Haltere determination and mutations at the bithorax locus

Mutations at the bithorax locus transform anterior haltere tissue into anterior wing. These transformations could in principle be due to the mutations altering either the expression or cell heredity functions of determination. I have studied two alleles of the bithorax locus bx³ and bx^34e using disc culture techniques and found that both produce their transformations by altering the expression of the determined state. I have also found that the expression of the temperature-sensitive allele, bx^34e, can be altered by temperature shifts during the culture period. Evidence has been obtained that suggests that such changes in expression do not require growth or cell division.     

Preblastoderm mosaics of mutants of the bithorax-complex

Summary InDrosophila melanogaster, segmental specification takes place in groups of cells around the blastoderm stage. This segmental specification requires the function of the genes of the bithorax-complex. We have studied preblastoderm mosaics (gynandromorphs) of mutant (bx ³,pbx, Ubx, Ubx ⁸⁰) and wildtype (heterozygotes for these alleles) cells. The results show a total cell autonomy in the differentiation of both wildtype and homoeotially transformed cells. However, several unexpected phenotypes were found. They are discussed in terms of the function of the bithorax genes and early interactions between mutant and wildtype territories.     

How do single homeotic genes control multiple segment identities?

Each of the homeotic genes of the bithorax complex of Drosophila defines the identities of more than one body segment. The mechanisms by which this occurs have been elusive. In a recent report, Castelli-Gair and Akam⁽¹⁾ analyze in detail the control of parasegment 5 and parasegment 6 identities by the bithorax complex gene Ubx. Their results indicate that differences in the spatial and temporal expression patterns of Ubx are critical in determining differences between these parasegments. However, dose effects observed by others indicate that parasegment-specific differences in the level of Ubx expression are also important. For the other BX-C genes, parasegment-specific expression of protein isoforms, or combinatorial control dependent on the expression patterns of other spatially restricted regulators, may also play a role.     

Dietary fatty acid effects on morphogenesis in bithorax mutants

Three bithorax alleles of Drosophila melanogaster were tested to determine if dietary additions of fatty acids would alter their gene expression. For the bx¹ allele, myristic, oleic, and linoleic acids were all effective in reducing gene expression while fatty acid supplementation was ineffective with the bx³ and bx^34e alleles. For bx¹ the nutritionally sensitive period was found to occur in the first 48 h of larval life.     

Determination of the shape of the operculum by the bithorax complex in Drosophila melanogaster

Summary We have studied the course of the operculum line in the larval hypoderm of several bithorax complex mutants of Drosophila melanogaster. The bifurcation of the line, a characteristic of the first abdominal segment in wild-type (A₁), can also appear in the metathoracic (T₃) and other abdominal segments (A₂, A₃) depending on mutations within the bithorax complex. Therefore, we concluded that the course of the operculum line and thus the shape of the operculum is not determined by a suprasegmental gradient of positional information but by the functional state of the genes of the bithorax complex in each metamere. The dorsal and ventral branches of the operculum line react differently, the dorsal branch being more sensitive to the effect of loss of function mutations (bxd, iab-2 ^k), the ventral branch more affected by gain of function mutations (Hab). In some cases the effects of the mutations on the operculum line differed from those in the adult, suggesting a difference in sensitivity of larval hypodermal cells and histoblast cells to the functional gene products of the bithorax complex.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

Positional information in imaginal discs transformed by homoeotic mutations

Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx ³ orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.     

Mutational dissection of gene expression in the abdominal region of the bithorax complex of Drosophila in imaginal tissue

Summary The phenotypic effects in imaginal hypodermal tissue of a number of Abdominal-B mutations of the bithorax complex are described. Evidence is given from complementation analysis that the phenotypic heterogeneity in both the spatial limits and the nature of the homeotic transformations produced is not an arbitrary classification of allelic differences that we find. We have used genetic mosaic analysis to support the interpretation that the Abdominal-B genetic unit can exist in a number of alternative functional states of expression during development and that individual Abdominal-B mutations may abolish some states whilst leaving others unaffected.     

Role of Zeste in transvection at the Bithorax locus of Drosophila

Summary We have re-examined the effect of the mutation z^a in the zeste locus on the phenotype of the bx ^34e /Ubx trans combination of bithorax mutations, in the presence and absence of rearrangements which presumably affect homologous pairing in the bithorax region. Our observations suggest the possibility of the zeste ⁺gene product being needed for transvection at the bithorax locus     

TheUbx syndrome ofDrosophila: the prothoracic transformation (ppx) is independent ofbx,bxd andpbx

Summary If, early in development, theUbx ⁺ gene is removed by mitotic recombination from cells of the meso-and metathoracic leg primordia, theseUbx ^– cells develop as in the posterior prothoracic leg. We show that this transformation, termedpostprothorax, is a discrete genetic function that is independent of other homeotic transformations such asbx, pbx orbxd, which also result from the inactivation of theUbx gene.     

