Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.     

Polymorphism of the Hereditary Sigma Virus in Natural Populations of DROSOPHILA MELANOGASTER

Previous studies have shown that, in natural French populations of Drosophila melanogaster, 10 to 20% of the flies are infected by the noncontagious, hereditary rhabdovirus sigma responsible for CO₂ sensitivity. These populations are also polymorphic for two alleles [ref(2)P^o and ref(2)P^p] of a gene for resistance to the sigma virus. Evidence is given here that two viral genetic types, differing in their response to the ref(2)P^p allele, are present in these populations of flies; the most common type is only slightly sensitive to the ref(2)P^p allele.     

Drosophila genes which intervene in multiplication of sigma virus (author''s transl)]

Summary Distinction between Drosophila strains, differing their capacity for supporting multiplication of sigma virus, arises essentially from comparison of the incubation time after inoculation of a viral suspension. This is the most general and the most useful characteristic. By this mean five allelic differences with the reference Drosophila strain Oregon have been found. Corresponding genes, ref(1)H, ref(2)M, ref(2)P, ref(3)O and ref(3)D are located all over Drosophila chromosomes. The specific spectra of viral strains sensitive to the one or the other allele was determined for each gene.Some characteristic properties of flies in which the virus has been brought by injection or heredity were compared between heterozygotes and homozygotes for the permissive and for the non permissive allele:time of incubation as a function of the size of the inoculum,probability of initiating infection,kinetics of the virus multiplication in inoculated fly,efficiency of a viral genome brought by a spermatozoa in infecting an egg,perpetuation of the carrier state of sigma virus in germ line cells of stabilized females or males and in somatic cells.The properties concerning the perpetuation of sigma virus carrier state allow to distinguish two classes of viral functions in which the considered ref gene product can intervene: 1) functions necessary for viral genome replication and, of course, for perpetuation of carrier state, 2) other functions, (late functions — necessary for maturation - and functions necessary for cell penetration of inoculated virus).Homozygotes for each of the two alleles of a gene which acts on incubation time can show no difference in one property which is specific of a differenciated cell type only because the considered gene is not expressed in the cell type involved. Conversely genes can exist which act on such a property and which have no action on incubation time. Probably such a gene has been discovered; it intervenes in the transmission of sigma virus by stabilized males; this gene is named ref(3)V.Discussion of all the properties of flies homozygotes for each allele permits us to conclude that ref(1)H, ref(2)M, ref(2)P, ref(3)D and possibly ref(3)V genes (if this last gene intervenes directly in sigma's physiology) are involved in a function necessary for replication. No conclusive evidence has been found for ref(3)O, still it seems to intervene in a late function. Problems of functional interactions between products of the first five ref genes have been mentioned.     

Perpetuation of the hereditary sigma virus in populations of its host,Drosophila melanogaster. Geographical analysis of correlated polymorphisms

In natural populations of Drosophila melanogaster, about 10% of the individuals are infected by a virus, sigma, which is not contagious but is transmitted through gametes. These populations are also regularly polymorphic for two alleles, O and P, of a locus ref(2)P; the P allele interferes with the multiplication of the virus. Two viral Types are found in populations, differing in their sensitivity to the P allele. Many samples of flies have been collected in different parts of the world and for each of them, the P frequency has been measured and the viral Type determined. A clear geographical differentiation appears for both these traits; they present a mutual adaptation leading to relatively low frequencies of infected flies in natural populations. Most viruses are only known from highly selected laboratory strains. The observations reported in this paper give evidence of the self restraint exercised by the sigma virus at the population level; they indicate that the characteristics of wild viral clones are likely to differ from those of laboratory strains and also from one population to another.The sigma virus is comparable to other genetical elements, that can be more efficiently transmitted than a mendelian allele, such as transposable elements. The discussion illustrates some of the factors involved in the perpetuation of such elements in a population and points out the difficulty of taking them all into consideration in theoretical models dealing with their perpetuation.     

Disease association mapping in Drosophila can be replicated in the wild

Association and linkage mapping have become important tools in understanding the genetics of complex traits, including diseases in humans. As the success of association mapping is reduced by small effect sizes and limited power, linkage studies in laboratory-based model systems are still heavily used. But whether the results of these studies can be replicated in natural populations has been questioned. Here, we show that a polymorphism in the gene ref(2)P, which had previously been linked to sigma virus resistance in Drosophila melanogaster under laboratory conditions, also provides resistance against the virus in female flies in a wild population in the field. This genetic association is thus upheld in spite of a known genotype-by-genotype interaction and environmental variation.     

