Transmission ratio distortion in mouse t-haplotypes is due to multiple distorter genes acting on a responder locus

Transmission ratios of male mice heterozygous for various combinations of partial t-haplotypes provide evidence in support of a model for the genetic basis of ratio distortion, involving two or more distorter genes acting on a responder locus. The t form of the responder locus, Tcr, in the medial part of the haplotype, must be present and heterozygous for distortion to occur. When the responder alone is present, as in t^low haplotypes, the chromosome carrying it is transmitted in a low ratio (<50%). The t forms of the distorter loci act additively, in cis or trans, to raise the transmission of whichever chromosome carries Tcr. Identified distorter loci are Tcd-1, in the proximal part of the haplotype, Tcd-2, distal to Tcr, and probably Tcd-3, lying between Tcr and Tcd-2. In the absence of Tcr the distorters are transmitted normally. The system is compared with the SD system of Drosophila.     

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.     

Limits of the distal inversion in the t complex of the house mouse: Evidence from linkage disequilibria

The suppression of crossing-over and the consequent linkage disequilibrium of genetic markers within the t complex of the house mouse is caused by two large and two short inversions. The inversions encompass a region that is some 15 centiMorgans (cM) long in the homologous wild-type chromosome. The limits of the proximal inversions are reasonably welldefined, those of the distal inversions much less so. We have recently obtained seven new DNA markers (D17Tu) which in wild-type chromosomes map into the region presumably involved in the distal inversions of the t chromosomes. To find out whether the corresponding loci do indeed reside within the inversions, we have determined their variability among 26 complete and 12 partial t haplotypes. In addition, we also tested the same collection of t haplotypes for their variability at five D17Leh, Hba-ps4, Pim-1, and Crya-1 loci. The results suggest that the distal end of the most distal inversion lies between the loci D17Leh467 and D17Tu26. The proximal end of the large distal inversion was mapped to the region between the D17Tu43 and Hba-ps4 loci, but this assignment is rather ambiguous. The loci Pim-1, Crya-1, and the H-2 complex, which have been mapped between the Hba-sp4 and Grr within the large distal inversion, behave as if they recombine from time to time with their wildtype homologs.     

Detailed physical and genetic mapping in the region of plasminogen,D17Rp17e,and quaking

We present here a detailed physical map encompassing over 600 kb of mouse Chromosome (Chr) 17 in the region of plasminogen, D17Rp17e, and quaking. This region is cloned in yeast artificial chromosomes (YACs). We have identified several CpG islands within this region from pulsed field gel mapping of mouse genomic DNA and YAC DNA. Five new DNA probes have been generated. One, D17Leh514, is a minimum of about 90 kb distal to plasminogen. Four, D17Leh513, D17Leh512, D17Leh511, and D17Leh510, are proximal to D17Rp17e, the closest previously described genetic marker to quaking^viable and quaking^lethal-1 mutations. We have genetically mapped D17Leh511 to within 0.15 cM of these mutations. The genetic distance to D17Rp17e from D17Leh511 is also 0.15 cM; the physical distance of less than 360 kb (minimum 200 kb) is consistent with an approximation of 2 Mbp per cM.     

Molecular characterization of four intra-t mouse recombinants

Recombination in the proximal region of mouse chromosome 17 is greatly reduced in heterozygotes carrying the wild-type and thet complex-type chromosomes. The reason for this is the presence of two non-overlapping inversions in thet complex. Rare crossing-over does, however, occur within thet complex of thet/+ heterozygotes. Here we characterize four such exceptional intra-t recombinants,t ^Tu1 throught ^Tu4 . To map the positions of the genetic exchange in these four recombinants, we analyzed them with DNA probes specific for 16 loci distributed over thet complex. The analysis revealed that in three of the four recombinants, an equal crossing-over occurred in the short region between the two inversions, producing chromosomes carrying either the proximal inversion only (t ^Tu1 andt ^Tu4 ) or the distal inversion only (t ^Tu2 ). In the fourth recombinant (t ^Tu3 ), unequal crossing-over occurred within the proximal inversion between lociD17Leh119 andD17Leh66, producing a chromosome in which the region containing lociTcp-1, T, andD17Tu5 has been duplicated. The duplication of theBrachyury locus leads to the suppression of the tail-shortening effect normally produced by the interaction of the dominant (T) and recessive (tct) alleles at this locus so that theT/t ^Tu3 mice have normal tails.     

