Mapping and cloning recombinant breakpoints demarcating the Hybrid Sterility 6-specific sperm tail assembly defect

Variants of the mouse t complex known as t haplotypes (t) express factors that perturb sperm differentiation, resulting in the non-Mendelian transmission of t from +/t heterozygous males and the sterility of t/t homozygous males. Previous studies of mice carrying heterospecific combinations of the t complex have revealed a 1-cM candidate locus, Hst6, for the distal-most of these factors, Tcd/Tcs2. Males heterozygous for the M. spretus allele of Hst6 and a t haplotype (Hst6 ^s /t) are sterile, expressing an abnormality in sperm flagellar curvature (``curlicue') indistinguishable from one exhibited by sperm from t/t homozygotes. Hst6 <sup>s</sup> /Hst6 <sup>s</sup> males are also sterile; however, sperm produced by these males are completely immotile owing to the absence of assembled flagella. Recent studies have shown that the complete presentation of ``curlicue' derives from expression of at least two factors within the locus, Curlicue a (Ccua) proximally and Curlicue b (Ccub) distally, with a factor affecting sperm-oolemma penetration, Stop1p, mapping between them. In the present report, we have examined expression of the Hst6-specific flagellar assembly phenotype in sperm from mice homozygous for M. spretus–M. m. domesticus recombinant Chr 17 homologs whose breakpoints map within the Hst6 locus. SSLP analysis of these homologs has demonstrated that the flagellar assembly defect maps to less than 0.2 cM between D17Mit61 and D17Mit135, coincident with Ccua. SSR content analysis of 23 BACs mapping to four contigs within the Hst6 locus has resulted in isolation of proximal and distal recombinant breakpoints circumscribing the flagellar assembly phenotype/Ccua factor. In addition, we have provided increased high-resolution mapping of the Stop1p and Ccub factors. These new data enhance our ability to isolate and characterize candidates for Tcd/Tcs2. Received: 12 August 1998 / Accepted: 1 October 1998     

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as ``curlicue' is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized ``curlicue' and a flagellar developmental defect, ``whipless', to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both ``curlicue' and ``whipless' to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both ``whipless' and ``curlicue' at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or ``whipless' animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility. Received: 13 July 1999 / Accepted: 25 August 1999     

A novel partial t haplotype with a Brachyury-independent effect on tail phenotype

The btm (brachyury-interacting tail length modifier) mutation was discovered in strain MOL-NIS derived from Japanese wild mice (Mus musculus molossinus) as an autosomal recessive mutation. Homozygotes for this mutation show a short tail phenotype and, moreover, this mutation causes the tailless character by interacting with the T (brachyury) gene on Chromosome (Chr) 17. Our linkage tests and RFLP analyses suggest that btm is located within the t complex on Chr 17 and represents a new partial t haplotype.     

The mouse plasminogen locus maps to the recombination breakpoints of the t Lub2and Tt Orlpartial t haplotypes but is not at the t w73locus

The mouse plasminogen (Plg) locus maps to a region of chromosome (Chr) 17 which is inverted in the t haplotype Chromosomal variant. Here we investigate the genomic organization of the Plg locus in structurally variant forms of Chr 17; wild-type (+), t haplotype (t), and two partial t haplotypes Tt ^Orland t ^Lub2which arose by recombination between + and t chromosomes. Our analysis suggests that the t haplotype chromosomal variant contains extra, inverted copies of the Plg locus, and that a single locus is present in the wild-type variant. Changes in the Plg locus in Tt ^Orland t ^Lub2suggest that they arose by homologous recombination across elements in the Plg locus having the same orientation in the wild-type and t haplotype chromosomes. One hundred ten kb around the wild-type Plg genomic locus have been cloned and the proximal breakpoint of a deletion in the t ^Lub2chromosome has been localized to a fragment 30 kb downstream of the Plg gene. The t ^Lub2deletion has been shown to delete a gene named t ^w73that affects blastocyst implantation, a process probably requiring proteases such as plasminogen. However, the mapping of Plg relative to the t ^Lub2deletion and mRNA analysis of plasminogen in t ^w73heterozygotes suggests that Plg does not lie at the t ^w73locus.     

