Characterization of the Drosophila melanogaster ribosomal proteome

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry to identifying proteins represented in a 2-D gel database of Drosophila melanogaster ribosomes. First, we purified ribosomes from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 58 Coomassie blue stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF. Using this strategy we identified 52 ribosomal spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF. The database can be used to analyze Minute mutants of Drosophila.     

Characterization of the Drosophila melanogaster mitochondrial proteome

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of Drosophila melanogaster mitochondria. First, we purified mitochondria from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 231 silver-stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF analysis. Using this strategy, we identified 66 mitochondrial spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis. In addition, we generated antibodies against two of the mitochondrial proteins as tools for characterizing the organelle.     

Patterns of protein synthesis in imaginal discs of Drosophila melanogaster.

Patterns of polypeptide synthesis in wing, leg and eye-antenna imaginal discs and in whole larvae of wild-type and and mutant Drosophila melanogaster have been examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. After 2 hr of labeling with 35S during the third larval instar, the synthesis of more than 318 polypeptides has been detected in imaginal discs. Of these, 268 are present in similar amounts in all three disc types. The remaining polypeptides detected in the three imaginal disc types fall into two categories: those unique to a particular disc type, and those specific for a particular pair of disc types. These results are discussed in relation to the spectrum of gene expression in imaginal discs.     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster

We have identified the initiation factor 4A (eIF4A) in a two-dimensional protein database of Drosophila wing imaginal discs. eIF4A, a member of the DEAD-box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5'-UTR of mRNAs during translational initiation. Two-dimensional gel electrophoresis and microsequencing allowed us to purify eIF4A, and generate specific polyclonal antibodies. A combination of immunoblotting and labelling with [(35)S]methionine + [(35)S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7-9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3'-untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A-related gene CG7483. We detected eIF4A protein expression in the wing imaginal discs of several Drosophila species, and in haltere, leg 1, leg 2, leg 3, and eye-antenna imaginal discs of D. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. We observed ubiquitous expression of eIF4A mRNA and protein during Drosophila embryogenesis. Yeast two-hybrid analysis demonstrated the in vivo interaction of Drosophila eIF4G with the N-terminal third of eIF4A.     

Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster: a comparison between different discs and stages

High-resolution two dimensional gel electrophoresis has been used to study the patterns of protein synthesis in imaginal discs of Drosophila melanogaster. In this paper we first compare the patterns of protein synthesis in wing, haltere, leg 1, leg 2, leg 3 and eye antenna imaginal discs of late third instar larvae. We have detected only quantitative changes: differences in 17 proteins among the different imaginal discs. In addition, we have analysed the variations in pattern of proteins in the wing disc of the last larval stage and early pupae as well as in wing discs cultured in vivo for 6 days. Variations in these patterns affect more than 20% of the proteins and involve both qualitative and quantitative changes. Some of the changes may correspond to protein phosphorylation. Correlations of these changes between discs and through development are also discussed. Correspondence to: F. Santaren     

The effects of 20-hydroxyecdysone on the metabolic labeling of membrane proteins in Drosophila imaginal discs

20-Hydroxyecdysone induces evagination of imaginal discs of Drosophila melanogaster cultured in vitro. The possible involvement of cell-surface proteins in this process has prompted us to study the synthesis of membrane proteins in imaginal discs. A procedure is reported for the isolation of membrane vesicle fractions from discs that are enriched for the plasma membrane enzyme, Na+/K+-ATPase, and that label with the surface-labeling reagent [125I]iodosulfanilic acid. 20-Hydroxyecdysone alters the pattern of [35S]methionine incorporation into polypeptides in these membrane vesicle fractions. Increased and decreased incorporation as well as changes in migration on two-dimensional gels of specific polypeptides are caused by the hormone. These changes parallel in time the onset and the continuation of evagination.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Identification of newly synthesized wing imaginal disc proteins induced by 20-hydroxyecdysone inGalleria mellonella

Summary Wing imaginal discs from 7th instarGalleria mellonella L. larvae evaginate and exhibit tracheolar elongation when exposed to 20-hydroxyecdysone in vitro. This response was elicited within 24 h of treatment as was a greater than fourfold stimulation of the incorporation of [³H]leucine into disc proteins. Autoradiographic analyses of [³⁵S]methionine labeled polypeptides separated on two-dimensional gels, however, revealed no differences in protein profiles between control and treated discs until 48 h following exposure to molting hormone. At this time, wing imaginal discs exposed to 1 μg/ml 20-hydroxyecdysone synthesized four unique polypeptides not detected either in controls or in discs treated for 24 h. These four new proteins were also found to be synthesized by imaginal discs that had evaginated in vivo. These results suggest that these proteins are normally synthesized subsequent to evagination and do not play a role in the morphological events necessary for evagination. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA. S. G. M. is employed through a cooperative agreement between the Insect Attractants, Behavior and Basic Biology Laboratory and the Department of Entomology, University of Florida.     

Protein synthetic patterns during differentiation of imaginal discs in vitro.

Leg, wing, and eye-antennal imaginal discs of Drosophila melanogaster were obtained by dissection from late-third instar larvae and cultured in vitro in Schneider's medium containing β-ecdysone. Differentiation of adult structures was obtained in more than 90% of all cultures. Differentiation was somewhat slower than normal in vivo development, but synchronous, repeatable, and reasonably completed structures were obtained. Our initial efforts at analyzing the molecular events of imaginal disc differentiation in culture have been to study the protein synthetic pattern which occurs throughout the culture period. Discs were pulse labeled with [³⁵S]methionine, and the proteins were separated by SDS-gel electrophoresis. Analysis of the synthetic pattern was done by autoradiography of these gels using X-ray film. In all three disc types, pronounced changes in protein synthetic patterns occurred throughout the culture period. These changes appeared to be under strict temporal control. Although disc-specific differences could be seen, a comparison of the three discs types revealed a striking similarity in the changes which occurred in the patterns of protein synthesis during the 5-day culture period. In general, the protein synthetic patterns of different imaginal discs at the same period during differentiation showed greater similarities than the patterns of a single disc type at different periods. These results are consistent with a view of differentiation as a tightly controlled program of gene activation and deactivation operating throughout the differentiation process.     

