Deficiency Breakpoints Research Articles

Use of SNPs to determine the breakpoints of complex deficiencies, facilitating gene mapping in Caenorhabditis elegans

BackgroundGenetic deletions or deficiencies have been used for gene mapping and discovery in various organisms, ranging from the nematode Caenorhabditis elegans all the way to humans. One problem with large deletions is the determination of the location of their breakpoints. This is exacerbated in the case of complex deficiencies that delete a region of the genome, while retaining some of the intervening sequence. Previous methods, using genetic complementation or cytology were hampered by low marker density and were consequently not very precise at positioning the breakpoints of complex deficiencies. The identification of increasing numbers of Single Nucleotide Polymorphisms (SNPs) has resulted in the use of these as genetic markers, and consequently in their utilization for defining the breakpoints of deletions using molecular biology methods.ResultsHere, we show that SNPs can be used to help position the breakpoints of a complex deficiency in C. elegans. The technique uses a combination of genetic crosses and molecular biology to generate robust and highly reproducible results with strong internal controls when trying to determine the breakpoints of deficiencies. The combined use of this technique and standard genetic mapping allowed us to rapidly narrow down the region of interest in our attempts to clone a gene.ConclusionUnlike previous methods used to locate deficiency breakpoints, our technique has the advantage of not being limited by the amount of starting material. It also incorporates internal controls to eliminate false positives and negatives. The technique can also easily be adapted for use in other organisms in which both genetic deficiencies and SNPs are available, thereby aiding gene discovery in these other models.

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster.

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.

Genetic and molecular analysis in the 70CD region of the third chromosome of Drosophila melanogaster.

A collection of lethal and semi-lethal P-element insertions in the 70CD region of chromosome 3 of Drosophila melanogaster was used to investigate genes and gene arrangements by a combination of genetic, cytological, functional and molecular methods. The 12 lethal insertions studied fall into seven complementation groups of six genes. Lethal phases, expression patterns and other phenotypic aspects of these genes were determined. The genes and additional available sequences were placed on cloned genomic DNA fragments and arranged in an EcoRI map of 150kb that covers approximately the bands 70C7-8 to 70D1. Determination of deficiency breakpoints links the genetic, physical and molecular data. The sequences adjacent to seven independent P-element insertions were established after plasmid rescue or polymerase chain reaction. Similarity searches allowed the assignment of the P-element insertions to known mutations, expressed sequence tags, sequence tagged sites, or homologous genes of other species. Among these were identified a putative transacylase, a putative cell cycle gene, and the gene responsible for the dominant Polycomb-suppressor phenotype of devenir. The genomic sequence of the l(3)70Ca/b gene reveals a novel heat shock protein (hsc70Cb). l(3)70Da was identified as a member of the CDC48/PEX1 ATPase family and its coding sequence was determined.

An essential cell division gene of Drosophila, absent from Saccharomyces, encodes an unusual protein with tubulin-like and myosin-like peptide motifs.

Null mutations at the misato locus of Drosophila melanogaster are associated with irregular chromosomal segregation at cell division. The consequences for morphogenesis are that mutant larvae are almost devoid of imaginal disk tissue, have a reduction in brain size, and die before the late third-instar larval stage. To analyze these findings, we isolated cDNAs in and around the misato locus, mapped the breakpoints of chromosomal deficiencies, determined which transcript corresponded to the misato gene, rescued the cell division defects in transgenic organisms, and sequenced the genomic DNA. Database searches revealed that misato codes for a novel protein, the N-terminal half of which contains a mixture of peptide motifs found in alpha-, beta-, and gamma-tubulins, as well as a motif related to part of the myosin heavy chain proteins. The sequence characteristics of misato indicate either that it arose from an ancestral tubulin-like gene, different parts of which underwent convergent evolution to resemble motifs in the conventional tubulins, or that it arose by the capture of motifs from different tubulin genes. The Saccharomyces cerevisiae genome lacks a true homolog of the misato gene, and this finding highlights the emerging problem of assigning functional attributes to orphan genes that occur only in some evolutionary lineages.

Mapping a mutator, mu2, which increases the frequency of terminal deletions in Drosophila melanogaster.