On the maintenance of the polymorphism at the ref(2)P locus in populations of Drosophila melanogaster

Polymorphism for two alleles of the ref(2)P locus is a very constant feature of French natural populations of Drosophila melanogaster. One of these alleles interferes with the multiplication of the hereditary sigma virus in the fly. An equilibrium, quite similar to the natural one, has been observed previously in experimental populations, whether the sigma virus is present or not. Evidence is given that one of the selection components involved in the maintenance of this equilibrium affects adult stages when flies have not suffered severe larval competition. In conditions of severe larval competition, a maternal effect seems to be involved in the differential egg-to-adult viability of heterozygotes.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at?>50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency.     

The age and evolution of an antiviral resistance mutation in Drosophila melanogaster

What selective processes underlie the evolution of parasites and their hosts? Arms-race models propose that new host-resistance mutations or parasite counter-adaptations arise and sweep to fixation. Frequency-dependent models propose that selection favours pathogens adapted to the most common host genotypes, conferring an advantage to rare host genotypes. Distinguishing between these models is empirically difficult. The maintenance of disease-resistance polymorphisms has been studied in detail in plants, but less so in animals, and rarely in natural populations. We have made a detailed study of genetic variation in host resistance in a natural animal population, Drosophila melanogaster, and its natural pathogen, the sigma virus. We confirm previous findings that a single (albeit complex) mutation in the gene ref(2)P confers resistance against sigma and show that this mutation has increased in frequency under positive selection. Previous studies suggested that ref(2)P polymorphism reflects the progress of a very recent selective sweep, and that in Europe during the 1980s, this was followed by a sweep of a sigma virus strain able to infect flies carrying this mutation. We find that the ref(2)P resistance mutation is considerably older than the recent spread of this viral strain and suggest that—possibly because it is recessive—the initial spread of the resistance mutation was very slow.     

Differential mitotic rates and patterns of growth in compartments in the Drosophila wing

In the wing disks of Drosophila slowly dividing cells of Minute mutations are progressively eliminated from Minute/Minute⁺ mosaic compartments by a process known as cell competition. From a study of two different Minutes we show here that the intensity of competition is greater in the more extreme Minute with the slowest rate of cell division. The way in which the more rapidly growing Minute⁺ clones grow and overcome the surrounding Minute cells is described and cell competition is shown to be a result of local interactions between slow- and faster-growing cells.     

A new strain of Drosophila that inhibits the development of symptoms in imagoes infected with sigma virus

A strain of Drosophila melanogaster bearing the mutant gene ebony has been found to slow the development of symptoms (carbon dioxide sensitivity) in adult flies inoculated with sigma virus, a member of the rhabdovirus group. This inhibition is made evident by comparing mean incubation times of the virus in ebony and wild-type (Oregon) flies. The increase in mean incubation time in ebony flies has ranged from about 3 to 8 days, depending on the virus strain, amount of virus injected, and the age of the flies at the time of inoculation. This delay in development of symptoms appears to be due to a dominant autosomal gene, although further work is needed to confirm this. When accumulation of infectious virus after inoculation is compared in ebony and Oregon flies, there seems to be no inhibition of multiplication in ebony at the level of the entire fly. The relationship of this work to current theories on the mechanism of symptom production by sigma virus is discussed.     

Localization of domains within the Drosophila Ref(2)P protein involved in the intracellular control of sigma rhabdovirus multiplication.

The ref(2)P gene of Drosophila melanogaster interferes with sigma rhabdovirus multiplication. This gene is highly variable, and the different alleles are considered permissive or restrictive according to their effects on virus replication. In all cases, the mechanisms involve intracellular interactions between the sigma virus and Ref(2)P proteins. We showed that the N-terminal domain of the Ref(2)P protein was required for its activity in vivo. The protein was inactive in the null p(od)2 mutant when its first 82 amino acids were deleted. The p delta n gene was constructed so that the first 91 amino acids coded for by the restrictive alleles could be expressed in vivo. It was active in a transformed line. This sequence was sufficient to impart a restrictive phenotype to an adult D. melanogaster fly after it was injected with the virus. However, the truncated protein expressed by p delta n did not have an effect on the hereditary transmission of the sigma virus to the offspring of the infected flies, even though it contained the restriction site. The native Ref(2)P protein has been previously shown to have conformation-dependent epitopes common with some of those of the viral N protein. We demonstrated the following. (i) These epitopes were found in a domain of the Ref(2)P protein distinct from the site involved in restriction. (ii) They were modified in the N protein of the haP7 sigma virus mutant selected as being adapted to the restrictive alleles of the ref(2)P gene; only one mutation in the N gene, leading to an amino acid substitution, distinguished the haP7 mutant from the original virus. (iii) The virus strains partially or totally adapted to the effects of the full restrictive protein expressed by pp were always found to multiply to a lesser extent in the presence of the protein expressed by p delta n. These data suggest that two distinct domains of the Ref(2)P protein are involved in the control of sigma virus multiplication.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at >50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency. Received: 20 January 1997 / Accepted: March 3 1997     

