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• W1969154002 abstract "Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr PG33 mutants do not survive past the early larval stage. These mutants have a P-element insertion within exon 1B upstream of the initiation ATG, which greatly reduces mRNA levels of all forms of CPD. Both svr 1 and svr poi mutants are viable, with a silvery body color and pointed wings. The wing shape is generally similar between these two mutants, although svr poi mutants have smaller wings. The svr 1 gene has a three-nucleotide deletion in exon 6, removing a leucine in a region of the protein predicted to function as a folding domain for the second CP-like domain. svr poi has a 1072-bp duplication of the gene that introduces a stop codon into the open reading frame, causing the truncation of the protein in the middle of the second CP-like domain. Both deletions eliminate enzyme activity of the second CP-like domain and appear to cause the misfolding of the protein. This greatly reduces the levels of the long forms of CPD protein but do not affect the levels of the short forms. Taken together, these findings suggest that lethal and viable svr alleles differ in which protein forms are affected. Flies that retain the short form are viable, whereas flies that are missing all forms of CPD do not survive past the early larval stages. Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr PG33 mutants do not survive past the early larval stage. These mutants have a P-element insertion within exon 1B upstream of the initiation ATG, which greatly reduces mRNA levels of all forms of CPD. Both svr 1 and svr poi mutants are viable, with a silvery body color and pointed wings. The wing shape is generally similar between these two mutants, although svr poi mutants have smaller wings. The svr 1 gene has a three-nucleotide deletion in exon 6, removing a leucine in a region of the protein predicted to function as a folding domain for the second CP-like domain. svr poi has a 1072-bp duplication of the gene that introduces a stop codon into the open reading frame, causing the truncation of the protein in the middle of the second CP-like domain. Both deletions eliminate enzyme activity of the second CP-like domain and appear to cause the misfolding of the protein. This greatly reduces the levels of the long forms of CPD protein but do not affect the levels of the short forms. Taken together, these findings suggest that lethal and viable svr alleles differ in which protein forms are affected. Flies that retain the short form are viable, whereas flies that are missing all forms of CPD do not survive past the early larval stages. Metallocarboxypeptidases (CPs) 3The abbreviations used are: CP, metallocarboxypeptidase; CPD and CPE, carboxypeptidase D and E, respectively; CNS, central neural system; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BMP, bone morphogenic protein. 3The abbreviations used are: CP, metallocarboxypeptidase; CPD and CPE, carboxypeptidase D and E, respectively; CNS, central neural system; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BMP, bone morphogenic protein. perform many physiological functions, ranging from the digestion of food to the biosynthesis of neuropeptides (1Aviles F.X. Vendrell J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 851-853Google Scholar, 2Auld D.S. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 812-821Google Scholar, 3Auld D.S. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 821-825Google Scholar, 4Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 840-844Google Scholar, 5Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 848-851Crossref Scopus (14) Google Scholar, 6Reznik S.E. Fricker L.D. Cell Mol. Life Sci. 2001; 58: 1790-1804Crossref PubMed Scopus (134) Google Scholar, 7Skidgel R.A. Erdos E.G. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 837-840Google Scholar, 8Skidgel R.A. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 851-854Google Scholar, 9Springman E.B. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 828-830Google Scholar, 10Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 844-846Google Scholar). CPs are divided into three subfamilies based on amino acid analysis: the A/B subfamily (1Aviles F.X. Vendrell J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 851-853Google Scholar, 2Auld D.S. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 812-821Google Scholar, 3Auld D.S. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 821-825Google Scholar, 9Springman E.B. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 828-830Google Scholar), the N/E subfamily (4Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 840-844Google Scholar, 5Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 848-851Crossref Scopus (14) Google Scholar, 6Reznik S.E. Fricker L.D. Cell Mol. Life Sci. 2001; 58: 1790-1804Crossref PubMed Scopus (134) Google Scholar, 7Skidgel R.A. Erdos E.G. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 837-840Google Scholar, 8Skidgel R.A. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 851-854Google Scholar, 10Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 844-846Google Scholar), and a recently discovered subfamily that includes Nna1 and related proteins but has not yet been demonstrated to have CP activity (11Harris A. Morgan J.I. Pecot M. Soumare A. Osborne A. Soares H.D. Mol. Cell Neurosci. 2000; 16: 578-596Crossref PubMed Scopus (76) Google Scholar). Within each subfamily, the members show 35-60% amino acid sequence identity, but between subfamilies, there is only 15-25% amino acid sequence conservation. The A/B subfamily is primarily involved in protein digestion, either in the digestive track or elsewhere in the body. In contrast, the N/E subfamily plays more of a biosynthetic role by selectively removing specific residues from peptide processing intermediates, and this step often affects the biological properties of the substrate. In humans and mice, there are eight members of this N/E family, of which five show enzymatic activity (CPE, CPN, CPM, CPZ, and CPD); the remaining members of this subfamily (CPX1, CPX2, and AEBP-1/ACLP) are not active toward standard substrates (4Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 840-844Google Scholar, 5Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 848-851Crossref Scopus (14) Google Scholar, 6Reznik S.E. Fricker L.D. Cell Mol. Life Sci. 2001; 58: 1790-1804Crossref PubMed Scopus (134) Google Scholar, 7Skidgel R.A. Erdos E.G. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 837-840Google Scholar, 8Skidgel R.A. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 851-854Google Scholar, 10Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 844-846Google Scholar, 12Xin X. Day R. Dong W. Lei Y. Fricker L.D. DNA Cell Biol. 1998; 17: 897-909Crossref PubMed Scopus (47) Google Scholar, 13Lei Y. Xin X. Morgan D. Pintar J.E. Fricker L.D. DNA Cell Biol. 1999; 18: 175-185Crossref PubMed Scopus (45) Google Scholar, 14He G.P. Muise A. Li A.W. Ro H.S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar, 15Layne M.D. Yet S.F. Maemura K. Hsieh C.M. Bernfield M. Perrella M.A. Lee M.E. Mol. Cell. Biol. 2001; 21: 5256-5261Crossref PubMed Scopus (76) Google Scholar). In contrast, Drosophila contains only two members of this subfamily, one that has high homology to CPM and another that is a CPD homolog (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). CPD is unique among CPs in that it contains multiple CP-like domains, a transmembrane domain, and a short cytosolic tail (5Fricker L.D. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego2004: 848-851Crossref Scopus (14) Google Scholar). Humans, rats, mice, duck, and Drosophila CPD all contain three CP-like domains, of which the first two are enzymatically active and the third is missing key catalytic residues but still appears to retain the basic CP-like structure (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 17Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Fan X. Qian Y. Fricker L.D. Akalal D.B. Nagle G.T. DNA Cell Biol. 1999; 18: 121-132Crossref PubMed Scopus (14) Google Scholar, 19Song L. Fricker L.D. J. Biol. Chem. 1995; 270: 25007-25013Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Song L. Fricker L.D. J. Biol. Chem. 1996; 271: 28884-28889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Xin X. Varlamov O. Day R. Dong W. Bridgett M.M. Leiter E.H. Fricker L.D. DNA Cell Biol. 1997; 16: 897-909Crossref PubMed Scopus (75) Google Scholar, 22Tan F. Rehli M. Krause S.W. Skidgel R.A. Biochem. J. 1997; 327: 81-87Crossref PubMed Scopus (61) Google Scholar, 23Kuroki K. Eng F. Ishikawa T. Turck C. Harada F. Ganem D. J. Biol. Chem. 1995; 270: 15022-15028Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Ishikawa T. Murakami K. Kido Y. Ohnishi S. Yazaki Y. Harada F. Kuroki K. Gene (Amst.). 