The genetic map around the tail kinks (tk) locus on mouse Chromosome 9

Tail kinks (tk) is a classical mouse skeletal mutation, located on Chromosome (Chr) 9. As the first step for the positional cloning of the tk gene, we have established a genetic map of a region surrounding the tk locus by generating a backcross segregating for tk. From this backcross, 1004 progeny were analyzed for the coat-color phenotype of the proximally located dilute (d) gene and for the distally flanking microsatellite marker, D9Mit12. Fifty-six recombinants between d and tk and 75 recombinants between tk and D9Mit12 were identified, completing a panel of 130 recombinants including one double recombinant. This panel allowed us to map five microsatellite loci as well as d and Mod-1 with respect to tk. We show that one of the microsatellite markers mapped, D9Mit9, does not recombine at all with tk in our backcross. This indicates that the D9Mit9 locus will serve as a good starting point for a chromosomal walk to the tk gene.     

Linkage analysis of the Bcg gene on mouse chromosome 1. Identification of a tightly linked marker

We have mapped and determined the gene order of five cloned genes in the vicinity of the murine host resistance gene Bcg on mouse chromosome 1. For this, we have used a RFLP-type analysis in panels of 43 recombinant inbred strains, 3 congenic mouse strains, and 186 segregating backcross progeny derived from inbred strains of Bcgr and Bcgs genotypes. The Bcg alleles of segregating animals were established by in vivo infection with Mycobacterium bovis (Bacillus Calmette-Guérin) strain Montreal. Genomic DNA prepared from progenitor mouse strains was isolated, digested with restriction endonucleases, and analyzed by Southern blotting to identify strain-specific RFLP for each DNA marker tested. Among a number of DNA markers tested, Len2, Fn, Vil, Alpi, and Achrg were found to co-segregate with Bcg in mouse strains congenic for this locus. Detailed segregation analysis of the five markers and Bcg showed that Vil was extremely close to Bcg with no recombinant identified, whereas Fn and Len2 were located 4.5 and 9 cM proximal of Bcg, respectively. Alpi and Achrg mapped 5 and 5.5 cM distal from Bcg, respectively. Pedigree analysis in the recombinant inbred strains and backcross animals indicated the gene order: centromere-Len2-Fn-Vil,Bcg-Alpi-Achrg. The tightly linked Vil marker can now be used as an entry point in recombinant genomic DNA libraries to clone sequences overlapping Bcg. This group of five genes flanking Bcg on mouse chromosome 1 is precisely conserved on the telomeric end of the long arm of human chromosome 2q. Our results suggest that a likely location for a putative human homologue to the murine host resistance gene Bcg is the long arm of human chromosome 2 (2q32-qter).     

Localization of insulin-2 (Ins-2) and the obesity mutant tubby (tub) to distinct regions of mouse chromosome 7.

A DNA mapping panel derived from an interspecific backcross was used to position the mouse insulin-2 locus (Ins-2) on Chromosome 7, near H19 (0/114 recombinants) and Th (1/114 recombinants). Ins-2 is part of a human-mouse conserved linkage group that includes Th, H19, and Igf-2. Analysis of segregation in the F2 generation from the cross C57BL/6J-tub/tub x CAST/Ei demonstrated that Ins-2 and the obesity mutant tubby (tub) are distinct loci, thus eliminating Ins-2 as a candidate gene for tub. These results also refine the estimated genetic distance between tub and Hbb to 2.4 +/- 1.4 cM. The predicted location for a human homolog of tubby is HSA 11p15.     

