Targeted Mutagenesis of a Candidate T Complex Responder Gene in Mouse T Haplotypes Does Not Eliminate Transmission Ratio Distortion

Transmission ratio distortion (TRD) associated with mouse t haplotypes causes +/t males to transmit the t-bearing chromosome to nearly all their offspring. Of the several genes involved in this phenomenon, the t complex responder (Tcr(t)) locus is absolutely essential for TRD to occur. A candidate Tcr(t) gene called Tcp10b(t) was previously cloned from the genetically defined Tcr(t) region. Its location, restricted expression in testis, and a unique postmeiotic alternative splicing pattern supported the idea that Tcp10b(t) was Tcr(t). To test this hypothesis in a functional assay, ES cells were derived from a viable partial t haplotype, and the Tcp10b(t) gene was mutated by homologous recombination. Mutant mice were mated to appropriate partial t haplotypes to determine whether the targeted chromosome exhibited transmission ratios characteristic of the responder. The results demonstrated that the targeted chromosome retained full responder activity. Hence, Tcp10b(t) does not appear to be Tcr(t). These and other observations necessitate a reevaluation of genetic mapping data and the actual nature of the responder.     

Segregation distortion of mouse t haplotypes the molecular basis emerges

The t haplotype is an ancestral version of proximal mouse chromosome 17 that has evolved mechanisms to persist as an intact genomic variant in mouse populations. t haplotypes contain mutations that affect embryonic development, male fertility and male transmission ratio distortion (TRD). Collectively, these mutations drive the evolutionary success of t haplotypes, a phenomenon that remains one of the longstanding mysteries of mouse genetics. Molecular genetic analysis of TRD has been confounded by inversions that arose to lock together the various elements of this complex trait. Our first molecular glimpse of the TRD mechanism has finally been revealed with the cloning of the t complex responder (Tcr) locus, a chimeric kinase with a genetically cis active effect. Whereas + sperm in a +/t male have impaired flagellar function caused by the deleterious action of trans-active, t-haplotype-encoded 'distorters,' the mutant activity of Tcr counterbalances the distorter effects, maintaining the motility and fertilizing ability of t sperm.     

The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance

The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+)-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr), a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa.     

A candidate gene family for the mouse t complex responder (Tcr) locus responsible for haploid effects on sperm function

The mouse t complex responder (Tcr) locus plays a central haploid-specific role in the transmission ratio distortion phenotype expressed during germ cell differentiation in t-carrying males. The accumulated data map Tcr to a region of less than 500 kb. Over 400 kb of this region has been cloned and consists entirely of sequences associated with a clustered family of large cross-hybridizing elements of 30 kb to 70 kb in size. We have characterized a gene family within this region that is expressed uniquely in male germ cells with a complex pattern of RNA processing. Antibodies produced against a product of the putative open reading frame recognize a testes-specific polypeptide. Genetic data support the hypothesis that this polypeptide(s) functions to effect the Tcr phenotype.     

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.     

Mouse Hus1, a homolog of the Schizosaccharomyces pombe hus1+ cell cycle checkpoint gene.

Cell cycle checkpoints are regulatory mechanisms that arrest the cell cycle or initiate programmed cell death when critical events such as DNA replication fail to be completed or when DNA or spindle damage occurs. In fission yeast, cell cycle checkpoint responses to DNA replication blocks and DNA damage require the hus1+ gene. Mammalian homologs of hus1+ were recently identified, and here we report a detailed analysis of mouse Hus1. An approximately 4.2-kb full-length cDNA encoding the 32-kDa mouse Hus1 protein was isolated. The genomic structure and exon-intron boundary sequences of the gene were determined, and mouse Hus1 was found to consist of nine exons. Mouse Hus1 was mapped to the proximal end of chromosome 11 and is therefore a candidate gene for the mouse mutation germ cell deficient, which maps to the same genomic region. Finally, mouse Hus1 was found to be expressed in a variety of adult tissues and at several stages of embryonic development.     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

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     

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.     

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).     

A Multi-Megabase Copy Number Gain Causes Maternal Transmission Ratio Distortion on Mouse Chromosome 2

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2^WSBdel1) that eliminates the majority of the R2d2^WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2^WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2^WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2^WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2^WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.