Positive and negative cis-regulatory elements in the bithoraxoid region of the Drosophila Ultrabithorax gene

Summary The Ultrabithorax (Ubx) gene is required during embryogenesis and larval development to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid (bxd) region, a 40 kb DNA stretch upstream of the Ubx promoter, affect cis-regulatory elements responsible for the ectodermal expression of the Ubx gene in the posterior compartment of the third thoracic segment and anterior compartment of the first abdominal segment. Our genetic data and the available molecular information are used to map the adult epidermal cis-regulatory elements within the bxd region. Genetic combinations involving mutations affecting the bxd region show that (1) redundant or cooperatively acting sequences are required for Ubx gene expression in the anterior compartment of the first abdominal segment, and (2) the expression of Ubx in the posterior compartment of the third thoracic segment is modulated by positive and negative cis-regulatory elements.The Wellcome Trust CRC Institute for Cancer Research and Developmental Biology, Tennis Court Road Cambridge, CB2 1QR, UKDivision de Genética, Departamento de Genética Molecular y Microbiología, Campus de San Juan, Apdo. 374, 03080 Alicante, Spain     

Interactions of thePolycomb group of genes with homeotic loci ofDrosophila

Summary Members of thePolycomb (Pc) group of genes are required for the correct determination of segment identity, and are thought to be negative regulators of thebithorax andAntennapedia complexes. This hypothesis has been tested molecularly for only some members of thePc group. Here, we examine the distribution ofUltrabithorax (Ubx),Antennapedia (Antp), andSex combs reduced (Scr) proteins in the epidermis, central nervous system, and midgut of embryos homozygous for mutations in tenPc group genes. We show that zygotic loss of mostPc group genes causes ectopic expression ofUbx andAntp, but that there are differences in time and tissue-specificity. FivePc group mutations lack midgut constrictions and also exhibit ectopic or suppressedUbx expression and suppression ofAntp expression. Distribution ofAntp is upset earlier than distribution ofUbx in the central nervous system of everyPc group mutant affecting both genes. Loss of the zygotic products ofPolycomb, extra sex combs, andAdditional sex combs cause ectopic expression ofScr in epidermis, and allPc group genes exceptPsc have suppressedScr expression in the nervous system. These results are discussed with respect to the function of thePc group.     

Lethal bithorax complex mutations of Drosophila melanogaster show no germ line maternal effects

Three Ultrabithorax (Ubx) alleles and three different deficiencies of the bithorax complex (BX-C) of Drosophila melanogaster have been tested for maternal effects in the germ line. The dominant female sterile technique was used. The Ubx alleles and a deletion of the abdominal region of the BX-C are homozygous viable in germ line clones and show no maternal effects. Two deletions which lack the proximal portion of the BX-C are lethal in the female germ line indicating either that these deficiencies lack genes apart from the BX-C that are necessary for fertility or that there are BX-C genes that are essential for normal maternal germ line function. The significance of the bias in the isolation of only zygotic mutations at the BX-C are discussed with respect to these results.     

Ethanol-hypersensitive and ethanol-dependent cdc? mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^– mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^– mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^– phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Novel genes influencing the expression of the yellow locus and mdg4 (gypsy) in Drosophila melanogaster

Summary We used a system with a mobilized Stalker transposable element, sometimes in combination with P-M hybrid dysgenesis, in the search for new mutations interfering with the y ² mutation induced by mdg4 (gypsy) insertion into the yellow locus. A novel gene, modifier of mdg4, was detected in chromosome 3. The mutation mod(mdg4) either enhanced or suppressed phenotypic changes in different mutations induced by mdg4 insertions. Thus, mod(mdg4) seems to be involved in the control of mdg4 expression. Six other loci designated as enhancers of yellow were also detected. The e(y) ⁿ (with n from 1–6) mutations enhanced the expression of several y mutations induced by different insertions into the yellow locus. The major change is a damage of bristle and hair pigmentation which is not suppressed by su(Hw) mutations. On the other hand, e(y) ⁿ alleles do not interact with mdg4 induced mutations in other loci. All e(y) ⁿ genes are located in different regions of the X chromosome. One may speculate that e(y) ⁿ genes are involved in trans-regulation of the yellow locus and possibly of some other loci.     

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm

In this study we describe the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein^gürtelchen (hkb^gurt) mutant embryos.It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. Our analyses of the visceral phenotype of hkb^gurt mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.     

Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12

Summary Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA ⁺ gene product. The requirement of SDR for recA ⁺ can be suppressed by rin mutations (for recA⁺-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA ⁺-dependent SDR seen in rnh ^- rin⁺ lexA⁺ strains, recA ⁺-independent in rnh ^- rin^- lexA⁺, and recA ⁺-independent in rnh ^- rin⁺ lexA(Def). The expression of SDR in rin ^- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA ⁺ requirement by rin mutations was shown to depend on some new function of the recF ⁺ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF ⁺. The lexA3 mutation inhibited recA ⁺-dependent SDR via reducing the amount of recA ⁺ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh ^- rin^- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA ⁺-regulated gene product in the recA ⁺-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA ⁺, rin⁺ and recF ⁺ genes.     

Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: Implications for cdc2+ protein structure and function

Summary The cdc2 ⁺ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2 ⁺ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughtout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2 ⁺ activity and regulation. These include regions which may be involved in the interaction of the cdc2 ⁺ gene product with the proteins encoded by the wee1 ⁺, cdc13 ⁺ and suc1 ⁺ genes.     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.