New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin.

The dystrophin gene encodes several tissue-specific protein isoforms that are generated by alternative splicing and by transcription from at least three separate promoters. We have characterized the mutation in a new strain of mdx mice that results in aberrant splicing of both the 14 and 4.8 kilobase dystrophin mRNAs and disrupts expression of the muscle and brain 427K and nonmuscle 70K isoforms of dystrophin. In contrast, we have determined that expression of the 70K isoform is normal in the original mdx mutant. We have cloned the unique 5' exon of the murine 4.8 kb mRNA and have analysed the tissue distribution and aberrant splicing of this transcript in the mdx3Cv mutant. This new mdx mutant will provide an improved model system for functional studies of the dystrophin C-terminus in muscle and nonmuscle tissues.

Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M.

Normal mammalian development requires a diploid combination of both haploid parental genomes. Uniparental disomy for certain segments of specific chromosomes results in aberrant development or prenatal lethality, indicating that the parental genomes have undergone modifications during gametogenesis. These modifications result in parent-of-origin specific expression for some genes, a phenomenon called genomic imprinting. Recent work with DNA methyltransferase deficient mice showed that differential methylation is the probable basis of the imprinted character of several genes. Screening for endogenous imprinted loci using restriction landmark genomic scanning with methylation sensitive enzymes (RLGS-M) identified eight imprinted RLGS (Irigs) candidate loci. Molecular analysis of the genomic region of one of the loci (Irigs2) resulted in the discovery of the paternally imprinted U2afbp-rs gene within a previously identified imprinted region on mouse chromosome 11 (refs 5, 7). This paper describes the characterisation of a novel imprinted RLGS-M locus, Irigs3, on mouse chromosome 9 (ref. 6). Within this locus we identified the Grf1 (also called Cdc25Mm) gene, which is homologous to the RAS-specific guanine nucleotide exchange factor gene, CDC25, in Saccharomyces cerevisiae. Grf1 is located about 30 kb downstream of the methylation imprinted site, identified by RLGS-M, and shows paternal allele specific expression in mouse brain, stomach and heart. Our results indicate that imprinting may have a role in regulating mitogenic signal transduction pathways during growth and development.

The murine Xe169 gene escapes X-inactivation like its human homologue.

Among a number of genes that escape X-chromosome inactivation in humans, three have been evaluated in mice and unexpectedly all three are subject to X-inactivation. We report here the cloning and expression studies of a novel mouse gene, Xe169, and show that it escapes X-inactivation like its human homologue. Xe169 was assigned to band F2/F3 on the mouse X chromosome by fluorescent in situ hybridization and Southern analysis indicates that the gene is located outside the pseudoautosomal region. Homologous, but divergent, sequences exist on the Y chromosome. In vitro and in vivo studies show that Xe169 is expressed from both the active and the inactive X chromosomes. Xe169 is the first cloned non-pseudoautosomal gene that escapes X-inactivation in mice.

The mouse genome: an overview.

A genetic map with one molecularly marked locus per cM will be available for the mouse in the near future. A map of this density should provide molecular reference points that connect genetic and physical maps, identify sites to initiate positional cloning studies for the molecular characterization of mutant loci, and define homologous regions of mouse and human genomes.

CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene.

Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.

Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

Detailed genetic mapping of the A-raf proto-oncogene on the mouse X chromosome.

The transcribed murine A-raf proto-oncogene has been localized to the proximal region of the mouse X chromosome, within the context of four other active genes in this region which together constitute a conserved linkage group between mouse and man. This localization has been accomplished using species-specific restriction fragment length variation and DNAs from a previously defined informative subset of progeny representative of a set of 100 progeny from an interspecific backcross between inbred C57BL/6JRos and wild-derived Mus spretus. This new data regionally orders the mouse A-raf locus relative to the 24 X-linked markers previously examined in this backcross. We find that A-raf co-localizes with two other active genes, tissue inhibitor of metalloproteinases (Timp) and synapsin (Syn-1), 4.0 +/- 2.0 cM distal to the Otc gene at the proximal end of the mouse X chromosome, for a partial gene order in this region of: centromere-Cybb-Otc-Timp/A-raf/Syn-1-Xlr-1-Hprt.

Close physical linkage of the FLT1 and FLT3 genes on chromosome 13 in man and chromosome 5 in mouse.

Receptor-type tyrosine kinases (RTK) with five or seven immunoglobulin-like domains in their extracellular region are encoded by genes grouped in clusters. In human, two such clusters have been individualized, in chromosomal regions 4q11-q12 and 5q33-qter respectively. We define here a third cluster located on chromosome 13q and containing two contiguous RTK genes, FLT1 and FLT3. The former has recently been shown to encode a RTK of a new class while the latter codes for a hematopoietic receptor closely related to the products of the FMS and KIT genes. The physical linkage is also evidenced in mouse, where the two genes appear to lie within a 350 kb Mlu I fragment, on mouse chromosome 5.

