Inhibition of NK cells by murine CMV-encoded class I MHC homologue m144

Murine cytomegalovirus (CMV)-encoded protein m144 is homologous to class I MHC heavy-chain and is thought to regulate NK-cell-mediated immune responses in vivo. To examine the effects of m144 on Nh cytotoxicity in vitro, various cell lines were transfected with wild-type m144 or a chimeric construct in which the cytoplasmic domain of m144 was replaced with green fluorescence protein. Burkitt lymphoma line Raji expressed a significant level of m144 as determined by anti-m144 mAb binding or the green fluorescence of the fusion protein. The level of m144 expression was relatively low compared with that of transfected murine class I MHC Dd. However, m144 on Raji cells partially inhibited antibody-dependent cell-mediated cytotoxicity of IL-2-activated NK cells. NK cells from the CMV-susceptible BALB/c as well as those from the resistant C57BL/6 mice were inhibited by m144. Antibodies against the known murine NK inhibitory receptors Ly-49A, C, G, and I did not affect the inhibitory effect of m144. These results suggest that the murine CMV class I MHC homologue m144 partially inhibits MZ cells by interacting with a novel inhibitory receptor. (C) 1999 Academic Press.

Mouse Sox8 is located between, not within, the t-complex deletions t(w18) and t(h20) on chromosome 17

The SOX family of developmental transcription factors is known to play critical roles in cell lineage specification, fate determination and differentiation during development in diverse phyla. Their importance is underscored by their involvement in a number of human diseases and mouse mutants, and by targeted mutation in mice. SOX8 is broadly expressed during development and is located on human chromosome 16p and within the t-complex on mouse chromosome 17, in the vicinity of two mutations t(w18) and t(h20). Here we analyse mutant genomic DNA to show that the Sox8 gene locus lies outside the deletion regions of both t(w18) and t(h20) and between these deletions. These data exclude Sox8 from contributing to the t(w18) and t(h20) phenotypes, and provide an additional marker for structural characterization of this complex genomic region. Copyright (C) 2001 S. Karger AG, Basel.

Human twinning is not linked to the region of chromosome 4 syntenic with the sheep twinning gene FecB

The tendency to dizygotic (DZ) twinning is inherited in both humans and sheep, and a fecundity gene in sheep (FecB) maps to sheep chromosome 6, syntenic with human 4q21-25. Our aim was to see whether a gene predisposing to human DZ twinning mapped to this region. DNA was collected from 169 pairs and 17 sets of 3 sisters (trios) from Australia and New Zealand who had each had spontaneous DZ twins, mostly before the age of 35, and from a replication sample of 111 families (92 affected sister pairs) from The Netherlands. Exclusion mapping was carried out after typing 26 markers on chromosome 4, of which 8 spanned the region Likely to contain the human homologue of the sheep FecB gene. We used nonparametric affected sib pair methods for linkage analysis [ASPEX 2.2, Hinds and Risch, 1999]. Complete exclusion of linkage (lod < -2) of a gene conferring a relative risk for sibs as low as 1.5 ((s) > 1.5) was obtained for all but the p terminus region on chromosome 4. Exclusion in the syntenic region was stronger, down to lambda (s) = 1.3. We concluded that if there is a gene influencing DZ twinning on chromosome 4, its effect must be minor. (C) 2001 Wiley-Liss, Inc.

Genomewide significant linkage to migrainous Hheadache on chromosome 5q21

Familial typical migraine is a common, complex disorder that shows strong familial aggregation. Using latent-class analysis (LCA), we identified subgroups of people with migraine/severe headache in a community sample of 12,245 Australian twins (60% female), drawn from two cohorts of individuals aged 23-90 years who completed an interview based on International Headache Society criteria. We report results from genomewide linkage analyses involving 756 twin families containing a total of 790 independent sib pairs ( 130 affected concordant, 324 discordant, and 336 unaffected concordant for LCA-derived migraine). Quantitative-trait linkage analysis produced evidence of significant linkage on chromosome 5q21 and suggestive linkage on chromosomes 8, 10, and 13. In addition, we replicated previously reported typical-migraine susceptibility loci on chromosomes 6p12.2-p21.1 and 1q21-q23, the latter being within 3 cM of the rare autosomal dominant familial hemiplegic migraine gene (ATP1A2), a finding which potentially implicates ATP1A2 in familial typical migraine for the first time. Linkage analyses of individual migraine symptoms for our six most interesting chromosomes provide tantalizing hints of the phenotypic and genetic complexity of migraine. Specifically, the chromosome 1 locus is most associated with phonophobia; the chromosome 5 peak is predominantly associated with pulsating headache; the chromosome 6 locus is associated with activity-prohibiting headache and photophobia; the chromosome 8 locus is associated with nausea/vomiting and moderate/severe headache; the chromosome 10 peak is most associated with phonophobia and photophobia; and the chromosome 13 peak is completely due to association with photophobia. These results will prove to be invaluable in the design and analysis of future linkage and linkage disequilibrium studies of migraine.