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.     

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.     

Transmission Ratio Distortion,Sterility, and Control of the t-Complex Function in Sperm

Mouse t-complex located on chromosome 17 contains genes affecting only male fertility. Some genes of this complex are recessive lethals; nonetheless, the high frequency of the t-complex carriers in a population is maintained due to a mechanism referred to as transmission ratio distortion (TRD), i.e., after crosses with wild-type females, males heterozygous for the t-complex transmit the t-bearing chromosome to nearly all their offspring, which suggests that the t-complex genes control sperm function. Analysis of this phenomenon shows that the resultant TRD is determined by the ratio between the distorter genes (Tcd) and a responder gene (Tcr) located within the t-complex region. Many authors believe that two to six distorter genes currently known have an additive effect. A genetic model of the non-Mendelian inheritance in the progeny of heterozygous male mice specifically explains sterility of animals carrying the t-complex with complementary lethal genes. The model suggests that some distorter gene products interacting with the responder gene have a selective effect on motility of both mutant and wild-type sperm. Insufficient sperm motility and/or their unsuccessful capacitation result in poor if any fertilization. Information on the t-complex genes is necessary for understanding the biological mechanisms of male sterility and may be used in medical practice.     

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.     

BAC clones and STS markers near the distal breakpoint of the fourth t-inversion, In(17)4d, in the H2-M region on mouse Chromosome 17

The H2-M region is the most distal part of the mouse major histocompatibility complex (Mhc) and is likely to include the distal breakpoint of the fourth t-inversion, In(17)4d. The conserved synteny breakpoint between mouse and human is located in the H2-M region between D17Leh89, a putative olfactory receptor gene, and Pgk2 (phosphoglycerate kinase 2). To analyze the H2-M region, we screened a mouse bacterial artificial chromosome (BAC) library, using the D17Mit64, D17Tu49, D17Leh89, D17Leh467, and Pgk2 markers. Thirty-eight BAC clones were obtained and mapped in five clusters, and 25 sequence-tagged site (STS) markers were newly developed. The regions surrounding D17Tu49 and D17Leh467 are abundant in L1 repeat sequences and may, therefore, be candidates for the breakpoints of conserved synteny and t-inversion. D17Leh89 was linked to D17Mit64 by two contiguous BAC clones. The Aeg1 (acidic epididymal glycoprotein 1) and Aeg2 genes were mapped close to Pgk2, on the same BAC clones. The genetic length between D17Leh89–D17Mit64 and Pgk2–Aeg can be estimated as 0.5–0.7 centiMorgan (cM), and the most distal class I gene, H2-M2, can be placed 0.3–1.0 cM proximal to the t-inversion breakpoint. A recombinational hotspot is suggested to be located between Aeg and Tpx1 in an interspecific cross of (C57BL/6J ×Mus spretus). Received: 23 July 1997 / Accepted: 13 November 1997     

Proximity of the CTLA-1 serine esterase and Tcr α loci in mouse and man

The serine esterase CTLA-1 gene was shown by in situ hybridization to map to the D segment of mouse chromosome 14, the same localization as a member of the immunoglobulin super family, Tcr . To further demonstrate the proximity of CTLA-1 and Tcr , genetic linkage was tested in mouse using restriction fragment length polymorphisms and a backcross progeny, and no recombination was observed in the 100 backcross products studied. Recombination events between Tcr /CTLA-1 and the markers Gdh-X and NP-1 show that the most probable order of these loci in the mouse 14D region is NP-1-Tcr /Ctla-1-Gdh-X. In man, the human homologue of CTLA-1 was shown by in situ hybridization to map on chromosome 14, at 14q11-q12, where Tcr also maps. Using the human cell line SUP-Tl, bearing the inversion inv(14)(q11;q32), we further demonstrated the loci order in man to be centromere-NP-1-Tcr -CTLA-1. To complement the cytogenetic and genetic mapping data, we tried to determine the physical distance between the two genes by pulsed field gel electrophoresis (PFGE). DNA prepared from various cell types, both mouse and human, were digested with a panel of rare cutter enzymes and hybridized first with CTLA-1, then with Tcr probes. None of the bands identified hybridized with both Tcr and CTLA-1 probes for either mouse or human cells. Although the physical mapping by PFGE is inconclusive, the cytogenetic and genetic data support close linkage of the Tcr and CTLA-1 genes in both mouse and man, suggesting homology between the D region of mouse chromosome 14 and the q11–q12 region of human chromosome 14, encompassing the Tcr and CTLA-1 loci. These findings also provide another example of proximity of genes coding for a member of the Ig super-family and a serine esterase.     