Altered turnover of hypoxanthine phosphoribosyltransferase in erythroid cells of mice expressing Hprt a and Hprt b alleles.

We have previously shown that mice expressing Hprt a allele(s) have erythrocyte hypoxanthine phosphoribosyltransferase (HPRT) levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than in mice that express the Hprt b allele (Mus musculus domesticus; C57BI/6J; C3H/HeHa), and that these differences in erythrocyte HPRT levels are due to differences in the turnover rates of the HPRT A and B proteins as reticulocytes mature to erythrocytes. We show here that: the taxonomic subgroups of the genus Mus are essentially monomorphic for the occurrence of either the Hprt a or the Hprt b allele, with Hprt a being common in the aboriginal species (M. spretus, Mus hortulanus and Mus abbotti) and in several commensal species (Mus musculus musculus, M. m. castaneus, Mus musculus molossinus), while Hprt b is common in feral M. m. domesticus populations as well as in all inbred strains of mice tested; in all these diverse Mus subgroups there is a strict association of Hprt a with high and Hprt b with low levels of erythrocyte HPRT; and, the association between the occurrence of the Hprt a allele and elevated erythrocyte HPRT levels is retained following repeated backcrosses of wild-derived Hprt a allele(s) into the genetic background of inbred strains of mice with the Hprt b allele. Collectively, these observations indicate that the elevated and low levels of erythrocyte HPRT are specified by differences in the Hprt a and b structural genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate oxaloacetate transaminase (got) genetics in the mouse: polymorphism of got-1.

We have examined a polymorphism for the soluble glutamate oxaloacetate (GOT-1) isozyme system which was found in the Asian mouse Mus castaneus. Variants of GOT-1 segregate as though they are controlled by codominant alleles for a single autosomal locus which we have designated Got-1. No close linkage of genes for soluble and mitochondrial forms of the enzyme, GOT-1 and GOT-2 respectively, was observed. Furthermore, no close linkage of Got-1 and the loci c, Gpi-1, Mod-2, Mod-1, Ld-1, Gpd-1, Pgm-1 or Gpo-1 was observed. Our results demonstrate the utility of sampling Mus from diverse populations to extend the repertoire of polymorphic loci and the genetic linkage map.

Electrophoretic variation for x-chromosome-linked phosphoglycerate kinase (pgk-1) in the mouse.

Electrophoretic variation for X-chromosome-linked phosphoglycerate kinase (PGK-1) has been found as a polymorphism in feral mice in Denmark. Males from feral sampling or from a variety of genetic crosses have only a single-banded phenotype of the variant PGK-1A type or of the PGK-1B type commonly found among inbred mice. By contrast, three phenotypes were observed among females; two homozygous single-banded types and a heterozygous double-banded type. The X-chromosome linkage of the Pgk-1 locus was determined from the mode of inheritance in F(1) and backcross generations and confirmed by the linkage of Pgk-1 and the X-linked markers Hq, Ta and Mo. Pgk-1 showed 29/122 recombinations with Hq, 5/185 with Ta and 0/108 recombinants with Mo. Based on these recombination data, a gene order of Hq-Ta-Pgk-1-Mo is suggested.

Electrophoretic variation for X chromosome-linked hypoxanthine phosphoribosyl transferase (HPRT) in wild-derived mice.

An electrophoretic variation for hypoxanthine phosphoribosyltransferase, HPRT, has been identified in samples of Mus spretus, a field mouse from southern Europe and in M. m. castaneus, a house mouse from southeast Asia. These mice will interbreed with laboratory mice to produce viable, fertile F1 progeny. The variation for HPRT segregates as an X chromosome gene in F1 and backcross progeny. Linkage analysis involving the markers Pgk-1 and Ags indicated a gene order of centromere--Hprt--Pgk-1--Ags in crosses involving both stocks of wild mice.

Direct determination of NotI cleavage sites in the genomic DNA of adult mouse kidney and human trophoblast using whole-range restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a method for visualizing restriction landmarks, employing direct labeling of restriction sites of genomic DNA and high-resolution two-dimensional electrophoresis. We determined the conditions for both the first and second dimensions of RLGS that define all of the restriction fragments which carry the NotI landmark. Using this system, we determined the number of cleavable NotI sites of genomic DNA from the mouse kidney (C57BL/6) and from the human placenta. The mouse and human genomes were cleaved at 2,380 +/- 80 sites (4,760 +/- 160 spots) and 3,240 +/- 110 sites (6,480 +/- 220 spots), respectively with NotI.

Identification and characterization of Zic4, a new member of the mouse Zic gene family.

