A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing.

Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.

A metric map of humans: 23,500 loci in 850 bands

High-resolution maps integrated with the enhanced location data base software (ldb+) give improved estimates of genetic parameters and reveal characteristics of cytogenetic bands. Chiasma interference is intermediate between Kosambi and Carter–Falconer levels, as in Drosophila and the mouse. The autosomal genetic map is 2832 and 4348 centimorgans in males and females, respectively. Telomeric T-bands are strikingly associated with male recombination and gene density. Position and centromeric heterochromatin have large effects, but nontelomeric R-bands are not significantly different from G-bands. Several possible reasons are discussed. These regularities validate the maps, despite their high resolution and inevitable local errors. No other approach has been demonstrated to integrate such a large number of loci, which are increasing at about 45% per year. The maps and the data and software from which they are constructed are available through the Internet (http://cedar.genetics.soton.ac.uk/public_html). Successive versions of this location data base may also be accessed on CD-ROM.

Double strand breaks at the HIS2 recombination hot spot in Saccharomyces cerevisiae

Double strand breaks (DSBs) have been found at several meiotic recombination hot spots in Saccharomyces cerevisiae; more global studies have found that they occur at many places along several yeast chromosomes during meiosis. Indeed, the number of breaks found is consistent with the number of recombination events predicted from the genetic map. We have previously demonstrated that the HIS2 gene is a recombination hot spot, exhibiting a high frequency of gene conversion and associated crossing over. This paper shows that DSBs occur in meiosis at a site in the coding region and at a site downstream of the HIS2 gene and that the DSBs are dependent upon genes required for recombination. The frequency of DSBs at HIS2 increases when the gene conversion frequency is increased by alterations in the DNA around HIS2, and vice versa. A deletion that increases both DSBs and conversion can stimulate both when heterozygous; that is, it is semidominant and acts to stimulate DSBs in trans. These data are consistent with the view that homologous chromosomes associate with each other before the formation of the DSBs.

A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11

Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for “modifier of VWF.” Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1-Cbx·rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is ≈0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.

High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2 nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) × Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

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.

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.

The optimal measure of allelic association

Allelic association between pairs of loci is derived in terms of the association probability ρ as a function of recombination θ, effective population size N, linear systematic pressure v, and time t, predicting both ρrt, the decrease of association from founders and ρct, the increase by genetic drift, with ρt = ρrt + ρct. These results conform to the Malecot equation, with time replaced by distance on the genetic map, or on the physical map if recombination in the region is uniform. Earlier evidence suggested that ρ is less sensitive to variations in marker allele frequencies than alternative metrics for which there is no probability theory. This robustness is confirmed for six alternatives in eight samples. In none of these 48 tests was the residual variance as small as for ρ. Overall, efficiency was less than 80% for all alternatives, and less than 30% for two of them. Efficiency of alternatives did not increase when information was estimated simultaneously. The swept radius within which substantial values of ρ are conserved lies between 385 and 893 kb, but deviation of parameters between measures is enormously significant. The large effort now being devoted to allelic association has little value unless the ρ metric with the strongest theoretical basis and least sensitivity to marker allele frequencies is used for mapping of marker association and localization of disease loci.

Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain.

The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens. To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E. coli K-12 strain (chi)289. The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E. coli chromosome. A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E. coli strains. To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E. coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence. The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens. Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens. One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype. The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map. Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis.

We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed.

Mapping the mouse genome: current status and future prospects.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

Importance of epistasis as the genetic basis of heterosis in an elite rice hybrid

The genetic basis of heterosis was investigated in an elite rice hybrid by using a molecular linkage map with 150 segregating loci covering the entire rice genome. Data for yield and three traits that were components of yield were collected over 2 years from replicated field trials of 250 F2:3 families. Genotypic variations explained from about 50% to more than 80% of the total variation. Interactions between genotypes and years were small compared with the main effects. A total of 32 quantitative trait loci (QTLs) were detected for the four traits; 12 were observed in both years and the remaining 20 were detected in only one year. Overdominance was observed for most of the QTLs for yield and also for a few QTLs for the component traits. Correlations between marker heterozygosity and trait expression were low, indicating that the overall heterozygosity made little contribution to heterosis. Digenic interactions, including additive by additive, additive by dominance, and dominance by dominance, were frequent and widespread in this population. The interactions involved large numbers of marker loci, most of which individually were not detectable on single-locus basis; many interactions among loci were detected in both years. The results provide strong evidence that epistasis plays a major role as the genetic basis of heterosis.

Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis

Autoimmune diseases such as systemic lupus erythematosus are complex genetic traits with contributions from major histocompatibility complex (MHC) genes and multiple unknown non-MHC genes. Studies of animal models of lupus have provided important insight into the immunopathogenesis of disease, and genetic analyses of these models overcome certain obstacles encountered when studying human patients. Genome-wide scans of different genetic crosses have been used to map several disease-linked loci in New Zealand hybrid mice. Although some consensus exists among studies mapping the New Zealand Black (NZB) and New Zealand White (NZW) loci that contribute to lupus-like disease, considerable variability is also apparent. A variable in these studies is the genetic background of the non-autoimmune strain, which could influence genetic contributions from the affected strain. A direct examination of this question was undertaken in the present study by mapping NZB nephritis-linked loci in backcrosses involving different non-autoimmune backgrounds. In a backcross with MHC-congenic C57BL/6J mice, H2z appeared to be the strongest genetic determinant of severe lupus nephritis, whereas in a backcross with congenic BALB/cJ mice, H2z showed no influence on disease expression. NZB loci on chromosomes 1, 4, 11, and 14 appeared to segregate with disease in the BALB/cJ cross, but only the influence of the chromosome 1 locus spanned both crosses and showed linkage with disease when all mice were considered. Thus, the results indicate that contributions from disease-susceptibility loci, including MHC, may vary markedly depending on the non-autoimmune strain used in a backcross analysis. These studies provide insight into variables that affect genetic heterogeneity and add an important dimension of complexity for linkage analyses of human autoimmune disease.

An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases β-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes β-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene β-cyclase, an enzyme that converts lycopene to β-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that β-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.