stimulator cells at theH-2K, D, andI regions were lysed efficiently.     

Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.     

A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence analysis because it was polymorphic in Southern blot analyses of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa-2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with β2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for either a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolated genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.     

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.     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.     

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.     

Phylotype diversity in a benthic cyanobacterial mat community on King George Island, maritime Antarctica

Cyanobacterial 16S ribosomal RNA gene diversity was examined in a benthic mat on Fildes Peninsula of King George Island (62o09′54.4′′S, 58o57′20.9′′W), maritime Antarctica. Environmental DNA was isolated from the mat, a clone library of PCR-amplified 16S rRNA gene fragments was prepared, and amplified ribosomal DNA restriction analysis (ARDRA) was done to assign clones to seven groups. Low cyanobacterial diversity in the mat was suggested in that 83% of the clones were represented by one ARDRA group. DNA sequences from this group had high similarity with 16S rRNA genes of Tychonema bourrellyi and T. bornetii isolates, whose geographic origins were southern Norway and Northern Ireland. Cyanobacterial morphotypes corresponding to Tychonema have not been reported in Antarctica, however, this morphotype was previously found at Ward Hunt Lake (83oN), and in western Europe (52oN). DNA sequences of three of the ARDRA groups had highest similarity with 16S rDNA sequences of the Tychonema group accounting for 9.4% of the clones. Sequences of the remaining three groups (7.6%) had highest similarity with 16S rRNA genes of uncultured cyanobacteria clones from benthic mats of Lake Fryxell and fresh meltwater on the McMurdo Ice Shelf.     

The Sec-1 locus on the short arm of chromosome 1R of rye (Secale cereale)

This paper describes a detailed sequence analysis of the ω-secalin gene array at theSec-1 locus on the short arm of chromosome 1 of rye. The analysis shows that the genes are separated by 8 kb of spacer sequence and that the gene/spacer units are arranged in a head to tail fashion. The boundaries of the array are identified, and a fragment containing the majority of the genes in the array is separated by PFG analysis. The sequence data of one 9.2 kb gene unit have been determined, and because of the similarity of the gene units within the array these data provide a detailed sequence analysis of 140 kb of theSec-1 locus. Fluorescence in situ hybridization, using lambda clones isolated for the structural analysis, identifies the position of the array on the rye chromosomes relative to the 5S rRNA genes. Edited by: W. Hennig     

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5 and 3 termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3 untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3UT regions of clones 2.1 and 5.1, respectively, Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of and homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the] w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.