A high-resolution genetic map of mouse Chromosome 15 encompassing the Dominant megacolon (Dom) locus

Dominant megacolon (Dom) is one of four mutations in the mouse that can produce a phenotype similar to Hirschsprung disease in human. The Dom gene product is not known, and no candidate region has been defined for a possible human homolog. In this publication we report mapping the Dom locus with high definition, using several intra- and interspecific crosses and a set of 16 Chr 15-specific microsatellites flanking this locus.     

Mapping of an immotile short tail sperm defect in the Finnish Yorkshire on porcine Chromosome 16

An immotile short tail sperm defect has recently been identified as a hereditary disorder present within the Finnish Yorkshire pig population. The syndrome is inherited as an autosomal recessive disease exclusively expressed in male individuals as shorter sperm tail length and immotile spermatozoa. Based on the assumption of a recent common origin of the disease-causing mutation, a genome-wide search was performed with 228 evenly spaced microsatellites by homozygosity mapping of affected and unaffected DNA pools. One locus, SW2411 on Chr 16, demonstrated a significantly skewed allele distribution between the two pools. Linkage analysis of five markers in this region mapped the disease-causing gene within a 6-cM confidence interval region with a highest LOD score of 7.7 at marker SW419. It appears that three-marker haplotypes can be used for marker-assisted selection within analyzed pedigrees. Furthermore, future fine mapping may reveal a more precise population-wide associated haplotype and facilitate identification of a new gene affecting sperm tail development. Received: 5 July 2001 / Accepted: 13 September 2001     

Genetic analysis of resistance to Type-1 Diabetes in ALR/Lt mice,a NOD-related strain with defenses against autoimmune-mediated diabetogenic stress

ALR mice are closely related to type-1 diabetes mellitus (T1DM)-prone NOD mice. The ALR genome confers systemically elevated free radical defenses, dominantly protecting their pancreatic islets from free radical generating toxins, cytotoxic cytokines, and diabetogenic T cells. The ALR major histocompatibility complex (MHC) (H2^gx haplotype) is largely, but not completely identical with the NOD H2^g7 haplotype, sharing alleles from H2-K through the class II and distally into the class III region. This same H2^gx haplotype in the related CTS strain was linked to the Idd16 resistance locus. In the present study, ALR was outcrossed to NOD to fine map the Idd16 locus and establish chromosomal regions carrying other ALR non-MHC-linked resistance loci. To this end, 120 (NOD×ALR)×NOD backcross progeny females were monitored for T1DM and genetic linkage analysis was performed on all progeny using 88 markers covering all chromosomes. Glucosuria or end-stage insulitis developed in 32 females, while 88 remained both aglucosuria and insulitis free. Three ALR-derived resistance loci segregated. As expected, one mapped to Chromosome 17, with peak linkage mapping just proximal to H2-K. A novel resistance locus mapped to Chr 8. A pairwise scan for interactions detected a significant interaction between the loci on Chr 8 and Chr 17. On Chr 3, resistance segregated with a marker between previously described Idd loci and coinciding with an independently mapped locus conferring a suppressed superoxide burst by ALR neutrophils (Susp). These results indicate that the Idd16 resistance allele, defined originally by linkage to the H2^gx haplotype of CTS, is immediately proximal to H2-K. Two additional ALR-contributed resistance loci may be ALR-specific and contribute to this strain's ability to dissipate free-radical stress.     

Genetic linkage analysis of X-ray hypersensitivity in the LEC mutant rat

The LEC rat has been reported to exhibit X-ray hypersensitivity and deficiency in DNA double-strand break (DSB) repair. The present study was performed to map the locus responsible for this phenotype, the xhs (X-ray hypersensitivity), as the first step in identifying the responsible gene. Analysis of the progeny of (BN × LEC)F₁× LEC backcrosses indicated that the X-ray hypersensitive phenotype was controlled by multiple genetic loci in contrast to the results reported previously. Quantitative trait loci (QTL) linkage analysis revealed two responsible loci located on Chromosomes (Chr) 4 and 1. QTL on Chr 4 exhibited very strong linkage to the X-ray hypersensitive phenotype, while QTL on Chr 1 showed weak linkage. The Rad52 locus, mutation of which results in hypersensitivity to ionizing radiation and impairment of DNA DSB repair in yeast, was reported to be located on the synteneic regions of mouse Chr 6 and human Chr 12. However, mapping of the rat Rad52 locus indicated that it was located 23 cM distal to the QTL on Chr 4. Furthermore, none of the radio-sensitivity-related loci mapped previously in the rat chromosome were identical to the QTL on Chrs 4 and 1 in the LEC rat. Thus, it seems that X-ray hypersensitivity in the LEC rat is caused by mutation(s) in as-yet-undefined genes. Received: 14 February 2000 / Accepted: 17 May 2000     

