Genetic interactions between the Polycomb locus and the Antennapedia and Bithorax complexes of Drosophila

Summary We have studied the embryonic and adult phenotypes of genetic combinations between Polycomb (Pc), Regulator of bithorax (Rg-bx) and the genes of the Bithorax complex (BX-C) and the Antennapedia complex (ANT-C). The products of Pc and Rg-bx genes act antagonistically, their mutant combinations leading to the ectopic expression of genes of the BX-C and ANT-C. The genetic analysis of the Pc locus suggests it is a complex gene. Pc⁺ products behave as members of a regulatory set that negatively control the expression of BX-C and ANT-C genes. Genetic combinations between different doses of Pc, Rg-bx and the genes of the BX-C and ANT-C have phenotypes which may be interpreted as resulting from ectopic derepression of posterior selector genes repressing selector genes of anterior segments. The transformation phenotypes of certain genetic combinations differ in embryos and adults. A model of regulation of the BX-C and the ANT-C genes during the imaginal cell proliferation is presented, in which the specification state is maintained by self-activation of a given selector gene and down modulation of other selector genes in the same cell.     

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.     

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.     

Maternal and zygotic requirement for thepolyhomeotic complex genetic locus inDrosophila

Summary The complex genetic locuspolyhomeotic (ph) is a member of thePolycomb (Pc)-group of genes and as such is required for the normal expression of ANT-C and BX-C genes. It also has probably other functions since amorphicph alleles display a cell death phenotype in the ventral epidermis of 12-h-old embryos. Here it is shown that lethal alleles ofph (amorph and strong hypomorph) show transformation of most of their segments towards AB8. Theph ⁺ product is required autonomously in imaginal cells. The total lack ofph ⁺ function prevents viability of the cuticular derivatives of these cells.ph has a strong maternal effect on segmental identity and epidermal development that can not be rescued by one paternally supplied dose ofph ⁺ in the zygote. These phenotypes differ substantially from those of previously describedPc-group genes. AmongPc-group genes,ph seems to be the only one that is strongly required both maternally and zygotically for normal embryonic development.     

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.     

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos

Summary spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)⁺ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)⁺ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)⁺ RNA. Mosaic females containing spz germ cells surrounded by spz ⁺ tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line.     

Effects of All-trans retinoic acid on germ cell development of embryos and larvae of the giant freshwater prawn, Macrobrachium rosenbergii

Embryos of the giant freshwater prawn, Macrobrachium rosenbergii, were treated with 1, 10 or 50 μg ml⁻¹ all-trans retinoic acid (AtRA) for 2 days. Survival and hatching rates were not affected. However, an increase in the number of primordial germ cells (PGCs), progenitors of gametes, and a slightly more advanced stage of the gonads were found in those treated with 10 or 50 μg ml⁻¹ AtRA. Newly hatched larvae were treated with 0.1, 0.5 or 1 μg ml⁻¹ AtRA for 2 days. Survival rates were lower in those treated with 0.5 or 1 μg ml⁻¹ AtRA; nevertheless, the gonads were slightly more developed. The results indicated that AtRA, an active metabolite of vitamin A, affected germ cell and gonad development of embryos and the larvae of giant freshwater prawn.     

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

When the control is lacking—the role of tumour suppressor genes in cancer development

The potential to genetically dissect tumorigenesis provides the major reason to study this process in the fruit flyDrosophila. Over the last 30 years genetic analysis has identified some 55 genes in which recessive mutations cause the appearance of specific tumours during development in tissues such as the imaginal discs, the brain hemispheres, the hematopoietic organs or the gonads, Since the normal allele acts dominantly over the mutated allele, these genes are designated as tumour suppressor genes. The estimate of the number of genes that can be mutated to tumour formation may be, however, much higher ranging between I00 to 200. The challenge before this field is how best to identify these genes and elucidate their function. Current molecular procedures, such as mutagenesis mediated by P-element transposon, provide new ways for tagging any gene of interest inDrosophila and thus for cloning it rapidly. Function of the gene product can be inferred by comparing its amino acid sequence with sequences of proteins with known function or can be determined by histochemical and biochemical investigations. Progress in the understanding of tumour suppression inDrosophila is most advanced in the case of genes regulating cell growth in imaginal discs. The imaginal discs are small groups of cells displaying a strong apical-basal polarity and form folded sacs of epithelia which grow throughout the larval life and give rise to the adult tegument during metamorphosis. Tumour suppressor genes regulating cell growth of imaginal discs, such as thelethal(2)giant larvae (l(2)g1),lethal(1)discs large-1 andexpanded genes, were found to encode proteins localized in domains of cell to cell contact on the plasma membrane and were thus thought to maintain cell adhesion. However, recent studies of l(2)gl have revealed that the l(2)gl protein is a component of the normal cytoskeleton which can participates to the cytoskeletal matrix underlaying the plasma membrane. These findings indicate that the changes in cell shape and the loss of apical-basal polarity in imaginal disc cells result primarily from alterations in the cytoskeleton structure. Furthermore the neoplastic growth of the mutated cells may be caused by the disorganization of an intracellular communication system that ultimately controls cell proliferation and/or cell differentiation.     

A gene that regulates the bithorax complex differentially in larval and adult cells of Drosophila

Loss-of-function mutations of a new homeotic gene, sxc, in Drosophila cause transformations of body segments, suggesting inappropriate expression of BX-C and ANT-C genes. I present evidence that sxc+ is required during embryogenesis for the selective repression of the BX-C in different larval segments and show that this requirement may be met entirely by maternally derived gene product. sxc+ is also required later in development to ensure the appropriate expression of ANT-C and BX-C genes in adult thoracic and abdominal segments. Absence of sxc+ in the mesothorax apparently results in the ectopic expression of the bx+ (or Ubx+) function in both the anterior and posterior compartments; this suggests that pbx mutations may define a regulatory rather than a structural function.     

