Comparative mapping of mouse and rat chromosomes by fluorescence in situ hybridization

Comparative fluorescence in situ hybridization mapping using DNA libraries from flow-sorted mouse chromosomes and region-specific mouse BAC clones on rat chromosomes reveals chromosomal homologies between mouse (Mus musculus, MMU) and rat (Rattus norvegicus, RNO). Each of the MMU 2, 3, 4, 6, 7, 9, 12, 14, 15, 16, 18, 19, and X chromosomes paints only a single rat chromosome or chromosome segment and, thus, the chromosomes are largely conserved between the two species. In contrast, the painting probes for MMU chromosomes 1, 5, 8, 10, 11, 13, and 17 produce split hybridization signals in the rat, disclosing evolutionary chromosome rearrangements. Comparative mapping data delineate several large linkage groups on RNO 1, 2, 4, 7, and 14 that are conserved in human but diverged in the mouse. On the other hand, there are linkage groups in the mouse, i.e., on MMU 1, 8, 10, and 11, that are disrupted in both rat and human. In addition, we have hybridized probes for Nap2, p57, Igf2, H19, and Sh3d2c from MMU 7 to RNO 1q and found the orientation of the imprinting gene cluster and Sh3d2c to be the same in mouse and rat. Hybridization of rat genomic DNA shows blocks of (rat-specific) repetitive sequences in the pericentromeric region of RNO chromosomes 3-5, 7-13, and 20; on the short arms of RNO chromosomes 3, 12, and 13; and on the entire Y chromosome.     

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization

By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24-->q25, 18p12-->p11, 8q32.1 and 2q22.1-->q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues for rat Smad2 (Madh2) and Smad4 (Madh4) genes were assigned to Chinese hamster chromosomes (CGR) 5q28, 2q17, 4q26, 2p29-->p27, 2q112-->q113 and 2q112-->q113, respectively. The chromosome assignments of homologues of Ctse and Cspg5 reinforced well-known homologous relationships among mouse chromosome (MMU) 1, RNO 13 and CGR 5q, and among MMU 9, RNO 8 and CGR 4q, respectively. The chromosome locations of homologues for Madh2, Madh4 and Pcdh13 genes suggested that inversion events were involved in chromosomal rearrangements in the differentiation of MMU 18 and RNO 18, whereas most of MMU 18 is conserved as a continuous segment in CGR 2q. Furthermore, the mapping result of Myo10 and homologues suggested an orthologous segment of MMU 15, RNO 2 and CGR 2.     

Polymorphisms distinguishing different mouse species and t haplotypes.

Three anonymous chromosome 17 DNA markers, D17Tu36, D17Tu43, and D17Le66B, differentiate between house mouse species and/or between t chromosomes. The D17Tu36 probe, which maps near the Fu locus and to the In(17)4 on t chromosomes, identifies at least 15 haplotypes, each haplotype characterized by a particular combination of DNA fragments obtained after digestion with the Taq I restriction endonuclease. Ten of these haplotypes occur in Mus domesticus, while the remaining five occur in M. musculus. In each of these two species, one haplotype is borne by t chromosomes while the other haplotypes are present on non-t chromosomes. The D17Tu43 probe, which maps near the D17Leh122 locus and to the In(17)3 on t chromosomes, also identifies at least 15 haplotypes in Taq I DNA digests, of which nine occur in M. domesticus and six in M. musculus. One of the nine M. domesticus haplotypes is borne by t chromosomes, the other haplotypes are borne by non-t chromosomes; two of the six M. musculus haplotypes are borne by t chromosomes and the remaining four by non-t chromosomes. Some of the D17Tu43 haplotypes are widely distributed in a given species, while others appear to be population-specific. Exceptions to species-specificity are found only in a few mice captured near the M. domesticus-M. musculus hybrid zone or in t chromosomes that appear to be of hybrid origin. The D17Leh66B probe, which maps to the In(17)2, distinguishes three haplotypes of M. domesticus-derived t chromosomes and one haplotype of M. musculus-derived t chromosomes. Because of these characteristics, the three markers are well suited for the study of mouse population genetics in general and of t chromosome population genetics in particular. A preliminary survey of wild M. domesticus and M. musculus populations has not uncovered any evidence of widespread introgression of genes from one species to the other; possible minor introgressions were found only in the vicinity of the hybrid zone. Typing of inbred strains has revealed the contribution of only M. domesticus DNA to the chromosome 17 of the laboratory mouse.     

Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25

Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.     

Assignment of the rat genes coding for DOPA decarboxylase (DDC) and glutamic acid decarboxylases (GAD1 and GAD2)

By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5-phosphate (PLP)-dependent decarboxylases—namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)—were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)-Tpl2-Gad2 on the rat Chr 17.     

Assignment of the rat genes coding for phenylalanine hydroxylase (PAH), tyrosine aminotransferase (TAT), and pyruvate kinase (PKL) to chromosomes 7, 19, 2, respectively

A panel of hybrid clones segregating rat chromosomes in a mouse background was used to determine the chromosomal localization of three genes specifically expressed in hepatocytes. The phenylalanine hydroxylase, tyrosine aminotransferase, and pyruvate kinase genes were assigned to rat chromosomes 7, 19, and 2, respectively.     

The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17

The human gene for cystathionine beta-synthase (CBS), the enzyme deficient in classical homocystinuria, has been assigned to the subtelomeric region of band 21q22.3 by in situ hybridization of a rat cDNA probe to structurally rearranged chromosomes 21. The homologous locus in the mouse (Cbs) was mapped to the proximal half of mouse chromosome 17 by Southern analysis of Chinese hamster X mouse somatic cell hybrid DNA. Thus, CBS/Cbs and the gene for alpha A-crystalline (CRYA1/Crya-1 or Acry-1) form a conserved linkage group on human (HSA) chromosome region 21q22.3 and mouse (MMU) chromosome 17 region A-C. Features of Down syndrome (DS) caused by three copies of these genes should not be present in mice trisomic for MMU 16 that have been proposed as animal models for DS. Mice partially trisomic for MMU 16 or MMU 17 should allow gene-specific dissection of the trisomy 21 phenotype.     

Assignment of the rat genes coding for α1-antitrypsin (PI), phosphoenolpyruvate carboxykinase (PEPCK), alcohol dehydrogenase (ADH), and fructose-1, 6-bisphosphatase (FDP)

The chromosomal localization of four genes which are expressed mainly in the liver has been undertaken for the rat. Using a panel of hybrid clones segregating rat chromosomes, and Southern blot analysis, 1-AT (PI), PEPCK, ADH and FDP are assigned to rat Chromosomes (Chr) 6, 3, 2 and 17, respectively. Groups of synteny among rat, mouse and human species are discussed in relationship to the new assignments.     

Generation of Five High-Complexity Painting Probe Libraries from Flow-Sorted Mouse Chromosomes

Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.     

Assignment of the rat genes coding for medium-chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and the beta subunit of propionyl-CoA carboxylase to chromosomes 2, 3, and 8, respectively

From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat genes coding for the enzymes medium-chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and the beta-subunit of propionyl-CoA carboxylase have been assigned to chromosomes 2, 3, and 8, respectively.     

Localization of genes on fractionated rat chromosomes by molecular hybridization

Chromosomes derived from rat kidney cells were separated in specially designed sedimentation chambers by velocity sedimentation at 30 g. The DNA of the chromosomal fractions was used in molecular hybridization experiments to localize single-copy genes on the fractionated rat chromosomes. By cross-hybridization with a mouse immunoglobulin light chain kappa c-DNA probe, the rat immunoglobulin genes were detected only on the DNA of chromosomal fractions highly enriched for chromosomes 3, 4 and 5. The rat albumin gene was detected on fractions greatly enriched for chromosomes 11, 13 and 14. The described method allows the localization of structural genes or introduced DNA sequences on the chromosomal level especially in those cell systems in which no suitable somatic cell hybrids are available.     

Gene mapping in the Chinese hamster and conservation of syntenic groups and Q-band homologies between Chinese hamster and mouse chromosomes

Using Chinese hamster/mouse somatic cell hybrids segregating hamster chromosomes, we assigned 15 enzyme genes to six different Chinese hamster autosomes. Of the 15 loci, three genes, HK1, PEPC, and SORD, were newly assigned to chromosomes 1, 5, and 6, respectively, while ENO1, PGD, and PGM1 were assigned to the long arm of chromosome 2, in the segment 2q113----qter. The locations of the following loci were confirmed: ESD, NP, and PEPB on chromosome 1, ME1 and MPI on chromosome 4, AK1 on chromosome 6, and GPI and PEPD on chromosome 9. Comparative mapping of Chinese hamster and laboratory mouse chromosomes revealed conservation of syntenic groups and extensive banding homology between the Chinese hamster and mouse chromosomes on which homologous enzyme markers have been mapped.     