Identification and characterization of a novel conserved DNA repeat

Homozygous In(10)17Rk mice contain a paracentric inversion within Chromosome (Chr) 10 and exhibit a pygmy phenotype, suggesting that the distal inversion breakpoint is within the pygmy (pg) locus. In order to obtain the pygmy gene by positional cloning procedures, In(10)17Rk DNA was subjected to RFLP analysis with single-copy probes derived from the wild-type pygmy locus. This analysis identified a DNA polymorphism in the DBA/2J mouse strain on which the In(10)17Rk mutation was originally induced. A detailed characterization of this polymorphism revealed the presence of a novel, tandemly repeated DNA element. Copy number estimation experiments indicate that there are approximately 100,000 copies of this element in the haploid DBA/2J genome. PCR typing studies revealed the presence of the repeat at the pygmy locus of 6 of the 18 Mus domesticus strains analyzed. The absence of the repeat from the pygmy locus of 12 strains of the M. domesticus species and from the M. caroli, M. spretus, M. castaneus, and M. molossinus species suggests that the repeat could serve as a strain-specific hybridization probe in genetic mapping studies. Finally, the novel tandem DNA repeat is conserved in both rat and human genomes as indicated by Southern hybridization experiments.     

Transgenic rescue of the mouse t complex haplolethal locus Thl1

Chromosomal deletions can uncover haploinsufficient or imprinted regions of the genome. Previously, the haploinsufficient locus t haplolethal 1 (Thl1) was identified and localized to a 1.3-Mb region using overlapping deletions around the Sod2 and D17Leh94 loci of the mouse t complex on Chr 17. Germline chimeric mice, produced from embryonic stem (ES) cells containing radiation-induced deletions of the Thl1 locus, never produced viable deletion-bearing progeny when mated to C57BL/6J (B6) females. However, deletion-bearing offspring could be obtained by mating to females of other strains. In this article we describe a transgenic approach to narrow the critical region for Thl1. BAC clones were introduced into a deletion-bearing ES cell line and one was shown to rescue the Thl1 phenotype, reducing the critical region to 140 kb. Analysis of the gene content of this region suggests two strong Thl1 candidates, Pdcd2 and a novel SET domain-containing gene termed Tset1. A more detailed analysis using mice carrying overlapping deletions identified subregions that influence the phenotypic characteristics of Thl1 hemizygotes.     

Identification of genetic determinants of IGF‐1 levels and longevity among mouse inbred strains

The IGF‐1 signaling pathway plays an important role in regulating longevity. To identify the genetic loci and genes that regulate plasma IGF‐1 levels, we intercrossed MRL/MpJ and SM/J, inbred mouse strains that differ in IGF‐1 levels. Quantitative trait loci (QTL) analysis of IGF‐1 levels of these F2 mice detected four QTL on chromosomes (Chrs) 9 (48 Mb), 10 (86 Mb), 15 (18 Mb), and 17 (85 Mb). Haplotype association mapping of IGF‐1 levels in 28 domesticated inbred strains identified three suggestive loci in females on Chrs 2 (13 Mb), 10 (88 Mb), and 17 (28 Mb) and in four males on Chrs 1 (159 Mb), 3 (52 and 58 Mb), and 16 (74 Mb). Except for the QTL on Chr 9 and 16, all loci co‐localized with IGF‐1 QTL previously identified in other mouse crosses. The most significant locus was the QTL on Chr 10, which contains the Igf1 gene and which had a LOD score of 31.8. Haplotype analysis among 28 domesticated inbred strains revealed a major QTL on Chr 10 overlapping with the QTL identified in the F2 mice. This locus showed three major haplotypes; strains with haplotype 1 had significantly lower plasma IGF‐1 and extended longevity (P < 0.05) than strains with haplotype 2 or 3. Bioinformatic analysis, combined with sequencing and expression studies, showed that Igf1 is the most likely QTL gene, but that other genes may also play a role in this strong QTL.     

