Identification of Differentially Expressed Proteins during Larval Molting of Helicoverpa armigera Xiao-Fan Zhao,* Hong-Juan He, Du-Juan Dong, and Jin-Xing Wang School of Life Sciences, Shandong University, Jinan 250100, China Received July 27, 2005
Abstract: Insect molting involves many molecular processes, such as protein degradation and protein synthesis in the epidermis. Various proteins have been implicated in these processes. The differentially expressed proteins during larval molting of Helicoverpa armigera were investigated using two-dimensional electrophoresis (2-DPAGE) and matrix-assisted laser desorption/ionizationtime-of-flight-mass spectrometry (MALTI-TOF-MS). Four larval tissues sampled during molting and feeding were examined. Seventy-seven differentially expressed proteins were identified in these tissues, including 20 proteins from the fifth-molting epidermis (fifth instar molting to sixth instar), 36 proteins from the fifth-molting hemolymph, and 21 from the fifth-molting fat bodies. No obviously different spots were identified from the fifth-molting midgut under these experimental conditions. After application of MALTI-TOF-MS and similarity analysis comparing results to a Drosophila protein database, 30 proteins were identified: 10 proteins from the fifthmolting epidermis, 11 proteins from the hemolymph, and 9 proteins from fat bodies. These proteins were separated into 5 groups according to their probable functions, such as enzymes, regulators, protein hydrolases, receptors, and proteins with unknown functions. These differentially expressed proteins were proposed to be involved in the Helicoverpa molting cascade. Keywords: Helicoverpa armigera • molting • differential protein expression • two-dimensional electrophoresis • MALTI-TOF-MS
Introduction Insect molting is a common phenomenon in nature. It is controlled primarily by three hormones: neurosecretory hormone prothoracicotropic hormone (PTTH), 20-hydroxyecdysone (20E) and juvenile hormone (JH). PTTH expression is directed by body size, photoperiod, and ambient temperature. PTTH then controls the precise timing of molting by stimulating the prothoracic gland to synthesize ecdysteroids.1 Ecdysteroids orchestrate the molting process and JH determines the nature of the molt. JH is normally present during the larval stages to enable growth and progression from one larval stage to the next until the larva attains the appropriate size for metamorphosis. * To whom correspondence should be addressed. Tel: 086-531-88364620. E-mail:
[email protected].
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The upstream genes that regulate the molting cascade have been well clarified, including transcription regulators such as EcR (ecdyson receptor), USP (ultraspiracal protein), HR3 (hormone receptor 3), E75 and E74 (ecdysone-induced transcription factors 74 and 75), and βFTZ-F1 (Fushi Tarazu-factor 1), which are normally transcription factors.2 However, many of the downstream genes involved in the molting cascade, which are regulated by the transcription factors, have not been thoroughly investigated. Larval molting includes many dramatic events. The first is apolysis, the process of separating the cuticle from the epidermal cells. During this process, the proteins and chitins in the old epidermis are degraded, and the substances are reused in building the new epidermis. Followed is ecdysis, the process of shedding the old exoskeleton off to make way for the formation of the new cuticle. It is known that chitinases degrade cuticule chitins during molting.3 Additionally, proteases degrade old epidermis proteins. A serine protease was recently found to degrade cuticle proteins in the epidermis during molting.4 Carboxypeptidase A (MF-CPA) appears to be involved in pupal ecdysis in Bombyx mori.5 However, most other proteins which participate in the molting cascade have not been well clarified, especially on the level of whole protein expression profiles in various tissues. Two-dimensional electrophoresis (2D-PAGE) combined with matrix assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALTI-TOF-MS) offer high throughput techniques by which differentially expressed proteins in various physiological processes can be identified. For example, Giavalisco and colleagues used 2D-PAGE and MALTI-TOF-MS for a large-scale