Assignment of snRNA gene sequences to the large chromosomes of rat kangaroo and Chinese hamster isolated by flow cytometric sorting

Chromosomes from a rat kangaroo (Potorous tridactylus) cell line (PtK2) and from a Chinese hamster (Cricetulus griseus) cell line (CHV79) were isolated by means of fluorescence activated flow cytometric sorting. DAPI (4-6-diamino-2-phenylindole) was used as the DNA specific fluorescent dye. The karyotype of the PtK2 cells which exhibits 13 chromosomes was separated into 6, and the 22 chromosomes of the CHV79 cells were resolved into 11 fractions. DNA extracted from these chromosomal fractions was used for restriction enzyme digestion and blotting on nitrocellulose filters. The blots were challenged with gene probes corresponding to ribosomal RNA (18S and 28S) and small nuclear RNA (U1-snRNA) genes. The rRNA genes were exclusively assigned to chromosomes containing the nucleolus organizing region (in PtK2: X chromosome; in CHV79: chromosomes 4, 5, 6, and 11). — Solely the largest chromosomes in both cell lines hybridized with U1-snRNA indicating that these gene sequences are located on those chromosomes only. Further possible genetic and biochemical applications of this experimental system are discussed.     

Chromosomal assignment of four rat genes coding for the spermatid-specific proteins proacrosin (ACR), transition proteins 1 (TNP1) and 2 (TNP2), and protamine 1 (PRM1)

The genes for proacrosin, protamines, and transition proteins are exclusively expressed in haploid spermatogenic cells. From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat gene for proacrosin (ACR) was assigned to chromosome 7, that for transition protein 1 (TNP1) to chromosome 9, and the genes for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 10.     

Chromosomal assignment of human genes for gastrin, thyrotropin (TSH)-beta subunit and C-erbB-2 by chromosome sorting combined with velocity sedimentation and Southern hybridization

Human genes for gastrin, thyrotropin (THS)-beta subunit and c-erbB-2 were assigned to specific chromosomes using a single-laser cell sorter. For this purpose, condensed human chromosomes prepared from a karyotypically normal lymphoblastoid cell line were preliminarily fractionated by velocity sedimentation, and then sorted using a fluorescence-activated cell sorter. DNA was then extracted from the chromosomes, cleaved by restriction enzymes, and subjected to Southern hybridization using gene-specific radioactive probes. When the assignment of specific chromosomes was not possible due to chromosomal size overlapping, sorted chromosomes from cell lines carrying chromosomal translocation or from hybrid cells carrying known human chromosomes were used in addition. The results indicate that human genes for gastrin, TSH-beta, and c-erbB-2 are located on chromosomes 17, 1 and 17, respectively.     

A Comprehensive Genetic Map of Murine Chromosome 11 Reveals Extensive Linkage Conservation between Mouse and Human

Interspecific backcross animals from a cross between C57BL/6J and Mus spretus mice were used to generate a comprehensive linkage map of mouse chromosome 11. The relative map positions of genes previously assigned to mouse chromosome 11 by somatic cell hybrid or genetic backcross analysis were determined (Erbb, Rel, 11-3, Csfgm, Trp53-1, Evi-2, Erba, Erbb-2, Csfg, Myhs, Cola-1, Myla, Hox-2 and Pkca). We also analyzed genes that we suspected would map to chromosome 11 by virtue of their location in human chromosomes and the known linkage homologies that exist between murine chromosome 11 and human chromosomes (Mpo, Ngfr, Pdgfr and Fms). Two of the latter genes, Mpo and Ngfr, mapped to mouse chromosome 11. Both genes also mapped to human chromosome 17, extending the degree of linkage conservation observed between human chromosome 17 and mouse chromosome 11. Pdgfr and Fms, which are closely linked to II-3 and Csfgm in humans on chromosome 5, mapped to mouse chromosome 18 rather than mouse chromosome 11, thereby defining yet another conserved linkage group between human and mouse chromosomes. The mouse chromosome 11 linkage map generated in these studies substantially extends the framework for identifying homologous genes in the mouse that are involved in human disease, for elucidating the genes responsible for several mouse mutations, and for gaining insights into chromosome evolution and genome organization.     

