Variance and covariance of actual relationship between relatives at one locus.

The relationship between pairs of individuals is an important topic in many areas of population and quantitative genetics. It is usually measured as the proportion of thegenome identical by descent shared by the pair and it can be inferred from pedigree information. But there is a variance in actual relationships as a consequence of Mendelian sampling, whose general formula has not been developed. The goal of this work is to develop this general formula for the one-locus situation,. We provide simple expressions for the variances and covariances of all actual relationships in an arbitrary complex pedigree. The proposed method relies on the use of the nine identity coefficients and the generalized relationship coefficients; formulas have been checked by computer simulation. Finally two examples for a short pedigree of dogs and a long pedigree of sheep are given.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13

Inbred 129 strain mice are predisposed to developing male germ cell tumors (GCTs) of the testes. The inherent genetic defects that underlie male GCT susceptibility in the 129 mouse strain are unknown. GCT incidence is increased in 129 strain males that lack functional p53 protein, and we have used this finding to facilitate the generation of panels of GCT-bearing intercross and backcross mice for genetic mapping analysis. A 129 strain locus, designated pgct1, that segregates with the male GCT phenotype has been identified on chromosome 13 near D13Mit188. This region of murine chromosome 13 may be syntenic to a portion of human chromosome 5q that is implicated in male GCT susceptibility in humans.

Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.)

For many agronomically important plant genes, only their position on a genetic map is known. In the absence of an efficient transposon tagging system, such genes have to be isolated by map-based cloning. In bread wheat Triticum aestivum, the genome is hexaploid, has a size of 1.6 × 1010 bp, and contains more than 80% of repetitive sequences. So far, this genome complexity has not allowed chromosome walking and positional cloning. Here, we demonstrate that chromosome walking using bacterial artificial chromosome (BAC) clones is possible in the diploid wheat Triticum monococcum (Am genome). BAC end sequences were mostly repetitive and could not be used for the first walking step. New probes corresponding to rare low-copy sequences were efficiently identified by low-pass DNA sequencing of the BACs. Two walking steps resulted in a physical contig of 450 kb on chromosome 1AmS. Genetic mapping of the probes derived from the BAC contig demonstrated perfect colinearity between the physical map of T. monococcum and the genetic map of bread wheat on chromosome 1AS. The contig genetically spans the Lr10 leaf rust disease resistance locus in bread wheat, with 0.13 centimorgans corresponding to 300 kb between the closest flanking markers. Comparison of the genetic to physical distances has shown large variations within 350 kb of the contig. The physical contig can now be used for the isolation of the orthologous regions in bread wheat. Thus, subgenome chromosome walking in wheat can produce large physical contigs and saturate genomic regions to support positional cloning.

Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2′-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase–PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.

Germ line deletion of the CD1 locus exacerbates diabetes in the NOD mouse

Quantitative and qualitative defects in CD1-restricted natural killer T cells have been reported in several autoimmune-prone strains of mice, including the nonobese diabetic (NOD) mouse. These defects are believed to be associated with the emergence of spontaneous autoimmunity. Here we demonstrate that both CD1d-null NOD and CD1d-null NOD/BDC2.5 T cell receptor transgenic mice have an accelerated onset and increased incidence of diabetes when compared with CD1d+/− and CD1d+/+ littermates. The acceleration of disease did not seem to result from changes in the T helper (Th)1/Th2 balance because lymphocytes purified from lymphoid organs and pancreatic islets of wild-type and CD1d-null mice secreted equivalent amounts of IFN-γ and IL-4 after stimulation. In contrast, the pancreata of CD1d-null mice harbored significantly higher numbers of activated memory T cells expressing the chemokine receptor CCR4. Notably, the presence of these T cells was associated with immunohistochemical evidence of increased destructive insulitis. Thus, CD1d-restricted T cells are critically important for regulation of the spontaneous disease process in NOD mice.

Detection of a major gene for resistance to fusiform rust disease in loblolly pine by genomic mapping.

Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analysis over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated, organisms, where conventional genetic analysis often is limited or not feasible.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

GSP-1 genes are linked to the grain hardness locus (Ha) on wheat chromosome 5D.

