In situ analysis of centromere segregation in C57BL/6 x Mus spretus interspecific backcrosses

The analysis of major satellite sequence differences between Mus spretus and laboratory mice provides a robust method for analyzing the centromere location for the genetic maps of each mouse chromosome. Fluorescence in situ hybridization (FISH) of a genomic probe, pMR196, for the laboratory mouse major satellite sequences was used to identify C57BL/6Ros (B6) pericentromeric heterochromatin in progeny of reciprocal backcross matings. These included 80 (B6xM. spretus)F₁xM. spretus progeny (BSS) and 70 (B6xM. spretus)F₁xB6 (BSB) progeny. FISH analysis of pericentromeric heterochromatin was conducted on the same metaphase spreads that were karyotypically analyzed for chromosomespecific banding patterns. Analysis of chromosomal segregation suggested that there was not primary deviation from random assortment during meiosis in the interspecific hybrid female, because nearly all of the 190 pair-wise comparisons did not deviate from expected and because there was no consistent pattern of deviation of the same chromosomes in the reciprocal backcross progeny from similar (C57BL/6xM. spretus)F₁ hybrid females. These results affirm the value of using the major satellite to genetically mark pericentromeric heterochromatin in the analysis of the segregation and assortment of centromeres in Mus interspecific crosses.     

Cytological and molecular characterization of centromeres in Mus domesticus and Mus spretus

We have applied EM in situ hybridization (EMISH) and pulsed field gel electrophoresis (PFGE) to samples from diploid primary cell cultures and an established cell line to examine in detail the relative organization of the major and minor satellite DNAs and telomere sequences in the genomes of Mus domesticus and Mus spretus. EMISH localizes the Mus domesticus minor satellite to a single site at the centromere-proximal end of each chromosome. Double label hybridizations with both minor satellite and telomere probes show that they are in close proximity and possibly are linked. In fact, PFGE of M. domesticus DNA digested with Sal I and Sfi I reveals the presence of fragments which hybridize to both probes and is consistent with the physical linkage of these two sequences. The M. domesticus minor satellite is the more abundant satellite in Mus spretus. Its distribution in M. spretus is characterized by diffuse labeling with no obvious concentration near chromosome ends. In addition to this repeat the M. spretus genome contains a small amount of DNA that hybridizes to a M. domesticus major satellite probe. Unlike the M. domesticus minor satellite, it is not telomere proximal but is confined to a domain at the border of the centromere and the long arm. Thus, although both species possess all three sequences, except for the telomeres, their distribution relative to one another is not conserved. Based on the results presented, we propose preliminary molecular maps of the centromere regions of Mus domesticus and Mus spretus.     

New polymorphic markers in the vicinity of the pearl locus on mouse Chromosome 13

We have used a Mus domesticus/-Mus spretus congenic animal that was selected for retention of Mus spretus DNA around the pearl locus to create a highly polymorphic region suitable for screening new markers. Representation difference analysis (RDA) was performed with either DNA from the congenic animal or C57BL/6J as the driver for subtraction. Four clones were identified, characterized, and converted to PCR-based polymorphic markers. Three of the four markers equally subdivide a 10-cM interval containing the pearl locus, with the fourth located centromeric to it. These markers have been placed on the mouse genetic map by use of an interspecific backcross panel between Mus domesticus (C57BL/6J) and Mus spretus generated by The Jackson Laboratory. Received: 12 June 1995 / Accepted: 17 August 1995     

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.     

Sequence of centromere separation: influence of pericentromeric heterochromatin (repetitive DNA) inMus

A relationship between the sequence of centromere separation and quantity of pericentromeric constitutive heterochromatin was studied using bone marrow cells ofMus musculus molossinus and three cell lines, viz., SEWA-Rec 4, brain tumor and L-cells, ofM. m. domesticus origin. The timing of separation of a centromere into two daughter centromeres is related to the quantity of pericentromeric heterochromatin. In these genomes, having qualitatively uniform DNA in their heterochromatin fraction, the chromosomes with none or small quantities of heterochromatin separate first. These are followed by those chromosomes which have increasingly larger quantities of heterochromatin. It appears that one function of repetitive DNA (pericentromeric heterochromatin) is to regulate the timing of separation of centromeres.     

