The genetic basis for susceptibility to Rift Valley fever disease in MBT/Pas mice.

The large variation in individual response to infection with Rift Valley fever virus (RVFV) suggests that host genetic determinants play a role in determining virus-induced disease outcomes. These genetic factors are still unknown. The systemic inoculation of mice with RVFV reproduces major pathological features of severe human disease, notably the hepatitis and encephalitis. A genome scan performed on 546 (BALB/c × MBT) F2 progeny identified three quantitative trait loci (QTLs), denoted Rvfs-1 to Rvfs-3, that were associated with disease susceptibility in MBT/Pas mice. Non-parametric interval-mapping revealed one significant and two suggestive linkages with survival time on chromosomes 2 (Rvfs-1), 5 (Rvfs-3) and 11 (Rvfs-2) with respective logarithm of odds (LOD) scores of 4.58, 2.95 and 2.99. The two-part model, combining survival time and survival/death, identified one significant linkage to Rvfs-2 and one suggestive linkage to Rvfs-1 with respective LOD scores of 5.12 and 4.55. Under a multiple model, with additive effects and sex as a covariate, the three QTLs explained 8.3% of the phenotypic variance. Sex had the strongest influence on susceptibility. The contribution of Rvfs-1, Rvfs-2 and Rvfs-3 to survival time of RVFV-infected mice was further confirmed in congenic mice.

Innate resistance to flavivirus infections and the functions of 2'-5' oligoadenylate synthetases.

Mouse susceptibility to experimental infections with flaviviruses is significantly influenced by a cluster of genes on chromosome 5 encoding a family of proteins with enzymatic properties, the 2'-5' oligoadenylate synthetases (OAS). Positional cloning of the locus in question has revealed that susceptibility of laboratory inbred strains to this class of virus is associated with a nonsense mutation in the gene encoding the OAS1B isoform. Analysis of the molecular structure of the cluster in different mammalian species including human indicates that the cluster is extremely polymorphic with a highly variable number of genes and pseudogenes whose functions are not yet completely established. Although still preliminary, a few recent observations also substantiate a possible role for OAS1 in human susceptibility to viral infections (West Nile virus, SARS, etc.) and its possible involvement in some other diseases such as type 1 diabetes and multiple sclerosis. Finally, convergent observations indicate that the molecules encoded by the 2 '-5' OAS cluster might be involved in other fundamental cellular functions such as cell growth and differentiation, gene regulation, and apoptosis.

The spastic mouse: aberrant splicing of glycine receptor beta subunit mRNA caused by intronic insertion of L1 element.

Mice homozygous for the spastic mutation (spa) suffer from a complex motor disorder resulting from reduced CNS levels of the adult glycine receptor isoform GlyRA, which is composed of ligand-binding alpha 1 and structural beta polypeptides. The beta subunit-encoding gene (Glyrb) was mapped near the spa locus on mouse chromosome 3. In spa/spa mice, aberrant splicing of the beta subunit pre-mRNA strikingly diminishes the CNS contents of full-length transcripts, whereas truncated beta subunit mRNAs accumulate. This is a result of exon skipping, which causes translational frameshifts and premature stop codons. Intron 5 of the spa Glyrb gene contains an L1 transposable element that apparently is causal for the aberrant splicing of beta subunit transcripts.

The murine vik gene (chromosome 9) encodes a putative receptor with unique protein kinase motifs.

Receptor tyrosine kinases are involved in the regulation of cell growth and may play a central role in embryonic development. We recently developed a polymerase chain reaction (PCR)-based gene cloning procedure that allows selective isolation of genes that encode novel transmembrane tyrosine kinases in tissues of embryonic origin. By employing this protocol on mRNA from a 12.5 day post-coitum mouse placenta, we identified a gene for a putative receptor protein kinase. The deduced amino acid sequence predicts the existence of an approximately 200 amino acid long extracellular domain that shows no similarity to known proteins. The cytoplasmic portion contains a core sequence that is structurally homologous to the tyrosine kinase family. However, a few highly conserved short blocks of sequences, shared by all protein kinases, display variations in the isolated gene. These include the glycine-rich block at the nucleotide-binding cleft and the Asp-Phe-Gly triplet at the substrate recognition site. On the basis of these variations, we named the gene vik for variant in the kinase. Northern analysis revealed two widely expressed transcripts of vik with molecular weights of 3 and 2.5 kb. Chromosomal mapping using restriction fragment length polymorphism localized the gene to murine chromosome 9. The unique structural landmarks of vik at both the extracellular and the cytoplasmic domains suggest novel ligand as well as substrate specificity of the presumed receptor.

Chromosomal localization of the three members of the jun proto-oncogene family in mouse and man.

