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.     

Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains

In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH- terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus.     

Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus

Summary The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) were analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long period (31–39 h), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. This is a unique phenotype that had not previously been observed for mutants at the per locus. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms. The clock phenotypes of 3B1-2 chromosome aberrations, the three per mutant alleles, and per ⁺ duplications suggest that mutations at the per locus shorten, lengthen, or eliminate periodicity by respectively increasing, decreasing, or eliminating per activity.     

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.     

The identification and characterization of two dominant r1 haplotype-specific inhibitors of aleurone color in Zea mays

We report the identification and characterization of two novel dominant inhibitors of aleurone color in Zea mays that interact with specific haplotypes of the r1 locus. One inhibitor locus, inr1 (inhibitor of r1 aleurone color 1), maps to the long arm of chromosome 10, distal to the TB-10L19 breakpoint and tightly linked to dull1, and the second inhibitor locus, inr2 (inhibitor of r1 aleurone color 2), maps to the long arm of chromosome 9. Dominant inhibitory alleles of inr1 and inr2 act by suppressing aleurone color conditioned by certain r1 haplotypes. Two haplotypes, R1-ch:Stadler and R1-Randolph, exhibit nearly complete suppression of aleurone color in the presence of inhibitory alleles of inr1 or inr2. Two members of the R1-d class of haplotypes, R1-d:Catspaw and R1-d:Arapaho, show partial suppression. Other haplotypes tested were not visibly affected. The response of r1 haplotypes to inhibitory inr1 and inr2 alleles provides another means of analyzing the complex behavior of the seed color components of r1 haplotypes. Possible mechanisms of action of inr1 and inr2 are discussed.     

Effect of mutations on developmental stability and canalization in morphological traits in Drosophila ananassae

The present study was designed to determine the effects of visible mutations of large effect on developmental stability and canalization in different morphological traits, namely, sternopleural bristle number, wing length, wing to thorax ratio, ovariole number, and sex comb tooth number (SCTN) in Drosophila ananassae. We have compared the mean trait size, fluctuating asymmetry (FA) (as an index of developmental stability), and morphological variation (as an index of canalization) of different mutant strains (yellow body color, y; claret eye color, ca; plexus wing, px; spread wing, spr; ebony body and sepia eye color, e se; yellow body and claret eye color, y ca; and cardinal eye color, curled wing, and ebony body color, cd cu e) with wild-type strain. The mean trait size of all morphological traits differs significantly among the wild-type and mutant strains. The wild-type and mutant strains vary significantly for the morphological variation and also for the levels of the FA in different morphological traits. However, we have found no increase in either the variance or in the degree of FA with the increase of the mutations (except in SCTN in y mutant). The plausible reasons for the variation in wild-type and mutant strains with particular reference to developmental stability and canalization have been discussed.     

Separation of meiotic and mitotic effects of claret non-disjunctional on chromosome segregation in Drosophila.

The claret (ca) locus in Drosophila encodes a kinesin-related motor molecule that is required for proper distribution of chromosomes in meiosis in females and in the early mitotic divisions of the embryo. Here we demonstrate that a mutant allele of claret non-disjunctional (ca(nd)), non-claret disjunctional Dominant (ncdD), causes abnormalities in meiotic chromosome segregation, but is near wild-type with respect to early mitotic chromosome segregation. DNA sequence analysis of this mutant allele reveals two missense mutations compared with the predicted wild-type protein. One mutation lies in a proposed microtubule binding region of the motor domain and affects an amino acid residue that is conserved in all kinesin-related proteins reported to date. This region of the motor domain can be used to distinguish meiotic and mitotic motor function, defining an amino acid sequence criterion for classifying motors according to function. ncdD's mutant meiotic effect, but near wild-type mitotic effect, suggests that interactions of the ca motor protein with spindle microtubules differ in meiosis and mitosis.     

Determining the order of genes,centromeres, and rearrangement breakpoints inNeurospora by tests of duplication coverage

Nontandem segmental duplications provide a useful alternative to conventional recombination mapping for determining gene order in a haploid organism such asNeurospora. When an insertional or terminal rearrangement is crossed by Normal sequence, a class of progeny is produced that have a precisely delimited chromosome segment duplicated. In such Duplication progeny, a recessive gene in the Normal-sequence donor chromosome may or may not be masked (“covered”) by its dominant wild-type allele in the translocation-sequence recipient chromosome. Coverage depends upon whether the gene in question is left or right of the rearrangement breakpoint. The recessive gene will be heterozygous and covered (not expressed) if its locus is within the duplicated segment, but it will be haploid and expressed if the locus is outside the segment. Not only genes but also centromeres can be mapped by means of duplications, because genes included in. the same viable duplication must reside in the same chromosome arm. - Numerous sequences in the current genetic maps ofN. crassa have been determined using duplications. Gene order in the albino region and in the centromere region of linkage group I provide examples. Over 50 insertional or terminal rearrangements are available from which nontandem duplications of defined content can be obtained at will; collectively these cover about 75% of the genome. - Intercrosses between partially overlapping chromosome rearrangements also produce Duplication progeny containing two copies of regions between the breakpoints. The 180 mapped reciprocal translocations and inversions include numerous overlapping combinations that can be used for duplication mapping.     