A mutator, mu2, in Drosophila melanogaster has been identified recently that potentiates the recovery of terminal deficiencies. The deleted chromosomes behave as if they had been capped; that is, they are protected from degradation and from fusion with other chromosome fragments. The mutator maps near the telomere on the left arm of chromosome 3. Using the selectable marker Aprt, 150 deficiencies for region 62 of the cytological map have been recovered. These deficiencies identify the map position of mu2 as 62B11-C1. A yeast artificial chromosome spanning this region has been subcloned into lambda phage, and the positions of deficiency breakpoints on either side of the mu2 gene have been identified within the subclones. These positions limit the location of the left end of the gene to a 23 kb region. In the course of these experiments, three additional, presumptive mutant alleles were identified, suggesting that other mutator alleles remain undiscovered in many standard laboratory stocks.

Molecular and genetic analyses of Drosophila Prat, which encodes the first enzyme of de novo purine biosynthesis.

The Drosophila Prat gene encodes phosphoribosylamidotransferase (PRAT), the enzyme that performs the first committed step of the de novo purine nucleotide biosynthesis pathway. Using information from amino acid sequence alignments of PRAT from other organisms, a polymerase chain reaction-based approach was employed to clone Prat. Amino acid sequence alignment of Drosophila PRAT with PRAT from bacteria, yeast, and vertebrates indicates that it is most identical (at least 60%) to the vertebrate PRATs. It shares putative amino-terminal propeptide and iron-binding domains seen only in Bacillus subtilis and vertebrate PRATs. Prat was localized to the right arm of chromosome 3 at polytene band 84E1-2. Owing to the fact that this region had been well characterized previously, Prat was localized to a 30-kilobase region between two deficiency breakpoints. By making the prediction that Prat would have a similar "purine syndrome" phenotype as mutations in the genes ade2 and ade3, which encode enzymes downstream in the pathway, five alleles of Prat were isolated. Three of the alleles were identified as missense mutations. A comparison of PRAT enzyme activity with phenotype in three of the mutants indicates that a reduction to 40% of the wild-type allele's activity is sufficient to cause the purine syndrome, suggesting that PRAT activity is limiting in Drosophila.

Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster.

The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.

Metaphase I pairing of deficient chromosomes and genetic mapping of deficiency breakpoints in common wheat

Metaphase I pairing of deficient chromosomes was analyzed in a set of 'Chinese Spring' (CS) wheat (Triticum aestivum L. em. Thell.) plants with varying lengths of deficiencies in the long arm of chromosome 4A (6, 8, 11, 17, 23, 34, 36, 39, and 50% missing), the long arm of chromosome 5B (49% missing), and the long arm of chromosome 2B (33% missing). Pairing in homologous chromosomes between deficient and complete arms was greatly reduced even by small differences in arm length. In deficiency homozygotes and in an isochromosome derived from a deficient 4AL arm, pairing of the two deficient arms was high and approached that of two complete arms. In plants where deficient and complete arms competed for pairing partners, pairing was exclusively between arms of the same length. These results suggest that in wheat, pairing initiation sites are distributed throughout at least the distal halves of the arms and that the alignment of telomeres may be critical for pairing success. Genetic mapping of the deficiency breakpoints was confounded by misdivision of unpaired chromosomes and abnormal transmission rates. Genetic distances between centromeres and breakpoints appeared to be proportional to metaphase I pairing frequencies.Key words: bread wheat, deficiency, chromosome pairing competition, mapping, telomere, pairing initiation.

Genetic and fine structure analysis of unc-26(IV) and adjacent regions in Caenorhabditis elegans

The genetic organization of unc-26(IV) and adjacent regions was studied in Caenorhabditis elegans. We constructed a fine structure genetic map of unc-26(IV), a gene that affects locomotion and pharyngeal muscle movement but not muscle structure. Eleven alleles were positioned relative to each other recombinationally and were classified according to phenotypic severity. The unc-26 gene spans at least 0.026 map units, which is exceptionally large for a C. elegans gene. All but one allele, e205, are amorphic alleles. Interestingly, e205 is hypomorphic but also suppressible by the amber suppressor sup-7. Nineteen lethal mutations in the unc-26 region were isolated and characterized. The unc-26 region is subdivided into four zones by five deficiency breakpoints. These mutations fall into 15 complementation groups. The stages of development affected by these mutations were determined.

Chromosome I duplications in Caenorhabditis elegans.

