The Rp3 disease resistance gene of maize: mapping and characterization of introgressed alleles

The Rp3 locus of maize conditions race-specific resistance to a fungal rust pathogen, Puccinia sorghi. Both morphological and DNA markers were employed to characterize alleles of Rp3 and to accurately position Rp3 on the maize genetic map. DNA marker polymorphisms distinctive to each Rp3 allele were identified, allowing the identification of specific Rp3 alleles in cases where rust races that differentiate particular alleles are not available. In a population of 427 progeny, Rp3 and Rg1 were found to be completely linked, while Lg3 was approximately 3 cM proximal on the long arm of chromosome 3. In this same population, 12 RFLP markers were mapped relative to Rp3; the closest markers were UMC102 (about 1cM distal to Rp1) and NPI114 (1–2 cM proximal). These and additional DNA probes were used to characterize the nature and extent of flanking DNA that was carried along when six different Rp3 alleles were backcrossed into a single background. Depending upon the allele investigated, a minimum of 2–10cM of polymorphic DNA flanking the Rp3 locus was retained through the introgression process. In addition, many of the probes that map near Rp3 were found to detect an additional fragment in the Rp3 region, indicating that portions of this chromosomal segment have been tendemly duplicated. The materials and results generated will permit marker-assisted entry of Rp3 into different maize backgrounds and lay the foundation for the eventual map-based cloning of Rp3.     

Human homologs of two testes-expressed loci on mouse chromosome 17 map to opposite arms of chromosome 6

Our laboratory has recently cloned and characterized two testes-expressed loci--the Tcp-10 gene family cluster and the D17Si11 gene--that map to the proximal portion of mouse chromosome 17. Human homologs of both loci have been identified and cloned. Somatic cell hybrid lines have been used to map the human homolog of D17Si11 to the short arm of chromosome 6 (p11-p21.1) along with homologs of other genes from the (Pim-1)-(Pgk-2) region of the mouse chromosome. The human TCP 10 locus maps to the long arm of chromosome 6 (q21-qter) along with homologs of other genes from the mouse chromosome 17 region between the centromere and Pim-1. The mapping of large portions of the mouse t haplotype to unlinked regions on human chromosome 6 rules out the possibility that a t-haplotype-like chromosome could exist in humans.     

Genetic Exchange across a Paracentric Inversion of the Mouse T Complex

Mouse t haplotypes are distinguished from wild-type forms of chromosome 17 by four nonoverlapping paracentric inversions which span a genetic distance of 20 cM. These inversion polymorphisms are responsible for a 100-200-fold suppression of recombination which maintains the integrity of complete t haplotypes and has led to their divergence from the wild-type chromosomes of four species of house mice within which t haplotypes reside. As evidence for the long period of recombinational isolation, alleles that distinguish all t haplotypes from all wild-type chromosomes have been established at a number of loci spread across the 20-cM variant region. However, a more complex picture emerges upon analysis of other t-associated loci. In particular, "mosaic haplotypes" have been identified that carry a mixture of wild-type and t-specific alleles. To investigate the genetic basis for mosaic chromosomes, we conducted a comprehensive analysis of eight t complex loci within 76 animals representing 10 taxa in the genus Mus, and including 23 previously characterized t haplotypes. Higher resolution restriction mapping and sequence analysis was also performed for alleles at the Hba-ps4 locus. The results indicate that a short tract of DNA was transferred relatively recently across an inversion from a t haplotype allele of Hba-ps4 to the corresponding locus on a wild-type homolog leading to the creation of a new hybrid allele. Several classes of wild-type Hba-ps4 alleles, including the most common form in inbred strains, appear to be derived from this hybrid allele. The accumulated data suggest that a common form of genetic exchange across one of the four t-associated inversions is gene conversion at isolated loci that do not play a role in the transmission ratio distortion phenotype required for t haplotype propagation. The implications of the results pose questions concerning the evolutionary stability of gene complexes within large paracentric inversions and suggest that recombinational isolation may be best established for loci residing within a short distance from inversion breakpoints.     

An unusually proximal deletion on the short arm of chromosome 3 in a patient with small cell lung cancer.

