Genomic evolution of the distal Mhc class I region on mouse Chr 17

A 5-Mb YAC contig, partly supplemented with BAC contigs, was created from the distal Mhc class I region on mouse Chr 17. The gene order of Znf173-Tctex5-Mog-D17Tu42-D17Leh 89 is conserved between mouse and human but not the physical distance, supporting the independent expansion of Mhc class I genes in the so-called accordion model of Mhc evolution. The distal H2-M region includes the breakpoint of conserved synteny between mouse and human as well as the In(17)4 t-inversion. The H2-M region is rich in L1 repeats, implying that the insertion of L1 repeats may be associated with the evolutionary flexibility to break a chromosome.     

Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include beta2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.     

Localization and characterization of the 86- and 84-kDa heat shock proteins in Hepa 1c1c7 cells

The central region of the mouse MHC harbors a recombinational hot spot area. Most recombinations in this part of the complex take place between the Hsp70.1 gene and the G7 gene. This interval is of interest since structurally indistinguishable recombinant haplotypes do differ in functional behavior. Susceptibility to experimental allergic orchitis, which is controlled by the Orch-1 locus, is one example. We have analyzed the hot spot region at the molecular level in order to understand the molecular organization of this chromosomal segment. From a C57BL genomic library we constructed a cosmid contig bridging the interval between Hsp70.1 and G7. The Orch-1 gene maps to a 60-kb segment of DNA in which we found a new Hsp70 homologue, Hsp70.3. Thus, as in the human MHC, the central region of the mouse MHC harbors a cluster of three Hsp70 genes; Hsp70.1, Hsp70.3, and Hsc70t. Two other genes are located in this critical interval (G7b and G7a/Bat-6), and there might still be other undetected genes present in the region. Heat shock proteins play an important role in a large number of physiological processes and it is tempting to speculate that Hsc70t, which exhibits testis-specific expression, may be identical to Orch-1.     

The human histone gene cluster at the D6S105 locus

The sequences and organization of the histone genes in the histone gene cluster at the chromosomal marker D6S105 have been determined by analyzing the Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (YAC) 964f1. The insert of the YAC was subcloned in cosmids. In the established contig of the histone-gene-containing cosmids, 16 histone genes and 2 pseudogenes were identified: one H1 gene (H1.5), five H2A genes, four H2B genes and one pseudogene of H2B, three H3 genes, and three H4 genes plus one H4 pseudogene. The cluster extends about 80 kb with a nonordered arrangement of the histone genes. The dinucleotide repeat polymorphic marker D6S105 was localized at the telomeric end of this histone gene cluster. Almost all human histone genes isolated until now have been localized within this histone gene cluster and within the previously described region of histone genes, about 2 Mb telomeric of the newly described cluster or in a small group of histone genes on chromosome 1. We therefore conclude that the data presented here complete the set of human histone genes. This now allows the general organization of the human histone gene complement to be outlined on the basis of a compilation of all known histone gene clusters and solitary histone genes.     

Two Mhc class I and two Mhc class II genes map to the chicken Rfp-Y system outside the B complex

Gene sequences highly similar to major histocompatibility complex (Mhc) class I and class II genes were recently recognized as mapping to a site in the genome of the chicken separate from the Mhc class I, class II, and B-G genes of the major histocompatibility (B) complex. The present study was undertaken to see whether this complex of Mhc-like genes designated as restriction fragment pattern Y (Rfp-Y) might reside in one of three clusters of cosmid clones contained within the molecular map of chicken Mhc genes, since only two of the three clusters can be assigned to the B system. To determine whether the third cluster (cluster II/IV) might contain Rfp-Y, a subclone (18.1) from within cluster II/IV near a polymorphic lectin gene was used to analyze the DNA of families in which Rfp-Y haplotypes are known to be segregating. The restriction fragment polymorphisms revealed by the 18.1 probe were found to segregate in parallel with the restriction fragment polymorphisms defining the Rfp-Y haplotypes, thus establishing the location of Rfp-Y within cosmid cluster II/IV. Two of six Mhc class I genes and two of five Mhc class II genes map to cosmid cluster II/IV, so a substantial fraction of chicken Mhc genes, including at least one that may be expressed, are located in a chromosomal region separate from the B system. In further linkage analyses, Rfp-Y was found to assort independently from more than 400 markers in the present linkage map of the chicken genome.     

