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     

The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23

Microsatellite markers are useful tools for understanding the evolutionary history of discrete segments of the mammalian genome. We used the microsatellite marker D17Mit23 to study the portion of the mouse genome known as the t complex, a naturally occurring variant of Chromosome 17. We identified an allelic variant of D17Mit23, which is shared by two forms of the t complex, the t haplotypes t ^w2 and t ^Lub2 . Polymerase chain reaction (PCR) analysis of DNA samples from mice that were heterozygous for either haplotype resulted in gel patterns with prominent bands of higher molecular weight in addition to the bona-fide D17Mit23 alleles. The appearance of these higher molecular weight bands, although consistent with heteroduplex formation, was not diminished through the use of reconditioning PCR. We used a modified form of asymmetric PCR, called “unilateral PCR”, to show that the higher molecular weight bands are heterodu-plexes and to identify their constituent strands. Certain microsatellite motifs may be especially prone to the production of prominent heteroduplex products, and this may lead to the erroneous genotyping of DNA samples.     

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.     

Functional analysis of a t complex responder locus transgene in mice

Transmission ratio distortion (TRD) of mouse t haplotypes occurs through the interaction of multiple distorter loci with the t complex responder (Tcr) locus. Males heterozygous for a t haplotype will transmit the t-bearing chromosome to nearly all of their offspring. This process is mediated by the production of functionally inequivalent gametes: wildtype meiotic partners of t spermatozoa are rendered functionally inactive. The Tcr locus, which is required for TRD to occur, is thought to somehow protect its host spermatid from the sperm-inactivating effects of linked distorter genes (Lyon 1984). In previous work, Tcr was mapped to a small genetic interval in t haplotypes, and a candidate gene from this region was isolated (Tcp-10b ^t). In this work, we further localize Tcr to a 40-kb region that contains the 21-kb Tcp-10b ^t gene. A cloned genomic copy of Tcp-10b ^t was used to generate transgenic mice. The transgene was bred into a variety of genetic backgrounds to test for non-Mendelian segregation. Abberrant segregation was observed in some mice carrying either a complete t haplotype or a combination of certain partial t haplotypes. These observations, coupled with those of Snyder and colleagues (in this issue), provide genetic and functional evidence that the Tcp-10b ^t gene is Tcr. However, other genotypes that were predicted to produce distortion did not. The unexpected data from a variety of crosses in this work and those of our colleagues suggest that elements to the TRD system and the Tcr locus remain to be identified.     

High-resolution mapping of sperm function defects in the t complex fourth inversion

Structural variants of the mouse Chr 17-specific t complex, known as t haplotypes, express factors that alter the ability of sperm to carry out their roles in the normal fertilization process. In previous studies of males carrying heterospecific combinations of the t complex, we discovered a unique M. spretus/t haplotype phenotype of male sterility. In additional studies with mice carrying a series of M. spretus–M. m. domesticus recombinant Chr 17 homologs and a complete t haplotype (S-+/t), we monitored physiological aspects of sperm function to map a locus (Hst6) responsible for expression of the t-specific ``curlicue' sperm flagellar curvature phenotype to 1 cM within the fourth inversion of the t complex. In the present report, we quantitatively analyze the in vitro capability of sperm from mice with similar S-+/t Chr 17 genotypes to fertilize zona pellucida-free mouse eggs. The results identify a locus, Stop1, mapping distal to Pim1, with acute effects on the ability of sperm to penetrate the oolemma. The data suggest that Stop1 is a complex locus consisting of at least two genetic elements, a proximal one overlapping the Hst6 locus, and another, distal to the Hst6 locus. Further quantitative analyses of the ``curlicue' phenotype produced by sperm derived from these same animals indicate that expression of this chronic flagellar curvature phenotype also derives from at least two elements, both mapping within the Hst6 locus. Thus, these studies provide higher resolution mapping of the molecular basis of t haplotype-specific sperm dysfunction emanating from In(17)4. Received: 22 May 1998 / Accepted: 17 June 1998     

Characterization of a Recombinant Mouse t Haplotype That Expresses a Dominant Lethal Maternal Effect