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.     

Five novel elements involved in the regulation of mitosis in fission yeast

Summary Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 weel ^ts cdc25^ts triple mutant strains. These genes were designated wisl ⁺ –wis5⁺for win suppressing, and do not correspond to winl ⁺ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25 ^ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wisl ⁺ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4 ⁺ appears to be a specific suppressor of the winl-1 mutation. wis2 ⁺ and wis3 ⁺ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1 ^ts and cdc25 ^ts, suggesting that the wis2 ⁺ and wis3 ⁺ products may interact with elements central to the mitotic control.     

低H~+_-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析

【目的】筛选H~+_-ATPase活性降低的植物乳杆菌突变菌,比较其与亲本菌基因表达水平的差异,进一步探索H~+_-ATPase的调控机制。【方法】利用硫酸新霉素诱变、筛选突变菌,并对亲本菌(ZUST)和突变菌(ZUST-1、ZUST-2)进行生长、产酸能力及H~+_-ATPase活性的测定。分别提取亲本菌和突变菌的基因组DNA,扩增H~+_-ATPase全部编码基因并测序。通过荧光定量PCR对H~+_-ATPase全部编码基因进行相对定量分析。【结果】突变菌的生长和产酸能力均低于亲本菌,突变菌ZUST-1和ZUST-2的H~+_-ATPase活性比亲本菌分别降低了10.1%和28.8%。突变菌ZUST-1和ZUST-2的atp A基因均有22个位点发生突变,而ZUST-2的atp C基因有6个位点发生突变。突变菌ZUST-1和ZUST-2的atp A在对数期基因表达水平分别比亲本菌ZUST下调了41.1%和35.7%,在稳定期分别下调了43.6%和14.2%;ZUST-1的atp C基因在对数期的表达水平比ZUST略高,在稳定期比ZUST上调了30%,而ZUST-2的atp C基因未表达。【结论】突变菌H~+_-ATPase活性减弱会导致其全部编码基因在稳定期表达水平上调(除ZUST-2的atp C不表达外),而且atp A和atp C基因突变导致的基因表达水平的差异是影响H~+_-ATPase活性的主要因素,此研究结果为进一步研究植物乳杆菌中H~+_-ATPase的调控机制奠定了基础。     

Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes

Summary Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod^-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix⁺ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix^- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod^- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix⁺ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.     

Polyhomeotic: A gene of Drosophila melanogaster required for correct expression of segmental identity

Summary A new locus in Drosophila melanogaster that is required for the correct expression of segmental identity has been discovered. The new locus, termed polyhomeotic (ph), is X-linked and maps cytologically to bands 2D2-3. Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANT-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb (Pc), Polycomblike (Pcl), and extra sex comb (esc), and acts as a dominant enhancer of Pc. The expression of ph depends on the ANT-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph ² show that the ph ⁺ product is required during embryogenesis and larval development. We propose that ph mutants in some way disrupt the normal expression of the ANT-C and BX-C, and, therefore, that ph ⁺ is needed for maintenance of segmental identity.     

Genetic interactions and dosage effects of Polycomb group genes of Drosophila

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc ⁺ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.     

The temperature-sensitive mutation vir ts(virilizer) identifies a new gene involved in sex determination of Drosophila

Summary When XX animals homozygous for the temperature-sensitive mutation vir ^tsof virilizer (2–103.9) are raised at the restrictive temperature of 29° C, they are transformed into sterile intersexes with a morphology comparable to XX flies mutant at the sex-determining gene doublesex (dsx). The gonads of the vir ^tsintersexes are ovaries in which the germ cells undergo abortive oogenesis. At the permissive temperature of 25° C or below, XX vir ^tsanimals develop into marginally fertile females. The temperature-sensitive period of vir ^tsis within the third larval instar. XY males are not affected by the mutation. Animals that are homozygous for vir ^tsand either transformer (tra) or tra2 develop as pseudomales; on the other hand, constitutive expression of a female-specific tra product rescues XX animals from the effect of vir ^ts, but these females are sterile. The data show that tra and tra2 are epistatic to vir. Animals with only one wildtype copy of either tra or tra2 and mutant for vir ^tsare already transformed into intersexes at 25° C. Conversely, the presence of three copies of the tra ⁺ gene largely prevents the effect of vir ^tsat 29° C; such flies are practically female, but sterile. Animals homozygous for vir ^tsand heterozygous for dsx ^D/+, raised at 29° C, are transformed into severely masculinized intersexes or almost pseudomales. The observations suggest that vir acts above and via tra and tra2 to achieve proper female-specific expression of the dsx gene in XX zygotes. Offprint requests to: R. Nöthiger     

Larval sex identification in the paper wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis> (Vespidae,Hymenoptera)

Identifying the sex of larvae is important in social Hymenoptera. Until now for Polistes wasps it has been necessary to genotype larvae at microsatellite loci, and assign their sex based on homozygosity at these loci. In our study on the paper wasp Polistes dominulus we have found morphological differences between larval sexes that can be used for larvae from the 3^rd instar on to easily and cheaply identify larval sex: the external gonopore and the shape and size of larval gonads. The robustness of these indicators was supported by genotype data at four microsatellite loci. Using gonopore and gonad features for sex assignment will assign diploid males as males, unlike techniques based on genetic loci or chromosomes. Received 12 July 2006; revised 4 January 2007; accepted 5 February 2007.