The three members of the jun proto-oncogene family c-jun, jun b and jun D were mapped on the mouse chromosome by in situ hybridization. The c-jun locus is on chromosome 4 subregion C5----C7, whereas jun B and jun D are co-localized on chromosome 8 subregion C. RFLP analysis of interspecific hybrids confirmed the mapping of jun B and D and showed that they are situated about 7.3 +/- 3.5 cM apart. Thus despite their possible origin from a single ancestral gene they are not closely linked on the chromosome. Using the same probes, we showed that the human genome also contains sequences homologous to the mouse jun B and jun D. They are located on human chromosome 19 p13.2, a region that may be involved in chromosomal translocation in acute lymphocytic leukemia (ALL), acute nonlymphocytic leukemia (ANLL) and malignant melanoma (MEL). Finally, the present data identify a new segmental homology between mouse and human chromosomes.

Isolation of Vgr-2, a novel member of the transforming growth factor-beta-related gene family.

A cDNA clone, Vgr-2, with homology to certain members of the transforming growth factor-beta superfamily has been isolated from a mouse embryo cDNA library. The encoded protein shows significant similarity to members of the Vg-1/decapentaplegic/bone morphogenetic protein subgroup of the transforming growth factor-beta family. Within this group, Vgr-2 is more similar to Xenopus Vg-1 than to any other member so far isolated. The gene is expressed at highest levels during midgestation mouse development, and transcripts are localized by in situ hybridization to the osteogenic zone of developing bone. Vgr-2 is expressed in F9 teratocarcinoma cells, and its RNA levels are down-regulated within 24 h after differentiation with retinoic acid. The genomic organization of Vgr-2 and its location on mouse chromosome 6 are reported.

Chromosomal localization of the mouse genes coding for alpha 2, alpha 3, alpha 4 and beta 2 subunits of neuronal nicotinic acetylcholine receptor.

The chromosomal localization of four neuronal nicotinic acetylcholine receptor subunit genes was performed by following the mendelian segregation of their corresponding alleles in backcrosses involving the mouse species Mus spretus and the laboratory strains C57BL/6 or BALB/c. A similar analysis previously performed with muscle nicotinic acetylcholine receptor subunits revealed that the genes coding for the alpha and beta subunits are respectively located on chromosome 2 and 11, whereas the gamma and delta subunit coding genes are linked and located on mouse chromosome 1. In this study, we show that the genes coding for the neuronal nicotinic acetylcholine receptor alpha 2, alpha 3 and beta 2 subunits are dispersed on three different mouse chromosomes, viz. 14, 9 and 3 respectively. Moreover, the alpha 4 subunit gene is located on chromosome 2 but is not genetically linked to the alpha 1 subunit gene.

Recent evolutionary origin of the expression of the glial fibrillary acidic protein (GFAP) in lens epithelial cells. A molecular and genetic analysis of various mouse species.

We have investigated the phylogenetic distribution of the glial fibrillary acidic protein (GFAP) in lens epithelial cells (LEC) of various mouse species within the genus Mus. We have shown that lens GFAP is expressed in mice of the Mus musculus complex and in Mus spicilegus and Mus macedonicus species (L.GFAP(+) phenotype) while it is absent in Mus spretus, Mus caroli and Mus cooki species (L.GFAP(-) phenotype). Our results argue in favour of one of the phenograms illustrating the probable phylogenetic relationships between these species in the genus Mus. In animals where lens GFAP was immunodetected, Northern blots of lens RNA extracts hybridized with a mouse GFAP cDNA probe, revealed a single 2.7 kb band. Comparative Northern blot analysis of lens tissue from L.GFAP(+) mice or of brain tissue from L.GFAP(+) or L.GFAP(-) mice did not show any size heterogeneity of the GFAP mRNA. The pattern of the GFAP immunostaining of astroglial cells in brain was identical in both L.GFAP phenotypes. Analysis of interspecific crosses showed that the L.GFAP(+) character is transmitted in a dominant fashion and seems to be linked to the Mus musculus Gfap gene. In this study we have also confirmed the localization of the mouse Gfap gene on chromosome 11.

Wobbler, a mutation affecting motoneuron survival and gonadal functions in the mouse, maps to proximal chromosome 11.

The wobbler mouse (genotype wr/wr) has been considered as an animal model for human neurodegenerative disorders. In the homozygous condition, the autosomal mutation wobbler (wr) causes a motoneuron disease and gonadal dysfunction. We have genetically mapped the wr gene, using an interspecific backcross between the laboratory strain C57BL/6J (wr/+) and Mus spretus. The expected percentage of wobbler progeny were obtained, but heterogeneous expression of the wobbler phenotype indicated the existence of modifier genes in the M. spretus genetic background. The segregation of DNA markers of known chromosomal location among wobbler progeny and unaffected mice was scored. Close linkage of wr was obtained with Erbb and Rel on chromosome 11 and the gene order cen-Nfh-Erbb-wr-Rel-Hba-Il-3 was established. Closely linked markers like Erbb provide tools for a prognostic DNA diagnosis of the wobbler disease, and thereby for its analysis by descriptive and experimental embryology.

