A polymorphism in New Zealand inbred mouse strains that inactivates phosphatidylcholine transfer protein

New Zealand obese (NZO/HlLt) male mice develop polygenic diabetes and altered phosphatidylcholine metabolism. The gene encoding phosphatidylcholine transfer protein (PC-TP) is sited within the support interval for Nidd3, a recessive NZO-derived locus on Chromosome 11 identified by prior segregation analysis between NZO/HlLt and NON/Lt. Sequence analysis revealed that the NZO-derived PC-TP contained a non-synonymous point mutation that resulted in an Arg120His substitution, which was shared by the related NZB/BlNJ and NZW/LacJ mouse strains. Consistent with the structure-based predictions, functional studies demonstrated that Arg120His PC-TP was inactive, suggesting that this mutation contributes to the deficiencies in phosphatidylcholine metabolism observed in NZO mice.     

Y-encoded, species-specific DNA in mice: evidence that the Y chromosome exists in two polymorphic forms in inbred strains

We have investigated the structure of the murine Y chromosome by first developing a novel method for specifically cloning Y-encoded DNA and then generating a library enriched for Y-specific DNA sequences. Three randomly chosen Y DNA clones were studied and found to share several interesting properties: all three are members of small Y-specific multisequence families; all three are mouse-specific; and all three probes detect Y-encoded restriction fragments that are polymorphic. Examination of polymorphic Y chromosome restriction fragments in male DNA from nine different inbred strains suggests that only two polymorphic forms of Y chromosomal DNA exist among inbred strains of mice.     

Longitudinal fundus and retinal studies with SD-OCT: a comparison of five mouse inbred strains

Spectral domain optical coherence tomography (SD-OCT) has recently been established as a method for in vivo imaging of fundus and retina in the mouse. It enables more effective studies of retinal diseases including investigations of etiopathologic mechanisms. In order to learn more about longitudinal fundus development and to enable recognition of disease-associated irregularities, we performed confocal scanning laser ophthalmoscopy (cSLO) and SD-OCT measurements in the inbred strains C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129S2/SvJ when they were between 2 and 6 months of age. In general, cSLO and SD-OCT data did not reveal sex-specific or unilateral differences. C3HeB/FeJ and FVB/NCrl mice showed diffuse choroidal dysplasia. Choroidal vein-like structures appeared as dark fundus stripes in C3HeB/FeJ. In FVB/NCrl, fundus fleck accumulation was found. In contrast, only minor time-dependent changes of fundus appearance were observed in C57BL/6J, BALB/cByJ, and 129S2/SvJ. This was also found for individual fundic main blood vessel patterns in all inbred strains. Vessel numbers varied between 6 and 13 in C57BL/6J. This was comparable in most cases. We further found that retinae were significantly thicker in C57BL/6J compared to the other strains. Total retinal thickness generally did not change between 2 and 6 months of age. As a conclusion, our results indicate lifelong pathologic processes in C3HeB/FeJ and FVB/NCrl that affect choroid and orbital tissues. Inbred strains with regular retinal development did not reveal major time-dependent variations of fundus appearance, blood vessel pattern, or retinal thickness. Consequently, progressive changes of these parameters are suitable indicators for pathologic outliers.     

Assignment of a processed mouse Aprt pseudogene to the same chromosome as the functional gene

A novel genetic system has been used to demonstrate that a processed adenine phosphoribosyltransferase (Aprt) pseudogene is located on mouse chromosome 8, which is the same chromosome that carries the functional Aprt gene. A restriction fragment length polymorphism associated with the pseudogene was found to segregate concordantly with chromosome 8 in APRT- mutants of a near-diploid cell line that had lost one copy of the chromosome.     

The identification of a repeated DNA sequence involved in the karyotype polymorphism of the human Y chromosome

We show that individual men are polymorphic for the amount of two different repeated DNA sequences. The amount of one of these sequences is proportional to the length of the brightly fluorescent heterochromatin on the Y chromosome. There are no detectable alterations in sequence between polymorphic individuals. Female DNA contains sequences complementary to those found on the Y, but at a much reduced level.     

Deep short-read sequencing of chromosome 17 from the mouse strains A/J and CAST/Ei identifies significant germline variation and candidate genes that regulate liver triglyceride levels

Genome sequences are essential tools for comparative and mutational analyses. Here we present the short read sequence of mouse chromosome 17 from the Mus musculus domesticus derived strain A/J, and the Mus musculus castaneus derived strain CAST/Ei. We describe approaches for the accurate identification of nucleotide and structural variation in the genomes of vertebrate experimental organisms, and show how these techniques can be applied to help prioritize candidate genes within quantitative trait loci.     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

The mouse Ly-17 locus identifies a polymorphism of the Fc receptor

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab)₂ fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2AG2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.Abbreviations used in this paper Ig immunoglobulin - FcR receptor for the Fc portion of Ig - TNP trinitrophenyl - Fab antigen-binding fragment - pA Protein A - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - PBS phosphate-buffered saline - BSA bovine serum albumin - SAMIg sheep antimouse Ig - SRBC sheep red blood cells - C complement - FITC fluorescein isothiocyanate - CNBr cyanogen bromide - EA antibody-sensitized erythrocytes     

A polymorphism in randomly amplified DNA that differentiates the Y chromosomes of Bos indicus and Bos taurus