Protein synthesis and accumulation in Drosophila melanogaster imaginal discs: Identification of a protein with a nonrandom spatial distribution

Proteins from Drosophila imaginal discs and disc fragments were analyzed on two-dimensional electrophoretic gels following labeling in vitro with [³⁵S]methionine. The protein synthetic pattern in autoradiograms is very complex and parallels the pattern of protein accumulation visualized in silver-stained gels. We find no reproducible qualitative differences in the proteins synthesized or accumulated by different disc types. Additionally, analysis of the proteins synthesized by different fragments of wing and haltere discs has resulted in the identification of a polypeptide which is synthesized preferentially in homologous regions of these two imaginal discs. Scanning densitometry of our autoradiograms corroborates these findings. This protein, therefore, has some of the properties one would predict for a molecule involved in the imaginal disc positional information system.     

Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs

The salivary glands and other tissues from Drosophila melanogaster were dissected at various times throughout the prepupal period, as well as after heat shocks and ecdysterone treatments, and the proteins labelled by incubating the isolated tissues with [³⁵S]methionine were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel. The labelled band patterns from salivary gland, as seen on the autoradiograph of the gel, showed striking variations, in a manner remarkably similar to variations in puff patterns during the same prepupal period. In proteins from Malpighian tubes, the pattern of bands varied to a lesser extent and in brain only a few components were modified.Heat shock brought about the appearance of a number of new bands, while others were reduced in intensity. This effect was observed with all the tissues examined, salivary glands, brain and Malpighian tubes, as well as wing imaginal discs, tissue lacking polytene chromosomes. The six most heavily labelled bands induced by heat shock represent about 30%, and one component alone represents over 15%, of the total label in the sample, as seen in salivary glands, brain and Malpighian tubes. The synthesis of RNA at puff sites was investigated after heat shock by [³H]uridine labelling. By correlating the amount of [³H]uridine in some puffs with the level of [³⁵S]methionine in some bands a tentative relation is suggested in a few instances.The effect of ecdysterone treatment was also studied in the salivary glands. Changes in a number of protein bands were noticed, though they were much less pronounced than those following heat shock.     

Polypeptides differentially expressed in imaginal discs define the peroxiredoxin family of genes in Drosophila.

2D gel electrophoresis followed by microsequencing has been used to purify and identify a protein (catalogued in the database as SSP5111) from Drosophila wing imaginal discs of third instar larvae that showed significant differences in their level of expression when compared with other imaginal discs of the same age. The microsequence data showed identity with amino acids encoded by the human proliferation association gene, pag, which is a thiol-specific antioxidant. By virtue of this homology we have cloned and sequenced two cDNAs that appear to define the peroxiredoxin family of Drosophila. One of them, Jafrac1, encodes the SSP5111 protein searched, had 194 amino acids and mapped in the region 11E in the X chromosome. The other, Jafrac2, encodes a protein of 242 amino acids and mapped in the region 62F in the 3 L chromosome. Both new peroxidases contain two conserved cysteines and share homology with other peroxidases that extends over the entire sequence and ranges between 47% and 76%. An antiserum raised against the SSP5111 protein showed significant changes in the amount of protein in different stages of Drosophila development, being a major product in early embryos. In 2D gels the antibody not only recognizes the SSP5111 polypeptide but also a related one (catalogued in the database as SSP6107) that exhibits identical amino-acid sequence over at least 85% of its sequence. The data also suggest that the SSP5111 polypeptide could be a maternal-effect product.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

Imaginal disc ribosomal proteins of D. melanogaster

Summary The ribosomal proteins from undifferentiated imaginal discs of Drosophila melanogaster were analyzed by two-dimensional gel electrophoresis and compared with the ribosomal protein pattern of adult flies. It is shown that the ribosomal proteins from these discs are qualitatively identical with those of adult flies except that two acidic proteins are missing in the discs. This heterogeneity is discussed in terms of the functional roles these two proteins may carry in connection with disc differentiation.Paper No. 6 of the present series. Paper No. 5 is by Lambertsson, A. G. Molec. gen. Genet. 139, 145–156 (1975)     

Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Transdetermination of imaginal wing disc fragments of Drosophila melanogaster

Fragments of the imaginal wing disc of Drosophila melanogaster were cultured in adult hosts before transfer to larvae for metamorphosis. Transdetermination occurred only after at least 2 weeks of culture in vivo, producing structures of the leg, antenna, head, and thoracic spiracle. Details of the transdetermined structures and their locations with respect to normal wing disc structures are reported. We present evidence suggesting that regulation can occur between the wing and the second leg imaginal discs, and we propose that many transdeterminations which involve neighboring discs may result from such interdisc regulation.     

Cell-free translation of Drosophila imaginal disc mRNA

Total RNA derived from the imaginal discs of Drosophila melanogaster was translated in vitro, and the polypeptide products electrophoresed on two-dimensional gels. In agreement with previously published examinations of imaginal disc protein synthesis and content, we can detect no reproducible differences in abundant mRNA populations between different disc types (foreleg and wing). Differences can be found, however, between imaginal discs and other tissues. We also present evidence for a nonuniformly distributed wing disc mRNA.     

Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.