New insights into the t-complex and control of sperm function

The mouse t-complex, located on chromosome 17, contains genes known to influence male, but not female, fertility. Although some t-complex genes are recessive lethals, t-chromosomes are maintained in the population by transmission ratio distortion. When male mice heterozygous for the t-chromosome mate with wild-type females, most offspring will possess the t-chromosome, indicating a link between t-complex genes and sperm function. Several proteins coded for by t-complex genes have been localised in the sperm flagellum, suggesting roles relating to motility. Another t-complex protein appears able to regulate the adenylyl cyclase/cAMP signal transduction pathway, known to play an important role in capacitation. Defective motility and/or failure to capacitate (“switch on”) would result in poorly fertile or infertile spermatozoa. Given the existence of human homologues for many genes in the t-complex and the prevalence of “male factor” infertility, information obtained about the t-complex not only will provide insight into basic biological mechanisms but may be of future clinical relevance as well. BioEssays 21:304–312, 1999. © 1999 John Wiley & Sons, Inc.     

Male sterility of the mouse t-complex is due to homozygosity of the distorter genes

Evidence is presented that the male sterility produced by the mouse t-complex is due to interaction of at least three sterility factors. These factors are carried in the same partial haplotypes as the three distorter genes, Tcd-1, Tcd-2, and Tcd-3 and are suggested to be identical with them. When heterozygous, the distorter/sterility genes act on the wild-type form of the responder gene, rendering sperm carrying it nonfunctional, thus leading to high transmission of the t form of the responder. When homozygous, the harmful effects of the distorter genes are stronger and affect both forms of the responder, leading to sterility. If homozygous sterility is an inescapable part of ratio distortion, then the t-lethals confer a selective advantage in removing sterile males from the population. Thus, the relationship between the various properties of the t-complex can now be understood.     

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.     

Structure of the murine E-selectin ligand 1 (ESL-1) gene and assignment to Chromosome 8

A 150-kDa glycoprotein designated in the mouse as E-selectin ligand-1 (ESL-1; gene symbol Selel) was first isolated based on its ability to function as a ligand for E-selectin. The gene appears equivalent to that for membrane glycoprotein MG160 encoded in the human by the locus for Golgi apparatus protein 1 (GLG1). ESL-1 is also highly homologous to the chicken cysteine-rich fibroblast growth factor receptor (CFR). We describe the genomic structure and chromosomal localization of the Selel locus. The gene is encoded by 27 exons and extends over approximately 75 kb. It maps to murine Chromosome (Chr) 8 in a region homologous to human Chr 16q where the GLG1 locus maps, further indicating that Selel and GLG1 are mouse and human equivalents of the same gene. Received: 21 April 1999 / Accepted: 12 July 1999     

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     

Gene content of the 750-kb critical region for mouse embryonic ectoderm lethal <Emphasis Type="Italic">tcl-w5</Emphasis>

Mice homozygous for the t^w5 allele arrest at gastrulation from defects associated with embryonic ectoderm development. The mutated gene has been genetically closely linked to the H-2K locus in the mouse MHC region, flanked by markers H-2Pb and D17Mit147. Aiming at the positional cloning of the mutated gene, we constructed a BAC contig spanning about 1 Mb of the genomic region. On the basis of our mapping and sequencing analysis of the BACs combined with public genome data, EST database searches, and gene prediction programs, we delimit the 1.06 cM of the t^w5 critical region to 750 kb, and infer 36 genes (1/20 kb) encoded in the interval. All of the 33 genes tested were confirmed as expressed in embryonic tissues by RT-PCR analyses, and in many cases by EST expression profiles as well. Thus, this highly gene-rich region is essentially totally transcribed during early development and provides priority candidates to be screened for the t^w5 embryonic lesion.     

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.