Genetic control of red cell osmotic fragility

Among normal mouse strains, natural genetic variation offers the potential to investigate the structure and function of cell membranes. One such polymorphism between C57BL/6J and DBA/2J is a difference in erythrocyte sensitivity to osmotic lysis. The genetic basis for erythrocyte osmotic fragility differences between mouse strains C57BL/6 and DBA/2 was examined through analyses of their serial backcross progeny, recombinant inbred (ri) strains (BXD), and congenic C57BL/6 strains with allelic differences at Hbb or Fv-2. The data indicate that the fragility difference between C57BL/6 and DBA/2 is the result of allelic differences at a minimum of two segregating loci. One of these might be linked to, but is not identical with the gene encoding the beta chain of hemoglobin (Hbb). Allelic differences at Fv-2, a gene known to control the proportion of erythroid precursors in the S phase, and at Hba, the structural locus of hemoglobin alpha chain also appear to exert no major influence on red cell osmotic fragility. Furthermore, the fact that red cells from one of the RI strans (BXD-31) are strikingly more resistant than those from the resistant parental strain DBA/2 leads to the conclusion that the degree of resistance/susceptibility for either strain is determined by the combined contributions of gene effects not all of which act in the same direction. We also found that red cells from strans C57BL/6 and DBA/2 differ in their uptake of 51Cr. This result suggests the possibility that red cell osmotic fragility differences may be due in part to differences in ion metabolism or membrane transport.     

Sequencing of the olfactory marker protein gene in normal and shaker-1 mutant mice

The mouse olfactory marker protein gene (Omp) maps close to the deafness mutation shaker-1 (sh-1) and has been considered a candidate gene for both sh-1 and its potential human homolog, the deaf-blind syndrome Usher Type I. Using primers devised from the available rat Olfactory Marker Protein gene sequence, we have determined the coding sequence of the mouse gene in both control inbred strains and six shaker-1 mutants. The coding sequence of the mouse Omp gene shows 97% nucleotide identity and 98% amino acid identity with the rat gene sequence. No sequence variants were detected in the coding region of any of the sh-1 mutants, ruling out Omp as the shaker-1 gene.     

Analysis of recombination in the centromere region of mouse chromosome 7 using ovarian teratoma and backcross methods

Recombination near the centromere of mouse chromosome 7 was studied using data obtained from ovarian teratomas and backcrosses. The recombination percentage for the centromere-Gpi-1 (glucose phosphate isomerase-1) interval was 13.4 +/- 2.6 using the ovarian teratoma mapping method. In a backcross using the Robertsonian translocation Rb(7.18)9Lub (Rb9) as the centromeric marker, the centromere-Gpi-1 recombination percentage was 4.5 +/- 1.3, demonstrating that Rb9 suppresses recombination near the centromere of chromosome 7. The recombination percentage for the Gpi-1-Ldh-1 (lactate dehydrogenase-1) interval was estimated on the LT/Sv mouse genetic background to be 19.0 +/- 2.9 using the ovarian teratoma mapping method, a value comparable to the 15.5 +/- 4.8 reported earlier. On the same genetic background in a backcross segregating for Rb9, the Gpi-1-Ldh-1 recombination percentage was 7.1 +/- 1.6. Another backcross, without the Rb9 translocation but utilizing a different genetic background, produced a recombination percentage for the Gpi-1-Ldh-1 interval of 10.7 +/- 1.5, a value similar to that obtained in the Rb-containing cross. These results suggest that either the recombination suppression in the centromere area caused by Rb9 does not extend to the Gpi-1-Ldh-1 genetic region or, if it does, that the differing genetic backgrounds of these two crosses influence recombination. No recombinants were detected among 410 offspring produced from a backcross mating segregating for Ldh-1 and ru-2 (ruby-eye-2). Thus, the gene order of Ldh-1 and ru-2 on chromosome 7 remains uncertain.     

The genetic relationship between the Hbb locus and body size in a population of mice divergently selected for six-week body weight.

Both pleiotropy and linkage were examined as possible explanations for the fixation of the Hbb3 allele in the six Large lines of a population of mice divergently selected for six-week body weight (six replicates in each direction and six controls). A survey of over 1200 individuals in the lines still segregating at the Hbb locus excluded pleiotropy as a possible explanation. The results showed a nonsignificant effect of haemoglobin genotype on body weight. The linkage relationship of the Hbb locus was examined using a backcross mating system. The Hbb3 c+ region (chromosome 7) of a Large line was backcrossed into its corresponding Small line (Hbb4 c). The resultant difference in body weight between the two segregants (Hbb8 c+ [Hbb4 c; Hbb4 c/Hbb4 c) was measured. The results suggested linkage as the most plausible explanation for the fixation of the Hbb4 allele in the six Large lines.     