The mouse Zic genes encode zinc-finger (Zf) proteins expressed only in the cerebellum of the adult brain. The genes are the vertebrate homologues of the Drosophila pair-rule gene, odd-paired (opa). We identified a novel gene, Zic4, which belongs to the Zic gene family, through a genomic and cDNA cloning study. Zic4 is highly similar to Zic1, Zic2 and Zic3, especially in its Zf motif. An analysis of the genomic organization of Zic4 showed that the gene shares a common exon-intron boundary with Zic1, Zic2, Zic3 and opa. The chromosomal location of Zic4 was determined to be mouse chromosome 9 in the vicinity of Zic1, using an interspecific backcross panel. An RNase protection study showed that Zic4 is expressed only in the cerebellum during the adult stage, as are the other Zic genes. The temporal profile of mRNA expression in the developing cerebellum is similar to that of Zic3 which has a peak on postnatal day 5. These findings suggest that Zic4 is a gene which works cooperatively with other Zic genes during cerebellar development.

mdxCv3 mouse is a model for electroretinography of Duchenne/Becker muscular dystrophy.

PURPOSE: To identify an animal model for the abnormal scotopic electroretinogram found in a majority of Duchenne and Becker muscular dystrophy patients. METHODS: Ganzfeld electroretinograms were recorded in dark-adapted normal C57BL/6 mice, and two strains of mice with different X-linked muscular dystrophy mutations (mdx and mdxCv3). Responses for the right eye were averaged and the amplitudes and implicit times of the a-wave and b-wave were measured. The electroretinogram was digitally filtered to extract the oscillatory potentials. Statistical analyses included one-way analysis of variance and the Scheffé S test. RESULTS: While the electroretinogram in mdx was normal, in mdxCv3 the scotopic b-wave was markedly reduced and the oscillatory potentials were delayed, similar to changes observed in Duchenne and Becker muscular dystrophy patients. Some of the mdxCv3 animals demonstrated negative configuration electroretinograms, with the b-wave amplitude reduced compared to that of the a-wave. CONCLUSIONS: Abnormalities found in the electroretinograms of Duchenne and Becker muscular dystrophy patients led to the identification of dystrophin in human retina and the discovery that dystrophin is required for normal retinal electrophysiology. These results indicate that mdxCv3 is a model for elucidating the role of dystrophin in retina and suggest that dystrophin isoforms, consisting of only the C-terminal domains of the full-length protein, may be important to the development of normal retinal electrical potentials.

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2.

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.

DNA methylation patterns and sequence transitions of the CpG island of mouse Hprt during early embryogenesis.

The timing and pattern of methylation of the CpG island in the X-chromosome gene, Hprt, was examined using bisulfite methods to assess the occurrence of DNA cytosine methylation at the onset of X-chromosome inactivation (XCI). The Hprt sequences were extensively methylated in 4.5 dpc blastocysts, and the levels of methylation progressively decreased to 7.5 dpc. Adult patterns of methylation were established in the embryonic tissues after 7.5 dpc. By contrast, methylation continued to decrease in extraembryonic lineages and at 13.5 days was not detectable on the paternal Hprt sequences in the yolk sac endoderm. In the bisulfite-treated coding strand, numerous G to A transitions and CpTpG methylations were observed that were unique to the methylated or nonmethylated Hprt sequences, respectively, in early development.

Comparison of baseline sister-chromatid exchanges (SCE), cyclophosphamide-, ethylnitrosourea (ENU)-induced SCE, ENU-induced cell-cycle delay and chromosome aberrations between Peru and laboratory mice.

The baseline sister-chromatid exchange (SCE) frequency and sensitivity to the effects of the mutagens cyclophosphamide (CPP) and ethylnitrosourea (ENU) in bone-marrow cells of descendants of wild mice trapped from Rimac valley in Peru (Peru mice) were studied and compared to the same effects in laboratory mice. Baseline SCE of the Peru mice were significantly higher than those of the C57BL/6J and DBA/2 mice. The average SCE/cell of 4 Peru mice was 5.4 (range 3.8-7.6), while the average of SCE/cell of either 4 C57BL or 5 DBA mice was 3.2 (range 3.0-3.4). The variation of SCE/cell among Peru mice studied was statistically significant whereas among C57BL or DBA mice it was not. SCE frequencies of primary cultures derived from the ear tissue of 10 Peru (mean SCE/cell = 8.5) were also significantly higher than those of 6 C57BL mice (mean SCE/cell = 7.4). CPP treatment resulted in a dose-dependent increase of SCE frequencies in bone-marrow cells of all the mice. However, some of Peru mice treated with CPP had significantly higher SCE than the other Peru mice and than all of the C57BL and DBA mice treated with equivalent dose. ENU induced increased SCE frequencies in Peru and C57BL mice. Again some of Peru mice either had significantly higher SCE, greater extent induced cell-cycle delay or chromosome aberrations (CA) than other Peru mice and than of all the C57BL mice treated with equivalent dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthroscopic lavage and debridement for osteoarthritis of the knee.

We measured the effect of arthroscopic lavage and debridement of the osteoarthritic knee by comparing objective measurements of thigh muscle function before and after operation. There was some improvement in quadriceps isokinetic torque at six and 12 weeks after joint lavage but not after debridement. Neither method significantly relieved the patients' symptoms.