The dynamic of the t-haplotype in wild populations of the house mouse Mus musculus domesticus in Israel

The t-haplotype, a variant of the proximal part of the mouse chromosome 17, is composed of at least four inversions and is inherited as a single genetic unit. The haplotype causes embryonic mortality or male sterility when homozygous. Genes within the complex are responsible for distortion of Mendelian transmission ratio in males. Thus, the t-haplotype in heterozygous males is transferred to over 95% of the progeny. We examined the dynamic and behavior of the t-haplotype in wild populations of the house mouse in Israel. The Israeli populations show high frequency (15%–20%) of both partial and complete t-carrying mice, supporting the suggestion that the t-complex evolved in the M. domesticus line in the Israeli region. In one population that had the highest frequency of t-carrying individuals, we compared the level of gene diversity between t-carrying and normal mice in the marker’s loci: H-2 locus of the major histocompatibility complex (MHC) on the t-haplotype of chromosome 17, three microsatellites on other chromosomes, and the mitochondrial D-loop. Genetic variability was high in all tested loci in both t and (+) mice. All t mice carried the same chromosome and showed the same H-2 haplotype. While t-carrying mice showed significant H-2 heterozygotes access, (+) mice expressed significant H-2 heterozygote deficiency. There were no differences in the level of gene diversity between t and (+) mice in the other loci. Heterozygosity level at the MHC may be an additional factor in the selective forces balancing the t-haplotype polymorphism.     

Polymorphism in hybrid male sterility in wild-derived Mus musculus musculus strains on proximal chromosome 17

The hybrid sterility-1 (Hst1) locus at Chr 17 causes male sterility in crosses between the house mouse subspecies Mus musculus domesticus (Mmd) and M. m. musculus (Mmm). This locus has been defined by its polymorphic variants in two laboratory strains (Mmd genome) when mated to PWD/Ph mice (Mmm genome): C57BL/10 (carrying the sterile allele) and C3H (fertile allele). The occurrence of sterile and/or fertile (wild Mmm × C57BL)F₁ males is evidence that polymorphism for this trait also exists in natural populations of Mmm; however, the nature of this polymorphism remains unclear. Therefore, we derived two wild-origin Mmm strains, STUS and STUF, that produce sterile and fertile males, respectively, in crosses with C57BL mice. To determine the genetic basis underlying male fertility, the (STUS × STUF)F₁ females were mated to C57BL/10 J males. About one-third of resulting hybrid males (33.8%) had a significantly smaller epididymis and testes than parental animals and lacked spermatozoa due to meiotic arrest. A further one-fifth of males (20.3%) also had anomalous reproductive traits but produced some spermatozoa. The remaining fertile males (45.9%) displayed no deviation from values found in parental individuals. QTL analysis of the progeny revealed strong associations of male fitness components with the proximal end of Chr 17, and a significant effect of the central section of Chr X on testes mass. The data suggest that genetic incompatibilities associated with male sterility have evolved independently at the proximal end of Chr 17 and are polymorphic within both Mmd and Mmm genomes.     

Localization of the locus causing Spider Lamb Syndrome to the distal end of ovine Chromosome 6

Spider Lamb Syndrome (SLS) is a semi-lethal congenital disorder, causing severe skeletal abnormalities in sheep. The syndrome has now been disseminated into several sheep breeds in the United States, Canada, and Australia. The mode of inheritance for SLS is autosomal recessive, making the identification and culling of carrier animals difficult due to their normal phenotype. Two large pedigrees segregating for the SLS mutation were established, and a genome scan with genetic markers from previously published genome maps of cattle and sheep was used to map the locus causing SLS. Genetic linkage between SLS and several microsatellite markers, OarJMP8, McM214, OarJMP12, and BL1038, was detected, thereby mapping the SLS locus to the telomeric end of ovine Chromosome (Chr) 6. Alignment of ovine Chr 6 with its evolutionary ortholog, human Chr 4, revealed a positional candidate gene, fibroblast growth factor receptor 3 (FGFR3). Received: 10 June 1998 / Accepted: 23 September 1998     

Microsatellite mapping of the bovine roan locus: A major determinant of White Heifer Disease

In the Belgian Blue Cattle breed, coat color variation is mainly under the influence of a single autosomal locus, the roan locus, characterized by a pair of codominant alleles: r ⁺ (black) and R (white). Heterozygous r ⁺ R animals have intermingled black and white hairs, yielding the ``blue' phenotype typical of the breed. Major interest for the roan locus stems from its pleiotropic effect on fertility, owing to the critical role of the R allele in the determinism of White Heifer Disease. We describe the linkage mapping of the roan locus to bovine Chromosome (Chr) 5, in the interval between microsatellite markers BPI and AGLA293, with an associated lodscore of 11.2. Moreover, we map a candidate gene, the Steel locus coding for the mast cell growth factor, to bovine Chr 5. Received: 30 May 1995 / Accepted: 6 September 1995     