The Interaction of Two Complex Loci, Zeste and Bithorax in DROSOPHILA MELANOGASTER

It has been found that certain alleles of the zeste locus (z(a) 1-1.0) have no phenotype of their own, but interact with certain alleles at the bithorax locus (bx 3-58.8). This interaction takes the form of an enhancement of the homeotic bx phenotype to a more extreme form-i.e., the metathorax is transformed into mesothorax in varying degrees depending on the bx allele used. This enhancement is somewhat reminiscent of the transvection effect described by Lewis (1954). The characterization of the interaction thus far has shown that the enhancement only effects bx alleles which arise spontaneously, whereas the origin of the z(a) allele is unimportant. The gene claret nondisjunctional was used for the production of gynandromorphs which showed that the enhancing ability of z(a), like the eye pigment change caused by z, is autonomous. The enhancement of one specific allele (bx(34e)), which is temperature-sensitive, has allowed a delineation of the temperature-sensitive period of the bithorax locus to a period extending from the middle of the second larval instar to the middle of the third larval instar. These results, as well as those of other enhancer and suppressor systems in Drosophila, have revealed the possibility of the involvement of heterocyclic compounds in the control of cell determination and fate in Drosophila melanogaster.     

Tissue-Specific and Complex Complementation Patterns in the Punch Locus of DROSOPHILA MELANOGASTER

Mutations in the Punch locus result in loss of GTP cyclohydrolase activity, but all mutations do not affect the enzyme in the same way. There are at least three classes of Punch mutations. One class results in a dominant eye color, recessive lethal phenotype. A second class of mutations also causes a recessive lethal phenotype, but heterozygous mutants have normal eye color. They show loss of GTP cyclohydrolase function in all tissues where activity can be measured. Alleles comprising a third class are recessive eye color mutations that are homozygous viable. Individuals with this third type of mutation show loss of enzyme activity in the eye, but show normal or near-normal activity elsewhere. In order to examine the organization and function of this locus further, we have performed interallelic complementation tests on 25 Punch mutations, monitoring viability and enzyme activity in prepupae and adults. Most allele combinations are lethal. Those that complement do so in ways that are tissue-or stage-specific and unpredictable. Tests of mutants with tissue-specific phenotypes and of individuals mutant for complementing Punch lethal alleles lead us to conclude that Punch is a complex locus, both with respect to its organization and to its products.     

w4-Mutable line in soybean

An unstable mutation for anthocyanin pigmentation in soybean (Giycine max [L.] Merr.) was identified in 1983. The mutability is conditioned by an allele at the w4 locus that is recessive to wild type. The population containing the mutable allele is known as the w4-mutable line. Most plants in the line have chimeric flowers with purple sectors on a near-white background. The mutable allele yields germinal revertants at a rate that varies from 5 to 10% per generation, and the revertant alleles are stable. Approximately 1% of the progenies derived from germinal revertant plants contain mutations at other loci These features, as well as the occurrence of pale flower phenotypes and changes of state, suggest that a transposable element system is producing the unstable phenotype. Several new mutants were isolated in an experiment designed to tag loci. The first three chlorophyll-deficient mutants found (CD-1, CD-2, and CD-3) are inherited as single-gene recessives. Each of the mutants lacks the same two mitochondrial malate dehydrogenase (MDH) bands. No recombination has been detected between the MDH phenotype and the chlorophyll-deficient phenotype. Genetic data indicate that the three mutants are allelic, and additional evidence suggests that each of the CD mutants is the result of a deletion. In the CD-1, CD-2, and CD-3 mutants, the deletions result in the silencing of an MDH locus, atypical chloroplast development, and an altered chlorophyll composition. Additional mutants for root necrosis, partial and near sterility, chlorophyll deficiency, and flower color isolated from the transposon tagging study have provided material for future research.     