Molecular cloning and characterization of the mouse Na+ sulfate cotransporter gene (Slc13a4): structure and expression

Sulfate is an essential ion required for numerous functions in mammalian physiology. Due to its hydrophilic nature, cells require sulfate transporters on their plasma membranes to allow entry of sulfate into cells. In this study, we identified a new mouse Na+-sulfate cotransporter (mNaS2), characterized its tissue distribution and determined its cDNA and gene (Slc13a4) structures. mNaS2 mRNA was expressed in placenta, brain, lung, eye, heart, testis, thymus and liver. The mouse NaS2 cDNA spans 3384 nucleotides and its open frame encodes a protein of 624 amino acids. Slc13a4 maps to mouse chromosome 6131 and contains 16 exons, spanning over 40 kb in length. Its 5'-flanking region contains CART- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, MTF-1, STAT6 and HNF4 consensus sequences. This is the first study to define the tissue distribution of mNaS2 and resolve its cDNA and gene structures, which will allow us to investigate mNaS2 gene expression in vivo and determine its role in mammalian physiology.

Molecular cloning reveals that the p160 myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

HLA and Genomewide Allele Sharing in Dizygotic Twins

Gametic selection during fertilization or the effects of specific genotypes on the viability of embryos may cause a skewed transmission of chromosomes to surviving offspring. A recent analysis of transmission distortion in humans reported significant excess sharing among full siblings. Dizygotic (DZ) twin pairs are a special case of the simultaneous survival of two genotypes, and there have been reports of DZ pairs with excess allele sharing around the HLA locus, a candidate locus for embryo survival. We performed an allele-sharing study of 1,592 DZ twin pairs from two independent Australian cohorts, of which 1,561 pairs were informative for linkage on chromosome 6. We also analyzed allele sharing in 336 DZ twin pairs from The Netherlands. We found no evidence of excess allele sharing, either at the HLA locus or in the rest of the genome. In contrast, we found evidence of a small but significant (P = .003 for the Australian sample) genomewide deficit in the proportion of two alleles shared identical by descent among DZ twin pairs. We reconciled conflicting evidence in the literature for excess genomewide allele sharing by performing a simulation study that shows how undetected genotyping errors can lead to an apparent deficit or excess of allele sharing among sibling pairs, dependent on whether parental genotypes are known. Our results imply that gene-mapping studies based on affected sibling pairs that include DZ pairs will not suffer from false-positive results due to loci involved in embryo survival.

A gene (Cargl) that regulates tissue resistance to Candida albicans maps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeast Candida albicans is genetically regulated. Analysis of the strain distribution pattern of the C. albicans resistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant for Carg1, Tcra and Rib1. Therefore, Carg1 is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes. (C) 1998 Academic Press.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo

S100A8 (also known as CP10 or MRP8) was the first member of the S100 family of calcium-binding proteins shown to be chemotactic for myeloid cells. The gene is expressed together with its dimerization partner S100A9 during myelopoiesis in the fetal liver and in adult bone marrow as well as in mature granulocytes. In this paper we show that S100A8 mRNA is expressed without S100A9 mRNA between 6.5 and 8.5 days postcoitum within fetal cells infiltrating the deciduum in the vicinity of the ectoplacental cone. Targeted disruption of the S100A8 gene caused rapid and synchronous embryo resorption by day 9.5 of development in 100% of homozygous null embryos. Until this point there was no evidence of developmental delay in S100A8(-/-) embryos and decidualization was normal. The results of PCR genotyping around 7.5-8.5 days postcoitum suggest that the null embryos are infiltrated with maternal cells before overt signs of resorption. This work is the first evidence for nonredundant function of a member of the S100 gene family and implies a role in prevention of maternal rejection of the implanting embryo. The S100A8 null provides a new model for studying fetal-maternal interactions during implantation.

Extraordinary and extensive karyotypic variation: A 48-fold range in chromosome number in the gall-inducing scale insect Apiomorpha (Hemiptera : Coccoidea : Eriococcidae)

Chromosome number reflects strong constraints on karyotype evolution, unescaped by the majority of animal taxa. Although there is commonly chromosomal polymorphism among closely related taxa, very large differences in chromosome number are rare. This study reports one of the most extensive chromosomal ranges yet reported for an animal genus. Apiomorpha Rubsaamen (Hemiptera: Coccoidea: Eriococcidae), an endemic Australian gall-inducing scale insect genus, exhibits an extraordinary 48-fold variation in chromosome number with diploid numbers ranging from 4 to about 192. Diploid complements of all other eriococcids examined to date range only from 6 to 28. Closely related species of Apiomorpha usually have very different karyotypes, to the extent that the variation within some species- groups is as great as that across the entire genus. There is extensive chromosomal variation among populations within 17 of the morphologically defined species of Apiomorpha indicating the existence of cryptic species-complexes. The extent and pattern of karyotypic variation suggests rapid chromosomal evolution via fissions and (or) fusions. It is hypothesized that chromosomal rearrangements in Apiomorpha species may be associated with these insects' tracking the radiation of their speciose host genus, Eucalyptus.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.