Molecular probes define different regions of the mouse t complex

Four genomic clones obtained from microdissected fragments of the proximal portion of mouse chromosome 17 have been used to identify a series of t-haplotype-specific restriction fragments. Their specificity is defined by presence in eight complete t haplotypes and absence from 18 inbred strains of wild-type mice. Partial t haplotypes contain subsets of the t-specific fragments, and each can be classified according to the t-specific fragments it contains. This is the first molecular evidence that independent partial t haplotypes contain different lengths of t haplotype DNA. Recombination studies indicate that partial t haplotypes suppress recombination in proportion to the extent of t haplotype DNA they contain. Molecular analysis of partial t haplotyes shows that the t-specific fragments map to and thus define different regions of the t complex. Certain regions of t haplotype DNA defined by t-specific restriction fragments can be correlated with loci involved in the control of transmission ratio distortion.     

QTL mapping in a mouse model of cardiomyopathy reveals an ancestral modifier allele affecting heart function and survival

The progression from myocardial hypertrophy to heart failure is a complex process, involving genetic and environmental factors. Elucidating the genetic components contributing to heart failure has been difficult, largely because of the heterogeneity of human populations. We have employed a strategy to map genetic loci that modify the heart failure phenotype in a transgenic mouse model of cardiomyopathy caused by cardiac-specific overexpression of calsequestrin. Strain-specific differences in both cardiac function and survival are observed when the transgene is moved into different inbred mouse strains. We have previously reported linkage results from mapping in reciprocal backcrosses between C57/BL6 (BL6) and DBA/2J (DBA) and a backcross between DBA/AKR and AKR. Here we report the results of a genome-wide linkage scan in the reciprocal backcross between DBA/AKR and DBA. We identified one novel locus on Chromosome (Chr) 18 that affects heart function and a second on Chr 3 that shows significant linkage to both survival and heart function. Intriguingly, the Chr 3 allele of AKR shows a susceptibility effect on phenotype, whereas the overall effect of the AKR genetic background is protective. The Chr 3 locus also completely overlaps the Hrtfm2 locus, which was previously mapped in crosses between DBA and BL6. Mapping the same QTL in two different crosses allowed us to use ancestral haplotypes to narrow the candidate gene interval from 9 to 2 Mb. Identification of the genes at these QTLs in the mouse will provide novel candidate genes that can be evaluated for their role in human heart failure.     

Genetic analysis of the organic cation transporter genes Orct2/Slc22a2 and Orct3/Slc22a3 reduces the critical region for the t haplotype mutant t w73 to 200 kb

Here we report an analysis of two candidate genes for the t ^w73 implantation mutation. The t ^w73 gene maps to a 20-cM region of mouse Chromosome (Chr) 17 known as the t-complex, which exists in a wild-type and t haplotype form in present-day mice. The t haplotype variants contain several mutant alleles affecting male fertility and embryonic viability and offer the opportunity to identify genes critical for these processes. t ^w73 homozygous embryos are defective in trophoblast production and fail to implant adequately, with death occurring at approximately 7.5 days post coitum (pc). Two recently described organic cation transporter genes, Slc22a2 (Orct2) and Slc22a3 (Orct3), fulfill criteria predicted for t ^w73 candidate genes, since both map to the previously defined 500-kb t ^w73 minimal region and both are also expressed in 7.5 days pc post-implantation embryos. The genomic locus of the Orct2 gene appears similar in wild-type and t ^w73 chromosomes. In contrast, the genomic locus of Orct3 is amplified and displays an altered expression profile in all t haplotype variant chromosomes tested. In addition, Orct3 shows a t ^w73 specific polymorphism. To test whether either Orct2 or Orct3 is involved in the t ^w73 phenotype, we have performed a genetic rescue experiment using YAC transgenes overexpressing Orct2, and genetic complementation with an allele in which the Orct3 gene was inactivated by homologous recombination. The results eliminate both Orct2 and Orct3 as candidates and further reduce the critical region containing the t ^w73 mutant from 500 kb to 200 kb. Received: 8 February 2001 / Accepted: 1 May 2001