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.     

Cloning and analysis of the plasmid-borne genes encoding the Bsp6I restriction and modification enzymes

The Bsp6I restriction and modification (R-M) system has been localized on the plasmid pXH13, naturally occurring in the Bacillus sp. strain RFL6. The genes coding for the Bsp6I R-M system, a Fnu4HI isoschizomer recognizing the sequence GCNGC, have been cloned in Escherichia coli by two steps. The nucleotide sequence of a 2126-bp region containing the genes for restriction endonuclease (ENase; bsp6IR) and DNA methyltransferase (MTase; bsp6IM) has been determined. The genes are separated by 99 bp and are arranged tandemly with bsp6IR preceding bsp6IM. The DNA sequence predicts an ENase of 174 amino acids (aa) (19.9 kDa) and a MTase of 315 aa (36.3 kDa). M.Bsp6I contains all the conserved aa sequence motifs characteristic for m5C-MTases. In addition, its variable region exhibits a slight similarity to the 5'-GCNGC-3'-specific target-recognition domain (TRD) from M.phi 3T. No aa sequence similarity was found between R.Bsp6I and M.Bsp6I, nor among R.Bsp6I and other known ENases. We have tested recombinant plasmids carrying the complete R-M system for their ability to transform native and pre-methylated Escherichia coli hosts. The results indicate that pre-methylation increases the efficiency of establishment of the complete R-M system. In addition, we have obtained orientation-dependent differences in transformation efficiency.     

The COI mitochondrial gene encodes a minor histocompatibility antigen presented by H2-M3

We found that (LP x C57BL/6)F1 mice could raise a CTL response against parental C57BL/6 cells. These CTLs recognized a maternally transmitted, H2-M3wt-restricted, minor histocompatibility Ag (MiHA) that is widely distributed among many strains of mice and encoded by the COI mitochondrial gene. The wild-type MiHA is the COI N-terminal hexapeptide. Sequencing the 5' end of the COI gene in LP and C57BL/6 mice showed that the LP allele arose by a T-->C transition in the third codon, which caused substitution of threonine for isoleucine. Molecular characterization of this MiHA and the demonstration that it is presented exclusively by H2-M3: 1) support the concept that differential expression of MiHA in MHC-identical animals is caused by polymorphism of the MiHA gene proper; 2) expand our knowledge of the repertoire of self-peptides naturally presented by H2-M3 and show that this MHC class I molecule can present short endogenous peptide ligands; and 3) suggest that mitochondrial DNA mutations that modify the repertoire of H2-M3-associated mitochondrial peptides are representative of mitochondrial DNA mutations in general.     

Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b)

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.     

Chromosome mapping of rat histone genes H1fv, H1d, H1t, Th2a and Th2b

Chromosome assignment of the rat histone genes H1t, H1d (H1.4), H1fv (H10), Th2a and Th2b is described. The testicularly expressed histone genes H1t, Th2a and Th2b could be assigned to rat chromosome (RNO) 17 by PCR analysis of somatic cell hybrid DNAs. The H1d gene was mapped to RNO17p12-->p11 by FISH. These genes might form a histone gene cluster homologous to that found on HSA6p21.3 in humans and MMU13A2-3 in mice. The rat histone H1fv gene was assigned to RNO7 by PCR. This result allows the inclusion of rat H1fv to an established conserved group of syntenic genes in rat, mouse and human on chromosomes RNO7, MMU15 and HSA22, respectively.     