proteome analysis of Arabidopsis thaliana.6 Vensel and colleagues studied the developmental changes in the metabolic protein profiles of wheat endosperm.7 Paskewitz and Shi investigated the hemolymph proteome of Anopheles gambiae and identified a phenoloxidase and two chitinase-like proteins.8 Wang and colleagues identified 5 lipopolysaccharide induced polypeptides.9 However, there have been no reports on the proteome involved in insect molting. This absence of information limits our understanding of the protein network involved in the molting cascade. Helicoverpa armigera belongs to the Noctuidae family in the Lepidopteran order. Its molting process may be compared with that of other lepidopteran insects, such as Bombix mori and Manduca sexta. Particularly, H. armigera has more in common with many agriculture pests in noctuidae, such as Spodoptera exigua and Spodoptera litura than with Drosophila, Bombyx or Manduca. Investigating the proteins expressed during molt10.1021/pr0502424 CCC: $33.50
2006 American Chemical Society
technical notes ing may provide a better understanding of the molting cascade. Meanwhile, identification of new downstream genes may facilitate investigations of the function of upstream transcription regulators in the molting cascade. Furthermore, this knowledge may provide new targets for the design of pest control agents. Therefore, 2-D-PAGE and MALTI-TOF-MS were performed to investigate molting-related proteins on a large scale.
Materials and Methods Reagents. The following products of Amershame Biosciences_(Uppsala Sweden) were used: Nonlinear immobilized pH gradient buffer (NL 3-10 IPG buffer), nonlinear immobilized pH gradient strip (NL 3-10 IPG strip), ExcelGel XL SDS 12-14 polyacrylamide gel and the low molecular weight standard proteins (hen egg white conalbumin type I 76.0 kDa, bovine serum albumin 66.2 kDa, bovine muscle actin 43.0 kDa, rabbit musle glyceraldehydes dehydrogenase 36.0 kDa, bovine carbonic anhydrase 31.0 kDa, soybean trypsin inhibtor 21.5 kDa and equine myoglobin 17.5 kDa). Mass Spectrometry Grade Trypsin Gold was purchased from Promega Corporation (Madison, USA). Matrix R-cyano-4-hydroxycinnamic acid (CHCA) was purchased from the Aldrich Company. Animals. Cotton bollworms (H. armigera) were reared in our laboratory on a light/dark schedule of 14:10 h. They were fed an artificial diet made from wheat and soybeans. Preparation of Protein Samples. The fifth molting larvae were identified as the larvae with head capsule splicing when they were molting to sixth instar. The sixth feeding larvae were defined as the larvae that had lost their old cuticles and had grown and were feeding for 48 h. Four kinds of tissues: epidermis, hemolymph, fat bodies and midguts were separated from 5∼10 molting and feeding larvae, respectively. Every 0.25 g sample of tissue was homogenized in 1 mL sample buffer (40 mM Tris, 3 mM EDTA, and 1 mM PMSF) on ice. After centrifugation at 10 000 × g at 4 °C for 10 min, the supernatant was separated to 300 µL and freeze-dried and stored at -80 °C. Total protein levels were determined by the Bradford method.10 2D-PAGE. The dried protein samples were resolved in rehydration solution containing 8 M urea, 2% CHAPS, 0.002% Bromophenol blue, 2% (v/v) NL 3-10 IPG buffer and DTT (7 mg/2.5 mL rehydration solution). One-hundred to fourhundred milligrams of protein (depended on different tissues) were loaded on an 18 cm rehydrated IPG strip (NL pH 3-10) via cup loading. A Multiphor II Electrophoresis Unit (Amersham Biosciences) was used for the first-dimensional electrophoresis. Focusing was performed at 500 V for 5 h, 3500 V for 5 h and 3500 V for 10 h. The low molecular mass standard proteins were used as internal standards for pI and for determining the molecular weight of the identified proteins on the 2D-PAGE gel. Detailed descriptions of procedures for 2-D-PAGE were described in the handbook of 2D Electrophoresis using immobilized pH gradients (Amersham Biosciences). After equilibration of the focused IPG strip with SDS-PAGE equilibration solution, second-dimensional electrophoresis was carried out on the same apparatus with ExcelGel XL SDS 1214 polyacrylamide gel (245 × 180 × 5 mm) at 20 mA for 45 min, and 40 mA for 2 h 50 min. The detailed protocol was described in the 2D Electrophoresis guide from Amersham Biosciences. Protein Staining. After two-dimensional electrophoresis, the gel was stained with a modified silver staining method, in which
