Analysis of chromosomal rearrangements induced by postmeiotic mutagenesis with ethylnitrosourea in zebrafish

Mutations identified in zebrafish genetic screens allow the dissection of a wide array of problems in vertebrate biology. Most screens have examined mutations induced by treatment of spermatogonial (premeiotic) cells with the chemical mutagen N-ethyl-N-nitrosourea (ENU). Treatment of postmeiotic gametes with ENU induces specific-locus mutations at a higher rate than premeiotic regimens, suggesting that postmeiotic mutagenesis protocols could be useful in some screening strategies. Whereas there is extensive evidence that ENU induces point mutations in premeiotic cells, the range of mutations induced in postmeiotic zebrafish germ cells has been less thoroughly characterized. Here we report the identification and analysis of five mutations induced by postmeiotic ENU treatment. One mutation, snh(st1), is a translocation involving linkage group (LG) 11 and LG 14. The other four mutations, oep(st2), kny(st3), Df(LG 13)(st4), and cyc(st5), are deletions, ranging in size from less than 3 cM to greater than 20 cM. These results show that germ cell stage is an important determinant of the type of mutations induced. The induction of chromosomal rearrangements may account for the elevated frequency of specific-locus mutations observed after treatment of postmeiotic gametes with ENU.     

Introducing defined chromosomal rearrangements into the mouse genome

Chromosomal rearrangements have been instrumental in genetic studies in Drosophila. Visibly marked deficiencies (deletions) are used in mapping studies and region-specific mutagenesis screens by providing segmental haploidy required to uncover recessive mutations. Marked recessive lethal inversions are used as balancer chromosomes to maintain recessive lethal mutations and to maintain the integrity of mutagenized chromosomes. In mice, studies on series of radiation-induced deletions that surround several visible mutations have yielded invaluable functional genomic information in the regions analyzed. However, most regions of the mouse genome are not accessible to such analyses due to a lack of marked chromosomal rearrangements. Here we describe a method to generate defined chromosomal rearrangements using the Cre--loxP recombination system based on a published strategy [R. Ramirez-Solis, P. Liu, and A. Bradley, (1995) Nature 378, 720--724]. Various types of rearrangements, such as deletions, duplications, inversions, and translocations, can be engineered using this strategy. Furthermore, the rearrangements can be visibly marked with coat color genes, providing essential reagents for large-scale recessive genetic screens in the mouse. The ability to generate marked chromosomal rearrangements will help to elevate the level of manipulative mouse genetics to that of Drosophila genetics.     

Homologous Recombination between Episomal Plasmids and Chromosomes in Yeast

We have observed genetic recombination between ura3( -) mutations (among them extensive deletions) carried on "episomal" (i.e., 2micro DNA-containing) plasmids and other ura3( -) alleles present at the normal chromosomal URA3 locus. The recombination frequency found was comparable to the level observed for classical mitotic recombination but was relatively insensitive to sunlamp radiation, which strongly stimulates mitotic recombination. Three equally frequent classes could be distinguished among the recombinants. Two of these are the apparent result of gene conversions (or double crossovers) which leave the URA3(+) allele on the chromosome (class I) or on the plasmid (class II). The third class is apparently due to a single crossover that results in the integration of the plasmid into a chromosome. Plasmid-chromosome recombination can be useful in fine structure genetic mapping, since recombination between a chromosomal point mutation and a plasmid-borne deletion mutation only 25 base pairs distant was easily detected.     

Molecular and functional mapping of the piebald deletion complex on mouse chromosome 14

The piebald deletion complex is a set of overlapping chromosomal deficiencies surrounding the endothelin receptor B locus collected during the Oak Ridge specific-locus-test mutagenesis screen. These chromosomal deletions represent an important resource for genetic studies to dissect the functional content of a genomic region, and several developmental defects have been associated with mice homozygous for distinct piebald deletion alleles. We have used molecular markers to order the breakpoints for 20 deletion alleles that span a 15.7-18-cM region of distal mouse chromosome 14. Large deletions covering as much as 11 cM have been identified that will be useful for regionally directed mutagenesis screens to reveal recessive mutations that disrupt development. Deletions identified as having breakpoints positioned within previously described critical regions have been used in complementation studies to further define the functional intervals associated with the developmental defects. This has focused our efforts to isolate genes required for newborn respiration and survival, skeletal patterning and morphogenesis, and central nervous system development.     