A small number of west African Bos taurus cattle breeds, including the N'Dama, constitute a valuable genetic resource by virtue of their ability to remain productive under trypanosomiasis challenge. However, introgression of Bos indicus genes into the trypanotolerant breeds, particularly by introduction of zebu bulls, is a threat to this resource. This work describes the characterization and cloning of a bovine randomly amplified polymorphic DNA (RAPD) that is generated in polymerase chain reaction (PCR) with the 10 base primer ILO1065 from Bos indicus male templates, but not from B. taurus male templates or female templates of either type. Male-specific sequences with homology to the RAPD also occur in B. taurus breeds. This suggests that the polymorphism may be due to base substitution(s) in an ILO1065 priming site, or insertion/deletion events either affecting priming sites or occurring between sites on the cattle Y chromosome. We have shown that cattle, whether of B. indicus or B. taurus phenotype, which possess a typically B. indicus metaphase Y chromosome on the basis of QFQ banding, have a B. indicus ILO1065-generated genotype. The ILO1065-primed RAPD can be used in a simple dot blot assay as a probe of RAPD-PCR products, to provide a convenient, reliable and effective means of detecting introgression of zebu genes in B. taurus cattle populations.     

Common inbred strains of the laboratory mouse that are susceptible to infection by mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV

Laboratory mouse strains carry endogenous copies of the xenotropic mouse leukemia viruses (X-MLVs), named for their inability to infect cells of the laboratory mouse. This resistance to exogenous infection is due to a nonpermissive variant of the XPR1 gammaretrovirus receptor, a resistance that also limits in vivo expression of germ line X-MLV proviruses capable of producing infectious virus. Because laboratory mice vary widely in their proviral contents and in their virus expression patterns, we screened inbred strains for sequence and functional variants of the XPR1 receptor. We also typed inbred strains and wild mouse species for an endogenous provirus, Bxv1, that is capable of producing infectious X-MLV and that also contributes to the generation of pathogenic recombinant MLVs. We identified the active Bxv1 provirus in many common inbred strains and in some Japanese Mus molossinus mice but in none of the other wild mouse species that carry X-MLVs. Our screening for Xpr1 variants identified the permissive Xpr1(sxv) allele in 7 strains of laboratory mice, including a Bxv1-positive strain, F/St, which is characterized by lifelong X-MLV viremia. Cells from three strains carrying Xpr1(sxv), namely, SWR, SJL, and SIM.R, were shown to be infectable by X-MLV and XMRV; these strains carry different alleles at Fv1 and vary in their sensitivities to specific X/P-MLV isolates and XMRV. Several strains with Xpr1(sxv) lack the active Bxv1 provirus or other endogenous X-MLVs and may provide a useful model system to evaluate the in vivo spread of these gammaretroviruses and their disease potential in their natural host.     

Satellite DNA from the Y chromosome of the malaria vector Anopheles gambiae

Satellite DNA is an enigmatic component of genomic DNA with unclear function that has been regarded as "junk." Yet, persistence of these tandem highly repetitive sequences in heterochromatic regions of most eukaryotic chromosomes attests to their importance in the genome. We explored the Anopheles gambiae genome for the presence of satellite repeats and identified 12 novel satellite DNA families. Certain families were found in close juxtaposition within the genome. Six satellites, falling into two evolutionarily linked groups, were investigated in detail. Four of them were experimentally confirmed to be linked to the Y chromosome, whereas their relatives occupy centromeric regions of either the X chromosome or the autosomes. A complex evolutionary pattern was revealed among the AgY477-like satellites, suggesting their rapid turnover in the A. gambiae complex and, potentially, recombination between sex chromosomes. The substitution pattern suggested rolling circle replication as an array expansion mechanism in the Y-linked 53-bp satellite families. Despite residing in different portions of the genome, the 53-bp satellites share the same monomer lengths, apparently maintained by molecular drive or structural constraints. Potential functional centromeric DNA structures, consisting of twofold dyad symmetries flanked by a common sequence motif, have been identified in both satellite groups.     

Enhancer elements in the mouse CYP1A2 gene: a comparative sequencing among different inbred mouse strains

CYP1A2 expression is constitutively high in mouse liver and is well known for metabolizing several drugs and many procarcinogens to reactive intermediates that can cause toxicity or cancer. In the present study, the basal level of hepatic CYP1A2 activity was shown to vary among different inbred mouse strains. The highest methoxyresorufin-O-demethylase activity (261+/-52pmol/mgprotein/min) was registered in CC57BR and the lowest (82+/-11pmol/mgprotein/min) in C3H/a. We have tested the hypothesis that possible polymorphisms in regulatory elements in the 5'-upstream region of the mouse CYP1A2 gene could cause the differences in CYP1A2 enzyme activity among different inbred strains. We have performed a study on the CYP1A2 gene by sequencing the regulatory region from -4675 to -4204 where two enhancer elements were recently identified. The absence of mutation prescribing the phenotype in the CYP1A2 gene was found. The region studied seems to be a highly conserved in mice and not to be associated with interstrain differences in constitutive CYP1A2 enzyme activity.     

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.     

BglII identifies a frequent biallelic DNA polymorphism of the human RT6 gene

A genomic probe of the human RT6 gene detects a frequent biallelic BglII polymorphism. Allele A has a frequency of 63%, whereas that of allele B is 37%. This restriction fragment length polymorphism provides the first known genetic marker for this gene.