cDNA sequence of four purine nucleoside phosphorylase (Np) alleles in the mouse

The molecular basis of four electrophoretic and activity variants of purine nucleoside phosphorylase in the mouse was examined by amplification and sequence analysis of cDNA. Compared with the cDNA coding sequence for C3H/HeHa designated Np ^a , there were five nucleotide changes for C57BL/6J, Np ^b ; three for MOLF/Ei, Np ^c ; and five for SPRET-1, Np ^d . There was only a single codon change between Np ^a and Np ^b , the deduced substitution of threonine 176 by serine. Similarly, there was only a single codon change between Np ^a and Np ^c , resulting in substitution of methionine 258 by lysine. There were three codon changes between Np ^a and Np ^d , resulting in substitution of glutamate 22 by lysine, threonine 39 by alanine, and aspartate 152 by glutamate. These amino acid substitutions-neutral to neutral, neutral to basic, and acidic to basic—are in agreement with the electrophoretic properties of the gene products of Np ^a relative to Np ^b , Np ^c , and Np ^d previously described by isoelectric focusing. Codon differences were confirmed by PCRRFLP or single nucleotide primer extension analysis and extended to include the assignment of other strains as Np ^a : C3H/HeHa, DBA/2J, CLA, Posch-2; or Np ^b : C57BL/6J, C57L/J, C58/J. Both RFLP analysis of amplified genomic DNA and Southern analysis are consistent with single but unique Np alleles present in the C3H/HeHa and C57BL/6J genomes. As these data do not support the previous two-loci, Np-1 and Np-2, classification, we propose and employ a new single locus multiple allele classification for Np on the basis of the sequence analysis.     

Nocardia rhamnosiphila sp. nov., isolated from soil

In this study two actinomycete strains were isolated in Cape Town (South Africa), one from a compost heap (strain 202GMO^T) and the other from within the fynbos-rich area surrounded by the horseracing track at Kenilworth Racecourse (strain C2). Based on 16S rRNA gene sequence BLAST analysis, the strains were identified as members of the genus Nocardia. Phylogenetic analysis showed that the strains clustered together and are most closely related to Nocardia flavorosea NRRL B-16176^T, Nocardia testacea JCM 12235^T, Nocardia sienata IFM 10088^T and Nocardia carnea DSM 43397^T. This association was also supported by gyrB based phylogenetic analysis. The results of DNA–DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of both strains 202GMO^T and C2 from related species. However, their high DNA relatedness showed that they belong to the same species. Strain 202GMO^T was selected as the type strain to represent this novel species, for which the name Nocardia rhamnosiphila is proposed (=DSM 45147^T = NRRL B-24637^T).     

Monoclonal antibody 7D5 recognizes the R147 epitope on the gp91phox,phagocyte flavocytochrome b558 large subunit

Human phagocyte flavocytochrome b₅₅₈ (Cyt b), the catalytic center of nicotinamide adenine dinucleotide phosphate oxidase, consists of a heavily glycosylated large subunit (gp91^phox; Nox2) and a small subunit (p22^phox). Cyt b is a membrane‐spanning complex enzyme. Chronic granulomatous disease (CGD) is predominantly caused by a mutation in the CYBB gene encoding gp91^phox on the X‐chromosome. Because the phagocytes of patients with CGD are not able to generate the superoxide anion, these patients are susceptible to severe infections that can be fatal. It has been suggested that the extracellular region of gp91^phox is necessary for and critical to forming the epitope of mAb 7D5 and that 7D5 provides a useful tool for rapid screening of X‐linked CGD by FACS. To further elucidate the mAb 7D5 epitope on human gp91^phox, chimeric DNA expressed human and mouse gp91^phox recombinant protein were constructed. The fusion proteins were immunostained for mAb 7D5 and analyzed by FACS and western blot analysis. The ¹⁴³ELGDRQNES¹⁵¹ region was found to reside at the extracellular surface on human gp91^phox and to be an important epitope for the interaction with mAb 7D5, as analyzed by FACS analysis. In particular, amino acid R147 is a unique epitope on the membrane‐associated Cyt b for mAb 7D5. In conclusion, it is proposed that FACS analysis using mAb 7D5 is a valuable tool for early diagnosis of CGD.

