A haplolethal locus uncovered by deletions in the mouse T complex

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.     

Molecular cloning of the t complex responder genetic locus

Although mouse t haplotypes carry recessive mutations causing male sterility and embryonic lethality, they persist in wild mouse populations via male transmission ratio distortion (TRD). Genetic evidence suggests that at least five t-haplotype-encoded loci combine to cause TRD. One of these loci, called the t complex responder (Tcr), is absolutely required for any deviation from Mendelian segregation to occur. A candidate for the Tcr gene has previously been identified. Evidence that this gene represents Tcr is its localization to the appropriate genomic subregion and testis-specific expression pattern. Here, we report the molecular cloning of the region between recombinant chromosome breakpoints defining the Tcr locus. These results circumscribe Tcr to a 150- to 220-kb region of DNA, including the 22-kb candidate responder gene. This gene and two other homologs were created by large genomic duplications, each involving segments of DNA 10-fold larger than the individual genes.     

Recombination between two mouse t haplotypes (t w12 tf) and t Lub-1) : Mapping. of the H-2 complex relative to centromere and tufted (tf) locus

A monoclonal antibody known to recognize the H-2.m3 specificity is shown to react with the class I H-2 product of t ^Lub-1 but not t ^w12 tf mice. This reagent was used to study the segregation of the H-2 complex in the progeny of t ^Lub-1 +/t ^ww12 tf females. The most straightforward interpretation of the results presented here is that these t haplotypes carry an H-2 complex located between the centromere and tufted locus. Possible consequences of such a location with regard to the recombination between t haplotypes and chromosome 17 from laboratory mice are discussed.     

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.     

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.     

A Drosophila melanogaster gene encodes a protein homologous to the mouse t complex polypeptide 1

We have isolated and sequenced a cDNA from Drosophila melanogaster that is homologous to the mouse Tcp-1 gene encoding the t complex polypeptide 1, TCP-1. The Drosophila gene maps by in situ hybridization to bands 94B1-2 of the polytene chromosomes. It shares 66% nucleotide sequence identity with the mouse gene. The predicted Drosophila protein consists of 557 amino acids and shares 72% identity with the mouse polypeptide. The TCP-1 polypeptide appears to be highly conserved in evolution from mammals to simple eukaryotes because the Drosophila gene probe also detects related sequences in DNA from the yeast, Saccharomyces cerevisiae. The presence of TCP-1-related polypeptides in organisms such as Drosophila and yeast should facilitate biochemical and genetic analysis of its function.     

An alpha globin pseudogene is located within the mouse t complex

Two-dimensional chymotryptic peptide maps of Lyt-1.1 and Lyt-1.2 from Lyt-1 congenic thymocytes labeled with ¹²⁵I in the usual way before lysis did not significantly differ, but there was a characteristic difference between maps of Lyt-1.1 and Lyt-1.2 obtained from ¹²⁵I-labeled solubilized membrane fragments. We conclude that the Lyt-1 locus of chromosome 19 includes the protein-structural gene for Lyt-1. This conclusion is further supported by evidence with ³⁵S-cysteine-labeled thymocytes: Thus an early 62K intermediate form, and a 60K form from tunicamycin-treated cells devoid of N-linked and O-linked carbohydrates, were precipitated by Lyt-1 alloantibody, which implies that the allo-specificity of Lyt-1 glycoprotein (67K) resides partly or wholly in protein.     

Location of theSas-1 locus on mouse chromosome 1

Using three sets of recombinant inbred strains (BXD, BXH, and BXJ), we found the locus controlling an antigenic substance (Sas}-1) in murine serum to be closely linked to the Chromosome-1 marker,Dip-1. This linkage was confirmed by an analysis of backcross linkage. The BXD and backcross data suggest that the gene order isId-1-Dip-1-Sas-1-Mls. Data from the three sets of RI strains and the 32 backcross mice lead to the estimate that the recombination frequency betweenDip-1 andSas-1 is 0.030 ±0.015.     

Location ofMls locus on mouse chromosome 1

Linkage between theMls locus and the chromosome 1 markersDip-1 andald was detected using two sets of recombinant inbred strains. Linkage betweenMls andDip-1 was confirmed in the fifth and sixth backcross generations of an incipient congenic strain. The AKXL data indicate that the gene order isDip-1-ald-Mls. The recombination frequency betweenald andMls is estimated to be 0.07 ±0.05, based on the AKXL data. The recombination frequency betweenDip-1 andMls is estimated to be 0.18 ±0.04, based on all the available data.