Genetic Organization of the Unc-60 Region in Caenorhabditis Elegans

We have investigated the chromosomal region around unc-60 V, a gene affecting muscle structure, in the nematode Caenorhabditis elegans. The region studied covers 3 map units and lies at the left end of linkage group (LG) V. Compared to the region around dpy-11 (at the center of LGV), the unc-60 region has relatively few visible genes per map unit. We found the same to be true for essential genes. By screening simultaneously for recessive lethals closely linked to either dpy-11 or unc-60, we recovered ethyl methanesulfonate-induced mutations in 10 essential genes near dpy-11 but in only two genes near unc-60. Four deficiency breakpoints were mapped to the unc-60 region. Using recombination and deficiency mapping we established the following gene order: let-336, unc-34, let-326, unc-60, emb-29, let-426. Regarding unc-60 itself, we compared the effect of ten alleles (including five isolated during this study) on hermaphrodite mobility and fecundity. We used intragenic mapping to position eight of these alleles. The results show that these alleles are not distributed uniformly within the gene, but map to two groups approximately 0.012 map unit apart.     

Centromere-linked microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 of zebrafish (Danio rerio)

A large number of interesting mutations affecting development and organogenesis have been identified through genetic screens in zebrafish. Mapping of these mutations to a chromosomal region can be rapidly accomplished using half-tetrad analysis. However, knowledge of centromere-linked markers on every chromosome is essential to this mapping method. Centromeres on all 25 linkage groups have been mapped on the RAPD zebrafish genetic map. However, species specificity and the lack of codominance make RAPD markers less practical for mapping than microsatellite-based markers. On the microsatellite-based genetic map, centromere-linked markers have been identified for 19 linkage groups. No direct evidence has been published linking microsatellite markers to the centromeres of linkage groups 3, 4, 6, 7, 13, and 20. Therefore, we compared the microsatellite-based genetic map with the RAPD map to identify markers most likely linked to the centromeres of these 6 linkage groups. These candidate markers were tested for potential centromere linkage using four panels of half-tetrad embryos derived by early-pressure treatment of eggs from four different female zebrafish. We have identified microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 to within 1.7 cM of their centromeres. These markers will greatly facilitate the rapid mapping of mutations in zebrafish by half-tetrad analysis.     

Genomic analysis of gamma-ray-induced germ-cell mutations at the b locus recovered from the medaka specific-locus test.

To study how gamma-ray-induced germ-cell mutations are fixed at the early embryonic stage of the next generation, genomic alterations in the b locus mutants (colorless melanophores) detected during development in the medaka specific-locus test (SLT) were analyzed. First, nine anonymous DNA markers linked to the b locus were cloned and mapped into the region extending about 47cM surrounding the b locus. Next, losses of paternal alleles of these DNA markers were examined in each of the 51 gamma-ray-induced b locus mutants obtained after irradiation of sperm or spermatids. In these mutants, 47 were dominant lethals, three were semi-viable and one was viable. All the mutants examined had large deletions surrounding the b locus. One viable mutant had an interstitial deletion, while all the semi-viable and dominant lethal ones appeared to have terminal deletions. Deletions extending about 20-35cM were the most frequently observed in 18 of the 51 mutants examined. The largest one extended more than 40cM. These results suggest that most of the gamma-ray induced germ cell mutations recovered as total specific-locus mutants were accompanied by large genomic deletions, which eventually led the mutant embryos to dominant lethality.     

Analysis of fast neutron-generated mutants at the Arabidopsis thaliana HY4 locus

Ionizing radiation is expected to produce mutants with deletions or other chromosomal rearrangements. These mutants are useful for a variety of purposes, such as creating null alleles and cloning genes whose existence is known only from their mutant phenotype; however, only a few mutations generated by ionizing radiation have been characterized at the molecular level in Arabidopsis thaliana . Twenty fast neutron-generated alleles of the Arabidopsis HY4 locus, which encodes a blue light receptor, CRY1, were isolated and characterized. Nine of the mutant alleles displayed normal genetic behavior. The other 11 mutant alleles were poorly transmitted through the male gametophyte and were lethal in homozygous plants. Southern blot analysis demonstrated that alleles of the first group generally contain small or moderate-sized deletions at HY4 , while alleles of the second group contain large deletions at this locus. These results demonstrate that fast neutrons can produce a range of deletions at a single locus in Arabidopsis . Many of these deletions would be suitable for cloning by genomic subtraction or representational difference analysis. The results also suggest the presence of an essential locus adjacent to HY4 .     

