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.     

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.     

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.     

Structures of two genes coding for melanin-concentrating hormone of chum salmon

Two MCH genes coding for melanin-concentrating hormone (MCH) were isolated from a chum salmon liver DNA library and characterized. They were shown to be intronless genes with 0.63-kb exons, each of which commonly consisted of an about 80-bp 5'-untranslated region, a region coding for 132 amino acids (aa) MCH precursor protein and an approx. 160-bp 3'-untranslated region. About 20 bp upstream from the putative cap site, sequences were found corresponding to the TATA box. The two genes were 86% identical at the nucleotide sequence level. Sequences homologous to the chum salmon MCH genes were present in the genomes of other fish such as catfish, carp and Chinese grass carp, whereas no highly homologous sequence could be detected in other vertebrate genomes.     

Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

We have confirmed the result that chicken beta-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on beta-globin DNA, we have studied chromatin assembly of cloned chicken beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into chromatin possessing a regular 180 +/- 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the beta A gene and promoter region, but lacking the downstream intergenic region between the beta A and epsilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta A gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta A-epsilon intergenic region.     

Sequence and haplotype analysis supports HLA-C as the psoriasis susceptibility 1 gene

Previous studies have narrowed the interval containing PSORS1, the psoriasis-susceptibility locus in the major histocompatibility complex (MHC), to an approximately 300-kb region containing HLA-C and at least 10 other genes. In an effort to identify the PSORS1 gene, we cloned and completely sequenced this region from both chromosomes of five individuals. Two of the sequenced haplotypes were associated with psoriasis (risk), and the other eight were clearly unassociated (nonrisk). Comparison of sequence of the two risk haplotypes identified a 298-kb region of homology, extending from just telomeric of HLA-B to the HCG22 gene, which was flanked by clearly nonhomologous regions. Similar haplotypes cloned from unrelated individuals had nearly identical sequence. Combinatorial analysis of exonic variations in the known genes of the candidate interval revealed that HCG27, PSORS1C3, OTF3, TCF19, HCR, STG, and HCG22 bore no alleles unique to risk haplotypes among the 10 sequenced haplotypes. SPR1 and SEEK1 both had messenger RNA alleles specific to risk haplotypes, but only HLA-C and CDSN yielded protein alleles unique to risk. The risk alleles of HLA-C and CDSN (HLA-Cw6 and CDSN*TTC) were genotyped in 678 families with early-onset psoriasis; 620 of these families were also typed for 34 microsatellite markers spanning the PSORS1 interval. Recombinant haplotypes retaining HLA-Cw6 but lacking CDSN*TTC were significantly associated with psoriasis, whereas recombinants retaining CDSN*TTC but lacking HLA-Cw6 were not associated, despite good statistical power. By grouping recombinants with similar breakpoints, the most telomeric quarter of the 298-kb candidate interval could be excluded with high confidence. These results strongly suggest that HLA-Cw6 is the PSORS1 risk allele that confers susceptibility to early-onset psoriasis.     

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.     

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.     

Characterization of a new type of Bacteroides conjugative transposon, Tcr Emr 7853.

Results of previous investigations suggested that the conjugative transposons found in human colonic Bacteroides species were all members of a closely related family of elements, exemplified by Tcr Emr DOT. We have now found a new type of conjugative transposon, Tcr Emr 7853, that does not belong to this family. Tcr Emr 7853 has approximately the same size as the Tcr Emr DOT-type elements (70 to 80 kbp) and also carries genes encoding resistance to tetracycline (Tcr) and erythromycin (Emr); however, it differs from previously described conjugative transposons in a number of ways. Its transfer is not regulated by tetracycline and its transfer genes are not controlled by the regulatory genes rteA and rteB, which are found on Tcr Emr DOT and related conjugative transposons. Its ends do not cross-hybridize with the ends of Tcr Emr DOT-type conjugative transposons, and the Emr gene it carries does not cross-hybridize with ermF, the Emr gene found on all previously studied Bacteroides conjugative transposons. There is only one region with high sequence similarity between Tcr Emr 7853 and previously characterized elements, the region that contains the Tcr gene, tetQ. This sequence similarity ends 145 bp upstream of the start codon and 288 bp downstream from the stop codon. A 2-kbp region upstream of tetQ on Tcr Emr 7853 cross-hybridized with four additional EcoRV fragments of Bacteroides thetaiotaomicron 7853 DNA other than the one that contained tetQ. These additional cross-hybridizing bands were not part of Tcr Emr 7853, but one of them cotransferred with Tcr Emr 7853 in some matings. Thus, at least one of the additional cross-hybridizing bands may be associated with another conjugative element or with an element that is mobilized by Tcr Emr 7853. DNA that cross-hybridized with the upstream region was found in one clinical isolate of Bacteroides ovatus and four Tcr isolates of Prevotella ruminicola.     

Concerted evolution of the mouse Tcp-10 gene family: implications for the functional basis of t haplotype transmission ratio distortion.

The mouse Tcr locus is defined by its central role in the transmission ratio distortion phenotype characteristic of t haplotypes. A molecular candidate for Tcr has been identified in the form of a gene--Tcp-10b--expressed during spermatogenesis. Tcp-10b is one member of a multigene family present in two to four copies on different homologs of chromosome 17. The coding regions of the Tcp-10 genes present within two inbred strains were compared with those of the tw5 haplotype. The various gene family members are highly conserved relative to each other with a minimum nucleotide identity of 98.6% in all pairwise comparisons. Maximal parsimony analysis indicates that the Tcp-10 gene family has evolved in a concerted manner with the obliteration of nearly all individual gene-specific characteristics. As a consequence, the candidate for the full-length mutant Tcr gene product is distinguished by only a single, highly conservative, amino acid change. The data are consistent with the hypothesis that the effector of mutant Tcr activity is a second, alternatively spliced product that is expressed in a haploid- and allele-specific manner.     

Cloning, mapping, and expression of two novel actin genes, actin-like-7A (ACTL7A) and actin-like-7B (ACTL7B), from the familial dysautonomia candidate region on 9q31.

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.     

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.     

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.