A Repeated Segment on the Mouse Y Chromosome Is Composed of Retroviral-Related, Y-Enriched and Y-Specific Sequences

We report the isolation and characterization of two recombinant clones containing DNA derived from the Y chromosome of the C57BL/10 inbred mouse strain. Both clones were isolated from a lambda phage library derived from a partial EcoRI digest of C57BL/10 male DNA using the murine retrovirus M720. Characterization of these clones showed they were derived from a repeated segment present on the C57BL/10J Y chromosome that contains sequences found elsewhere in the genome. In addition, one clone contained a sequence, designated YB10, that is unique to the Y chromosome and present in approximately 500 copies on the C57BL/10J Y chromosome. Analysis of Southern blots containing DNAs prepared from females and males of representative species from four subgenera of Mus probed with pYB10 and the 3'LTR from one of the Y-associated retroviruses (MuRVY) revealed that, with the exception of a single fragment observed in both female and male DNA of Mus saxicola, hybridization to pYB10 was observed only to male DNA of the species Mus spretus, Mus hortulanus, Mus musculus, Mus domesticus and Mus abbotti. In addition, the pattern and intensity of hybridization to YB10 and the MuRVY-LTR indicated that sequence of divergence was followed by amplification of Y chromosome sequences containing YB10 and MuRVY. The divergence and amplification occurred separately in each of the ancestral lineages leading to M. spretus, M. hortulanus, M. abbotti, M. musculus and M. domesticus. We suggest that acquisition and amplification of DNA sequences by the mammalian Y chromosome has contributed to its evolution and may imply that the mammalian Y chromosome is evolving at a faster rate than the rest of the genome.     

Nearly identical members of the heterogeneous IAP gene family are expressed in thymus of different mouse strains.

Poly(A)RNAs prepared from the thymuses of C57BL/6J and DBA/2J mice were used to construct cDNA libraries in the bacterial expression vector lambda gt11. The libraries were scanned first for protein production with polyvalent antiserum prepared against the 73kDa gag protein of mouse intracisternal A-particles (IAP). Reactive plaques were crossed-screened by hybridization with an IAP-specific DNA probe. Two IAP-specific protein-producing plaques were obtained from the C57BL/6 library and 4 from the DBA/2 library. One C57BL/6 cDNA clone (B12) and two DBA cDNA clones (D8 and D20) were sequenced in their entirety. Clones B12 and D8 were remarkably similar, particularly when compared to the 6 other IAP elements that have been sequenced thus far. We discuss the evidence which leads us to suggest that these clones may be derived from allelic IAP elements expressed in mouse thymus.     

A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies

Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI-EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 x 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI-EcoRV-HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5' regions of known genes or mRNAs, and all 132 NotI-EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI-EcoRV library fragments in both orientations. Luciferase assays using NotI-EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.     

Cosmid clones derived from both euchromatic and heterochromatic regions of the human Y chromosome.

Clones containing sequences derived from the human Y chromosome have been isolated from cosmid libraries of a human-mouse hybrid cell line. These libraries were constructed in the new expression vectors Homer V and Homer VI. The collection of cosmids isolated is enriched for unique sequence DNA and only a few of the cosmids contain the tandemly repeated sequences which constitute a major portion of the Y chromosome. Three cosmids have been studied in detail. One cosmid shows extensive homology over at least 20 kb with the long arm of the X chromosome; this homology is outside the predicted homology region required for sex chromosome pairing. The other two clones contain unique sequences specific to the Y chromosome and both map to the heterochromatic region of the Y chromosome long arm.     

DNA sequence error rates in Genbank records estimated using the mouse genome as a reference.

We estimate DNA sequence error rates in Genbank records containing protein-coding and non-coding DNA sequences by comparing sequences of the inbred mouse strain C57BL/6J, sequenced as part of the mouse genome project and independently by other laboratories. C57BL/6J was produced by more than 100 generations of brother-sister mating, and can be assumed to be virtually free of residual polymorphism and mutational variation, so differences between independent sequences can be attributed to error. The estimated single nucleotide error rate for coding DNA is 0.10% (SE 0.012%), which is substantially lower than previous estimates for error rates in Genbank accessions. The estimated single nucleotide error rate for intronic DNA sequences (0.22%; SE 0.051%) is significantly higher than the rate for coding DNA. Since error rates for the mouse genome sequence are very low, the vast majority of the errors we detected are likely to be in individual Genbank accessions. The frequency of insertion-deletion (indel) errors in non-coding DNA approaches that of single nucleotide errors in non-coding DNA, whereas indel errors are uncommon in coding sequences.     