The tumors of patients with small cell lung carcinoma (SCLC) frequently exhibit the loss of alleles at polymorphic loci on the short arm of chromosome 3. We report the genotype analysis of six SCLC patients obtained using 15 chromosome 3 probes that identified 19 restriction fragment length polymorphisms (RFLPs). Five of the six patients were reduced to homozygosity in the tumor DNA at every informative 3p locus, and thus did not serve to delineate the deletion. However, the RFLP analysis of the tumor DNA of the sixth patient demonstrated both heterozygous and hemizygous loci on 3p and allowed the definition of an interstitial deletion that extends proximal to the D3S2 locus at 3p14.2-p21 to include at least 3p13-p14. The exclusion of the D3F15S2 locus from the deleted region, observed in this patient, is an uncharacteristic feature of SCLC deletions. This deletion includes the location of D3S30 and D3S4, and thus serves to map these loci within the proximal half of chromosome 3.     

Detection of a polymorphism within the pepsinogen C gene with PCR: construction of a linkage map around PGC from 6p11-6p21.3.

An insertion/deletion polymorphism between exons 7 and 8 of the pepsinogen C gene (PGC), previously detectable with Southern analysis, was formatted for detection with PCR. Alleles were rapidly typed by UV irradiation of ethidium bromide-stained agarose gels. Whereas Southern analysis revealed two alleles, the smaller fragments generated with PCR allowed the resolution of three alleles that were previously scored as a single allele and increased the heterozygosity of the system from 0.20 to 0.53. After a set of reference families was genotyped with the PCR-based polymorphism, a linkage map around the PGC gene on chromosome 6 was constructed. This included the HLA cluster and the highly informative D6S223 locus. PGC lies 22 cM proximal to HLA-DPB and between D6S5 and D6S4 at distances of 4.5 and 13.1 cM, respectively.     

Functional Analysis of Mutations of Murine Chromosome 17 with the Use of Tertiary Trisomy

Analysis of the functional nature of mutations can be based on comparisons of their manifestation in organisms with a deletion or duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H, it is feasible to produce mice with tertiary trisomy of the proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t complex tail interaction), and tcl (t complex lethal) that are specific to t haplotypes. The set of dominant and recessive mutations was assigned to two groups: one obligatory, manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes. It seeks to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.     

Gene dosage effects on transmission ratio distortion and fertility in mice that carry t haplotypes

Complete t haplotypes can be transmitted at distorted ratios from heterozygous +/t male mice as a consequence of t-specific alleles at a series of t complex distorter loci (Tcd-1t through Tcd-4t) and a t complex responder locus. Partial t haplotypes that lack the Tcd-2t allele cannot be transmitted at the very high ratios characteristic of complete t haplotypes. The breeding studies reported here tested the possibility that the absence of Tcd-2t could be compensated for by the presence of double doses of other Tcdt alleles. The results indicate that a double dose of Tcd-4t alone will not work, but that a double dose of both Tcd-1t and Tcd-4t can promote a very high transmission ratio in the absence of Tcd-2t. These results suggest that the extent to which transmission ratios are distorted is dependent upon the absolute level of expression of the individual Tcd genes. Further studies of genotypic effects on transmission ratio distortion, as well as fertility, lead to the suggestion of a fifth t complex distorter (Tcd-5) locus within t haplotypes.     

Differentiation between wheat chromosomes 4B and 4D.

A linkage map based on homoeologous recombination, induced by the absence of the Ph1 locus, between chromosome 4D of Triticum aestivum L. (genomes AABBDD) and chromosome 4B of T. turgidum L. (genomes AABB) was compared with a linkage map of chromosome 4Am of T. monococcum L. and a consensus map of chromosomes 4B and 4D of T. aestivum based on homologous recombination. The 4D/4B homoeologous map was only one-third the length of the homologous maps and all intervals were reduced relative to the 4B-4D consensus map. After the homoeologous map was corrected for this overall reduction in recombination, the distribution of recombination in the short arm was similar in both types of maps. In the long arm, homoeologous recombination declined disproportionally in the distal to proximal direction. This gradient was shown to be largely caused by severe segregation distortion reflecting selection against 4D genetic material. The segregation distortion had a maximum that coincided with the centromere and likely had a polygenic cause. Chromosomes 4D and 4B were colinear and recombination between them occurred in almost all intervals where homologous recombination occurred. These findings suggest that these chromosomes are not differentiated structurally and that the differentiation is not segmental. In the presence of Ph1, metaphase I chromosome pairing between chromosomes composed of homologous and differentiated regions correlated with the lengths of the homologous regions. No compensatory allocation of crossovers into the homologous regions was detected. In this respect, the present results are in dramatic contrast with the crossover allocation into the pseudoautosomal region in the mammalian male meiosis.     