Two new balancer chromosomes on mouse chromosome 4 to facilitate functional annotation of human chromosome 1p

To facilitate genetic screens to identify and maintain recessive mutations that map to the short arm of human chromosome 1, we have utilized chromosome engineering to generate two mouse strains that carry large inversions on the distal region of mouse chromosome 4. The inversion intervals are 16 and 22 cM in size together they cover approximately half of chromosome 4. Since recombination between the wild-type and inversion chromosomes does not occur within these inversion intervals, mutant alleles of genes mapping to this region can be identified and maintained. Therefore, these inversion chromosomes work as balancer chromosomes. These inversions have the additional advantage that they are tagged with genes encoding the visible coat color markers tyrosinase and agouti, and therefore the dosage of the inversion chromosome (+/+, Inv/+, Inv/Inv) can be visually recognized. These inversion strains will be extremely useful for mutagenesis screens that focus on functional annotation of human chromosome 1p.     

Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange

Sulfolobus acidocaldarius is so far the only hyperthermophilic archaeon in which genetic recombination can be assayed by conjugation and simple selections. Crosses among spontaneous pyr mutants were able to resolve closely spaced chromosomal mutations, identify deletions and rearrangements, and map mutations to a given deletion interval. Frameshift mutations in pyrE exerted polar effects that depressed orotidine-5'-monophosphate decarboxylase activity (encoded by pyrF), whereas base pair substitutions and an 18-bp deletion had no effect.     

The master sex-determination locus in threespine sticklebacks is on a nascent Y chromosome

BACKGROUND: Many different environmental and genetic sex-determination mechanisms are found in nature. Closely related species can use different master sex-determination switches, suggesting that these developmental pathways can evolve very rapidly. Previous cytological studies suggest that recently diverged species of stickleback fish have different sex chromosome complements. Here, we investigate the genetic and chromosomal mechanisms that underlie sex determination in the threespine stickleback (Gasterosteus aculeatus). RESULTS: Genome-wide linkage mapping identifies a single chromosome region at the distal end of linkage group (LG) 19, which controls male or female sexual development in threespine sticklebacks. Although sex chromosomes are not cytogenetically visible in this species, several lines of evidence suggest that LG 19 is an evolving sex chromosome system, similar to the XX female/XY male system in many other species: (1) males are consistently heterozygous for unique alleles in this region; (2) recombination between loci linked to the sex-determination region is reduced in male meiosis relative to female meiosis; (3) sequence analysis of X- and Y-specific bacterial artificial chromosome (BAC) clones from the sex-determination region reveals many sequence differences between the X- and Y-specific clones; and (4) the Y chromosome has accumulated transposable elements and local duplications. CONCLUSIONS: Taken together, our data suggest that threespine sticklebacks have a simple chromosomal mechanism for sex determination based on a nascent Y chromosome that is less than 10 million years old. Further analysis of the stickleback system will provide an exciting window into the evolution of sex-determination pathways and sex chromosomes in vertebrates.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. II. Complementation Analyses of Lethal Brown Deletions

Numerous new mutations at the brown (b) locus in mouse chromosome 4 have been recovered over the years in germ-cell mutagenesis experiments performed at the Oak Ridge National Laboratory. A large series of radiation- and chemical-induced b mutations known to be chromosomal deletions, and also known to be prenatally lethal when homozygous, were analyzed by pairwise complementation crosses as well as by pseudodominance tests involving flanking loci defined by externally visible phenotypes. These crosses were designed to determine the extent of each deletion on the genetic and phenotype map of the chromosomal region surrounding the b locus; the crosses also provided basic data that assigned deletions to complementation groups and defined four new loci associated with aberrancies in normal development. Specifically, the pseudodominance tests identified deletions that include the proximally mapping whirler (wi) and the distally mapping depilated (dep) genes, thereby bracketing these loci defined by visible developmental abnormalities with landmarks (deletion breakpoints) that are easily identified on the physical map. Furthermore, the complementation crosses, which were supplemented with additional crosses that allowed determination of the gross time of lethality of selected deletions, defined four new loci required for normal development. Homozygous deletion of one of these loci (b-associated fitness, baf) results in a runting syndrome evident during postnatal development; deletion of one locus [l(4)2Rn] causes death in the late gestation/neonatal period; and deletion of either of two loci [l(4)1Rn or l(4)3Rn] results in embryonic death, most likely in pre-, peri- or postimplantation stages. The placement of these new functionally defined loci on the evolving molecular map of the b region should be useful for continuing the analysis of the roles played in development by genes in this segment of chromosome 4.     