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glutaraldehyde was omitted in the sensitization step and formaldehyde was omitted in the silver step. Details of the procedure were described by Yan and colleagues.11 Micro Scale Protein Digestion. Gels were imaged and analyzed with ImageMaster 2D Platinum & Melanie analysis software from http://www.expay.ch. Protein spots of interest were excised with a scalpel and were subjected to tryptic digestion. The protocol recommended by the supplier of Trypsin Gold (Promega Company) was followed. The spot gel was washed with 200 µL of deionized water twice in a microcentrifuge tube, dehydrated for 5 min at room temperature in 100 µL 100% acetonitrile (ACN), dried by vacuum to remove ACN, then 5∼10 µL trypsin (1 µg/µL in 50 mM acetic acid and diluted in 40 mM NH4HCO3/10% ACN to 20 µg/mL) was added and samples were incubated on ice for 45 min. Excess trypsin was then removed and 5 µL of 40 mM NH4HCO3/ 10% buffer was added to cover the gel. The tube, with the spot gel and the buffer at the bottom, was inverted and incubated at 37 °C for 18 h. Then, 5 µL 50% ACN/5% TFA was added and the tube was incubated at room temperature for 1 h. The tube with the gel and the extract mixture was vortexed and briefly centrifuged. The supernatant was prepared for MALDI TOFMS. Application of MALDI-TOF-MS. The tryptic-digested extract was mixed with a matrix (50% ACN/0.1 TFA containing 10 mg/ mL CHCA), 3:3 µL, and spotted on the stainless steel sample plate, 2 µL per spot. After air-drying, peptide masses in the samples were detected using a Voyager Linear-DE/K, Perseptive Biosystems, Biospectrometry Workstation (Farmington, MA). MALDI TOF-MS was operated in the linear mode using delayed ion extraction (200 ns). Positively charged ions in the m/z range of 500∼82 705 were analyzed automatically. Other conditions were as follows: accelerating voltage 25 000, laser strength 2200∼2300 V and vacuum 5 × 10-7. Trypsin autodygests (842.18 and 2283.18 D) were used for calibration. First 20∼30 scans per detection were acquired for analysis. Peptide Mass Fingerprinting (PMF) results from MALDI-TOF-MS were kept as pkm files (mass peak table files) and were compared with the Drosophila melanogaster protein sequence database through an application of the Aldente Peptide Mass Fingerprinting tool search program at www.expay.ch.
Results and Discussion 2D-PAGE and Analysis of Differentially Expressed Proteins. The results of 2D-PAGE showed that the protein expression patterns varied between tissues. In epidermis, although proteins were distributed from the acidic pH region to the alkaline region, many of the spots in the acidic region could not be separated as well as those in neutral and alkaline pH regions (Figure 1, a and b). In hemolymph, proteins were distributed throughout the whole range of pH regions (3∼10). The number of protein spots was highest of the four examined tissues (Figure 1, parts c and d). In fat bodies, most spots were distributed in acidic to neutral pH regions and fewer protein spots were detected than other tissues, even though 400 µg of protein were loaded on the IPG strip. This difference reflects the special properties of proteins in fat bodies, which were not easily focused on the IPG strip (Figure 1, parts e and f). In midgut, proteins were distributed mostly in acidic and neutral pH regions (Figure 1, parts g and h). Only the proteins expressed during molting in the 4 tissues were analyzed by the ImageMaster 2D Platinum & Melanie analysis software. Twenty differentially expressed protein spots Journal of Proteome Research • Vol. 5, No. 1, 2006 165
Differentially Expressed Proteins during Larval Molting
technical notes
Figure 1. Maps of 2D-PAGE. Panels a and b are epidermis from fifth molting larvae and sixth feeding larvae, panels c and d are hemolymph from fifth molting larvae and sixth feeding larvae, panels e and f are fat bodies from fifth molting and sixth feeding larvae, panels g and h are midgut from fifth molting and sixth feeding larvae, respectively. The numbers in the panels indicate the spots which were differentially expressed during molting and were applied to MALDI TOF-MS.