A new strategy useful for rapid identification of microsatellites from DNA libraries with large size inserts.

Microsatellites are new powerful polymorphic markers used for gene mapping. Their characterization requires that all the sequence surrounding the repeat be known in order to be able to design primers for PCR amplification. However, when using DNA libraries with large cloned inserts, this sequence characterization is not immediately practicable. In this paper, we describe a new strategy, based both on the use of a microsatellite specific probing and on the creation of nested deleted clones with the Exonuclease III, in order to position microsatellites in a range allowing direct sequencing. This method was applied to the screening of a mouse chromosome 19 DNA specific library. In this way, thirteen clones were identified by specific probing and seven were submitted to the nested deletion strategy. Five of them presented microsatellite sequences in specific deleted subclones which were selected and sequenced. Primers were designed for each of them and polymorphism between the genomes of several inbred strain of mouse have been determined. These microsatellites were mapped, three of them to chromosome 19 and two to chromosome 11.

Cardiac and skeletal muscle troponin I isoforms are encoded by a dispersed gene family on mouse chromosomes 1 and 7.

We mapped the locations of the genes encoding the slow skeletal muscle, fast skeletal muscle, and cardiac isoforms of troponin I (Tnni) in the mouse genome by interspecific hybrid backcross analysis of species-specific (C57BL/6 vs Mus spretus) restriction fragment length polymorphisms (RFLPs). The slow skeletal muscle troponin I locus (Tnni1) mapped to Chromosome (Chr) 1. The fast skeletal muscle troponin I locus (Tnni2), mapped to Chr 7, approximately 70 cM from the centromere. The cardiac troponin I locus (Tnni3) also mapped to Chr 7, approximately 5-10 cM from the centromere and unlinked to the fast skeletal muscle troponin I locus. Thus, the troponin I gene family is dispersed in the mouse genome.

Gtl2lacZ, an insertional mutation on mouse chromosome 12 with parental origin-dependent phenotype.

We have produced a transgenic mouse line, Gtl2lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed.

Hst-3: an X-linked hybrid sterility gene.

A gene, Hst-3, responsible for sterility in F1 males from crosses between Mus spretus and laboratory strains of mice such as C57BL/6, has been localized on the distal part of the X chromosome, using both DNA probes and biochemical markers on a panel of F1(C57BL/6 x SEG) x C57BL/6 backcross males. This gene may be a model for studying mammalian hybrid sterility.

Comparative mapping of lipocalin genes in human and mouse: the four genes for complement C8 gamma chain, prostaglandin-D-synthase, oncogene-24p3, and progestagen-associated endometrial protein map to HSA9 and MMU2.

The lipocalin superfamily encompasses a large set of quite distantly related proteins that act as carriers for small, lipophilic molecules. The lipocalin genes coding for orosomucoid, the alpha 1-microglobulin/bikunin precursor, and the major urinary protein map to MMU4, while their human counterparts map to the homologous HSA9q34 area where three other lipocalin genes for complement C8 gamma chain (C8G), progestagen-associated endometrial protein (PAEP), and prostaglandin D synthase (PTGDS) are also located. By linkage analyses in an interspecific backcross progeny in mouse, the Lcn2 gene coding for the oncogenic lipocalin 24p3, as well as the 3 lipocalin genes for C8G, PTGDS, and PAEP, have now been assigned to MMU2. The first three genes map to proximal MMU2, which is known to be homologous to HSA9q34. Paep is more distally located, which extends the number of regions with conserved syntenies between HSA9q34 and MMU2. By in situ hybridization, the human LCN2 gene maps to HSA9q34. Our data indicate that the lipocalin locus arrangement in the human/mouse ancestor is closer to that found at HSA9q than to that in the MMU genome.

Etl2, a novel putative type-I cytokine receptor expressed during mouse embryogenesis at high levels in skin and cells with skeletogenic potential.

