In vitro growth of imaginal disks from Drosophila melanogaster.

Imaginal disks were cultured in a new medium in vitrol Optimal growth occurred when the medium was supplemented with insulin and a juvenile hormone analog and conditioned by larval fat body. The disks grow in vitro by normal cell division and maintain their capacity to differentiate into normal patterns of adult cuticular structures.

A deletion of the 3' end of the Drosophila melanogaster hsp70 gene increases stability of mutant mRNA during recovery from heat shock.

hsp40, an X-ray-induced deletion mutant of the major Drosophila melanogaster heat shock protein gene hsp70, was shown to be incorrectly regulated at the translational level. hsp40 protein synthesis persisted at a high level after the release from heat shock, whereas hsp70 protein production was rapidly repressed. This result was observed both in flies heterozygous for the hsp40 gene and in tissue culture cells transfected with the truncated gene. Analysis of the transcription of the hsp40 gene indicated that its mRNA, unlike hsp70 mRNA, was not actively destabilized after a return to control temperatures, permitting prolonged production of the mutant protein.

Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter.

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.

Mutations in the beta-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain.

Inactivation of both alleles of the fruit fly D. melanogaster brain tumor (brat) gene results in the production of a tumor-like neoplasm in the larval brain, and lethality in the larval third instar and pupal stages. We cloned the brat gene from a transposon-tagged allele and identified its gene product. brat encodes for an 1037 amino acid protein with an N-terminal B-boxl zinc finger followed by a B-box2 zinc finger, a coiled-coil domain, and a C-terminal beta-propeller domain with six blades. All these motifs are known to mediate protein-protein interactions. Sequence analysis of four brat alleles revealed that all of them are mutated at the beta-propeller domain. The clustering of mutations in this domain strongly suggests that it has a crucial role in the normal function of Brat, and defines a novel protein motif involved in tumor suppression activity. The brat gene is expressed in the embryonic central and peripheral nervous systems including the embryonic brain. In third instar larva brat expression was detected in the larval central nervous system including the brain and the ventral ganglion, in two glands - the ring gland and the salivary gland, and in parts of the foregut - the gastric caecae and the proventriculus. A second brat-like gene was found in D. melanogaster, and homologs were identified in the nematode, mouse, rat, and human. Accumulated data suggests that Brat may regulate proliferation and differentiation by secretion/transport-mediated processes.

The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function

The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.

Complementation analysis of late lethal mutants of Drosophila melanogaster.

Late larval and pupal lethal mutants of Drosophila define those gene functions which are essential for the development of pupae (metamorphosis) but not for embryonic or larval development. In a previous report the isolation of a large number of such mutants was outlined, and a description of the imaginal disc defects in those mutants was described. This report concerns genetic analysis of those mutants. 3746 different pairwise combinations of mutants have been tested for complementation. Only 10 pairs fail to complement. In all of the cases tested, the lethal mutation in each member of a non-complementing pair has a similar map location. In addition to the non-complementing pairs one group of seven partially-complementing mutants has been identified. Comparisons of the imaginal disc defects within the non-complementing pairs and the lethal hybrids formed by the respective pairs were made to test for uniformity of phenotype. No significant qualitative differences were detected between any non-complementing pairs or their respective hybrids.

The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related.

Mutations in the ash-1 and ash-2 genes of Drosophila melanogaster cause a wide variety of homeotic transformations that are similar to the transformations caused by mutations in the trithorax gene. Based on this similar variety of transformations, it was hypothesized that these genes are members of a functionally related set. Three genetic tests were employed here to evaluate that hypothesis. The first test was to examine interactions of ash-1, ash-2 and trithorax mutations with each other. Double and triple heterozygotes of recessive lethal alleles express characteristic homeotic transformations. For example, double heterozygotes of a null allele of ash-1 and a deletion of trithorax have partial transformations of their first and third legs to second legs and of their halteres to wings. The penetrance of these transformations is reduced by a duplication of the bithorax complex. The second test was to examine interactions with a mutation in the female sterile (1) homeotic gene. The penetrance of the homeotic phenotype in progeny from mutant mothers is increased by heterozygosis for alleles of ash-1 or ash-2 as well as for trithorax alleles. The third test was to examine the interaction with a mutation of the Polycomb gene. The extra sex combs phenotype caused by heterozygosis for a deletion of Polycomb is suppressed by heterozygosis for ash-1, ash-2 or trithorax alleles. The fact that mutations in each of the three genes gave rise to similar results in all three tests represents substantial evidence that ash-1, ash-2 and trithorax are members of a functionally related set of genes.