were identified in fifth-molting epidermis (fifth instar molting to sixth instar) (Figure 1, part a), 36 differentially expressed protein spots were seen in fifth-molting hemolymph (Figure 1, part c) and 21 differentially expressed protein spots were 166
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observed in fifth-molting fat bodies (Figure 1, part e). However, no obvious differentially expressed protein spots were identified in fifth-molting midgut under these conditions (Figure 1, parts g and h).
technical notes
Figure 2. Peptide mass fingerprinting by MALDI TOF-MS. Panels 1 and 2 are as follows: probable serine hydrolase (no. 10 in hemolymph) and activator 1 40 kDa subunit (no. 1 in fat body), respectively.
Figure 3. Chart summarizing the composition of the proteins identified during activation of the molting cascade.
MALTI-TOF-MS and Identification of the Proteins. All identified molting-specific protein spots were applied to MALTITOF-MS. Figure 2 exhibits examples of representative MALTITOF-MS results. The resulting PMFs were saved as pkm files and were used in a similarity comparison with the D. melanogaster protein sequence database. Ten proteins were identified in the fifth-molting epidermis, along with 11 proteins in the hemolymph and 9 proteins in fat bodies (Table 1). Other numbered spots in the pictures, were not identified by the similarity search because of no matched proteins, pooled PMFs, or contamination of keratin. The identified 30 proteins were divided into 5 groups according to probable functions, which were: regulators, enzymes, hydrolases, receptors, and unknown proteins (Figure 3). Analysis of the Roles of the Identified Proteins during Molting Cascade. The roles of most identified proteins have been investigated previously in Drosophila, which provides a good reference for understanding and annotating of the roles of these proteins in the molting cascade. In the enzyme group, both protein phosphatase No. 14 in epidermis and protein
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kinase No. 7 in hemolymph were identified by 2D-PAGE. In Drosophila, Serine/threonine phosphatases are important in reversible protein phosphorylation by opposing the actions of protein kinases.12 The protein kinases catalyze protein phosphorylation and the kinases themselves are dephosphorylated and deactivated by protein phosphatases. Both activation and inhibition of phosphatases could result in cell cycle arrest and/ or apoptosis. Ray and colleagues reported that protein phosphatase 2A regulated apoptosis.13 The mitogen-activated protein kinases play important roles in regulating many cellular events including cell-cycle related events. The phosphorylation and activity of mitogen-activated protein kinases were upregulated by PTTH which, thus, regulated ecdysteroid synthesis.1 These proteins were probably involved in reversible protein phosphorylation and resulted in the activation of some enzymes that were related to the molting cascade. In the regulator group, a circadian clock-controlled protein was found from the fifth molting epidermis and hemolymph (No. 1 and No. 21, respectively). The reason that two spots display different molecular masses and isoelectric points was not clear. This protein is abundantly expressed during pupation, just prior to eclosion, and then rapidly decays in Drosophila.14 Although the role of this protein is not clear, it appears to be a molting or metamorphosis-related protein and, therefore, may play a role in the molting cascade. Two calcium binding proteins, annexin X (No 4 in epidermis)15 and neurocalcin homologue (No 9 in epidermis)16 were also found in fifth molting epidermis. Because cytoplasmic Ca2+ mobilization and entry pathways were regulated