The t^wLub2 chromosome was generated by rare recombination between a complete t haplotype and a wild-type form of mouse chromosome 17. This recombinant chromosome expresses a dominant lethal effect in all embryos that inherit the mutant chromosome from their mothers. The phenotype of this maternal effect is indistinguishable from that expressed by the previously described T^hp deletion chromosome. It appears likely that the crossing over event that gave rise to t^wLub2 was unequal and resulted in the alteration or deletion of a gene (which is named the T-associated maternal effect locus, Tme) that must be inherited from the mother in order for normal development to proceed through late stages of gestation. The results presented here allow a mapping of the Tme locus between the quaking and tufted loci which are 3 cM apart within the proximal region of chromosome 17.     

Sequence of the t complex Tcp-10a t gene and examination of the Tcp-10 t gene family

Transmission ratio distortion (TRD) is a property of complete t haplotypes which results in the preferential transmission of the t haplotype chromosome from heterozygous t/+ males to the majority of the offspring. A candidate gene for one of the primary genetic elements in TRD, the t complex responder locus has recently been suggested to be Tcp-10b ^t. There are multiple, functional Tcp-10 ^t genes, but genetic data suggest the presence of the Tcp-10a ^t gene alone is compatible with normal transmission ratios. Here we present the complete sequence and genomic structure of the Tcp-10a ^t gene which is compared with sequence data from a number of cDNAs and genomic subclones representing all active Tcp-10 ^t family genes. A detailed table of all sequence variants discovered in the course of our investigation is presented, and we have clarified the extent of 5 untranslated alternative splicing patterns exhibited by this gene family. A 60 base pair (bp) in-frame deletion from the 5 end of exon 3 of the Tcp-10a ^t gene is also presented and compared with the equivalent region of Tcp-10b ^t and Tcp-10c ^t. A search of the University of Edinburgh database has revealed a significant homology between the Tcp-10b ^t open reading frame and several cytosolic filament proteins. Interestingly, the region of homology is involved in the deletion from the Tcp-10a ^t gene.     

A complex structural variant at the KIT locus in cattle with the Pinzgauer spotting pattern

A specific white spotting phenotype, termed finching or line‐backed spotting, is known for all Pinzgauer cattle and occurs occasionally in Tux‐Zillertaler cattle, two Austrian breeds. The so‐called Pinzgauer spotting is inherited as an autosomal incompletely dominant trait. A genome‐wide association study using 27 white spotted and 16 solid‐coloured Tux‐Zillertaler cattle, based on 777k SNP data, revealed a strong signal on chromosome 6 at the KIT locus. Haplotype analyses defined a critical interval of 122 kb downstream of the KIT coding region. Whole‐genome sequencing of a Pinzgauer cattle and comparison to 338 control genomes revealed a complex structural variant consisting of a 9.4‐kb deletion and an inversely inserted duplication of 1.5 kb fused to a 310‐kb duplicated segment from chromosome 4. A diagnostic PCR was developed for straightforward genotyping of carriers for this structural variant (KIT^PINZ) and confirmed that the variant allele was present in all Pinzgauer and most of the white spotted Tux‐Zillertaler cattle. In addition, we detected the variant in all Slovenian Cika, British Gloucester and Spanish Berrenda en negro cattle with similar spotting patterns. Interestingly, the KIT^PINZ variant occurs in some white spotted animals of the Swiss breeds Evolèner and Eringer. The introgression of the KIT^PINZ variant confirms admixture and the reported historical relationship of these short‐headed breeds with Austrian Tux‐Zillertaler and suggests a mutation event, occurring before breed formation.     