Germline transformation using a prune cDNA rescues prune/killer of prune lethality and the prune eye color phenotype in Drosophila.

Null mutations in the prune gene of Drosophila melanogaster result in prune eye color due to reductions in red pigment accumulation. When one copy of the awd(Killer of prune) mutant gene is present in a prune background, the animals die. The cause of prune/Killer of prune lethality remains unknown. The genomic region characterized for the prune locus is transcriptionally active and complex, with multiple and overlapping transcripts. Despite the transcriptional complexity of the genomic region of prune, accumulated evidence suggests that the prune locus is small and consists of a single transcription unit, since every prune allele to date exhibits both prune eye color and prune/Killer of prune lethality. A functional prune product from a single, full-length cDNA was identified in this study that can rescue both the eye phenotype and prune/Killer of prune lethality. The DNA sequences of several mutant prune alleles along with Western blot analysis of mutant proteins provide convincing evidence that prune mutations are nulls, and that the cDNA identified in this study encodes the only product of the prune locus.

Molecular genetic analysis of Drosophila ash2, a member of the trithorax group required for imaginal disc pattern formation.

The ash2 gene is a member of the trithorax group of genes whose products function to maintain active transcription of homeotic selector genes. Mutations in ash2 cause the homeotic transformations expected for a gene in this group but, in addition, cause a variety of pattern formation defects that are not necessarily expected. The ash2 gene is located in cytogenetic region 96A17-19 flanked by slowpoke and tolloid and is included in a cosmid that contains part of slowpoke. The ash2 transcript is 2.0 kb and is present throughout development. The ASH2 protein predicted from the nucleotide sequence of the open reading frame has a putative double zinc-finger domain, called a PHD finger, that is present not only in the products of other trithorax group genes such as TRX and ASH1, but also in the product of a Polycomb group gene, PCL. Polyclonal antibodies directed against ASH2 detect the protein in imaginal discs and in the nuclei of salivary gland and fat body cells. On immunoblots these affinity-purified antibodies detect a 70-kDa protein in larvae and a 53-kDa protein in pupae.

Mutations in polycombeotic, a Drosophila polycomb-group gene, cause a wide range of maternal and zygotic phenotypes.

The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycomb-group mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function.

Genetic analysis of two allelic temperature-sensitive mutants of Drosophila melanogaster both of which are zygotic and maternal-effect lethals.

After fertilization, the development of a zygote depends upon both gene products synthesized by its maternal parent and gene products synthesized by the zygote itself. To analyze genetically the relative contributions of these two sources of gene products, several laboratories have been isolating two classes of mutants of Drosophila melanogaster: maternal-effect lethals and zygotic lethals. This report concerns the analysis of two temperature-sensitive mutants, OX736hs and PC025hs, which were isolated as alleles of a small-disc mutant, l(3)1902. These alleles are not only zygotic lethals, but also maternal-effect lethals. They have temperature-sensitive periods during larval life and during oogenesis. Mutant larvae exposed continuously to restrictive temperature have small discs. One-or two-day exposures to the restrictive temperature administered during the third larval instar lead to a homeotic transformation of the midlegs and hindlegs to the pattern characteristic of the forelegs. Mutant females exposed to the restrictive temperature during oogenesis produce eggs that can develop until gastrulation, but do not hatch.--The existence of these mutants, and one that was recently described by another group, implies that there may be a class of genes, heretofore unrecognized, whose products are synthesized during oogenesis, are essential for embryogenesis and are also synthesized during larval stages within imaginal disc cells.