The genetics of a new mutable allele at the white locus in Drosophila melanogaster

Summary The genetics of a third case of high mutation frequency at the white locus in Drosophila melanogaster has been analyzed. The new mutable allele, w ^+u, mutates from a wild-type to a white-eyed phenotype in both males and females. The mutational event is 1) premeiotic, 2) not associated with crossingover, 3) sensitive to genetic modification, and 4) restricted to germinal tissue. The only mutants produced by w ^+u are deletions of the white locus. These deficiencies include subsites 4 and 5 of the white locus, but are cytologically unobservable. The mutable allele itself maps to subsite 4.The mutational properties of w ^+u are unlike those of the other highly mutable white alleles which have been interpreted in terms of phage-like controlling elements. Rather, the properties of w ^+u favor a model based on the premature termination of chromosome replication near the terminus of a replicon which leads to a chromosome deficient for the material between the point of premature termination and the end of the replicon.Supported by NIH predoctoral traineeship GM-150 and by NIH research grant GM-07428 to Dr. W. K. Baker.From a dissertation submitted to the Division of Biological Sciences of The University of Chicago in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Cytogenetic Localization of the Acid Phosphatase-1 Gene in DROSOPHILA MELANOGASTER

A translocation in which a segment of chromosome 3 is inserted into the Y chromosome was found to contain the acid phosphatase-1 gene (Acph-1). In flies hyperploid for that gene, acid phosphatase-1 levels are proportional to the dose of the gene. The locus is placed within the salivary chromosome subdivisions 99D and 99E on the basis of its inclusion in the translocated segment and on the previous placement of the claret locus. Several chromosomal rearrangements involving heterochromatic breakpoints and euchromatic breakpoints adjacent to 99D-99E were tested for possible postiion-effect variegation of acid phosphatase-1. No decrease in the synthesis of the electorphoretic subunit encoded by the relocated gene was observed within any of the rearrangements.     

Derivative Alleles of the Arabidopsis Gibberellin-Insensitive (gai) Mutation Confer a Wild-Type Phenotype

The gai mutation of Arabidopsis confers a dwarf phenotype resembling that of mutants defective in gibberellin (GA) biosynthesis. However, gai mutant plants differ from GA biosynthesis mutants because they fail to respond to exogenous GAs and accumulate endogenous GA species to higher (rather than lower) levels than found in wild-type controls. The gai mutation, therefore, identifies a gene that modulates the response of plant cells to GA. We have mapped gai with respect to visible and restriction fragment length polymorphism (RFLP) markers from chromosome 1. To observe the phenotype exhibited by individuals potentially lacking wild-type (GAI) function, we have also isolated novel irradiation-induced derivative alleles of gai. When homozygous, these alleles confer a revertant phenotype that is indistinguishable from the wild type. gai is a semidominant mutation that exerts its effects either because it is a gain-of-function mutation or because it is a loss-of-function or reduced-function mutation. The genetic and physiological properties of the derivative alleles are considered with reference to these alternative modes of dominance of gai. Because these alleles are potential deletion or rearrangement mutations, together with the closely linked RFLP markers identified in the linkage mapping experiments, they provide useful resources for the isolation of the gai locus via a map-based cloning approach.     

The Effects of Chromosomal Rearrangements on the zeste-white Interaction in Drosophila melanogaster

Three gene systems have been shown to exhibit proximity-dependent phenotypes in Drosophila melanogaster: bithorax (BX-C), decapentaplegic (DPP-C) and white (w). In structurally homozygous genotypes, specific allelic combinations at these loci exhibit one phenotype, while in certain rearrangement heterozygotes the same allelic combinations exhibit dramatically different phenotypes. These observations have led to the suggestion that, through the process of somatic chromosome pairing, such loci are brought into sufficient proximity to permit effective passage of molecular information between homologues; rearrangement heterozygosity would then displace the homologues relative to one another such that this trans-communication is obviated. The genetic properties of the proximity-dependent allelic complementation (termed transvection effects) at the BX-C and DPP-C, are quite similar. Chromosomal rearrangements which disrupt transvection possess a breakpoint in a particular segment of the chromosome arm bearing the transvection-sensitive gene (arm 2L for the DDP-C and 3R for the BX-C); this segment of each arm has been termed the critical region by Lewis (1954). As determined by cytogenetic analysis of transvection-disrupting rearrangements, the critical regions for the BX-C and DDP-C transvection effects extend proximally from these loci for several hundred polytene chromosome bands (Lewis 1954; Gelbart 1982). The interaction between the zeste and white loci appears to depend upon the proximity of the two w+ alleles. By use of insertional duplications, displacement of w+ homologues has been shown to interfere with the zeste-white interaction. In contrast to transvection at bithorax and decapentaplegic, however, only breakpoints in the immediate vicinity of the white locus can disrupt the zeste-white interaction (Gans 1953; Green 1967; Gelbart 1971; this report). In this report, we investigate the basis for the difference in the size of the BX-C and DPP-C critical regions from that of white. We test and eliminate the possibility that the difference is due to the presence near the white locus of a site which mediates somatic chromosome pairing. Rather, our evidence strongly suggests that the zeste-white interaction is, at the phenotypic level, much less sensitive to displacement of the homologous genes than is transvection at either the BX-C or DPP-C. We also show that many of the breakpoints in the vicinity of the white locus do not behave as if they are disrupting a critical region for somatic chromosome pairing. Given these results, we suggest that the zeste-white interaction and transvection are two different proximity-dependent phenomena.     

