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.     

Third chromosome suppressor of position-effect variegation loci in Drosophila melanogaster

Summary As a result of a genetic analysis of 63 third chromosome suppressor mutations of position-effect variegation 12 different loci showing dominant suppression have been identified and their map positions determined. A compilcation of the genetic data available for each suppressor locus is given. The strong suppressor effects of the mutations have been quantified by measurements of white variegation inw ^m4h /w ^m4h ,w ^m4h /Y andw ^m4h /O flies. Mutant alleles of three loci were found in these studies to dominate over the strong enhancer effect of complete loss of the Y chromosome. Most of the identified loci suppressing position-effect variegation represent essential genetic funtions; only three loci represent nonessential functions. Mutations of two loci display recessive butyrate sensitivity and lethal interaction with the heterochromatic Y chromosome suggesting that these genes affect chromosomal condensation. Studies with deficiencies and triploids revealed that most of the loci represent haplo-abnormal suppressor functions. The use of the isolated mutant material for genetic, developmental and molecular studies of processes connected with gene inactivation in position-effect variegation is discussed.Dedicated to Prof. H.J. Becker on the occasion of his 6th birthday     

Analysis of lethal mutations induced in a mutator strain that activates transposable elements in Caenorhabditis elegans

A screen was conducted for lethal mutations in the nematode Caenorhabditis elegans in a strain containing the mutator mut-4 (st700)I to examine the nature of mutator-induced lethal mutations within two large chromosomal regions comprising a total of 49 map units (linkage group IV (right) and linkage group V (left)). The genetic analysis of 28 lethal mutations has revealed that the mutator locus mut-4(st700)I causes both putative single-gene mutations and deficiencies. We have identified lethal mutations in three different genes, in addition to seven deficiencies. There is a mutational hot spot on linkage group V (left) around the lin-40 locus. Six mutations appear to be alleles of lin-40. In addition, 5 of 7 deficiencies have breakpoints at or very near lin-40. All seven deficiencies delete the left-most known gene on linkage group V (left) and thus appear to delete the tip of the chromosome. This is in contrast to gamma ray and formaldehyde induced deficiencies, which infrequently delete the closest known gene to the tip of a chromosome.     

Cytogenetic analysis of chromosome region 89A of Drosophila melanogaster: isolation of deficiencies and mapping of Po, Aldox-1 and transposon insertions

Summary We have initiated a cytogenetic analysis of chromosome region 89A of Drosophila melanogaster by isolating a set of radiation-induced mutations causing loss of function of P[(w)B]1-1, a transposon bearing the white locus inserted in 89A. Complementation tests and cytological examination of these chromosomes identified four new deficiencies (Df(3R)Po ², Df(3R)Po ³, Df(3R)Po ⁴ and Df(3R)c(3)G ² ). The new deficiencies and three previously identified deficiencies (Df(3R)sbd ²⁶, Df(3R)sbd ⁴⁵ and Df(3R)sbd ¹⁰⁵) were tested for the ability to complement mutations in the enzyme loci Po and Aldox-1, the indirect flight muscle genes Tm2 and act88F, the morphological mutations jvl, sbd ² and Sb, the vital loci srp, pnr and mor, and a newly described vital locus l(3)89Aa. We also used linkage analysis to determine the order and relative positions of P[(w)B]1-1 and an independent transposon insertion, P[w⁺]21, with respect to cv-c, Po, Aldox-1 and sbd ². Cytological examination of the deficiencies and analysis of the transformed lines by in situ hybridization permits the correlation of genetically defined regions with specific polytene chromosome bands. A revised cytogenetic map of the 8817–8913 region is presented.     

QTL mapping of naturally-occurring variation in flowering time of Arabidopsis thaliana

A segregating F₂ population of Arabidopsis thaliana derived from a cross between the late-flowering ecotype Hannover/Münden (HM) and the early-flowering ecotype Wassilewskija (WS) was analyzed for flowering time and other morphological traits. Two unlinked quantitative trait loci (QTLs) affecting days to first flower (DFF-a and DFF-b) mapped to chromosome 5. QTLs which affect node number (NN), leaf length at flowering (LLF), and leaf length at 35 days (LL35) also mapped to chromosome 5; LLF-a, LL35-a, NN-a map to the same region of chromosome 5 as DFF-a; LLF-b and LL35-bmap to the same region of chromosome 5 as DFF-b. Another QTL affecting leaf length at flowering (LLF-c) maps to chromosome 3. The proximity of DFF-a, LLF-a, LL35-a and NN-a, as well as the similarity in gene action among these QTLs (additivity), suggest that they may be pleiotropic consequences of a single gene at this locus. Similarly, LL35-b and LLF-b map near each other and both display recessive gene action, again suggesting the possibility of pleiotropy. DFF-b, which also maps near LL35-b and LLF-b, displays largely additive gene action (although recessive gene action could not be ruled out). This suggests that DFF-b may represent a different gene from LL35-b and/or LLF-b. DFF-a maps near two previously identified mutants: co (which also affects flowering time and displays gene action consistent with additivity) and flc. Similar map locations and gene actions of QTLs affecting the correlated traits DFF, LLF, LL35 and NN suggest that these genomic regions harbor naturally occurring allelic variants involved in the general transition of the plant from vegetative to reproductive growth.     