     

Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA

Application of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) for the analysis of cytokinins was examined. The fragmentation of cytokinins was studied using authentic trans-zeatin (t-Z), trans-zeatin riboside (t-ZR), isopentenyl adenine (i⁶Ade), isopentenyl adenosine (i⁶Ado), benzyl adenine (BAde), benzyl adenosine (BAdo), and kinetin. These cytokinins were effectively ionized by APCI in aqueous acetonitrile. t-Z, i⁶Ade, BAde, and kinetin showed prominent quasi-molecular ions of [M + H]⁺, and ribosylcytokinins clearly showed both [M + H]⁺ and a characteristic fragment ion ([M + H-ribose]⁺), giving some information about their structures. The qualitative and semi-quantitative analyses of cytokinins by LC/APCI-MS were validated in combination with enzyme-linked immunosorbent assay (ELISA) through the analysis of t-ZR in the teratoma of Nicotiana tobacum. t-ZR was conclusively identified and a semi-quantitative estimate of its endogenous levels were provided by the combination of LC/APCI-MS and ELISA. The quantified values obtained by LC/ APCI-MS (single ion detection) and ELISA are in close agreement.     

Lanthanum(III) Impacts on Metallothionein MTT1 and MTT2 from <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Metallothionein MTT1 and MTT2 from Tetrahymena thermophila are sulfydryl-rich proteins that can bind to and are inducible by heavy metals such as mercury, cadmium, zinc, and copper. However, little is known about the induction and binding of T. thermophila metallothionein by trivalent metals. In this study, we found that 10–80 μM La³⁺ can promote Tetrahymena cells proliferation, and fluorescence spectrum analysis showed that La³⁺ can enter T. thermophila cells. Real-time quantitative polymerase chain reaction showed La³⁺ induced the expression of MTT1 and MTT2. Furthermore, Fluorescence analysis indicated La³⁺ bind to MTT1 and MTT2. These results implied that La³⁺ could interact with MTT1 and MTT2 via aspartic or glutamic acid oxygen atoms.     

Biochemical genetics of TL antigens

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tla ^a, Tla ^d, Tla ^c), intermediate (Tla ^c, Tla ^f), and low (Tla ^b). (Relative amounts: 1000 : 100 : 1.) Some thymic leukemias arising in (Tla ^b, Tla ^c, Tla ^c) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.     

Population genetics of chimpanzee blood groups

A series of 60 chimpanzees (Pan troglodytes) were tested for their human-type A-B-O blood groups and for ten simian-type blood factors. Of the 60 chimpanzees four were group O and 56 group A; combining this with our previous results, among 274 chimpanzees there were 36 group O and 238 group A. Gene frequency analysis of the V-A-B types (determined by three antisera, anti- V ^c, anti- A ^c and anti- B ^c) of the 60 chimpanzees indicated inheritance by four allelic genes, namely, the amorphic gene v and the three additional alleles v^A, v^B and V. This theory allows for the existence of ten genotypes but only seven V-A-B phenotypes, since the type V.AB is excluded. Gene frequency analysis confirmed that C^c and c^c are contrasting antigens determined by corresponding allelic genes. The distribution of the C-c-E-F types among the 60 chimpanzees, as well as among 133 chimpanzees previously tested, is compatible with the postulation of five allelic genes, namely, the amorph c, and the alleles C^E, C^F, C^EF and the very rare allele C. The blood factor G ^c appears to define a separate blood group system, independent of the V-A-B and C-E-F systems. The newly defined blood factor Lindsay appears to be related to the V-A-B system, while factor H ^c may be related to the C-E-F system.     

Alcohol dehydrogenase polymorphism in Apis mellifera

A polymorphic system of ADH isozymes is described in the honeybee Apis mellifera. Three and six different electrophoretic patterns were found, respectively, in drone and worker pupae analysis. The data indicate that the ADH isozymes are controlled by three alleles, Adh-1 ¹, Adh-1², and Adh-1 ³. The frequency of the Adh-1 alleles is different in two analyzed subspecies, Apis mellifera adansonii (African bees) and Apis mellifera ligustica (Italian bees). In the African bees, the frequencies are 0.256 and 0.697 for Adh-1 ¹ and Adh-1², respectively. In the Italian bees, these values are shown to be 0.902 and 0.098, respectively. The allele Adh-1 ³ was not detected in the Italian bee population. The effect of NAD on the resolution of this system was investigated, and only one region of ADH activity was obtained in drone pupae analysis when NAD was used in the gels. However, two different regions of activity were observed in the same samples, in the absence of the coenzyme. ADH activity was not detected in young larvae, but it increased to a maximum in prepupal and white-eyed pupal phases. It then declined progressively to total absence in the emerging bees.This work was supported by the Brazilian Research Council (CNPq) and S~ao Paulo State Research Foundation (FAPESP). This study was performed by the senior author in partial fulfillment of the requirements for a master's degree and was directed by Dr. M. A. Mestriner, Genetics Department, University of São Paulo at Ribeirão Preto.     