A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants.

Previously we described the dose-response relationship for X-ray-induced mutation of the two homologous alleles of the thymidine kinase (tk) gene in a human lymphoblastoid cell line (Amundson and Liber, 1991). The two alleles were differentially mutable by X-rays, with one allele 6-10 times more mutable than the other. This difference was shown to be due to the virtual absence of the class of slow growth mutants from one allele. In the present report, restriction fragment length polymorphism (RFLP) analyses of informative markers along chromosome 17 have been used to delineate a region of chromosome 17 in which heterozygosity is lost with relatively high frequency among slow growth TK- mutants from the more mutable allele. However, loss of heterozygosity of this region has never been observed in normal growth mutants obtained from the more mutable allele, or in TK- mutants from the other, less mutable, allele. This may indicate the presence of a heterozygous essential gene on chromosome 17 distal to TK1.     

Chromosome Fragile Sites in Arabidopsis Harbor Matrix Attachment Regions That May Be Associated with Ancestral Chromosome Rearrangement Events

Mutations in the BREVIPEDICELLUS (BP) gene of Arabidopsis thaliana condition a pleiotropic phenotype featuring defects in internode elongation, the homeotic conversion of internode to node tissue, and downward pointing flowers and pedicels. We have characterized five mutant alleles of BP, generated by EMS, fast neutrons, x-rays, and aberrant T–DNA insertion events. Curiously, all of these mutagens resulted in large deletions that range from 140 kbp to over 900 kbp just south of the centromere of chromosome 4. The breakpoints of these mutants were identified by employing inverse PCR and DNA sequencing. The south breakpoints of all alleles cluster in BAC T12G13, while the north breakpoint locations are scattered. With the exception of a microhomology at the bp-5 breakpoint, there is no homology in the junction regions, suggesting that double-stranded breaks are repaired via non-homologous end joining. Southwestern blotting demonstrated the presence of nuclear matrix binding sites in the south breakpoint cluster (SBC), which is A/T rich and possesses a variety of repeat sequences. In situ hybridization on pachytene chromosome spreads complemented the molecular analyses and revealed heretofore unrecognized structural variation between the Columbia and Landsberg erecta genomes. Data mining was employed to localize other large deletions around the HY4 locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the BP/HY4 regions of A. lyrata and A. thaliana revealed that several chromosome rearrangement events have occurred during the evolution of these two genomes. Collectively, the features of the region are strikingly similar to the features of characterized metazoan chromosome fragile sites, some of which are associated with karyotype evolution.     

Characterization of New Punch Mutations: Identification of Two Additional Mutant Classes

The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis.     

reduced ocelli encodes the leucine rich repeat protein Pray For Elves in Drosophila melanogaster

The ocelli are three simple photoreceptors on the vertex of the fruit fly head. We sought to identify the gene encoded by the classical ocellar mutant, reduced ocelli (rdo). Deficiency and inversion breakpoint mapping and P-element induced male recombination analyses were performed and Pray For Elves (PFE; CG15151; Fbgn0032661) emerged as a promising candidate for the rdo phenotype. The PFE locus maps to polytene region 36E on chromosome 2L between elfless (Fbgn0032660) and Arrestin 1 (Fbgn0000120). FlyBase annotation predicts that PFE encodes a serine/threonine kinase, yet protein prediction programs revealed no kinase domain. These analyses suggest that PFE simply encodes a leucine rich repeat molecule of unknown function, but presumably functions in nervous system protein-protein interaction. Two classical spontaneous alleles of rdo, rdo(1) and rdo(2), were characterized and the underlying mutations result from a small deletion spanning exon 1/intron 1 and a B104/roo insertion into the 3'UTR of PFE, respectively. Transposase-mediated excisions of several P-elements inserted into the PFE locus revert the rdo phenotype and a full-length PFE cDNA is sufficient to rescue rdo. A Gal4 enhancer trap reveals a broad adult neural expression pattern for PFE. Our identification and initial characterization of the rdo locus will contribute to the understanding of neurogenesis and neural development in the simple photoreceptors of the Drosophila visual system.