Butyrate sensitive suppressor of position-effect variegation mutations in drosophila melanogaster

Summary Mutations at a locus on chromosome II of D. melanogaster suppressing position-effect variegation mutations have been identified which display recessive butyrate sensitivity. Survival of homozygous mutant flies is significantly reduced on medium containing sodium n-butyrate. The butyrate sensitive suppressor mutations are further characterized by recessive female sterility and reduced survival of homozygotes. Complementation analysis showed their allelism. The locus of these mutations, Su-var (2) 1, has been localized to 40.5±0.2 and, by using interstitial duplications, to region 31CD on the cytogenetic map. Moreover, the mutant alleles of the Su-var (2) 1 locus display a lethal interaction with the heterochromatic Y chromosome. The presence or absence of a Y chromosome in males or females has a strong influence on the viability of homozygous or transheterozygous suppressor flies. All the genetic properties of Su-var (2) 1 mutants suggest strongly that this locus affects chromosome condensation.     

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

Molecular analysis of two genes between let-653 and let-56 in the unc 22(IV) region of Caenorhabditis elegans

Summary A previous study of genomic organization described the identification of nine potential coding regions in 150 kb of genomic DNA from the unc-22(IV) region of Caenorhabditis elegans. In this study, we focus on the genomic organization of a small interval of 0.1 map unit bordered on the right by unc-22 and on the left by the left-hand breakpoints of the deficiencies sDf9, sDf19 and sDf65. This small interval at present contains a single mutagenically defined locus, the essential gene let-56. The cosmid C11F2 has previously been used to rescue let-56. Therefore, at least some of C11F2 must reside in the interval. In this paper, we report the characterization of two coding elements that reside on C11F2. Analysis of nucleotide sequence data obtained from cDNAs and cosmid subclones revealed that one of the coding elements closely resembles aromatic amino acid decarboxylases from several species. The other of these coding elements was found to closely resemble a human growth factor activatable Na⁺/H⁺ antiporter. Pairs of oligonucleotide primers, predicted from both coding elements, have been used in PCR experiments to position these coding elements between the left breakpoint of sDf19 and the left breakpoint of sDf65, between the essential genes let-653 and let-56.     

Genetic analysis of the X-chromosomal region 1E-2A of Drosophila melanogaster

Reversion mutagenesis of three single P elements located in the cytogenetic interval 1E-2A at the tip of the X chromosome of Drosophila melanogaster was used to recover new deletions in this chromosomal region. The deletions obtained include small aberrations within region 2A and larger lesions extending from 2A into 1E and 1B. All three screens also yielded terminal deficiencies. The new deficiencies, together with previously characterized rearrangements, were analyzed for their complementation behaviour with the maternal effect locus fs(1)Nasrat and lethal loci in the region. These analyses provide an overall genetic map of the interval 1E-2A. In addition, the smaller deletions were physically mapped within cloned genomic DNA of the 2A region.     

Chromosomal Mapping ofTmp(Emp1),Xmp(Emp2), andYmp(Emp3), Genes Encoding Membrane Proteins Related toPmp22

We have recently characterized a novel mammalian gene family, encoding membrane glycoproteins with four trans-membrane domains. This gene family includes the previously studiedPMP22,which is involved in the Charcot–Marie–Tooth neuropathy, and three novel genes:TMP, XMP,andYMP(HGMW-approved symbolsEMP1, EMP2andEMP3,respectively). TheTmp(tumor-associated membrane protein) gene was isolated from a c-mycinduced mouse brain tumor and is expressed in several highly proliferative cell types. We have now isolated cDNAs of the mouseXmpandYmpgenes and determined the chromosomal localization of mouseTmp, Xmp,andYmp. Tmpwas mapped to mouse chromosome 6,Xmpwas mapped to chromosome 16, andYmpwas mapped to chromosome 7.TmpandYmpmap to paralogous chromosomal regions, whereasXmpmaps to a chromosomal region that is putatively paralogous to a region on chromosome 11, to whichPmp22was previously mapped. These data suggest that this family of membrane glycoproteins evolved as a result of chromosomal duplications.     

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6–7 kb from C9 in a head-to-head or 5′ to 5′ orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.     

Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ⁻ mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome. Received: 29 July 1996 / Accepted: 27 August 1996     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

 The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26 000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy. Received: 25 April 1996 / Accepted: 26 June 1996     

A High-Resolution Genetic, Physical, and Comparative Gene Map of the Doublefoot (Dbf) Region of Mouse Chromosome 1 and the Region of Conserved Synteny on Human Chromosome 2q35

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0. 4-cM (±0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.     

Location of the ompT gene relative to that of appY on the physical map of the Escherichia coli chromosome

Results concerning the precise location of the ompT gene (encoding the outer membrane protease OmpT) on the Escherichia coli chromosome were obtained which disagree with published restriction sites in the gene. It is shown that the gene, together with appY, is present on a 3.075 PstI fragment, encompassing positions 596–598 of the E. coli physical map.     

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.