by PTTH17, the calcium binding proteins were likely involved in molting regulation. In the protein hydrolase group, aprobable serine hydrolase (No. 10) was identified from the fifth molting Helicoverpa hemolymph. In D. melanogaster, it is named kraken and it is expressed ubiquitously in the gut in early embryogenesis. During the third-instar larval stage, kraken is expressed at low levels in the gastric caeca and parts of the gut, and at higher levels in the fat bodies. Kraken played a role in detoxification and digestion during embryogenesis and larval development.18 The lysozyme X (1, 4-beta-N-acetylmuramidase X, No. 12) was identified in hemolymph. It is primarily expressed in the metamorphosing midgut of late larvae and early pupae, but not in the fat bodies or haemocytes of Drsophila.19 These two hydrolyases were first detected in the hemolymph in this study. The proteasome subunit alpha type 5 (No 2) was identified in fat bodies. In Drosophila, it encodes one of the proteasomal alpha subunits. Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most regulated intracellular protein breakdown in eukaryotic cells.20 The Caspase Nc subunit 1 (or DRONC, No. 4 and 12) was also identified in fat body. DRONC is involved in apoptosis in Drosophila. It exhibits substrate specificity similar to mammalian caspase-2. DRONC is ubiquitously expressed in Drosophila embryos during early stages of development. In late third instar larvae, dronc mRNA is dramatically up-regulated in salivary glands and midguts before histolysis of these tissues. DRONC is known an effecter of steroid-mediated apoptosis during insect metamorphosis.21 Of the four protein hydrolases described above, two are known to be related to the molting cascade, whereas further evidence is required to demonstrate a function in the molting cascade for the other two. In the receptor group, 3 proteins with substance binding properties were found. A general odorant-binding protein (OBP, No. 24) and a pheromone-binding protein (No. 31) were seen Journal of Proteome Research • Vol. 5, No. 1, 2006 167
technical notes
Differentially Expressed Proteins during Larval Molting Table 1. Identification of Differentially Expressed Proteins Based on the Drosophila Protein Database spot no.
1 4 7 9 10 11 13
14
2
10 12 17 18 21 22 24 31 34 1 2 4 12 5 6
14 15
20
Epidermis 13
function
3.7 × 10-4
expressed during pupation just prior to eclosion Ca2+ and phospholipid-binding proteins guiding the germ cells migrating toward the overlying mesoderm Calcium binding translation initiation factor
P22465 Q9V574
11 13
4.2 × 10-4 3.1 × 10-4
Neurocalcin homolog Eukaryotic translation initiation factor Glutathione S-transferase Probable ARP2/3 complex 34 kDa subunit
P42325 Q9GU68
12 25
8.3 × 10-5 2.4 × 10-4
Q9VG94 Q9VIM5
16 10
1.4 × 10-4 2.6 × 10-4
P12982
12
3.7 × 10-4
Glutathione S-transferase actin-related protein 2/3 (Arp2/3) complex plays a key role in microfilament formation protein phosphatase
9 × 10-5 1.2 × 10-4
ATP synthase Splicing of RNA
1.2 × 10-4
cytochrome 450
Activator 1 40 kDa subunit Proteasome subunit alpha type 5 Caspase Nc subunit 1 Fat body protein 2 Probable phosphormevalonate kinase Synaptosamal-associated protein 25 Peroxiredoxin 1 Mediator complex subunit TRAP18 Probable glucosamine 6-phosphate N-acetyltransferase
Q94516 Q02427-2 P82712
16 17 Hemolymph 9
P83104
12
8 × 10-5
O18391 P37161 P36975
11 41 21
3.5 × 10-4 5.5 × 10-5 1.9 × 10-5
P00334 O76879
16 16
2.5 × 10-4 2.7 × 10-5
P49906
20
9.2 × 10-6
hydrolase hydrolase involved in vesicle docking and membrane fusion in brain and ganglia dehydrogenase expressed during pupation, just prior to eclosion transcription initiation factor
Q8MKK0
23
9.3 × 10-5
receiptor
Q23970
19
8.1 × 10-4
receptor
1.2 × 10-4