A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2.

The proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of homeotic genes during Drosophila development. To identify new members of the trithorax group, we screened a collection of deficiencies for intergenic noncomplementation with a mutation in ash1, a trithorax group gene. Five of the noncomplementing deletions uncover genes previously classified as members of the Polycomb group. This evidence suggests that there are actually three groups of genes that maintain the expression pattern of homeotic genes during Drosophila development. The products of the third group appear to be required to maintain chromatin in both transcriptionally inactive and active states. Six of the noncomplementing deficiencies uncover previously unidentified trithorax group genes. One of these deficiencies removes 25D2-3 to 26B2-5. Within this region, there are two, allelic, lethal P-insertion mutations that identify one of these new trithorax group genes. The gene has been called little imaginal discs based on the phenotype of mutant larvae. The protein encoded by the little imaginal discs gene is the Drosophila homologue of human retinoblastoma binding protein 2.

The Drosophila ash1 gene product, which is localized at specific sites on polytene chromosomes, contains a SET domain and a PHD finger.

The determined state of Drosophila imaginal discs depends on stable patterns of homeotic gene expression. The stability of these patterns requires the function of the ash1 gene, a member of the trithorax group. The primary translation product of the 7.5-kb ash1 transcript is predicted to be a basic protein of 2144 amino acids. The ASH1 protein contains a SET domain and a PHD finger. Both of these motifs are found in the products of some trithorax group and Polycomb group genes. We have determined the nucleotide sequence alterations in 10 ash1 mutant alleles and have examined their mutant phenotype. The best candidate for a null allele is ash1. The truncated protein product of this mutant allele is predicted to contain only 47 amino acids. The ASH1 protein is localized on polytene chromosomes of larval salivary glands at > 100 sites. The chromosomal localization of ASH1 implies that it functions at the transcriptional level to maintain the expression pattern of homeotic selector genes.

Multiple Allele Approach to the Study of Genes in DROSOPHILA MELANOGASTER That Are Involved in Imaginal Disc Development.

The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.

Molecular genetic analysis of the Drosophila melanogaster gene absent, small or homeotic discs1 (ash1).

The absent, small or homeotic discs1 gene (ash1) is one of the trithorax set of genes. Recessive loss of function mutations in ash1 cause homeotic transformations of imaginal disc derived tissue which resemble phenotypes caused by partial loss or gain of function mutations in genes of the Antennapedia Complex and bithorax Complex. F2 screens were used to isolate P element insertion alleles and EMS-induced alleles of ash1, including one temperature-sensitive allele, and an F1 screen was used to isolate gamma-ray-induced alleles. Analysis of ash1 mutant flies that survive until the adult stage indicates that not only imaginal disc- and histoblast-derived tissues are affected but also that oogenesis requires ash1 function. Mutations in the gene brahma (brm) which also is one of the trithorax set of genes interact with mutations in ash1 such that non-lethal ash1 +/+ brm double heterozygotes have a high penetrance of homeotic transformations in specific imaginal disc- and histoblast-derived tissues. The cytogenetic location of ash1 was determined to be 76B6-11 by the breakpoint of a translocation recovered in the F1 screen. The gene Shal, which is located cytogenetically nearby ash1, was used to initiate an 84-kb genomic walk within which the ash1 gene was identified. The ash1 gene encodes a 7.5-kb transcript that is expressed throughout development but is present at higher levels during the embryonic and pupal stages than during the larval stages. During the larval stages the transcript accumulates primarily in imaginal discs. During oogenesis the transcript accumulates in the nurse cells of developing egg chambers.

Trans-regulation of thoracic homeotic selector genes of the Antennapedia and bithorax complexes by the trithorax group genes: absent, small, and homeotic discs 1 and 2.