1998; 215: 361-370Crossref PubMed Scopus (23) Google Scholar, 25Aloy P. Companys V. Vendrell J. Aviles F.X. Fricker L.D. Coll M. Gomis-Ruth F.X. J. Biol. Chem. 2001; 276: 16177-16184Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 26Gomis-Ruth F.X. Companys V. Qian Y. Fricker L.D. Vendrell J. Aviles F.X. Coll M. EMBO J. 1999; 18: 5817-5826Crossref PubMed Scopus (71) Google Scholar, 27Settle S.H.J. Green M.M. Burtis K.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9470-9474Crossref PubMed Scopus (43) Google Scholar). The functional significance of the three CP-like domains in CPD is not clear. One proposal is that the distinct pH optima of the first two domains enable CPD to be active throughout the secretory pathway, which ranges from neutral to acidic pH values (17Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Novikova E.G. Eng F.J. Yan L. Qian Y. Fricker L.D. J. Biol. Chem. 1999; 274: 28887-28892Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In both duck and Drosophila, the first domain is more active at neutral pH, whereas the second domain is optimally active at pH 5-6 (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 17Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Novikova E.G. Eng F.J. Yan L. Qian Y. Fricker L.D. J. Biol. Chem. 1999; 274: 28887-28892Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Protein containing both domains together showed a broader pH optimum (17Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Novikova E.G. Eng F.J. Yan L. Qian Y. Fricker L.D. J. Biol. Chem. 1999; 274: 28887-28892Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). There are several known mutations for CPD in Drosophila that are collectively known as the silver,or svr, mutants based on the silvery body color of the viable mutants (27Settle S.H.J. Green M.M. Burtis K.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9470-9474Crossref PubMed Scopus (43) Google Scholar, 29Walter M.F. Zeineh L.L. Black B.C. McIvor W.E. Wright T.R.F. Biessmann H. Arch. Insect. Biochem. Physiol. 1996; 31: 219-233Crossref PubMed Scopus (60) Google Scholar). In addition to the body color, the viable svr mutants have been reported to show altered wing shape and mating behavior in light (30Wright T.R. Adv. Genet. 1987; 24: 127-222Crossref PubMed Scopus (381) Google Scholar, 31Lindsley D.L. Grell E.H. Genetic Variations of Drosophila melanogaster. Carnegie Institute, Baltimore1968: 468-469Google Scholar), although these results have not been adequately described in the literature. In addition to the viable svr mutants, there are a number of svr mutants that have been reported to be embryonic lethal (27Settle S.H.J. Green M.M. Burtis K.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9470-9474Crossref PubMed Scopus (43) Google Scholar, 30Wright T.R. Adv. Genet. 1987; 24: 127-222Crossref PubMed Scopus (381) Google Scholar, 31Lindsley D.L. Grell E.H. Genetic Variations of Drosophila melanogaster. Carnegie Institute, Baltimore1968: 468-469Google Scholar). Through genetic complementation studies, the viable and lethal mutants were shown to represent the same gene, but the molecular basis of the defects was not characterized. The purpose of the present study was to first evaluate the phenotypic differences between CPD mutants and then to determine the molecular basis for each mutant. The results of these analyses provide insights into the function of the various domains of CPD. Drosophila Strains—Flies were maintained on standard cornmealagar Drosophila medium at 18 or 25 °C. Wild type stock (w-) and the mutants svr 1 and svr poi were obtained from Bloomington Stock Center. The mutant svr PG33 was obtained from Dr. Alain Vincent (32Bourbon H. Gonzy-Treboul G. Peronnet F. Alin M. Ardourel C. Benassayag C. Cribbs D. Deutsch J. Ferrer P. Haenlin M. Lepesant J. Noselli S. Vincent A. Mech. Dev. 2002; 110: 71-83Crossref PubMed Scopus (139) Google Scholar). The svr PG33 line was propagated as yw[PG33w+]/FM7. For selection of svr PG33 homozygous embryos, yw[PG33w+] flies were balanced over the FM7, act:GFP chromosome (obtained from Bloomington Stock Center). Embryos for RNA