unknown
4.9 × 10-4 1.4 × 10-4
replication factor Hydrolase Hydrolase unknown catalyzes an essential step in the mevalonate pathway vesicle docking and membrane fusion in brain and ganglia redox reaction coactivator to integrate promoterspecific activation signals to the basal transcription machinery transferase
Q9VSU7-2 P53034 Q95083
12 Fat Body 10 10
Q9XYF4 P54398 Q9VIT2
18 10 16
3.9 × 10-4 1.9 × 10-4 1.4 × 10-4
P36975
18
1.4 × 10-4
Q9V3P0 Q8IH24
20 23
5.4 × 10-5 6.8 × 10-4
Q9VAI0
18
6.5 × 10-5
in the hemolymph, and a synaptosomal-associated protein 25 (No. 11) was found in the fat bodies. OBP is thought to facilitate the delivery of hydrophobic odorants, such as sex pheromones or food odors, to receptors on sensory neurons in Drosophila. Increasingly, OBP family members are also being found in nonsensory tissues where they might carry other types of small hydrophobic molecules.22 The pheromone-binding protein (No. 31 in hemolymph) is also thought to be a member of a larger class of proteins, extending beyond the olfactory system.23 In Helicoverpa, these sensory receptors may carry some hydrophobic substances which are involved in the molting cascade. In this study, a microscale protein digestion method was developed. Inverting incubation tubes during overnight protein 168
p value
O76879
Probable cytochrome 450 12d1 Putative mitogen-activated protein kinase Probable serine hydrolase Lysozyme x Synaptosomal-associated protein 25 Alcohol dehydrogenase Circadian clock-controlled protein Transcription initiation factor TFIID General odorant-binding protein 57a Pheromone-binding proteinrelated protein Splice isoform 2 of Q9vsu7
7
covrage %
Circadian clock-controlled protein Annexin X Lethal (2) k 10201 protein
Serine/threonine protein phosphatase ATP synthase B Chain Splice isoform B of Q02427
17 20
11
identification
NCBI datebase no.
Journal of Proteome Research • Vol. 5, No. 1, 2006
protein kinase
digestion overcomes the problem of evaporation of digestioncovering buffer during incubation. When followed with use of a small volume extraction buffer, a high concentration of digested products were obtained and were directly applied to MALTI-TOF-MS. By this method, there is no need to concentrate the trypsin digestion products from a large volume. Therefore, this method is useful for operating a large scale investigation of a proteome. The modified silver stained gel can be digested by this method without silver destain. The PMF resulting from the linear model of MALTI-TOF-MS can be used to identify proteins by the Aldente Peptide Mass Fingerprinting tool program. Because the whole genome of H. armigera has not yet been sequenced, the proteins identified against D.
technical notes melanogaster showed lower amino acid coverage and p-values. Besides, the molecular masses and isoelectric points of some identified proteins are not identity to the proteins from Drosophila. This might because of the difference between the species, modification of the proteins or other unknown reasons. The true identities of these proteins still need to be verified in future study by Western blot with specific antibodies or via micro-sequencing.
Conclusion With the combination of a modified silver stain microscale protein digestion and MALTI-TOF-MS, 30 differentially expressed proteins were identified, which might be involved in the molting cascade. In which, the mitogen-activated protein kinases, the circadian clock-controlled protein, the lysozyme X, the caspase Nc, the calcium binding proteins annexin X and the neurocalcin homologue are known to be related to the molting cascade in Drosophila melanogaster. Further study is required for precise interpretations of the functions of all these proteins in the molting cascade.