Two genes controlling the expression of extended globoglycolipids in mouse kidney are closely linked to each other on chromosome 19

The gene (Gsl-5) controlling the expression of GL-Y (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6(Gal beta 1-3)Gb4Cer) in mouse kidney was suggested to be located near Ea-4 on mouse chromosome 19 by the results of glycolipid analysis of BXD/Ty recombinant inbred strains (Sekine et al. [1987] J. Biochem. 101, 563-568). In this study, Gsl-5 was mapped on mouse chromosome 19. Among 133 backcross progeny produced on mating between DBA/2 mice and (WHT/Ht x DBA/2)F1 mice, 10 recombinants between Lyt-1 and Gsl-5 were detected, indicating that Gsl-5 is located at 7.5 +/- 2.3 centimorgans (cM) from Lyt-1. While among 154 backcross progeny produced on mating between DBA/2 and (DBA/2 x Mus musculus castaneus)F1 mice, 39 recombinants between Got-1 and Gsl-5 were obtained, indicating that the distance between Got-1 and Gsl-5 is 25.3 +/- 3.5 cM and that Gsl-5 is telomeric to Lyt-1. In the latter mating experiment, we detected 3 recombinants between Gsl-5 and the gene (Gsl-6) controlling the expression of the Z1 ganglioside (NeuGc alpha 2-3Gal beta 1-3Gb4Cer) among the 154 backcross mice. These results indicate that these two genes, Gsl-5 and Gsl-6, are closely linked to each other, being 1.9 +/- 1.1 cM apart. This is the report of evidence that two genes controlling the expression of carbohydrates in glycoconjugates are closely linked and the first to suggest that some genes controlling the expression of carbohydrates may be clustered.(ABSTRACT TRUNCATED AT 250 WORDS)     

The properdin structural locus (Pfc) lies close to the locus for tissue inhibitor of metallothionine proteases (Timp) on the mouse X chromosome

We have confirmed the assignment of the structural locus of the complement factor properdin (Pfc) to the mouse X chromosome and mapped it between monoamine oxidase-A (Mao-a) and hypoxanthine phosphoribosyltransferase (Hprt) using a Mus spretus x Mus musculus interspecific backcross of 108 animals. The structural locus for murine tissue inhibitor of metallothionine proteases (Timp) could not be separated from properdin in a panel of 18 recombinant animals. By minimizing the number of double recombinants the following gene order was obtained: Otc-Mao-a-(Pfc, Timp)-Hprt-Cf-9. The implications for comparative mapping of human and mouse X chromosomes are discussed.     

Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.     

The Charles River "hairless" rat mutation maps to chromosome 1: allelic with fuzzy and a likely orthologue of mouse frizzy

Recent evidence has indicated that the recessive mutation affecting hypotrichosis in the Charles River (CR) "hairless" rat does not involve the hairless gene (hr) on rat chromosome 15. To determine if this mutation might be allelic (or orthologous) with any other previously mapped hypotrichosis-generating mutation in mammals, we have produced a panel of backcross rats segregating for the CR hairless rat mutation as well as numerous other markers from throughout the rat genome. Analysis of this panel has located the CR hairless rat's hypotrichosis-generating mutation on chromosome 1, near Myl2, where only the fuzzy mutation in rat (fz) and the frizzy mutation in mouse (fr) have been previously localized. Intercrossing fz/fz and CR hairless rats produced hybrid offspring with abnormal hair, showing that these two rat mutations are allelic. We suggest that the CR hairless rat mutation and fuzzy be renamed frizzy-Charles River (fr(CR)) and frizzy-Harlan (fr(H)), respectively, to reflect their likely orthology with the mouse fr mutation.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

Identification and mapping of six microdissected genomic DNA probes to the proximal region of mouse chromosome 1.