Functional analysis of mutations on murine chromosome 17 using tertiary trisomy

Analysis of the functional nature of mutations can be based on their manifestation in organisms with a deletion or a duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H it is feasible to produce mice with tertiary trisomy for proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t-complex tail interaction) and tcl (t complex lethal), that are specific for t haplotypes. The set of dominant and recessive mutations was assigned to two groups, one obligatory manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself, depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes which is to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.     

Molecular analysis of a t(7;14)(q35;q32) chromosome translocation in a T cell leukemia of a patient with ataxia telangiectasia

Molecular analysis of somatic cell hybrids derived from T cells carrying a t(7;14)(q35;q32) chromosomal translocation from a patient with ataxia telangiectasia and T cell leukemia indicates that the breakpoint on chromosome 14 is proximal to the IgH locus and to the D14S1 locus, while the breakpoint on chromosome 7 involves the T cell receptor beta chain locus immediately 5' to J beta 1.5 on chromosome 7. The separation of V beta and C beta observed in somatic cell hybrids defined the orientation of the T cell receptor beta chain locus on chromosome 7 where the V beta genes are centromeric and the C beta genes are telomeric. A novel chromosomal alteration, undetected cytogenetically, was revealed as being an inversion with duplication of the distal band of chromosome 14q32. The importance of the 14q32 region in the leukemogenic process is discussed.     

Complex duplications in maize lines

Rp1 is a disease resistance complex and is the terminal morphological marker on the short arm of maize chromosome 10. Several restriction fragment length polymorphisms (RFLPs), which map within 5 map units of Rp1, were examined to determine if they are also complex in structure. Two RFLP loci, which mapped distally to Rp1, BNL3.04 and PIO200075, existed in a single copy in all maize lines examined. These two loci cosegregated perfectly in 130 test cross progeny. Two RFLP loci that map proximally to Rp1 had unusual structures, which have not yet been reported for maize RFLPs; the loci were complex, with variable numbers of copies in different maize lines. One of the loci, NPI285, occasionally recombined in meiosis to yield changes in the number of copies of sequences homologous to the probe. The other proximal locus, detected by the probes NPI422, KSU3, and KSU4, was relatively stable in meiosis and no changes in the number of restriction fragments were observed. The similarity in map position between Rp1 and the complex RFLP loci indicate there may be genomic areas where variable numbers of repeated sequences are common. The structure of these complex loci may provide insight into the structure and evolution of Rp1.     

Overlapping deletions define novel embryonic lethal loci in the mouse t complex

Summary: The t complex region of mouse chromosome 17 contains genetic information critical for embryonic development. To identify and map loci required for normal embryogenesis, a set of overlapping deletions (D17Aus9(df10J), D17Aus9(df12J), and D17Aus9(df13J)) surrounding the D17Aus9 locus and one encompassing the T locus, Del(17)T(7J), were bred in various combinations and the consequences of nullizygosity in overlapping regions were examined. The results indicated that there are at least two functional units within 1 cM of D17Aus9. l17J1 is a peri-implantation lethal mutation within the region deleted in D17Aus9(df13J), whereas l17J2 is a later-acting lethal defined by the region of overlap between Del(17)T(7J) and D17Aus9(df12J). Del(17)T(7J)/D17Aus9(df12J) embryos die around 10.5 dpc. The development of the mutant embryos is characterized by lack of axial rotation, an abnormal notochord structure, and a ballooning pericardium. These studies demonstrate the value of overlapping deletion complexes, as opposed to individual deletion complexes, for the identification, mapping, and analysis of genes required for embryonic development.     

tctN-mutation causing taillessness in mice heterozygous for gene T

Screening of wild male mice trapped in Turkmenistan (Middle Asia) revealed a mouse with a mutation causing strong increase in expressivity of T mutation (Brachyury). A novel mutation was designated tctN-t complex tail interaction Novosibirsk. Compound heterozygotes T/tctN have tailless of extremely short tailed phenotype. Homozygotes tctN/tctN were completely viable and fertile. It was shown that the novel mutation was closely linked to T locus. Further genetic analysis showed that the chromosome with tct had no properties of t haplotypes: no lethal factors or the factors influencing fertility and segregation of homologoues; there was no effect on recombination frequency in the proximal part of chromosome 17 in tctN/+mice. The problems of t haplotype evolution are discussed.     