The regulatory effects of signaling proteins like hormones, growth factors, and cytokines are mediated by specific cell surface receptors which are grouped into distinct families on the basis of structural criteria. Here we report on the isolation and embryonic expression of a novel mouse gene, Etl2 (enhancer trap locus 2) which, based on its deduced amino acid sequence, constitutes a new member of the cytokine type-I receptor family. Among type-I receptors Etl2 is most similar to the alpha subunits of the human ciliary neurotrophic factor (CNTF) receptor and the mouse interleukin-6 (IL6) receptor with 32 and 30% identical amino acids, respectively. From Day 9 p.c. (postcoitum) onward low levels of Etl2 mRNA were detected in mesenchymal cells throughout the embryo and in parts of the nervous system, in particular in the ependymal linings of the spinal cord and the developing brain vesicles and in the neuronal layer of the retina. Highest levels of Etl2 expression were found on Day 12.5 p.c. in the craniofacial mesenchyme and during subsequent development in mesenchymal cells around all developing cartilages. At later stages, Etl2 transcripts were abundant in the dental papilla, the dermis, and hair follicles, as well as in the perichondrium and periost, i.e., in regions containing chondro and osteo progenitor cells. Etl2 mRNA was not detected, however, in mature odontoblasts, chondroblasts, osteoblasts, chondrocytes, and osteocytes. Our results suggest that Etl2 is a new orphan receptor belonging to the type-I cytokine receptor family and that Etl2 might have regulatory functions, particularly in the control of proliferation and/or differentiation of skeletogenic progenitor and other mesenchymal cells.

Re-localization of Actsk-1 to mouse chromosome 8, a new region of homology with human chromosome 1.

We present here the genetic mapping of the alpha-skeletal actin locus (Actsk-1) on mouse Chromosome (Chr) 8, on the basis of the PCR analysis of a microsatellite in an interspecific backcross. Linkage and genetic distances were established for four loci by analysis of 192 (or 222) meiotic events and indicated the following gene order: (centromere)-Es-1-11.7 cM-Tat-8.3 cM-Actsk-1-0.5 cM-Aprt. Mapping of ACTSK to human Chr 1 and of TAT and APRT to human Chr 16 demonstrates the existence of a new short region of homology between mouse Chr 8 and human Chr 1. Intermingling on this scale between human and mouse chromosomal homologies that occurred during evolution creates disorders in comparative linkage studies.

Polymorphisms revealed by PCR with single, short-sized, arbitrary primers are reliable markers for mouse and rat gene mapping.

Ten single, arbitrarily designed oligodeoxynucleotide primers, with 50-70% (G+C) content, were used to amplify by polymerase chain reaction (PCR) sequences with DNA templates from several mouse species (Mus spretus, Mus musculus musculus, and Mus musculus domesticus), as well as DNA from the laboratory rat (Rattus norvegicus). Eight of these ten primers, used either individually or associated in pairs, generated a total of 13 polymorphic products which were used as genetic markers. All of these polymorphic sequences but one were mapped to a particular mouse chromosome, by use of DNA panels prepared either from interspecific backcross progeny of the type (C57BL/6 x Mus spretus)F1 x C57BL/6 or DNA samples prepared from two sets of recombinant inbred (RI) strains (AKXL and BXD). Six rat-specific DNA segments were also assigned to a particular chromosome with DNA panels prepared from 18 rat/mouse somatic cell hybrids segregating rat chromosomes. From these experiments we conclude that, under precisely standardized PCR conditions, the DNA molecules amplified with these arbitrarily designed primers are useful and reliable markers for genetic mapping in both mouse and rat.

Mapping of the genes encoding tum- transplantation antigens P91A, P35B, and P198.

Tum- comprises a class of genes, mutation of which in P815 tumor cells has led to the acquisition of new cytotoxic T cell-recognized epitopes. The cells carrying the mutant alleles have impaired tumorigenicity compared with their progenitors due to in vivo induction of a cytotoxic T-cell response specific for tum- antigens. Two tum- genes, P91A and P35B, were found to be single copy loci mapping to chromosomes 11 and 15 respectively. A third, P198, was found to map to chromosome 7 and to be a member of a small gene family with other members on chromosomes 13, 14, and 15. Multiple P198-related sequences were found in other mammalian species suggesting the P198 related gene family is a general feature of mammalian genomes.

Cloning of human hepatic nuclear factor 1 (HNF1) and chromosomal localization of its gene in man and mouse.

HNF1 is a transcription factor that is required for hepatocyte-specific expression of several genes, including albumin and fibrinogen. Rat HNF1-encoding cDNAs have recently been cloned, revealing that this factor is a distant member of the homeoprotein family. We have now isolated HNF1 clones from a human liver cDNA library by using a rat HNF1 cDNA-derived probe. The longest clone, HCL20, contains a sequence corresponding to the intact rat HNF1-coding region followed by a 3' nontranslated region and a poly(A) tail, hence representing an almost full-length HNF1 cDNA. Alignment of the human and rat sequences shows that HNF1 is highly conserved between the two species. The HNF1 gene was mapped by in situ hybridization and by RFLP analysis of interspecific mouse backcrosses to chromosomes 12q24.3 and 5F in human and mouse, respectively, establishing a new segmental homology between these two chromosomes.