The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies

Insect mitochondrial cytochrome oxidase I (COI) genes are used as a model to examine the within-gene heterogeneity of evolutionary rate and its implications for evolutionary analyses. The complete sequence (1537 bp) of the meadow grasshopper (Chorthippus parallelus) COI gene has been determined, and compared with eight other insect COI genes at both the DNA and amino acid sequence levels. This reveals that different regions evolve at different rates, and the patterns of sequence variability seems associated with functional constraints on the protein. The COOH-terminal was found to be significantly more variable than internal loops (I), external loops (E), transmembrane helices (M) or the NH2 terminal. The central region of COI (M5-M8) has lower levels of sequence variability, which is related to several important functional domains in this region. Highly conserved primers which amplify regions of different variabilities have been designed to cover the entire insect COI gene. These primers have been shown to amplify COI in a wide range of species, representing all the major insect groups; some even in an arachnid. Implications of the observed evolutionary pattern for phylogenetic analysis are discussed, with particular regard to the choice of regions of suitable variability for specific phylogenetic projects.     

Activation of DNA synthesis and expression of the JE gene by catalase in mouse osteoblastic cells: possible involvement of hydrogen peroxide in negative growth regulation

Type I diabetes susceptibility genes have been identified within the major histocompatibility complex (MHC) on chromosome 6p21.3 and near the VNTR/insulin region on chromosome 11p15.5. We have used polymorphic dinucleotide repeat markers to search the human genome for additional susceptibility genes in 162 type I diabetic families with an affected sibling pair. We report that an additional susceptibility gene is located on chromosome 2q31 near HOXD8 (P < 10(-5), maximum logarithm of odds score = 4.8) in an analysis of affected sibling pairs having specific human leukocyte antigen (HLA) and hypervariable nucleotide tandem repeat (VNTR)/insulin gene haplotypes (absence of high-risk HLA-DR3/4 haplotypes and presence of homozygous high-risk class I VNTR alleles). These results suggest the interaction of a minimum of three genes in the pathogenesis of type I diabetes in humans.     

Antigen presentation and T cell development in H2-M-deficient mice

HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.     

A highly conserved major histocompatibility complex class I-related gene in mammals

We report here a cDNA sequence of a murine homolog of the human major histocompatibility complex (MHC) class I-related gene, MR1. The analyses revealed unprecedentedly high conservation of MR1 in the alpha1 and alpha2 domains (corresponding to the peptide-binding domains in the classical MHC class I molecules) between human and mouse (predicted amino acid identity: 90 and 89% for the alpha1 and alpha2 domain, respectively), compared to MHC class I and other class I molecules. On the other hand, conservation in the alpha3 domain (73%) is comparable to those of others, suggesting domain-specific conservation of MR1. The localization of the mouse MR1 gene was determined to be chromosome 1H1, which corresponds to the human chromosomal region where the human MR1 gene is located (chromosome 1q25). High conservation of MR1 among mammals suggests that MR1 may be involved in critical conserved biological function(s).     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Effect of prostaglandin F2 alpha treatment before norgestomet and estradiol valerate treatment on regression, formation, and function of corpora lutea in beef heifers

Despite the presence of several human disease genes on chromosome 11q13, few of them have been molecularly cloned. Here, we report the construction of a contig map encompassing 11q13.1-q13.3 using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The contig map comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and 1 YAC clone and spans a 3-Mb region from D11S480 to D11S913. The map encompasses all the candidate loci of Bardet-Biedle syndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5), one-third of the distal region for hereditary paraganglioma 2 (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 (IDDM4). In the process of map construction, 61 new sequence-tagged site (STS) markers were developed from the Not I linking clones and the termini of clone inserts. We have also mapped 30 ESTs on this map. This contig map will facilitate the isolation of polymorphic markers for a more refined analysis of the disease gene region and identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these bacterial clones.