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 (H2bc). 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 H2k, H2b, and H2bc, 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.     

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).     

Chromosomal localization of mouse Cenpa gene

Using a previously isolated mouse centromere protein A (Cenpa) probe, we have localized the gene to the proximal region of mouse Chromosome 5, between the known Il6 and Yes1 loci near [Adra2C-D5H4S43-Hdh]. Comparison of this localization with that of human CENPA, which maps to chromosome 2, is consistent with the presence of a new region of conserved synteny between the two species.     

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.     

Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler

The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the alpha2 domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21-25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths (1.3-2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (dN) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (dS), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken.     

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.     

Surgical treatment of obstructive jaundice

Genetically engineered structural variants of human beta2-microglobulin (beta2m) were produced by sequence exchange with mouse beta2m for the purpose of examining species-specific antigenic determinant expression. For aggregate mapping, mouse and human beta2m, which differ by 30% in their primary sequence of 99 amino acids, were prepared as chimeric (human X mouse) molecules and expressed in the FO-1 beta2m-null human melanoma cell line. A chimera containing residues 1-69 from human beta2m (and residues 70-99 from mouse beta2m) induced expression of the epitopes defined by the anti-beta2m monoclonal antibodies (mAb) BBM.1, NAMB-1, and L368; the reverse chimera did not, although HLA class I heavy chain was evident on the cell surface as determined with the TP25.99 mAb. For fine dissection of the epitopes defined by these mAbs, site-directed mutants of beta2m were prepared by replacement of individual amino acids in human beta2m with the dimorphic residue from mouse beta2m. Substitutions were made at each divergent residue between positions 1 and 66 and, as controls for COOH-terminal modification, a series of residues between positions 75 and 94. Replacement of amino acids 38, 44, and 45, but not 16 other dimorphic residues in the linear stretch from residue 1 to residue 66, resulted in the loss of, or gross reduction in, binding by mAbs BBM.1 and NAMB-1. A reduction in binding was also observed for mAb L368. These data provide strong evidence that the antigenic epitopes defined by these mAb map to a region including S3 and its adjacent intra-beta-strand turn of the three-stranded beta-pleated sheet of beta2m. The mapping of these epitopes is consistent with their accessibility in the assembled major histocompatibility complex class I molecule and indicates that the region from amino acid 38 to 45 is an important structural feature in the "foreignness" of human and mouse beta2m.     

Can a protein influence the fate of its own coding sequence?: the amino- and carboxyl-terminal regions of H1 histone

The amino- and carboxyl-terminal regions of H1 histone are afforded an opportunity to bind to their own coding segments. While such binding is of no consequence to the type of H1 histone that is expressed only in terminally differentiated cells (here referred to as H5), the same binding occurring in H1 of proliferating cells might affect the course of evolution of its amino- and carboxyl-terminal regions. With the above in mind, amino acid sequences of H5 from three species were compared with those of H1 from five species. The spherical core was more conserved in H1 than in H5, whereas the amino- and carboxyl-terminal regions of both were hypervariable. A distinct evolutionary pattern, however, was observed only in these regions of H1 but not of H5. Tandem repeats of short palindromes often generated nearly identical long palindromes in distantly related species. Thus, as far as the 37-residue-long amino-terminal region was concerned, the greatest homology was between the rat and human rather than between the rat and the mouse. With regard to the last 25 residues in the carboxyl-terminal region, the greatest homology was between the chicken and the rainbow trout; the next was between the mouse and the trout. Successions of long palindromes populating the amino- and carboxyl-terminal regions of H1 might be the direct consequence of the autologous binding between peptides and their coding sequence.     

Linkage of LMP, TAP, and RING3 with Mhc class I rather than class II genes in the zebrafish

The LMP2 and LMP7 genes code for subunits of the proteasome, a multimeric enzymatic complex that degrades proteins into peptides. The two subunits replace corresponding constitutively expressed subunits during the immune response. Some of the peptides generated by the proteasome in the cytosol are transported by the products of the TAP1 and TAP2 genes into the lumen of the endoplasmic reticulum and are loaded onto the assembling MHC class I molecules. In mammals, the LMP2, LMP7, TAP1, and TAP2 genes reside in the class II region of the Mhc, closely linked to the RING3 gene. In the present study we identified, cloned, and sequenced the LMP, TAP2, and RING3 genes of the zebrafish, Danio rerio. We identified variants of these genes and used them in a segregation analysis of haploid embryos derived from heterozygous mothers. The analysis revealed that in zebrafish, the LMP2, LMP7, TAP12, and RING3 loci are closely linked but, in contrast to mammals, the LMP/TAP/RING3 cluster resides not in the Mhc class II but in the class I region. We also confirmed that in the zebrafish, the class I and class II regions are not linked to each other. In this species, therefore, the LMP/TAP/RING3 genes are clustered with the class I genes on a chromosome that apparently does not contain any class II genes. The linkage of LMP/TAP/RING3/class I may be the original and the LMP/TAP/RING3/class II a derived arrangement of these genes.     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies.     

Chromosomal localization of the human and mouse hyaluronan synthase genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17.HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

Genetic mapping of the human and mouse phospholipase C genes

To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2. Two PLC genes, Plcd1 and Plcg2, mapped to distinct sites on Chr 8. We mapped the human homologs of eight of these genes to six chromosomes by analysis of human x rodent somatic cell hybrids. The map locations of seven of these genes were consistent with previously defined regions of conserved synteny; Plcd1 defines a new region of homology between human Chr 3 and mouse Chr 8.     

Murine Stim1 maps to distal chromosome 7 and is not imprinted

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects.     

Tctex3, related to Drosophila polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6.