H-2 typing of mutants of thet 6 haplotype in the mouse

Mutantt haplotypes derived from thet ⁶ haplotype were typed forH-2. The mutantt ^h2 that arose fromt ⁶ due to crossing over in the region betweenT andtf had, as expected, lost theH-2 haplotype characteristic oft ⁶. The haplotypest ^h17,t ^h18, andt ^p1, which also arose by recombination, but which represent the complementary crossover products, including the distal part of thet ⁶ haplotype, carried the sameH-2 type ast ⁶. This suggests that crossing over betweentf andH-2 is suppressed int ^h17 andt ¹⁸. This in turn suggests that mutantt haplotypes suppress crossing over for that part of thet chromatin that they still retain.The origin oft ^h7, which apparently did not include any crossover distal toT, and which retains the crossover-suppressing property oft ⁶, retains thet ⁶ H-2 type. Unexpectedly, J ^h20 , which expressestf and was at first thought to have arisen due to crossing over, also retains theH-2 type oft ⁶. This provides part of the evidence thatt ^h20 arises fromt ⁶ not by crossing over, but by a small deletion, and hence that duplication and deletion are possible modes of origin of mutantt haplotypes.Abbreviations used in this paper are t haplotype mutant haplotype of the chromosome 17, often designated J allele - T Brachyury mutant - T/+ short-tailed mouse - T/T lethal during embryogenesis - T-int T interaction (characteristic oft haplotypes that interact in heterozygotes withT to produce a tailless mouse) - tf locus homozygotes showing waves of hair loss - Kb knobby, which produces a knobbly tailed heterozygote, homozygous lethal - titer reciprocal of serum dilution giving 50% kill     

An unstable family of large DNA elements in the center of the mouse t complex

We have cloned 363 kb (× 10³ bases) from a novel, locally dispersed family of 11 large DNA elements, called T66 elements, within the center of complete mouse t haplotypes. Homologies among individual members of the T66 family are observed along a repeated unit of at least 75 kb in length. Individual T66 homology units are classified into three subfamilies through hybridization studies with a series of diagnostic subfamily-specific probes. The organization and number of elements in wild-type forms of chromosome 17 are very different from those found within t haplotype forms of this chromosome. The number of T66 elements present within individual chromosomes is highly polymorphic among both inbred strains of mice and among independently derived t haplotypes. Wild-type chromosomes have between five and nine T66 elements distributed between two loci that are separated by a genetic distance of at least three map units, whereas t haplotypes have between 9 and 11 T66 elements within a single cluster. Many of the rare recovered products of recombination between a t haplotype and a wild-type form of chromosome 17 have resulted from recombination within or near the T66 regions present on each chromosome. Molecular and genetic data lead to the speculation that portions of individual T66 homology units could be involved in t haplotype effects on sperm differentiation.     

Mapping of the Sod-2 locus into the t complex on mouse chromosome 17

A human DNA probe specific for the superoxide dismutase gene was used to identify the corresponding mouse gene. Under the chosen hybridizing conditions, the probe detected DNA fragments most likely carrying the mouse Sod-2 gene. Mapping studies revealed that the Sod-2 gene resides in the proximal inversion of the t complex on mouse chromosome 17. All complete t haplotypes tested showed restriction fragment length polymorphism which is distinct from that found in all wild-type chromosomes tested. The Sod-2 locus maps in the same region as some of the loci that influence segregation of t chromosomes in male gametes. The possibility that the Sod-2 locus is related to some of the t-complex distorter or responder loci is discussed. The data indicate that the human homolog of the mouse t complex has split into two regions, the distal region remaining on the p arm of human chromosome 6, while the proximal region has been transposed to the telomeric region of this chromosome's q arm.     

New haplotypes in the Bedlington terrier indicate complexity in copper toxicosis

Copper toxicosis (CT) is an autosomal recessive disorder common in Bedlington terriers. Previously, the CT locus was mapped to canine Chromosome (Chr) 10q26 through linkage to marker C04107. Diagnosis, traditionally based on liver biopsy, has recently shifted to interpretation of the C04107 microsatellite alleles where allele 2 segregates with the disease with 90–95% accuracy. Recently, CT has been attributed to a deletion of exon 2 in the MURR1 gene. We also identified a deletion of exon 2 of MURR1 in our collection of 2-2 homozygous affected terriers. However, our collection also included affected 1-1 homozygotes and 1-2 heterozygotes, and these dogs did not have the homozygous deletion. In addition to C04107, we analyzed an adjacent microsatellite (C04107B), and two novel SNPs, all within intron 1 of MURR1, and sequenced all exons and their intronic boundaries. Pedigree analysis indicates that there are two typical haplotypes, one normal and one affected, maintaining complete linkage disequilibrium between C04107 allele 2 and the deletion in most pedigrees. Most importantly, we identified a recombinant haplotype present in a North American pedigree, where allele 2 is not linked with the deletion, and a fourth haplotype containing a splice site variant. Although the splice site alteration appears to be a normal variant, it is present in two affected dogs, which do not carry homozygous deletions of MURR1.     