Six independent DNA probes, lambda Mm1C-150, lambda Mm1C-153, lambda Mm1C-156, lambda Mm1C-162, lambda Mm1C-163, and lambda Mm1C-165, have been isolated from a library of microdissected fragments from mouse chromosome 1, spanning cytogenetic bands C2 to C5. These DNA probes have been mapped by restriction fragment length polymorphism analysis with respect to 12 marker loci previously assigned to this portion of mouse chromosome 1, in a panel of 251 segregating Mus spretus x C57BL/6J interspecific backcross mice. The gene order and intergene distances were determined by segregation analysis to be centromere- lambda Mm1C-162-11.1 cM-Col3a1-8.8 cM-Len-2-2.6 cM-lambda Mm1C-163-1.6 cM-Fn-1-1.6 cM-Tp-1-0.8 cM-lambda Mm1C-165/Vil-0.4 cM-Inha-2.8 cM-lambda Mm1C-153-2.4 cM-lambda Mm1C-156-1.2 cM-Pax-3-5.6 cM-Akp-3-0.8 cM-Acrg-2.0 cM-Sag-0.5 cM-Col6a3-1.8 cM-lambda Mm1C-150-15.4 cM-Ren1,2. Four of these probes map within a chromosome 1 segment that is homologous to human chromosome 2q. Southern blotting analyses indicate that one of these anonymous probes, lambda Mm1C-165, detects DNA fragments highly conserved across species. These novel polymorphic probes should prove useful for linkage and physical mapping of this chromosomal region.     

Size of donor chromosome segments around introgressed loci and reduction of linkage drag in marker-assisted backcross programs.

This article investigates the efficiency of marker-assisted selection in reducing the length of the donor chromosome segment retained around a locus held heterozygous by backcrossing. First, the efficiency of marker-assisted selection is evaluated from the length of the donor segment in backcrossed individuals that are (double) recombinants for two markers flanking the introgressed gene on each side. Analytical expressions for the probability density function, the mean, and the variance of this length are given for any number of backcross generations, as well as numerical applications. For a given marker distance, the number of backcross generations performed has little impact on the reduction of donor segment length, except for distant markers. In practical situations, the most important parameter is the distance between the introgressed gene and the flanking markers, which should be chosen to be as closely linked as possible to the introgressed gene. Second, the minimal population sizes required to obtain double recombinants for such closely linked markers are computed and optimized in the context of a multigeneration backcross program. The results indicate that it is generally more profitable to allow for three or more successive backcross generations rather than to favor recombinations in early generations.     

Mapping Creb-1 to chromosome 1 in the mouse.

The gene CREB1 encoding the cyclic AMP response element DNA binding protein was previously assigned to human 2q32.3-q34. In this study, a panel of 207 backcross mice made between C57BL/10ScSn (=B10) females and (B10 x B10.L-Lsh)F1 males were used to map Creb-1 with respect to Cryg and Lsh/Vil on mouse chromosome 1. A reverse-transcribed, polymerase chain reaction-amplified cDNA probe covering bp 39 to 554 of the human sequence identified restriction fragment length polymorphisms with 7/18 restriction endonucleases used to digest whole genomic mouse DNA from the parental strains. BglII and DraI RFLPs for Creb-1 were scored on a subpanel of 16/207 known recombinants between Cryg and Lsh/Vil, yielding 2/16 recombinants between Cryg and Creb-1 and 14/16 recombinants between Creb-1 and Lsh/Vil. The 16/207 recombinants observed between Lsh/Vil and Cryg provide an estimated recombination frequency of 0.077 +/- 0.019, equivalent to a map distance of 7.7 +/- 1.9 cM. This is in good agreement with previously published map distances. The number of recombinants observed between Creb-1 and the other markers place Creb-1 approximately 1 cM distal to Cryg and 7 cM proximal to Lsh/Vil.     