Control of sigma virus multiplication by the ref(2)P gene of Drosophila melanogaster: an in vivo study of the PB1 domain of Ref(2)P

Ref(2)P has been described as one of the Drosophila proteins that interacts with the sigma virus cycle. We generated alleles to identify critical residues involved in the restrictive (inhibiting viral multiplication) or permissive (allowing viral multiplication) character of Ref(2)P. We demonstrate that permissive alleles increase the ability of the sigma virus to infect Drosophila when compared to null alleles and we confirm that restrictive alleles decrease this capacity. Moreover, we have created alleles unfunctional in viral cycling while functional for Ref(2)P fly functions. This type of allele had never been observed before and shows that fly- and virus-related activities of Ref(2)P are separable. The viral status of Ref(2)P variants is determined by the amino-terminal PB1 domain polymorphism. In addition, an isolated PB1 domain mimics virus-related functions even if it is similar to a loss of function toward fly-related activities. The evolutionary tree of the Ref(2)P PB1 domain that we could build on the basis of the natural allele sequences is in agreement with an evolution of PB1 domain due to successive transient selection waves.     

Association between HLA-DRB1, HLA-DRQB1 alleles,and CD4<Superscript>+</Superscript>CD28<Superscript>null</Superscript> T cells in a Chinese population with coronary heart disease

CD4⁺CD28^null T cells are present in increased numbers in the peripheral blood of patients with acute coronary syndrome. However, the triggers of expansion of these cells are unclear. Susceptibility to coronary heart disease (CHD) is strongly associated with alleles of human leukocyte antigen (HLA), but it is not equally strong in different human populations. The objective of the study was to investigate association between CD4⁺CD28^null T cells and HLA-DRB1 alleles. The HLA alleles were determined by polymerase chain reaction with sequence-specific primers (PCR-SSP) method, in a group of CHD patients and control subjects from the same area. The number of CD4⁺CD28^null T cells was measured using the flow cytometry technique. The HLA-DRB1*01 (RR = 4.705, P < 0.005) and DRB1*04 (RR = 3.554, P < 0.005) alleles showed the strongest association with CHD in the Chinese population, and increased numbers of CD4⁺CD28^null T cells were found in association with HLA-DRB1*04 (17.1%) and DRB*01 (12.9%), while decreased numbers of CD4⁺CD28^null T cells were present in subjects with DRB1*15 (0.8%). CHD in Chinese population is strongly associated with HLA-DRB1*01 and DRB1*04 haplotypes, and formation of CD4⁺CD28^null T cells was related to HLA-DRB1*01, DRB1*04, and DRB1*15 alleles.     

Extracellular spreading of Wingless is required for Drosophila oogenesis

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.     

Inactivation of Bacteriophage T4 by Ethyl Methanesulfonate: Influence of Host and Viral Genotypes

Inactivation of bacteriophage T4 by ethyl methanesulfonate (EMS) is a complex process which depends critically upon the conditions of treatment and upon both the viral and the host genotypes. EMS-inactivated particles are capable of multiplicity and cross-reactivation, indicating the need for caution in using EMS in certain types of mutation studies. The pyrimidine dimer excision systems of the phage and the host do not affect the EMS sensitivity of T4, but the T4x⁺y⁺ system does. Mutational defects in the deoxyribonucleic acid (DNA) ligase and the DNA polymerase systems both of the virus and of its host also affect viral EMS sensitivity.     

DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(o^c) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(o^c) alleles fully suppress the phenotype of constitutively SOS-“off” lexA(Ind⁻) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(o^c) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition.     

Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val¹⁰⁹Ile, Asp³³⁵Glu and Asn³⁸⁴Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

Immunological cross-reactions and interactions between the Drosophila melanogaster ref(2)P protein and sigma rhabdovirus proteins.

The ref(2)P gene is one of the Drosophila melanogaster genes involved in the inhibition of sigma rhabdovirus multiplication. The partial restriction of viral replication varies according to the ref(2)P alleles and virus strains and involves intracellular interactions between parasite and host products. We identified the protein encoded by the ref(2)P gene and produced polyclonal antibodies directed against the whole ref(2)P protein obtained from a recombinant baculovirus and against a part of the protein expressed as a fusion protein. These antibodies were used to study the interactions with sigma virus proteins by different immunoprecipitation techniques. We showed that the native ref(2)P protein shared conformation-dependent common epitopes with the viral structural genome-associated N protein. Furthermore, the cellular protein was found to be associated in complexes with the viral P protein required for RNA polymerase activity. The significance of these observations in the control of sigma virus multiplication by its host is discussed.