preparation were collected on agar-apple juice plates at 16-20 h after egg laying. Nonfluorescent svr PG33 homozygous embryos were selected using a Leica MZFLIII microscope with the GFP2 fluorescent filter. Wing Shape Analysis—Wings were cut from 3-4-day-old females and mounted in DPX mounting medium (Fluka BioChemika). Images were collected with a Nikon SMZ-U microscope with ×6 optical magnification. The relative length of each wing segment was determined by tracing the outline of the segment, as shown in Fig. 1, using ImageJ 1.33u (National Institutes of Health). The Effect of Light on Reproduction—Nine vials of each of the two viable silver mutants (svr 1, svr poi) and nine vials of wild type (w-) flies were used. Three vials of each strain were exposed to constant bright light by exposing the vials within 10-20 cm of a light box containing two 20-watt fluorescent bulbs. Three vials were wrapped in foil and kept in darkness for the same time period, and three vials were housed under regular conditions (ambient room light). Each vial contained 1-2 virgin males and 5-6 virgin females, which were put into the test within 1-8 h from eclosion. Flies were kept in vials for 2-5 days, the adults were discarded, and their progeny were counted. The entire experiment was repeated several times with comparable results. PCR and Sequencing—Genomic DNA for PCR amplification was prepared using the Qiagen DNeasy tissue kit. Pairs of oligonucleotides located 1-3 kb apart were used as PCR primers to determine if there were any large changes in the CPD gene in the svr mutants, compared with wild type flies. Each PCR product overlapped so that the entire 11-kb svr gene was covered. When an insert was identified within the svr poi mutant, several additional oligonucleotides were used to narrow down the insertion point. PCR product containing the insert was amplified, purified (Qiagen), introduced into the pCR4-TOPO vector (Invitrogen), and sequenced. For the svr 1 mutant, this analysis did not reveal any detectable insertion or deletion. Therefore, the PCR was repeated using the Roche Expand HiFi PCR system to amplify all of the exons and introns of the svr 1 gene. PCR products from three independent reactions were gel-purified (Qiagen), and sequenced. The sequences were analyzed by the MegAlign program (DNA-Star). Southern Blot—To confirm the position of the P-element insertion in yw[PG33w+]/FM7 flies, the Southern blot method was employed. DNA was prepared by overnight digestion of 50 adult animals at 55 °C with 20 mg/ml Proteinase K (Invitrogen) in TNE buffer (0.1 m EDTA, 0.4 m NaCl, 5 mm Tris, pH 7.5) followed by the addition of 5 m NaCl, extraction with phenol/chloroform, and ethanol precipitation. Approximately 20 μg of genomic DNA was digested with EcoRI or ClaI and fractionated on a 0.9% agarose gel. After photography of the ethidium bromide-stained gel, the DNA was transferred to a nitrocellulose membrane (Optitran; Scheicher and Schuell) and probed with a riboprobe corresponding to the 5′ end of fly CPD exon 1B. The production of the probe has been previously described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Approximately 3 × 106 cpm of exon 1B probe was hybridized with the nitrocellulose blot in 5× SSC (0.75 m NaCl, 75 mm sodium citrate), 50% formamide, 5× Denhardt's solution, 1% SDS, and 100 μg/ml denatured salmon sperm DNA at 50 °C overnight. After hybridization, the blot was washed with 2× SSC containing 0.1% SDS, 1× SSC containing 0.1% SDS, and then 0.1× SSC containing 0.1% SDS buffer at 50 °C. The blot was dried and exposed to x-ray film (X-Omat Blue XB-1; Eastman Kodak Co.) for 1 day at -80 °C with an intensifying screen. Northern Blot—Total RNA was prepared from embryos (16-20 h old), from larvae (third instar stage), and from adult w-, svr 1, and svr poi strains using the Qiagen RNeasy minikit. For yw[PG33w+]/FM7, act: GFP, only nonfluorescent embryos (16-20 h old) were used. About 7.5 μg of RNA was fractionated on an agarose gel containing 2% formaldehyde. After photography of the ethidium bromide-stained gel, the RNA was transferred to a nitrocellulose membrane (Optitran; Scheicher and Schuell) and probed with fly CPD riboprobes. The riboprobes and Northern blot procedure have been previously described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The blots probed with