The chromosomal distribution of the major and minor satellite is not conserved in the genusMus

The cytological distribution of the major and minor satellite first identified inMus musculus was studied in the karyotypes of three related subspecies and two other species of the genusMus. Both the major and minor satellite showed species dependent hybridization patterns. The major satellite is confined to the centromere region inM. musculus and related subspecies. However, inM spretus andM. caroli, the chromosomal arm regions contain this sequence class. In contrast the minor satellite is found at the kinetochore region inM. musculus and related subspecies but is distributed throughout the entire centromeric domain inM. spretus and appears to be excluded from the chromosomes ofM. caroli. There is an apparent correlation between the chromosomal location of these satellites and their phylogenetic relationship. Determination of the biological roles of the major and minor satellites fromM. musculus must take into account their differential chromosomal distribution in otherMus species.     

Genetic mapping of two DNA markers,D16Ros1 and D16Ros2, flanking the mutation site in the chakragati mouse,a transgenic insertional mutant

We present here the genetic mapping of two novel loci, D16Ros1 and D16Ros2, to mouse Chromosome (Chr) 16. The probes for these loci were genomic framents isolated from the chakragati mouse, a behavioural mutant resulting from insertional mutagenesis during the course of making transgenic mice. D16Ros1 and D16Ros2 were first mapped by recombinant inbred (RI) strain analysis and subsequently by the analysis of 145 progeny of two interspecific backcrosses between Mus domesticus and Mus spretus. These progeny had been typed for the centromere and this allowed mapping of D16Ros1 and D16Ros2 relative to the centromere. The other markers included in this study were Prm-1, Gap43 and Sod-1. The genetic map generated spanned 47.5 cM from the centromere to Sod-1, the most distal marker mapped here. The linkage data presented here should prove useful in mapping other loci relative to the centromere of Chr 16.     

Gene flow of unique sequences between Mus musculus domesticus and Mus spretus

Allelic diversity has been examined from a variety of Mus musculus subspecies and Mus spretus strains by sequencing at a 453-bp unique sequence locus. One M. m. domesticus classic inbred strain, C57BL/KsJ, contained a sequence identical to that in the M. spretus wild-derived inbred strain SEG, and other wild M. spretus isolates. Such a result should have been precluded by the expected divergence between the species unless there has been interspecies gene flow. Examination of C57BL/KsJ for M. spretus-specific repetitive sequences shows that it is neither a mis-identified spretus strain nor a domesticus/spretus hybrid. Thus, in addition to the previously reported presence of small amounts of Mus spretus-specific repetitive DNA in M. m. domesticus, there is a detectable flow of unique sequence between the two species. There was also ancestral polymorphism observed among the spretus alleles. The difficulty of distinguishing ancestral polymorphism from horizontal transfer is discussed. Received: 14 May 1999 / Accepted: 5 November 1999     

Evidence for the presence of two sympatric species of mice (genus Mus L.) in southern France based on biochemical genetics

Populations of mice established outdoors as well as indoors have been investigated at 24 loci using starch gel electrophoresis. Two reproductively isolated groups are recognized, one of which is referable to a house mouse subspecies, Mus musculus brevirostris, and the other to a different species, Mus spretus, contrary to the view of Schwarz and Schwarz that only one species of Mus is present in the Mediterranean Basin. The genetic distance between these two groups is larger than between any pair of investigated subspecies of M. musculus. M. m. brevirostris is biochemically almost indistinguishable from M. m. domesticus. On the other hand, M. spretus exhibits several allelic variants unknown or at most very infrequent in M. musculus, as for instance at the lactate dehydrogenase B-chain locus.This work was supported by research grants from the Centre National de la Recherche Scientifique (E.R.A. No. 261) and the Ecole Pratique des Hautes Etudes.     

The thrombospondin-4 gene

Thrombospondins are a family of extracellular, adhesive proteins that are widely expressed in vertebrates. Five distinct gene products, designated thrombospondin-1 through -4 and cartilage oligomeric matrix protein (COMP), have been identified. With the exception of thrombospondin-4, the structure and location of thrombospondin genes have been determined in the human and/or mouse genomes. In this study, the structure and location of the murine thrombospondin-4 gene and the location of the human thrombospondin-4 gene are reported. The murine thrombospondin-4 gene is approximately 4.5 kb in length and includes 22 exons. Interspecific backcross analysis of progeny derived from matings of (C57BL/6J ×Mus spretus)F₁× C57BL/6J mice indicates that the thrombospondin-4 gene is tightly linked to the Dhfr locus on murine Chromosome (Chr) 13. The human gene maps to Chr 5 in band q13 by in situ hybridization to human metaphase chromosomes. The thrombospondin-4 promoter is similar to promoters of some housekeeping, growth control, and other thrombospondin genes in that it contains multiple GC box sequences and lacks a CAAT box. The presence of multiple E-box sequence motifs is consistent with thrombospondin-4 expression in muscle and bone tissue. Received: 18 May 1998 / Accepted: 24 May 1999     