Structural and functional organization of the '1S0.8 gene-rich region' in the Triticeae

Wheat genes are present in physically small, gene-rich regions, interspersed by gene-poor blocks of retrotransposon-like repetitive sequences. One of the largest gene-rich regions is present around fraction length (FL) 0.8 of the short arm of wheat homoeologous group 1 chromosomes and is called `1S0.8 region'. The objective of this study was to reveal the structural and functional organization of the `1S0.8 region' in various Triticeae and other Poaceae species. Consensus genetic linkage maps of the `1S0.8 region' were constructed for wheat, barley, and rye by combining mapping information from 16, 11, and 12 genetic linkage maps, respectively. The consensus genetic linkage maps were compared with each other and with a consensus physical map of wheat homoeologous group 1. Comparative analyses localized 75 agronomically important genes to the `1S0.8 region'. This high-resolution comparison revealed exceptions to the rule of conserved gene synteny, established using low-resolution marker comparisons. Small rearrangements such as duplications, deletions, and inversions were observed among species. Proportion of chromosomal recombination occurring in the `1S0.8 region' was very similar among species. Within the gene-rich region, the extent of recombination was highly variable but the pattern was similar among species. Relative recombination among markers was similar except for a few loci where drastic differences were observed among species. Chromosomal rearrangements did not always change the extent of recombination for the region. Differences in gene order and relative recombination were the least between wheat and barley, and were the highest between wheat and oat.     

Chromosomal rearrangements differentiating the ryegrass genome from the Triticeae, oat, and rice genomes using common heterologous RFLP probes

An restriction fragment length polymorphism (RFLP)-based genetic map of ryegrass (Lolium) was constructed for comparative mapping with other Poaceae species using heterologous anchor probes. The genetic map contained 120 RFLP markers from cDNA clones of barley (Hordeum vulgare L.), oat (Avena sativa L.), and rice (Oryza sativa L.), covering 664 cM on seven linkage groups (LGs). The genome comparisons of ryegrass relative to the Triticeae, oat, and rice extended the syntenic relationships among the species. Seven ryegrass linkage groups were represented by 10 syntenic segments of Triticeae chromosomes, 12 syntenic segments of oat chromosomes, or 16 syntenic segments of rice chromosomes, suggesting that the ryegrass genome has a high degree of genome conservation relative to the Triticeae, oat, and rice. Furthermore, we found ten large-scale chromosomal rearrangements that characterize the ryegrass genome. In detail, a chromosomal rearrangement was observed on ryegrass LG4 relative to the Triticeae, four rearrangements on ryegrass LGs2, 4, 5, and 6 relative to oat, and five rearrangements on ryegrass LGs1, 2, 4, 5, and 7 relative to rice. Of these, seven chromosomal rearrangements are reported for the first time in this study. The extended comparative relationships reported in this study facilitate the transfer of genetic knowledge from well-studied major cereal crops to ryegrass.     

Fine Structure Mapping and Deletion Analysis of the Murine Piebald Locus

piebald (s) is a recessive mutation that affects the development of two cell types of neural crest origin: the melanocytes, responsible for pigment synthesis in the skin, and enteric ganglia, which innervate the lower bowel. As a result, mice carrying piebald mutations exhibit white spotting in the coat and aganglionic megacolon. Previously the gene had been localized to the distal half of mouse chromosome 14. To determine its precise location relative to molecular markers, an intersubspecific backcross was generated. Two anchor loci of chromosome 14, slaty and hypogonadal, in addition to simple sequence length repeat markers, were used to localize s to a 2-cM interval defined by the markers D14Mit38 and D14Mit42. The molecular markers were also used to characterize nine induced s alleles. Three of these mutations exhibited no deletions or rearrangements of the flanking markers, whereas the other six had two or more of these markers deleted. The extent of the deletions was found to be consistent with the severity of the homozygous phenotype. The location of deletion breakpoints in the induced alleles, coupled with the recombination breakpoints in the backcross progeny, provide useful molecular landmarks to define the location of the piebald gene.     

A Cytogenetic Analysis of the Punch-Tudor Region of Chromosome 2r in Drosophila Melanogaster

Eleven chromosomal deficiencies and several rearrangements in the Pu-tud region of chromosome 2R have been generated and examined cytologically. The Pu locus has been localized to chromosome bands 57C5-6 and tud to 57C7-8. Mutagenesis within the region defined by the deletion intervals has resulted in the isolation of 92 new lethal mutations. Seventy-six of these mutations have been separated into 16 complementation groups that have been ordered and placed cytologically by deletion mapping. All new alleles fully complement tud for both lethal and grandchildless phenotypes. The largest number of new mutations, a total of 25, are Pu alleles.     