Co-amplification of tail-to-tail copies of MuRVY and IAPE retroviral genomes on the Mus musculus Y Chromosome

We have isolated a clone from a C57BL/6 genomic library that contains both part of the Y Chromosome-specific 8.7 kbp MuRVY genome (Hutchinson and Eicher, J. Virol. 63, 4043, 1989) and a full-length 8.3 kbp Intracisternal A Particle genome (IAPE-Y), in a tail-to-tail organization. Although IAPs are encoded by a disperse multigene family (∼1000 copies per haploid genome), we present evidence that a significant proportion of the IAP-related sequences are present on the Y Chromosome (Chr) and that a >25 kbp genomic sequence, which contains the two proviral genomes, has been amplified on the Y Chr. Two discrete amplified families of MuRVY retroviral genomes distinguishable by a polymorphic restriction site were detected, suggestive that amplification occurred in incremental stages. The presence of MuRVY-related DNA sequences, but absence of IAPE-Y-related DNA sequences in Mus spretus suggests that the IAPE-Y retrotransposition event occurred after the evolutionary divergence of the lineages leading to Mus musculus and Mus spretus, and that the amplification of MuRVY occurred independently in the two lineages. Received: 28 July 1995 / Accepted: 1 September 1995     

A mouse Y Chromosome pseudogene is related to human ubiquitin activating enzyme E1

A 2041 bp DNA fragment isolated from the Sxr (sex reversed) region of the mouse Y Chromosome (Chr) was sequenced and characterized. The sequence, pY8/b, contains four exons that are highly similar to 525 contiguous bases from the cDNA of human ubiquitin activating enzyme El. Two of the exons contain stop codons, indicating that pY8/b is not part of a functional gene. Sequences related to pY8/b were amplified from the Y Chr of the inbred mouse strain, C57BL/6J. These sequences may be portions of the recently discovered functional equivalent of pY8/b. Despite a high degree of similarity with the human El gene, the functional equivalent of pY8/b is not the mouse El gene, because unlike El, the functional equivalent of pY8/b is expressed in a tissue-specific manner. These data are discussed with respect to theory on the evolution of the mammalian Y Chr, and in particular, to the prediction that functional genes on the Y Chr have a male-specific function.     

Novel Sxr(a) ES cell line offers hope for Y chromosome gene-targeted mice

A mouse targeted for a Y Chromosome gene has not been reported. Because the Y Chromosome is present in only one copy, and most of its genes are critical for germ cell development, such a mouse would likely be infertile. Thus, we describe a new reproductive strategy to enable transmission of targeted Y Chromosome genes to subsequent generations. The strategy uses two segregating copies of Y Chromosome genes to mimic the autosomal condition. To achieve this, we developed a new embryonic stem cell line from the XYSxr(a) mouse, which carries a duplication of the gene-rich Y Chromosome short arm. Importantly, we demonstrate germ line transmission of the YSxr(a) chromosome and describe this significant new tool as a practical solution to enable reproduction in mice targeted for Y Chromosome genes.     

A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits

The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.     

Normal Testis Determination in the Mouse Depends on Genetic Interaction of a Locus on Chromosome 17 and the Y Chromosome

We previously described a locus on chromosome (Chr) 17 of the mouse that is critical for normal testis development. This locus was designated "T-associated sex reversal" (Tas) because it segregated with the dominant brachyury allele hairpin tail (Thp) and caused gonads of C57BL/6J XY, Thp/+ individuals to develop as ovaries or ovotestes rather than as testes. To clarify the inheritance of Tas, we investigated the effects of T-Orleans (TOrl), another brachyury mutation, on gonad development. We found that gonads of C57BL/6J XY, Thp/+ and TOrl/+ mice develop ovarian tissue if the Y chromosome is derived from the AKR/J inbred strain, whereas normal testicular development occurs in the presence of a Y chromosome derived from the C57BL/6J inbred strain. From these observations we conclude that: (1) Tas is located in a region on Chr 17 common to the deletions associated with Thp, and TOrl, and (2) the Y-linked testis determining gene, Tdy, carried by the AKR/J inbred strain differs from that of the C57BL/6J inbred strain. We suggest that in mammals Tdy is not the sole testis determinant because autosomal loci must be genetically compatible with Tdy for normal testicular development.     