Laboratory and field evidence of paternal care in the Algerian mouse (Mus spretus)

Paternal care is generally, although not exclusively, associated with monogamy in mammals. The Algerian mouse, Mus spretus, is a nocturnal murid living in xeric habitats in northern Africa, the Iberian peninsula and the south of France. We compared the amount of paternal care induced by removing young in Mus spretus and in the polygamous house mouse M. musculus domesticus in terms of the evolution of paternal care during the first 10-days post-partum and absolute (father alone) and relative (both parents) values. We then recovered indirect evidence of young being cared for by males from field data. The results were unambiguous in showing that the two species differed dramatically in paternal behaviour, with no variation being observed during the initial pup development. Mus spretus dedicated half of its time to caring from the young against less than 10% in M. m. domesticus. The former species showed the same number of retrieving acts and direct care bouts both in the presence and in the absence of the mother. Merging these results with field observations of spatial association “male-young” until subadult age, we drew conclusions on the likely occurrence of paternal care in M. spretus. Taken together with our previous results on socio-spatial organisation and pair bonding, we propose that social monogamy is advanced for this species.     

The Gene Encoding Protein Kinase SEK1 Maps to Mouse Chromosome 11 and Human Chromosome 17

We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human–rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouseSerk1gene was mapped to chromosome 11, closely linked toD11Mit4,using genomic DNAs from a (C57BL/6J ×Mus spretus)F₁×M. spretusbackcross.     

Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR

Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3 end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.     

Mus and Peromyscus chromosome homology established by FISH with three mouse paint probes

Fluorescence-labeled DNA probes constructed from three whole house mouse (Mus domesticus) chromosomes were hybridized to metaphase spreads from deer mouse (Peromyscus maniculatus) to identify homologies between the species. Mus Chr 7 probe hybridized strongly to the ad-centromeric two-thirds of Peromyscus Chr 1q. Most of Mus 3 probe hybridized principally to two disjunct segments of Peromyscus Chr 3. Mus Chr 9 probe hybridized entirely to the whole Peromyscus Chr 7. Three Peromyscus linkage groups were assigned to chromosomes, based on linkage homology with Mus. The data also are useful in interpretation of chromosomal evolutionary history in myomorphic rodents. Received: 1 December 1998 / Accepted: 17 February 1999     

Localization of the murine cholecystokinin A and B receptor genes

We have determined the chromosomal locations of the two cholecystokinin (CCK) receptor genes in the mouse. Genetic localization utilized an interspecific backcross panel formed from the cross (C57BL/6J x Mus spretus) F₁ x Mus spretus. Genomic DNAs from 94 individuals in the backcross were analyzed by Southern hybridization with rat CCK_A and CCK_B receptor cDNA probes. Unique map positions were determined by haplotype analysis with 650 previously mapped loci in the mouse backcross. The CCK_A receptor gene (Cckar) mapped to mouse Chromosome (Chr) 5, in tight linkage with the DNA marker D5Bir8. The CCK_B receptor gene (Cckbr) mapped to mouse Chr 7, tightly linked to the -hemoglobin locus (Hbb). This localization places Cckbr in the same region as the mouse obesity mutation tubby (tub), which also maps near Hbb (2.4±1.4 cM). Since CCK can function as a satiety factor when administered to rodents, localization of Cckbr near the tub mutation identifies this receptor as a possible candidate gene for this obesity mutation.     