Acknowledgment. This work was supported by a grant from the National Natural Science Foundation of China (No. 30330070). Authors thank Professor H. Kondo and K. Shimizu in Kyushu Institute of Technology Iizuka, Japan for their kindly offering the conditions and facilitates for this study. References (1) Rybczynski, R.; Bell, S. C.; Gilbert, L. I. Mol. Cell. Endocrinol. 2001, 184, 1-11. (2) Riddiford, L. M.; Hiruma, K.; Zhou, X.; Nelson, C. A. Insect Biochem. Mol. Biol. 2003, 33, 1327-1338. (3) Zheng, Y.; Zheng, S.; Cheng, X.; Ladd, T.; Lingohr, E. J.; Krell, P. J.; Arif, B. M.; Retnakaran, A.; Fenng, Q. Insect Biochem. Mol. Biol. 2002, 32, 1813-1823.
Zhao et al. (4) Samuels, R. I.; Charnley, A. K.; Reynolds, S. E. Insect Biochem. Mol. Biol. 1993, 23, 607-614. (5) Ote, M.; Mita, K.; Kawasaki, H.; Daimon, T.; Kobayashi, M.; Shimada, T. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2005, 141, 314-322. (6) Giavalisco, P.; Nordhoff, E.; Kreitler, T.; Kloppel, K. D.; Lehrach, H.; Klose, J.; Gobom, J. Proteomics 2005, 5, 1902-1913. (7) Vensel, W. H.; Tanaka, C. K.; Cai, N.; Wong, J. H.; Buchanan, B. B.; Hurkman, W. J. Proteomics 2005, 5, 1594-1611. (8) Paskewitz, S. M.; Shi, L.Insect. Biochem. Mol. Biol. 2005, 35, 815824. Epub 2005 Apr 26. (9) Wang, Y.; Zhang, P.; Fujii, H.; Banno, Y.; Yamamoto, K.; Aso, Y. Biosci. Biotechnol. Biochem. 2004, 68, 1821-1823. (10) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (11) Yan, J.-X.; Wait, R.; Berkelman, T.; Harry, R. A.; Westbrook, J. A.; Wheeler, C. H.; Dunn, M. J. Electrophoresis 2000, 21, 3666-3672. (12) Berndt, N. Prog. Cell Cycle Res. 2003, 5, 497-510. (13) Ray, R. M.; Bhattacharya, S.; Johnson, L. R. J. Biol. Chem. 2005, Jul 1; [Epub ahead of print]. (14) Lorenz, L. J.; Hall, J. C.; Rosbash, M. Development 1989, 107, 869880. (15) Johnston, P. A.; Perin, M. S.; Reynolds, G. A.; Wasserman, S. A.; Sudhof, T. C. J. Biol. Chem. 1990, 265, 11382-11388. (16) Teng, D. H,; Chen, C. K.; Hurley, J. B. J. Biol. Chem. 1994, 269, 31900-31907. (17) Fellner, S. K.; Rybczynski, R.; Gilbert, L. I. Insect Biochem. Mol. Biol. 2005, 35, 263-275. (18) Edwin Chan, H. Y.; Harris, S. J.; O’Kane, C. J. Gene 1998, 222, 195-201. (19) Daffre, S.; Kylsten, P.; Samakovlis, C.; Hultmark, D. Mol. Gen. Genet. 1994, 242, 152-162. (20) Zaiss, D.; Belote, J. M. Gene 1997, 201, 99-105. (21) Dorstyn, L.; Colussi, P. A.; Quinn, L. M.; Richardson, H.; Kumar, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4307-4312. (22) Graham, L. A.; Davies, P. L. Gene 2002, 292, 43-55. (23) McKenna, M. P.; Hekmat-Scafe, D. S.; Gaines, P.; Carlson, J. R. J. Biol. Chem. 1994, 269, 16340-16347.
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