Genetic mapping of unstable chloramphenicol resistance determinant in Streptomyces coelicolor A3(2)

The results indicative of chromosomal localization of the unstable chloramphenicol resistance determinant in Streptomyces coelicolor A3(2) have been obtained. Independent mutations specifying chloramphenicol sensitivity in different strains of S. coelicolor A3(2), S18 and A617M are localized in the same region flanked by markers argA1 and cysD18 on the genetic map. Mutations restoring chloramphenicol resistance are also localized in this region. Different locations of the genetically unstable determinant of chloramphenicol resistance detected in various laboratories are discussed, in relation to the results showing that transfer to chloramphenicol sensitivity is due to a set of various rearrangements (deletions, amplifications, deamplifications, etc.), differing in separate variants.     

An intraspecific linkage map of the chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) genome based on sequence tagged microsatellite site and resistance gene analog markers

An intraspecific linkage map of the chickpea genome based on STMS as anchor markers, was established using an F(2) population of chickpea cultivars with contrasting disease reactions to Ascochyta rabiei (Pass.) Lab. At a LOD-score of 2.0 and a maximum recombination distance of 20 cM, 51 out of 54 chickpea-STMS markers (94.4%), three ISSR markers (100%) and 12 RGA markers (57.1%) were mapped into eight linkage groups. The chickpea-derived STMS markers were distributed throughout the genome, while the RGA markers clustered with the ISSR markers on linkage groups LG I, II and III. The intraspecific linkage map spanned 534.5 cM with an average interval of 8.1 cM between markers. Sixteen markers (19.5%) were unlinked, while l1 chickpea-STMS markers (20.4%) deviated significantly ( P < 0.05) from the expected Mendelian segregation ratio and segregated in favor of the maternal alleles. However, ten of the distorted chickpea-STMS markers were mapped and clustered mostly on LG VII, suggesting the association of these loci in the preferential transmission of the maternal germ line. Preliminary comparative mapping revealed that chickpea may have evolved from Cicer reticulatum, possibly via inversion of DNA sequences and minor chromosomal translocation. At least three linkage groups that spanned a total of approximately 79.2 cM were conserved in the speciation process.     

Molecular Mapping of the ROSY Locus in DROSOPHILA MELANOGASTER

The DNA from the chromosomal region of the Drosophila rosy locus has been examined in 83 rosy mutant strains. Several spontaneous and radiation-induced alleles were associated with insertions and deletions, respectively. The lesions are clustered in a 4-kb region. Some of the alleles identified on the DNA map have been located on the genetic map by fine-structure recombination experiments. The genetic and molecular maps are collinear, and the alignment identifies the DNA location of the rosy control region. A rosy RNA of 4.5 kb has been identified; its 5' end lies in or near the control region.     

Somatic events unmask recessive cancer genes to initiate malignancy

A heritable mutation predisposes an individual to certain childhood malignancies, such as retinoblastoma and Wilms' tumor. The chromosomal locations of the genes responsible for the predisposition are known by linkage with chromosomal deletions and enzyme markers. A study of these tumors in comparison to the normal constitutional cells of the patients, using enzyme and DNA markers near the predisposing genes, has shown that these genes are recessive to normal wild-type alleles at the cellular level. Expression of the recessive phenotype (malignancy) involves the same genetic events that were observed in Chinese hamster cell hybrids carrying recessive drug resistance genes. In both the experimental and clinical situations, the wild-type allele is most commonly eliminated by chromosome loss with duplication of the mutant chromosome. Simple chromosome loss and mitotic recombination have been documented in both systems. In the remaining 30% of cases, inactivation or microdeletion of the wild-type allele are assumed to be responsible for expression of the recessive phenotype. Osteosarcoma is a common second tumor in patients who have had retinoblastoma. Studies with markers in osteosarcoma show that these tumors also result from unmasking of the recessive phenotype by loss of the normal allele at the retinoblastoma locus, whether or not the patient had retinoblastoma. Subsequent chromosomal rearrangements and amplification of oncogenes that occur in these homozygous tumors provide progressive growth advantage. In other malignancies, in which studies have so far focused on oncogene amplification and chromosomal rearrangements, unmasking of recessive mutations may also be the critical initiating events.