HIF-1α involvement in low temperature and anoxia survival by a freeze tolerant insect

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11^p58 during postnatal development in mouse testes and examined the cellular localization of CDK11^p58 and cyclinD3, which was associated with CDK11^p58 in mammalian cells. Western blot analysis revealed that CDK11^p58 was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11^p58 was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11^p58 were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11^p58 expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11^p58 and cyclinD3 in the adult testeis was revealed by immunofluorescence analysis. (Mol Cell Biochem 270: 99–106, 2005)     

Characterization of a hemin-storage locus ofYersinia pestis

Summary The pigmentation phenotype (Pgm⁺) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm^– phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm⁺ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms⁺) inY. pestis spontaneous Pgm^– strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms⁺ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm⁺, spontaneous Pgm^–, and Hms^– transposon strains, as well as a spontaneous Pgm^– strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms⁺ phenotype.     

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina,an orchid mainly utilizing saprotrophic rhizoctonia

The majority of chlorophyllous orchids form mycorrhizal associations with so‐called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their ¹³C and ¹⁵N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in ¹³C but not so greatly in ¹⁵N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, ¹³C levels were depleted and those of ¹⁵N were indistinguishable from the co‐occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.     

Comparative genetic analysis of the Aegilops longissima and Ae. sharonensis genomes with common wheat

Aegilops longissima Schw. et Musch. (2n= 2x=14, S^lS^l) and Aegilops sharonensis Eig. (2n=2x=14, S^lS^l) are diploid species belonging to the section Sitopsis in the tribe Triticeae and potential donors of useful genes for wheat breeding. A comparative genetic map was constructed of the Ae. longissima genome, using RFLP probes with known location in wheat. A high degree of conserved colinearity was observed between the wild diploid and basic wheat genome, represented by the D genome of cultivated wheat. Chromosomes 1S^l, 2S^l, 3S^l, 5S^l and 6S^l are colinear with wheat chromosomes 1D, 2D, 3D, 5D and 6D, respectively. The analysis confirmed that chromosomes 4S^l and 7S^l are translocated relative to wheat. The short arms and major part of the long arms are homoeologous to most of wheat chromosomes 4D and 7D respectively, but the region corresponding to the distal segment of 7D was translocated from 7S^lL to the distal region of 4S^lL. The map and RFLP markers were then used to analyse the genomes and added chromosomes in a set of ’Chinese Spring’ (CS)/Ae. longissima chromosome additions. The study confirmed the availability of disomic CS/Ae. longissima addition lines for chromosomes 1S^l, 2S^l, 3S^l, 4S^l and 5S^l. An as yet unpublished set of Ae. sharonensis chromosome addition lines were also available for analysis. Due to the gametocidal nature of Ae. sharonensis chromosomes 2S^l and 4S^l, additions 1S^l, 3S^l, 5S^l, 6S^l and 7S^l were produced in a (4D)4S^l background, and 2S^l and 4S^l in a euploid wheat background. The analysis also confirmed that the 4/7 translocation found in Ae. longissima was not present in Ae. sharonensis although the two wild relatives of wheat are considered to be closely related. The phenotypes of the Ae. sharonensis addition lines are described in an Appendix. Received: 28 September 2000 / Accepted: 19 January 2001     

Comparison of crude lysate pellets from isogenic strains of yeast with different prion composition: Identification of prion-associated proteins

A new approach involving the comparative analysis of proteins of crude cell lysate pellets from isogenic strains of Saccharomyces cerevisiae distinguished by their prion composition permitted us to identify a large group of prion-associated proteins in yeast cells. 35 proteins whose aggregation state depends on prion content have been identified by 2D-electrophoresis followed by the MALDI analysis of a recipient [psi ⁻] strain and of [PSI ⁺] cytoductant. Approximately half of these proteins belong to functional groups of chaperones and enzymes involved in glucose metabolism. Other proteins are involved in translation, stress response and protein degradation. The data obtained are compared with the results of other groups who used different approaches to detect proteins involved in prion aggregates.     

Transformation by integration in Podospora anserina

Summary A new transformation system for spheroplasts of Podospora anserina has been developed. The recipient leu1-1 strain is auxotrophic for leucine. The plasmid DNA does not carry the wild-type allele leu ⁺.but a tRNA suppressor: su4-1 or su8-1. The following protocol for genetic analysis has been developed: the [leu ⁺transformants are crossed with another mutant strain, carrying the 193 mutation. This mutation prevents the pigmentation of the spores and is also suppressed by the cloned suppressor. Thus, the genetic analysis of the transformants can be performed directly on ordered tetrads by the observation of pigmentation restoration. The first application of the method is described comparing the integration points when different suppressors are used. Integration of the plasmid DNA in the homologous site was not the rule; in most cases the integration point was located elsewhere.     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.     

Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants

Two newly isolated obligate methanol-utilizing bacteria (strains Iva^T and Lap^T) with the ribulose monophosphate pathway of C₁ assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, motile rods multiplying by binary fission, mesophilic and neutrophilic, synthesize indole-3-acetate. The prevailing cellular fatty acids are straight-chain saturated C_16:0 and unsaturated C_16:1 acids. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia is assimilated by glutamate dehydrogenase. The DNA G+C contents of strains Iva^T and Lap^T are 54.0 and 50.5 mol% (T_m), respectively.Based on 16S rRNA gene sequence analysis and DNA–DNA relatedness (38–45%) with type strains of the genus Methylobacillus, the novel isolates are classified as the new species of this genus and named Methylobacillus arboreus Iva^T (VKM B-2590^T, CCUG 59684^T, DSM 23628^T) and Methylobacillus gramineus Lap^T (VKM B-2591^T, CCUG 59687^T, DSM 23629^T).The GenBank accession numbers for the 16S rRNA gene and mxaF gene sequences of the strains Iva^T and Lap^T are GU937479, GU937478 and HM030736, HM030735, respectively.     

Analysis of Ca2+ mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules

Host cell invasion, exit and parasite dissemination is critical to the pathogenesis of apicomplexan parasites such as Toxoplasma gondii and Plasmodium spp. These processes are regulated by intracellular Ca²⁺ signaling although the temporal dynamics of Ca²⁺ fluxes and down‐stream second messenger pathways are poorly understood. Here, we use a genetically encoded biosensor, GFP‐Calmodulin‐M13–6 (GCaMP6), to capture Ca²⁺ flux in live Toxoplasma and investigate the role of Ca²⁺ signaling in egress and motility. Our analysis determines how environmental cues and signal activation influence intracellular Ca²⁺ flux, allowing placement of effector molecules within this pathway. Importantly, we have identified key interrelationships between cGMP and Ca²⁺ signaling that are required for activation of egress and motility. Furthermore, we extend this analysis to show that the Ca²⁺ Dependent Protein Kinases–TgCDPK1 and TgCDPK3—play a role in signal quenching before egress. This work highlights the interrelationships of second messenger pathways of Toxoplasma in space and time, which is likely required for pathogenesis of all apicomplexan species.     

Cytogenetic characterization of the 4BC region on the X chromosome of Drosophila melanogaster: Localization of the mei-9, norpA and omb genes

Thirty genetic alterations, which involve the 4BC region of the Drosophila X chromosome, have been induced by ionizing radiation or by an endogenous mutator element. These mutations were recovered by screening for reversion of the dominant mutants Oce and Qd or for induction of the recessive mutants bi and rb. Among the 23 mutants generated by ionizing radiation, 20 have proven to be cytologically detectable chromosomal aberrations. Seven additional unique aberrations were generated in the Uc mutator strain. In total, 22 cytologically detectable deficiencies, 3 translocations, 1 inversion, 1 transposition, and 3 cytologically normal mutants have been recovered. Complementation analysis has permitted the cytogenetic localization of eight genes in the 4BC region. The mei-9 locus has been assigned to region 4B4-6, because this function is carried by Df (1)rb ⁴¹ but not by Df(1)bi ^D1. The norpA locus has been placed in the 4B6-C1 region based on its location between the distal breakpoints of Df(1)bi ^D2 and Df(1)rb ⁴¹. The genes lac, Qd, bi, and omb are localized to bands 4C5,6, rb to 4C6 and amb to 4C7,8. With one exception the complementation analysis has also permitted a determination of the linear sequence of these genes. This cytogenetic localization of these loci will facilitate the cloning and molecular analysis of genes controlling a key function in DNA repair and recombination (mei-9), and two fundamental neural functions (norpA and omb).     

Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.     

The dominant Drop eye mutations of Drosophila melanogaster define two loci implicated in normal eye development

The three existing dominant gain-of-function Drop alleles, Dr ¹, Dr ^Mio and Dr ^We , previously assumed to define a single locus, severely disrupt eye development. Genetic analysis of ethylmethanesulphonate (EMS) and irradiation-induced revertants revealed that the Drop mutations define two loci: the Drop locus, which is defined by the Dr ¹ and Dr ^Mio mutants, and a separate locus defined by the Dr ^We mutation, which has been renamed Wedge. The majority of the Dr ¹ and Dr ^Mio revertants are embryonic lethal in trans, mutant embryos exhibiting trachea that fail to join the Filzkörper, thus revealing a role for the Drop gene in embryogenesis. Clonal analysis of lethal revertant alleles suggests a role for both genes in eye development. In the Drop homozygous mutant clones, the outer photoreceptor cells R1–R6 develop aberrantly. Wedge, however, is not required by the developing photoreceptor cells but its absence does disrupt normal ommatidial alignment. Although the Drop and nearby string loci were shown to be genetically distinct, both Dr ¹ and Dr ^Mio were found to interact in trans with lesions at the string locus, causing loss and derangement of bristles and loss of neuromuscular coordination.