exons 1A and 1B were exposed for 1 week, and the blots probed with exons 3 and 6 were exposed for 3 days at -80 °C with an intensifying screen. Protein Extraction, Purification, and Western Blot—To obtain the membrane fraction for Western blot analysis, frozen adult flies were homogenized (Polytron; Brinkman) in 0.1 m Na2CO3 (pH 11-12) and centrifuged at 15,000-30,000 × g for 20-30 min, and the pellet was resuspended and heated at 95 °C in gel loading buffer containing 4% SDS. To examine the forms of CPD in whole tissue extracts, the central neural system (CNS) was dissected from third instar larvae. Dissections were done in 0.1 m phosphate buffer, pH 7.4, containing a protease inhibitor mixture (Roche Applied Science), transferred to loading buffer containing 4% SDS, sonicated, heated at 95 °C, and loaded onto a denaturing polyacrylamide gel. The gel was transferred to nitrocellulose and probed as described below. For purification of the soluble forms of CPD, adult frozen flies were homogenized (Polytron) in 20 mm NaAc, pH 7.4, filtered through mesh, and centrifuged at 800 × g for 5 min. The supernatant was centrifuged at 4 °C for 40 min at 40,000 × g. The second supernatant was adjusted to 100 mm NaAc, pH 5.5, and applied to a 0.5-ml p-aminobenzoyl-Arg-Sepharose affinity resin column as described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The column was first washed with 0.5 m NaCl and 1% Triton X-100 at pH 5.5 and then washed with 50 mm Tris containing 100 mm NaCl and 0.01% CHAPS at pH 8.0. CPD was eluted with 25 mm Arg in the Tris/NaCl, pH 8, wash buffer. Western Blot—Proteins were fractionated on SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membrane (Optitran; Scheicher and Schuell). The nitrocellulose blots were blocked as described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Blots were probed with 1:1000 dilutions of polyclonal rabbit or mouse antisera raised against peptides corresponding to the N-terminal region of exon 1A, the N-terminal region of exon 1B, or the C-terminal region of exon 8, which corresponded to the long tail 2 form of Drosophila CPD (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Following exposure of the blots to the primary antiserum, the blots were washed and exposed to horseradish peroxidase-labeled goat anti-rabbit antiserum or sheep anti-mouse antiserum (Amersham Biosciences). The enhanced chemiluminescence method (Pierce) was used for detection of bound antiserum. Expression Constructs—For expression of different forms of CPD in the baculovirus system, reverse transcription-PCR was used to generate the wild type or mutant form of domain 2. These were then attached to fly CPD1 containing either exon 1A or 1B; the creation of the 1A and 1B forms of CPD has been previously described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). CPD domain 2 was amplified as a 1.3-kb band for both wild type and svr 1 mutant flies using the Qiagen One Step reverse transcription-PCR kit. The 3′ oligonucleotide introduced a NotI restriction site (downstream of the coding region) to facilitate subsequent cloning steps. PCR products were digested with StuI and NotI, gel-purified (Qiagen), and ligated into StuI/NotI-digested CPD1A or CPD1B constructs, as described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). To generate the svr poi mutant form of CPD domain 2, PCR was used to introduce the break point and short insert found in the svr poi gene sequence into the corresponding region of fly CPD cDNA. The oligonucleotide used for the mutagenesis included a NotI site downstream of the coding region, and the CPD1A/2wt construct described above was used as a template. After PCR with Platinum Taq (Invitrogen), the 780-bp product was purified (Qiagen) and subcloned into pCR-Blunt II-TOPO cloning vector (Invitrogen). Then the resulting plasmid was digested with StuI and NotI, and the 780-bp band was ligated into StuI/NotI-digested CPD1A or CPD1B. All of the constructs were confirmed by sequencing in both directions. Baculovirus Expression and Protein Purification—Baculovirus expressing only the short 1B form or wild type domain 2 were previously created (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The new constructs containing either the 1A or the 1B form and one of three different sequences of domain 2 (wild type, svr 1,or