An important determinant of wheat grain quality is the hardness of the grain. The trait is controlled by a major locus, Ha, on the short arm of chromosome 5D. Purified starch granules from soft-grained wheats have associated with them 15-kDa polypeptides called grain softness proteins (GSPs) or "friabilins." Genes that encode one family of closely related GSP polypeptides - GSP-1 genes - were mapped using chromosome substitution lines to the group 5 chromosomes. An F2 population segregating for hard and soft alleles at the Ha locus on a near-isogenic background was used in a single-seed study of the inheritance of grain softness and of GSP-1 alleles. Grain softness versus grain hardness was inherited in a 3:1 ratio. The presence versus absence of GSPs in single seed starch preparations was coinherited with grain softness versus hardness. This showed that grain softness is primarily determined by seed, and not by maternal, genotype. In addition, no recombination was detected in 44 F2 plants between GSP-1 restriction fragment length polymorphisms and Ha alleles. Differences between hard and soft wheat grains in membrane structure and lipid extractability have been described and, of the three characterized proteins that are part of the mixture of 15-kDa polypeptides called GSPs, at least two, and probably all three, are proteins that bind polar lipids. The data are interpreted to suggest that the Ha locus may encode one or more members of a large family of lipid-binding proteins.

Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

Fine mapping of colon tumor susceptibility (Scc) genes in the mouse, different from the genes known to be somatically mutated in colon cancer.

The predisposition to colon cancer is multigenetically controlled in animals and probably also in humans. We have analyzed the multigenic control of susceptibility to 1,2-dimethylhydrazine-induced colon tumors in mice by using a set of 20 homozygous CcS/Dem recombinant congenic strains, each of which contains a different random subset of approximately 12.5% of genes from the susceptible strain STS/A and 87.5% of genes from the relatively resistant strain BALB/cHeA. Some CcS/Dem strains received the alleles from the susceptible strain STS/A at one or more of the multiple colon tumor susceptibility loci and are susceptible, whereas others are resistant. Linkage analysis shows that these susceptibility genes are different from the mouse homologs of the genes known to be somatically mutated in human colon cancer (KRAS2, TP53, DCC, MCC, APC, MSH2, and probably also MLH1). Different subsets of genes control tumor numbers and size. Two colon cancer susceptibility genes, Scc1 and Scc2, map to mouse chromosome 2. The Scc1 locus has been mapped to a narrow region of 2.4 centimorgans (90% confidence interval).

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

Physical mapping of the minimal region of loss in 5q- chromosome.

Acquired interstitial loss of all or part of the long arm of human chromosome 5 (5q-) is an anomaly that is seen frequently in patients with preleukemic myelodysplasia and acute myelogenous leukemia. Loss of a critical region of overlap at band 5q31.1 in all of these cases, with various cytogenetic breaks, signifies the existence of a key negative regulator of leukemogenesis. Previous studies have defined the proximal and distal ends of the critical region to reside between the genes for IL9 and EGR1, respectively. In this report, we describe a yeast artificial chromosome contig spanning this myeloid tumor suppressor locus. The combined order of the polymorphic loci is centromere-IL9-(D5S525-D5S558-D5S89-D5S526 -D5S393)-D5S399-D5S396-D5S414-EGR1 and telomere. The physical distance between the IL9 and EGR1 genes is estimated to be < 2.4 Mb. Here we report the utility of these polymorphic loci by detecting a submicroscopic deletion of 5q31; an acute myelogenous leukemia patient with a three-way translocation, t(5;18;17)(q31;p11;q11), as the sole anomaly revealed allele loss of the D5S399 and D5S396 loci.

Increased expression in adipocytes of ob RNA in mice with lesions of the hypothalamus and with mutations at the db locus.

The gene product of the recently cloned mouse obese gene (ob) is important in regulating adipose tissue mass. ob RNA is expressed specifically by mouse adipocytes in vivo in each of several different fat cell depots, including brown fat. ob RNA is also expressed in cultured 3T3-442A preadipocyte cells that have been induced to differentiate. Mice with lesions of the hypothalamus, as well as mice mutant at the db locus, express a 20-fold higher level of ob RNA in adipose tissue. These data suggest that both the db gene and the hypothalamus are downstream of the ob gene in the pathway that regulates adipose tissue mass and are consistent with previous experiments suggesting that the db locus encodes the ob receptor. In db/db and lesioned mice, quantitative differences in expression level of ob RNA correlated with adipocyte lipid content. The molecules that regulate expression level of the ob gene in adipocytes probably are important in determining body weight, as are the molecules that mediate the effects of ob at its site of action.