Characterization and mapping of the gene encoding mouse proteasome subunit DELTA (Lmp19)

The proteasome subunit DELTA is unusually closely related to the major histocompatibility complex (MHC)-linked proteasome subunit, LMP2. The sequence of a mouse cDNA for DELTA confirms that this 22 100 M _r proteasome subunit is highly conserved across species. Sequence analysis of the mouse gene encoding DELTA, designated Lmp19, indicates that it consists of six exons and five introns, similar to the Lmp2 gene. The 5 upstream region lacks a TATA regulatory sequence, which is also absent from proteasome genes isolated from Drosophila. BXD recombinant inbred (RI) mice were used to map the potential chromosomal location of Lmp19, and revealed that the DELTA subunit has related sequences present on two different mouse chromosomes, chromosomes 1 and 11. Typing of 89 progeny from a C57BL/6J X Mus spretus DNA backcross panel (BSS) confirmed the chromosome 1 assignment. Southern hybridization with a polymerase chain reaction-generated Lmp19 intron 2-specific probe indicates that the Lmp19 genomic clone corresponds to the sequence on chromosome 11, and further suggests that the chromosome 1 copy represents a processed pseudogene (Lmp19-ps1).     

Liver chromatin fractions inMus andAkodon

Summary The liver chromatin fromMus musculus andAkodon molinae was separated in 8 fractions by differential centrifugation. Like fractions from both species showed approximately similar contents of DNA, equivalent ratios of histone to non-histone proteins, corresponding template activities and equal amounts of positive C-banded material. On the other hand, heavy chromatin fractions ofMus were highly enriched in satellite DNA whereas no satellite DNA was found inAkodon chromatin. Heavy chromatin fractions isolated by differential sedimentation have been usually homologued with the constitutive heterochromatin. The properties of the constitutive chromatin are discussed and the validity of the foregoing concept is challenged. It is proposed to define the constitutive heterochromatin as those chromatin regions comprising highly repeated DNA sequences clustered in restricted areas of chromosomes and not transcribed (satellite DNA).     

A locus on the X Chromosome is linked to body length in mice

Linkage between body length (anus to nose (AN) length) and three markers on the mouse X Chromosome was found in an interspecific backcross ((C57BL/6J×Mus spretus) F1 ×C57BL/6J), designated BSB. A cross of 409 mice were scored for 148 genetic markers distributed on all chromosomes except the Y Chromosome. Statistical analysis revealed highly significant linkage (LOD score 5.5) between body length and a locus in the middle portion of the X Chromosome, the nearest markers being the microsatellite marker DXMit73 and a farnesyl pyrophosphate locus (Fpsl9) 3.1 cM proximal to DXMit73. The locus explains 10% of the variance in AN length and affects both males and females to about the same extent. Received: 17 April 1995 / Accepted: 13 October 1995     

Ifg,Gli, Mdm1, Mdm2, and Mdm3: candidate genes for the mouse pg locus

Various genes that mapped to the distal end of Chromosome (Chr) 10 were considered as possible candidates for the mouse pygmy (pg) locus. Probes derived from Ifg, Gli, Mdm1, Mdm2 and Mdm3 (Mdm2 and Mdm3 are genes that are coamplified with Mdm1 on the same double minute chromosomes in 3T3DM cells) were used for Southern analysis of DNA from wild-type mice and various pg mutants. In addition, the chromosomal locations of Ifg, Gli, Mdm1, Mdm2, and Mdm3 were determined by interspecific backcross analysis with progeny derived from matings of [(C57BL/6J x Mus spretus)F₁ x C57BL/6J] mice. The mapping data indicate that the Mdm loci are linked to each other and to Ifg, pg, and Gli in the distal region of mouse Chr 10. Both the mapping data and the Southern analysis confirm that mdm1, Mdm2, Mdm3, Ifg, and Gli are distinct from pg.     

Heterochromatins and band karyotypes in three species of salmonids

Summary The heterochromatins of rainbow trout (Salmo gairdneri R.), brown trout (Salmo trutta fario L.) and brook trout (Salvelinus fontinalis M.) were characterized by sequential chromomycin A₃/distamycin A/DAPI (CDD) and DAPI/actinomycin D (DAPI/AmD) fluorescence. On most biarmed chromosomes, an equilocal localization of prominent DAPI/AmD positive, chromomycin A₃ negative, AT-rich blocks at the centromeres were observed in all three species. Band karyotypes of the three species were established. In rainbow trout, several DAPI/AmD positive heterochromatin blocks behaved positive in a silver-staining method. Mitotic and interphase studies proved the presence of inter-individual NOR variation in brown trout. The NORs of brook trout were localized on chromosomes 5, 10, 14, 15 and 29.