svr poi) were generated using a similar procedure. For expression, 108 Sf9 cells growing in solution were infected with one of the baculovirus constructs. After 72 h, the cell suspensions were pelleted at 300 × g for 10 min, and the media were removed. Cells were resuspended in 50 mm NaAc, pH 7.4, and sonicated. Aliquots of media and cell homogenates were assayed for enzymatic activity, as described (16Sidyelyeva G. Fricker L.D. J. Biol. Chem. 2002; 277: 49613-49620Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), using 200 μm dansyl-Phe-Ala-Arg substrate in 100 mm Tris acetate at either pH 7.4 or 5.6. All enzyme assays were performed in triplicate, with variation among replicates typically less than 5%. Aliquots of media and cells were also analyzed on Western blots, as described above. The blots were probed with a 1:2000 dilution of a rabbit antiserum to purified duck CPD (AE178). For detection of bound antiserum, a 1:5000 dilution of IRDye 800-conjugated α-rabbit IgG (Rockland, Inc.) was used with an Odyssey® version 1.2 Infrared Imaging System (LI-COR Biosciences). To determine the solubility of the different CPD forms expressed in Sf9 cells, the cell pellets were sequentially extracted with 0.1 m Tris, pH 6.0; 0.1 m Tris, pH 6.0, containing 1 m NaCl; and 0.1 m Tris, pH 6.0, containing 1 m NaCl and 0.5% CHAPS. Each extraction step involved sonication of the pellet in the indicated buffer and centrifugation at 4 °C for 30 min at 24,000 × g. The final pellet remaining after all extractions was resuspended in Tris buffer. All fractions were assayed for carboxypeptidase activity at pH 5.6 and 7.4, as described above. Although the original Drosophila svr mutation was described many years ago and a number of other mutations in the svr gene have been reported (27Settle S.H.J. Green M.M. Burtis K.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9470-9474Crossref PubMed Scopus (43) Google Scholar), no previous reports have quantified the difference in wing shape between the viable svr mutants and wild type flies. To gain a more precise understanding of the phenotype of the viable svr mutants, the length of various wing veins was determined for svr 1, svr poi, and wild type Drosophila. Both of these viable svr mutants have pointed wings and show some common differences with wild type flies as well as several unique differences (Fig. 1). Both mutants have shorter proximal segments of the third and fourth longitudinal veins and longer distal segments of these veins (Fig. 1). Both mutants also have shorter anterior and posterior cross-veins (Fig. 1). Finally, both mutants have a longer wing edge between the second and third longitudinal veins and a shorter wing edge between the third and fourth longitudinal veins (Fig. 1). In general, the wings of the svr poi mutants are smaller than either the wild type or svr 1 wings, and many of the individual segments are shorter in the svr poi mutants compared with the other two. The above observations are consistent with the previous literature describing the phenotype of svr 1 and svr poi (27Settle S.H.J. Green M.M. Burtis K.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9470-9474Crossref PubMed Scopus (43) Google Scholar, 31Lindsley D.L. Grell E.H. Genetic Variations of Drosophila melanogaster. Carnegie Institute, Baltimore1968: 468-469Google Scholar). However, it is possible that another mutation has spontaneously arisen in these lines and that this contributes to the observed phenotype. To ensure that the wing shape is solely due to the svr mutation, we crossed homozygous viable svr mutants with svr PG33 to create svr 1/svr PG33 and svr poi/svrPG33 lines. The same general tendency was observed in these two lines as found in the homozygous lines: decreased length of proximal wing parts, increased length of distal parts of the third, fourth, and fifth longitudinal veins, and decreased length of cross-veins (data not shown). Previously, svr 1 males were reported to be unable to mate when exposed to constant light (30Wright T.R. Adv. Genet. 1987; 24: 127-222Crossref PubMed Scopus (381) Google Scholar)." @default.
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• W1969154002 title "Characterization of the Molecular Basis of the Drosophila Mutations in Carboxypeptidase D" @default.
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