Sex-restricted non-Mendelian inheritance of mouse Chromosome 11 in the offspring of crosses between C57BL/6J and (C57BL/6J × DBA/2J)F1 mice

We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J × DBA/2J)F₁ hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sex-specificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability. Received: 19 December 1997 / Accepted: 10 June 1998     

The alternative exons 1 of the mouse aromatase cytochrome P-450 gene

Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5′ regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5′ regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.     

Assays of testis development in the mouse distinguish three classes of domesticus-type Y chromosome

The Y chromosome of Mus musculus poschiavinus interacts with the autosomal recessive gene tda-1b of the C57BL/6J laboratory strain of the house mouse to cause complete or partial sex reversal. Ovaries or ovotestes develop in a substantial proportion of the XY fetuses. Several different Y-specific DNA probes distinguish two major types of Y chromosome in the house mouse and they are represented by M. m. domesticus and M. m. musculus. The poschiavinus Y chromosome appears identical to the domesticus Y. The developmental distribution of the gonad types was examined in the first backcross or N2 generation of fetuses in C57BL/6J with six different domesticus-type Y chromosomes and, as controls, three different musculus-type Y chromosomes. Gonadal hermaphrodites were found with three of the six domesticus-type Y chromosomes. Both overall frequency and phenotypic distribution of types of gonadal hermaphrodites identify three classes of domesticus-type Y chromosome by their differential interaction with the C57BL/6J genetic background.     

A malic enzyme probe detects cross-hybridizing sequences closely linked to loci encoding other metabolic enzymes.

The cytoplasmic malic enzyme (Mod-1) catalyzes the oxidative decarboxylation of malate: malate + NADP+----pyruvate + CO2 + NADPH + H+. Using a cDNA clone of Mod-1 as a probe, two new DNA markers not at the Mod-1 locus (restriction fragment length polymorphisms, RFLP) were detected by Southern blot analysis that showed extensive homology to Mod-1 sequences. Linkage of each restriction fragment length polymorphism to loci other than Mod-1 was assessed using the BXD (C57BL/6J x DBA/2J) recombinant inbred strains and confirmed by backcrosses. One polymorphic site, designated D9Rti1, was found to be closely linked to the phosphoglucomutase (Pgm-3) locus on Chromosome 9. The other hybridization site, designated D1Rti2, was closely linked to the isocitrate dehydrogenase (Idh-1) locus on Chromosome 1. The data presented imply that Mod-1 homologous sequences are tightly linked to three different metabolic enzymes.     

An interstitial telomere array proximal to the distal telomere of mouse chromosome 13

Lambda clones of mouse DNA from BALB/c and C57BL/10, each containing an array of telomere hexamers, were localized by FISH to a region close to the telomere of Chr 13. Amplification of mouse genomic DNA with primers flanking SSRs within the cloned DNA showed several alleles, which were used to type eight sets of RI strains. The two lambda clones contained allelic versions of the interstitial telomere array, Tel-rs4, which is 495 bp in C57BL/10 and which includes a variety of sequence changes from the consensus telomere hexamer. Comparison of the segregation of the amplification products of the SSRs with the segregation of other loci in an interspecies backcross (C57BL/6JEi × SPRET/Ei) F₁× SPRET/Ei shows recombination suppression, possibly associated with ribosomal DNA sequences present on distal Chr 13 in Mus spretus, when compared with recombination in an interstrain backcross, (C57BL/6J × DBA/J) F₁× C57BL/6J, and with the MIT F₂ intercross. Analysis of recombination in females using a second interstrain backcross, (ICR/Ha × C57BL/6Ha) F₁× C57BL/6Ha, also indicates recombination suppression when compared with recombination in males of the same strains, using backcross C57BL/6Ha × (ICR/Ha × C57BL/6Ha) F₁. Thus, more than one cause may contribute to recombination suppression in this region. The combined order of the loci typed was D13Mit37–D13Mit30–D13Mit148–(D13Rp1, 2, 3, 4, Tel-rs4)–D13Mit53–D13Mit196–D13Mit77–(D13Mit78, 35). Data from crosses where apparently normal frequencies of recombination occur suggest that the telomere array is about 6 map units proximal to the most distal loci on Chr 13. This distance is consistent with evidence from markers identified in two YAC clones obtained from the region. Received: 24 September 1996/Accepted: 20 January 1997     

Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays (¹³⁷Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose–response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.