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.     

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.     

Assignment of the TCP1 locus to the long arm of human chromosome 6 by in situ hybridization

TCP1, the human homolog of the Tcp-1 locus in the mouse, which is part of the murine t complex and codes for an abundant testicular germ-cell protein, has been mapped within the human genome by in situ hybridization. Using a cDNA probe for TCP1, pB1.4 hum, we assigned TCP1 to human chromosome region 6q23----qter, with the most likely localization being 6q25----q27.     

Sterility of Males Determined by Functional Features of the Mouse Spermatozoa Bearing t-Complex

The mechanisms underlying normal spermatogenesis and its pathology expressed as male sterility determined by t-complex located on chromosome 17 in mice are considered in this review. t-Complex is a very convenient model with diverse markers of expression of the genes involved in development of the functional features of the spermatozoa bearing t-complex. These features include defects of mobility, capacitation, and acrosome reactions, which determine full or partial male sterility. It has been proposed that the defects of capacitation are also inherent in humans and affect male fertility. This homology is confirmed by the presence of the male gene Tcp11 in humans and demonstration of the fact that the protein TCP11 plays a leading role in modulation of the capacitation of murine spermatozoa. Hence it follows that the defects of human genes leading to incomplete binding of the fertilization promoting peptide could play a certain role in a decreased male fertility. All this is essential not only for deeper understanding of the biology of spermatozoa, but also for development of new therapeutic methods of finding and treating the pathology of spermatozoa.     

Tctex3, related to Drosophila Polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6. Received: 10 April 1998 / Accepted: 22 June 1998     

Genetic polymorphism of a bovine t-complex gene (TCP1) linkage to major histocompatibility genes

Southern blot analysis of genomic cattle DNA was carried out using murine cDNA probes representing the Tcp-1 gene of the t complex. Excellent cross-hybridization was obtained, and the probes apparently hybridized to at least two bovine TCP1 genes. Two independent restriction fragment length polymorphisms, each composed of two allelic variants, were detected; the inheritance of the restriction fragment length polymorphisms was confirmed by family data. One of the restriction fragment length polymorphisms, designated TCP1B, was evidently due to a gene duplication and was revealed with any restriction enzyme used. The duplication was found in three different cattle breeds investigated. Family segregation data indicated that TCP1B is linked to major histocompatibility complex genes. The result was consistent with close linkage to the major histocompatibility complex class II DO beta gene, whereas a fairly high recombination frequency was indicated between TCP1B/DO beta and other major histocompatibility complex genes. The result assigns TCP1B to a bovine linkage group previously comprising major histocompatibility complex class I and class II genes and blood group locus M. The similarity between this linkage group and parts of mouse chromosome 17 (t-H-2) and human chromosome 6 (TCP1-HLA) is discussed.     

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.     

Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues

 LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western blot analysis with mouse LOK-specific antibody detected 130 000 M _r LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome 10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11. Received: 28 September 1998 / Revised: 2 November 1998     

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.     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.     

MappingTNNC1, the Gene That Encodes Cardiac Troponin I in the Human and the Mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding,Protein Kinase DYRK1A Binding,and Nuclear Accumulation

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.     

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase β-subunit gene in mouse and human: Tight linkage to the Huntington disease region (4p16.3)

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE β-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 ± 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.     

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.     

Fine mapping of Hyplip1 and the human homolog, a potential locus for FCHL

Familial combined hyperlipidemia (FCHL) is a common genetic dyslipidemia predisposing to premature coronary heart disease (CHD). We previously identified a locus for FCHL on human Chromosome (Chr) 1q21-q23 in 31 Finnish FCHL families. We also mapped a gene for combined hyperlipidemia (Hyplip1) to a potentially orthologous region of mouse Chr 3 in the HcB-19/Dem mouse model of FCHL. The human FCHL locus was, however, originally mapped about 5 Mb telomeric to the synteny border, the centromeric part of which is homologous to mouse Chr 3 and the telomeric part to mouse Chr 1. To further localize the human Hyplip1 homolog and estimate its distance from the peak linkage markers, we fine-mapped the Hyplip1 locus and defined the borders of the region of conserved synteny between human and mouse. This involved establishing a physical map of a bacterial artificial chromosome (BAC) contig across the Hyplip1 locus and hybridizing a set of BACs to both human and mouse chromosomes by fluorescence in situ hybridization (FISH). We narrowed the location of the mouse Hyplip1 gene to a 1.5-cM region that is homologous only with human 1q21 and within approximately 5–10 Mb of the peak marker for linkage to FCHL. FCHL is a complex disorder and this distance may, thus, reflect the well-known problems hampering the mapping of complex disorders. Further studies identifying and sequencing the Hyplip1 gene will show whether the same gene predisposes to hyperlipidemia in human and mouse. Received: 9 September 2000 / Accepted: 30 October 2000     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.