We have isolated and characterized 76 duplications of chromosome I in the genome of Caenorhabditis elegans. The region studied is the 20 map unit left half of the chromosome. Sixty-two duplications were induced with gamma radiation and 14 arose spontaneously. The latter class was apparently the result of spontaneous breaks within the parental duplication. The majority of duplications behave as if they are free. Three duplications are attached to identifiable sequences from other chromosomes. The duplication breakpoints have been mapped by complementation analysis relative to genes on chromosome I. Nineteen duplication breakpoints and seven deficiency breakpoints divide the left half of the chromosome into 24 regions. We have studied the relationship between duplication size and segregational stability. While size is an important determinant of mitotic stability, it is not the only one. We observed clear exceptions to a size-stability correlation. In addition to size, duplication stability may be influenced by specific sequences or chromosome structure. The majority of the duplications were stable enough to be powerful tools for gene mapping. Therefore the duplications described here will be useful in the genetic characterization of chromosome I and the techniques we have developed can be adapted to other regions of the genome.

Molecular cloning of the lethal(1)discs large-1 oncogene of Drosophila

We present a genetic, developmental and molecular analysis of lethal(1)discs large-1 [ l(1)d.lg-1; Stewart et al., 1972], an oncogene of Drosophila. Mutations in this gene cause the imaginal discs to grow by cell proliferation beyond their normal final size, transform into solid tumors, fuse with one another and the brain, and lose their ability to differentiate. The oncogene represents the only known complementation group between two deficiency breakpoints, and 15 recessive lethal alleles are available. Cloning of the DNA between the two deficiency breakpoints defines a region of 45 ± 2 kb. The l(1)d.lg-1 transcription unit is identified by both qualitative and quantitative effects of several l(1)d.lg-1 mutations on the RNA transcripts and by the presence of a DNA insert in one of the l(1)d.lg-1 alleles. It gives rise to at least five different transcripts ranging in size from 1.9 to 6.0 kb. Three other transcription units are present within this region, two 5′ to the l(1)d.lg-1 gene and one at the 3′ end. A near full-length cDNA from one of the larger transcripts of l(1)d.lg-1 has homology to genomic DNA spanning over 20 kb. A developmental profile of l(1)d.lg-1 transcription is presented. We discuss how mutations in this gene could disrupt epithelial structure and how this might be related to the excessive cell proliferation and interdisc fusion that is observed. We also compare this gene with another recessive oncogene of Drosophila, lethal(2)giant larvae, that has been cloned and characterized.

A genetic analysis of the rose-gespleten region (68C8-69B5) of Drosophila melanogaster.

We describe a genetic analysis of the region 68C8-69B5 defined by Df(3L)vin-7. We have induced 35 new lethal mutations in this region, which together with 20 existing lethal mutations, visible mutations, genes identified by protein products and one gene deduced from complementation data fall into 37 complementation groups in this 35-band interval. Using existing and newly induced deficiencies we have assigned these to 11 intervals defined by deficiency breakpoints. Those mutations which fell in the same breakpoint interval as the Lsp-2 gene, which codes for the abundant larval serum protein 2, were the subject of detailed study. None was rescued by the active Lsp-2 gene transformed on to chromosome II and we conclude that, as yet, we have no lethal mutations of Lsp-2.

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.

Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome.

Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3)14 maps in 86CD, Su-var(3)13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.

The sex-determining gene tra of Drosophila: molecular cloning and transformation studies.

In Drosophila, the primary signal for sex determination is given by the ratio of X chromosomes to sets of autosomes (X:A). The primary signal is read by a key gene (Sxl) and transmitted down to the differentiation genes by the subordinate control genes tra, tra-2, ix and dsx. Mutations in tra transform chromosomal females (X/X; tra/tra) into sterile males (pseudomales). We have cloned the tra region by microdissection and chromosomal walking. We identified the gene using deficiency breakpoints, DNA aberrations in three different alleles of tra and by P-mediated transformation. A 3.8-kb fragment perfectly rescued the mutant phenotype of X/X; tra/tra flies, showing that it contained all the necessary information to restore female-specific functions in the mutant flies. We present evidence that most of the function of tra can be provided by a subsegment of 2 kb that is differentially transcribed or processed in males and females.

The genetics of the dorsal-Bicaudal-D region of Drosophila melanogaster.

The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respectively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of kelch, two previously identified maternal-effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.