The length of the intact donor chromosome segment around a target gene in marker-assisted backcrossing

Recurrent backcrossing is an established procedure to transfer target genes from a donor into the genetic background of a recipient genotype. By assessing the parental origin of alleles at markers flanking the target locus one can select individuals with a short intact donor chromosome segment around the target gene and thus reduce the linkage drag. We investigated the probability distribution of the length of the intact donor chromosome segment around the target gene in recurrent backcrossing with selection for heterozygosity at the target locus and homozygosity for the recurrent parent allele at flanking markers for a diploid species. Assuming no interference in crossover formation, we derived the cumulative density function, probability density function, expected value, and variance of the length of the intact chromosome segment for the following cases: (1) backcross generations prior to detection of a recombinant individual between the target gene and the flanking marker; (2) the backcross generation in which for the first time a recombinant individual is detected, which is selected for further backcrossing; and (3) subsequent backcross generations after selection of a recombinant. Examples are given of how these results can be applied to investigate the efficiency of marker-assisted backcrossing for reducing the length of the intact donor chromosome segment around the target gene under various situations relevant in breeding and genetic research.     

The host resistance locus Bcg is tightly linked to a group of cytoskeleton-associated protein genes that include villin and desmin.

In the mouse, innate resistance or susceptibility to infection with a group of unrelated intracellular parasites which includes, Mycobacteria, Salmonella, and Leishmania is determined by the expression of a single dominant autosomal gene designated Bcg located on the proximal portion of chromosome 1. The gene is expressed at the level of the mature tissue macrophage and influences its capacity to restrict intracellular proliferation of the parasites. We have used restriction fragment length polymorphism analysis in segregating populations of inter- and intraspecific backcross mice and in recombinant inbred strains to position four new marker genes, transition protein 1 (Tp-1), desmin (Des), the alpha subunit of inhibin (Inha), and retinal S-antigen (Sag), in the vicinity of the host resistance locus, Bcg. The gene order for Tp-1, Des, Inha, and Sag was established in an eight-point testcross with respect to anchor loci previously assigned to that portion of mouse chromosome 1 and was found to be centromere-Fn-1-Tp-1-(Vil,Bcg)-Des-Inha-Akp-3-Acrg+ ++-Sag. Two of these new marker genes were found very tightly linked to Bcg: Des was located 0.3 +/- 0.3 cM distal from (Vil,Bcg) and 0.3 +/- 0.3 cM proximal to Inha. Tp-1 mapped 0.8 +/- 0.8 cM proximal and Sag 12.8 +/- 1.7 cM distal to (Vil,Bcg). Tp-1, Des, Inha, and Sag all fall within a large mouse chromosome 1 segment homologous with the telomeric region of the long arm of human chromosome 2 (2q). Our findings indicate that the two closest markers to the host resistance locus, Bcg, encode cytoskeleton-associated proteins which are capable of interaction with actin filaments.     

The isolation of evolutionarily conserved Eag I end-clones from mouse chromosome 17 using cloned DNA.

To isolate DNA markers from mouse chromosome 17, a genomic phage library was constructed from the mouse-hamster CMGT cell hybrid RcE-B52. This hybrid contains a chromosomal fragment from the distal end/flanking region of the t complex on mouse chromosome 17. Recombinants of mouse origin were identified by using a panel of mouse-specific repetitive sequences as a probe. A total of 1,500 mouse phage recombinants were isolated. These were found to represent 250-300 individual recombinants, comprising about 4 Mbp of cloned mouse DNA. The pooled mouse recombinant phages were used to construct an Eag I end-library. This was achieved by the specific insertion of a marker plasmid in Eag I recognition sites when present in the mouse inserts of the recombinant phages. The Eag I end-fragments were subsequently subcloned using a simple procedure taking advantage of the inserted plasmid. A total of 56 individual Eag I end-fragments were identified. These were found to contain recognition sites for rare cutting enzymes at high frequency. A large proportion (73%) were found to be evolutionarily conserved in human DNA. Furthermore, a significant fraction of these fragments, two of six tested, appears to detect specific cDNAs in a 8.5-day mouse embryo cDNA library.