Localization of the gene for plasma retinol binding protein to the distal half of mouse chromosome 19

A DNA polymorphism for the mouse retinol binding protein has been identified using the enzyme BamHI and a rat partial cDNA probe. Analysis of the polymorphism in DNA from 64 inbred mouse strains demonstrated the presence of a single gene with two alleles, Rbp-4b and Rbp-4d. Comparison of the segregation patterns of these alleles in three sets of recombinant inbred strains with allele segregation patterns of previously characterized loci shows that the Rbp-4 locus is closely linked to the locus for phenobarbital-inducible cytochrome P450-2c (Cyp-2c) that has been shown by in situ hybridization to lie on chromosome 19, bands D1-D2. The Rbp-4 locus is just proximal to Cyp-2c at the distal end of chromosome 19.     

Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17

A full-length cDNA coding for mouse plasminogen has been isolated and characterized. The cDNA is 2720 bp in length (excluding the poly(A) tail) and contains a 24-bp 5' noncoding region, an open reading frame of 2436 bp, and a 3' noncoding region of 257 bp. The open reading frame codes for 812 amino acids and includes a signal peptide that is likely 19 amino acids in length and the mature protein of 793 amino acids. The calculated Mr of mouse plasminogen is 88,706 excluding carbohydrate. There are two potential N-linked carbohydrate addition sites; one of which is glycosylated in human, bovine, and porcine plasminogens. Mouse plasminogen was found to contain two additional amino acids compared to the human protein. In addition, mouse and human plasminogens were found to be 79 and 76% identical at the protein and DNA levels, respectively. Analysis of the segregation of two allelic forms, Plgb and Plgd, of plasminogen DNA in three sets of recombinant inbred strains has allowed the localization of the mouse plasminogen gene to the proximal end of mouse chromosome 17 within the t complex and close to the locus D17Rp17. The Plg gene is deleted in the semidominant deletion mutant, hair-pintail (Thp).     

TheD17Tu5 locus in thet complex: implications for the origin oft haplotypes and inbred strains

The mouse × Chinese hamster cell line R4 4-1 contains only one mouse chromosome, the bulk of which corresponds toMus musculus chromosomes 17 and 18 (MMU17 and MMU18, respectively). A genomic library was prepared from the R4 4-1 DNA, and a mouse clone was isolated from the library, which—with the help of somatic cell hybrids-could be mapped to the MMU17. A locus defined by a 2.7-kb longBam HI probe from this clone was designatedD17Tu5 (Tu for Tübingen). The locus proved to be polymorphic among inbred strains and wild mice. By testing of recombinant inbred strains and partialt haplotypes, theD17Tu5 locus could be mapped to a position between theD17Leh66E andD17Rp17 loci within thet complex. Two alleles were found at this locus,D17Tu5 ^a andD17Tu5 ^b , defined byTaq I restriction fragment length polymorphism. Both alleles are present among inbred strains and wild mice of the speciesM. domesticus. All completet haplotypes tested carry theD17Tu5 ^a allele and all tested wild mice of the speciesM. musculus, with the exception of those bearingt haplotypes, carry theD17Tu5 ^b allele. Additional alleles are found in some populations of wild mice and in other species of the genusMus. The distribution of the two alleles among the inbred strains correlates well with their known or postulated genealogy. Their distribution between the two species ofMus and among the mice withT haplotypes suggests a relatively recent origin of thet haplotypes.     

Analysis of the Om(1d) Locus in Drosophila Ananassae

From the ca;px stock, which is the progenitor of Om mutants caused by insertions of the tom retrotransposon, 50 kb of genomic DNA including the Om(1D) locus was cloned by tom tagging and chromosome walking. Southern blot analyses of six Om(1D) mutants exposed one or two tom elements inserted at five nonrandom sites within an 18-kb distal segment of the restriction map; the phenotypic uniformity between these mutants was not affected by variations in the position, number or orientation of their inserts. Spontaneous revertants or more extreme derivatives of Om(1D) alleles were nonlinearly associated with losses or gains of tom inserts. Seven of eight radiation induced derivatives of Om(1D) mutants had one breakpoint of a chromosome rearrangement in polytene section 13A which includes the Om(1D) locus. Two Om(1D) derivatives, a spontaneous revertant and an induced extreme allele, were associated with overlapping deficiencies which define a region that is likely to contain the Om(1D) coding seguences proximal to the tom insertion sites. Incidental results confirm the previously indicated homology of the Om(1D) locus with the Bar locus of Drosophila melanogaster.     

Physical mapping of male fertility and meiotic drive quantitative trait loci in the mouse t complex using chromosome deficiencies

The t complex spans 20 cM of the proximal region of mouse chromosome 17. A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes. Transmission ratio distortion and sterility are associated with t and affect males only. It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility. One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion. To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region. We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1. YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1. We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity. These studies demonstrate the utility of chromosome deletions for complex trait analysis.