Intra-H-2 recombination in t haplotypes shows a hot spot and close linkage of l tw5 to H-2K

The embryonic lethal mutation in the t ^w5 haplotype is known to map near the H-2K region of the mouse major histocompatibility complex. Additional data obtained by classical genetic methods demonstrate that the t ^w5 lethal gene is effectively inseparable from H-2K. No recombinants were found between H-2K and t ^w5 in a sample representing over 1200 mice. On a statistical basis t ^w5 must be less than 250 kb from the H-2K gene. In the course of these mapping studies we obtained a set of 11 intra-H-2 recombinants. We have analyzed these and three others derived from another experiment to define their breakpoints as precisely as possible. Southern blot analysis with molecular probes to the D, S, I, and K regions of the H-2 complex defines seven recombinations between the D and S regions, two between S and I, none within the I region, and five events between I and K. The last category was studied in finer detail by developing unique copy probes to the I-K boundary region. Two of the five events occurred within probably less than 6 kb of each other: these two recombinants define the centromeric limit of the location of the t ^w5 gene within the H-2K region. The other three I-K recombinants occurred in at least two other nearby locations. Altogether at least three, and probably all five I-K recombinants fall within a 45 kb recombinational hot spot recently identified in Mus musculus castaneus.     

A deletion spanning the promoter and first exon of the hair cycle-specific ASIP transcript isoform in black and tan rabbits

Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the a^t mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an a^t allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit a^t allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 a^t-associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the a^t allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.     

The mouse Rsk3 gene maps to the Leh66 elements carrying the t-complex responder Tcr

A variant form of mouse Chromosome (Chr) 17, the t-haplotype, contains several loci responsible for transmission ratio distortion in males. Sperm carrying the responder locus (Tcr) have a high probability of fertilizing eggs at the expense of wild-type sperm, provided that distorter loci (Tcd-1 to Tcd-5) are expressed during spermatogenesis. Tcr has been mapped to the Leh66b region within a maximum of 155 kb. In the search for genes in the genomic region Leh66EI, we have identified the mouse homolog of human ribosome S6 kinase 3 (RSK3) on cosmid DNA. The complete mouse Rsk3 gene is encoded in the region Leh66a of t-haplotypes and Leh66EI of the wild-type chromosome. It consists of at least 13 exons spanning over more than 120 kb. Rsk3 is expressed in embryos and in several adult organs including testis. Cosmids covering 100 kb of the Leh66b region or 120 kb of the Leh66a region were isolated. Rsk3 covers about 65 kb of the Leh66b region and appears to be incomplete at its 5′-end. A correlation of the physical map provided here with the genetic mapping of Tcr reported previously suggests that Tcr is most likely encoded within a fragment of 30 kb upstream or 20 kb downstream of Rsk3. These data will facilitate the isolation of Tcr, a prerequisite for understanding transmission ratio distortion in mouse. Received: 21 January 1999 / Accepted: 16 April 1999     

Relationship of F9 antigen and genes of theT/t complex

Serological studies relating F9 antigen of embryonal carcinoma cells to at the murineT/t complex have been extended and confirm that only the lethal haplotype t¹²- and none of the other five lethal haplotypes-affects the quantitative expression of F9 antigen on sperm. Cytotoxicity tests on preimplantation embryos show that t¹² homozygotes are less susceptible to antiF9 serum than t^w5 homozygotes, and that using specific antimutant haplotype antisera prepared against sperm, t¹² antigen is detectable on morulae, whereas t^w5 antigen is not.     

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.     

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.     

Recombination within mouse <Emphasis Type="Italic">t</Emphasis> haplotypes has replaced significant segments of <Emphasis Type="Italic">t</Emphasis>-specific DNA

Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1–2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the “complete” t haplotypes t ^w2 , t ⁰ , t ^w32 , t ^w71 , and t ^w75 . We show that only t ^w32 is a true “complete” t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within t ^w71 and t ^w75 mice.