Genes of the trithorax group appear to be required for the maintenance of expression of the homeotic selector genes of the Antennapedia and bithorax complexes. According to genetic criteria, the Drosophila melanogaster genes absent, small, or homeotic discs 1 and 2 (ash1 and ash2) are members of the trithorax group. In this paper we examine the consequences of ash1 and ash2 mutations on the expression of homeotic selector genes in imaginal discs. The results of these experiments demonstrates that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed. Complete loss of ash2 activity causes the loss of expression of Sex combs reduced in first leg imaginal discs, loss of expression of Ultrabithorax in third leg discs, and a late-patterned loss of expression of Ultrabithorax within haltere discs, yet has no effect on engrailed or Antennapedia expression. These results suggest that the range and action of trithorax group genes is varied and complex and argue against any model in which all of the products of the trithorax group act together in a single mechanism or complex.

minidiscs encodes a putative amino acid transporter subunit required non-autonomously for imaginal cell proliferation.

Drosophila minidiscs mutant larvae have smaller imaginal discs than wild-type larvae. However, transplantation experiments have revealed that minidiscs mutant imaginal discs can grow if cultured in non-mutant hosts. These data suggest that minidiscs is required in one or more non-imaginal tissues for synthesis and/or secretion of a diffusible factor that stimulates imaginal cell proliferation. The 2. 3 kb minidiscs transcript accumulates in the larval fat body and encodes a protein containing 12 putative membrane spanning domains that is similar in sequence to amino acid transporter subunits from other eukaryotes, including humans. We propose that in response to amino acid uptake by the transporter encoded by minidiscs, the fat body secretes a diffusible factor required for imaginal disc proliferation.

Establishment of imaginal discs and histoblast nests in Drosophila.

In Drosophila the homeotic genes of the bithorax-complex (BX-C) and Antennapedia-complex (ANT-C) specify the identity of segments. Adult segment primordia are established in the embryo as the histoblast nests of the abdomen and the imaginal discs of the head, thorax and terminalia. We have used a molecular probe for the limb primordia and in vivo culture to describe the nature of the adult primordia in mutants in which the pattern of homeotic gene expression was altered. The results suggest that the histoblast or disc 'mode' of development is initiated by the extended germ band stage through activity of the BX-C and ANT-C and is relatively inflexible thereafter [corrected].

Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis.

OBJECTIVE: Oxidative stress is a significant contributing factor in the pathogenesis of alcoholic liver disease (ALD). In the murine models of chronic alcohol consumption, induction of oxidative stress results in increased peroxidation of polyunsaturated fatty acids to form highly reactive electrophilic α/ß unsaturated aldehydes that post-translationally modify proteins altering activity. Data are presented here suggesting that oxidative stress and the resulting carbonylation of hepatic proteins is an ongoing process involved in alcohol-induced cirrhosis. METHODS: Using age-matched pooled hepatic tissue obtained from healthy humans and patients with end stage cirrhotic ALD, overall carbonylation was assessed by immunohistochemistry and LC-MS/MS of streptavidin purified hepatic whole cell extracts treated with biotin hydrazide. Identified carbonylated proteins were further evaluated using bioinformatics analyses. RESULTS: Using immunohistochemistry and Western blotting, protein carbonylation was increased in end stage ALD occurring primarily in hepatocytes. Mass spectrometric analysis revealed a total of 1224 carbonylated proteins in normal hepatic and end-stage alcoholic cirrhosis tissue. Of these, 411 were unique to cirrhotic ALD, 261 unique to normal hepatic tissue and 552 common to both groups. Bioinformatic pathway analysis of hepatic carbonylated proteins revealed a propensity of long term EtOH consumption to increase post-translational carbonylation of proteins involved in glutathione homeostatic, glycolytic and cytoskeletal pathways. Western analysis revealed increased expression of GSTA4 and GSTπ in human ALD. Using LC-MS/MS analysis, a nonenaldehyde post-translational modification was identified on Lysine 235 of the cytoskeletal protein vimentin in whole cell extracts prepared from human end stage ALD hepatic tissue. CONCLUSIONS: These studies are the first to use LC-MS/MS analysis of carbonylated proteins in human ALD and begin exploring possible mechanistic links with end-stage alcoholic cirrhosis and oxidative stress.