Mouse-human orthology relationships in an olfactory receptor gene cluster

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. One of the best characterized OR clusters, located at human chromosome 17p13.3, has previously been studied by us in human and in other primates, revealing a conserved set of 17 OR genes. Here, we report the identification of a syntenic OR cluster in the mouse and the partial DNA sequence of many of its OR genes. A probe for the mouse M5 gene, orthologous to one of the OR genes in the human cluster (OR17-25), was used to isolate six PAC clones, all mapping by in situ hybridization to mouse chromosome 11B3-11B5, a region of shared synteny with human chromosome 17p13.3. Thirteen mouse OR sequences amplified and sequenced from these PACs allowed us to construct a putative physical map of the OR gene cluster at the mouse Olfr1 locus. Several points of evidence, including a strong similarity in subfamily composition and at least four cases of gene orthology, suggest that the mouse Olfr1 and the human 17p13.3 clusters are orthologous. A detailed comparison of the OR sequences within the two clusters helps trace their independent evolutionary history in the two species. Two types of evolutionary scenarios are discerned: cases of "true orthologous genes" in which high sequence similarity suggests a shared conserved function, as opposed to instances in which orthologous genes may have undergone independent diversification in the realm of "free reign" repertoire expansion.     

The interleukin-6 gene locus seems to be a preferred target site for retrotransposon integration

Recently, we have observed the insertion of a retrotransposon into the interleukin-6 (II-6) locus of a mouse somatic cell line. Here we report the characterization of Il-6 genomic regions from both mouse and rat. Restriction site analysis, DNA sequence analysis, and computer-assisted search revealed eight retrotransposon-like elements distributed over a 25 kilobase (kB) mouse Il-6 region. In the rat, five different retrotransposons have been identified within a 17 kb Il-6 region. The retrotransposons belong to the LINE-, Alu I or Alu II families, or to a rat specific class of retrotransposons. Some of the retrotransposons class of retrotransposons. Some of the retrotransposons exhibit characteristic features such as target site duplication and a poly A-tract. Remarkably, several retrotransposons map to different chromosomal locations in the mouse and rat. A genealogical tree of mouse, rat, and human Il-6 loci demonstrates a series of retrotranspositions that recently occurred in evolution. These results suggest that the Il-6 locus serves as a preferred target site for retrotransposon integration during evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M36993-4 (L.1.R3), M36995 (L1.R2), and M36996 (L1.M1/L1.M2).This work contains part of the doctoral thesis of Z. Qin and I. Schuller.     

The gene map of the Norway rat (Rattus norvegicus) and comparative mapping with mouse and man

The current status of the rat gene map is presented. Mapping information is now available for a total of 214 loci and the number of mapped genes is increasing steadily. The corresponding number of loci quoted at HGM10 was 128. Genes have been assigned to 20 of the 22 chromosomes in the rat. Some aspects of comparative mapping with mouse and man are also discussed. It was found that there is a good correlation between the morphological homologies detectable in rat and mouse chromosomes, on the one hand, and homology at the gene level on the other. For 10 rat synteny groups all the genes so far mapped are syntenic also in the mouse. For the remaining rat synteny groups it appears that the majority of the genes will be syntenic on specific (homologous) mouse chromosomes, with only a few genes dispersed to other members of the mouse karyotype. Furthermore, the data indicate that mouse chromosome 1 genetically corresponds to two rat chromosomes, viz., 9 and 13, equalizing the difference in chromosome number between the two species. Further mappings will show whether the genetic homology will prove to be as extensive as these preliminary results indicate. As might be expected from evolutionary considerations, rat synteny groups are much more dispersed in the human genome. It is clear, however, that many groups of genes have remained syntenic during the period since man and rat shared a common ancestor. One further point was noted. In two cases groups of genes were syntenic in the mouse but dispersed to two chromosomes in rat and man, whereas in a third case a group of genes was syntenic in the rat but dispersed to two chromosomes in mouse and man. This finding argues in favor of the notion that the original gene groups were on separate ancestral chromosomes, which have fused in one rodent species but remained separate in the other and in man.