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Differential expression of ras in organs and embryos of shrimp Penaeus monodon (Crustacea: Decapoda)
Comparative Biochemistry and Physiology Part B 125 (2000) 307 – 315 www.elsevier.com/locate/cbpb
Differential expression of ras in organs and embryos of shrimp Penaeus monodon (Crustacea: Decapoda) Chein-Fuang Huang a, Wen-Yi Chen a, Nin-Nin Chuang b,* a
Department of Zoology, National Taiwan Uni6ersity, Nankang 11529, Taipei, Taiwan b Institute of Zoology, Academia Sinica, Nankang 11529, Taipei, Taiwan
Received 12 May 1999; received in revised form 30 November 1999; accepted 6 December 1999
Abstract Total RNA from shrimp hepatopancreas of Penaeus monodon showed three prominent bands that react with the shrimp ras probe, a 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) on the northern blot. The R1 is the least abundant. Analyses of total RNA from gill and heart were similar to each other. The highest expression of Ras was observed in the gill, while a negligible signal was detected with the Ras probe in muscle. Ras expression is developmentally regulated in embryonic stages of shrimp. Messenger RNA levels of ras were increased from a minimum in the nauplius stage to a maximum in the post-larvae stage for R1 and R2. R3 showed a maximum at the protozoea stage. On the other hand, the activity of protein geranylgeranyltransferase I was increased significantly in the early nauplius stage. No correlative increase of prenylation activity by protein geranylgeranyltransferase I was observed with the transcription activity of ras. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ras; Protein geranylgeranyltransferase I; Shrimp; Embryo; Hepatopancreas; Gill; Heart; Mitogen; Aquaculture
1. Introduction Ras proteins are membrane-associated small guanine nucleotides binding proteins and are required at different stages of the cell cycle (Moodie and Wolfman, 1994). In particular, it has been shown that Ras is required for cell cycle progression from G0 to G1 (Dobrowolski et al., 1994) and in the G1/S transition (Stacey et al., 1991; Lu and Campisi, 1992; Howe et al., 1993). In mammals, four isoforms of Ras exist: H-Ras, N-Ras, KARas, and KB-Ras (Lowy and Willumsen, 1993). They are products of three genes, with KA-Ras and KB-Ras being splice variants of the same gene. Among them, the protein sequences are 80% * Corresponding author. Tel./fax: + 886-2-27899531. E-mail address: [email protected] (N.-N. Chuang)
identical with major differences residing in their carboxyl termini, including CAAX motif of prenylation (Del Villar et al., 1996). Lipid modification of Ras results in an increase in its intrinsic affinity for the plasma membrane where Ras protein can participate in signal transduction (Zhang et al., 1997). The addition to Ras of isoprenoid residues, such as geranylgeranyl (20carbon) and farnesyl residues (15-carbon), is determined by the X residue of the carboxyl terminal CAAX (C, cysteine; A, an aliphatic amino acid) of proteins. If X is leucine or phenylalanine, the protein is geranylgeranylated (Casey et al., 1991; Moores et al., 1991; Yokoyama et al., 1991); if X is methionine, serine, alanine or glutamine, the protein is farnesylated (Hancock et al., 1989; Schaber et al., 1990; Reiss et al., 1991). Ras is preferentially farnesylated in mammals
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(James et al., 1995), but geranylgeranylated in shrimp (Lin and Chuang, 1998; Huang and Chuang, 1999). Abolishing prenylation disrupts the association of Ras with membranes, thereby disrupting its function (Der and Cox, 1991; Kato et al., 1992). Inhibitors of prenylation are effective at suppressing the growth of tumor cells possessing oncogenic Ras (Hancock et al., 1989; Seabra et al., 1991; Sun et al., 1995; Lerner et al., 1997). Ras is a specific and interesting regulation target for the applications in aquaculture, as suggested by the fact that microinjection of oncogenic mammalian Ras proteins into Xenopus lae6is oocytes would induce cellular divisions (Pomerance et al., 1992). Since the cultivation of penaeid shrimp is a worldwide economically important industry. In this report, we undertake the characterization of Ras at early developmental stages in the tropical shrimp Penaeus monodon, and probe the correlation of prenylation activity with the Ras expression. We attempt in the future the transfer of Ras into developing embryos to probe its effect on growth. In addition, we plan to use Ras for further investigations of the precise mechanism by which the mitogenic signal induces maturation and releases the M-phase arrest in oocytes of shrimp, as suggested by the results of vertebrates (Rhodes et al., 1994, 1997).
2. Materials and methods
2.1. Materials All reagents used were of the highest grade available commercially.
2.2. Experimental animals Shrimp (P. monodon), collected off the coast of Taiwan, were kept at 18°C for less than 3 days before experiments in a recirculating seawater system. Organs were dissected out immediately after shrimp had been killed and frozen in liquid nitrogen and stored at −80°C.
2.3. Southern blotting of shrimp ras PCR products The Dig-11-UTP labeled human H-ras RNA fragment was synthesized by 1× transcription
buffer (40 mM Tris–HCl, pH 8.0, 6 mM MgCl2, 10 mM DTT, 2 mM spermidine), 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.65 mM UTP, 0.35 mM Dig-11-UTP, 30 units RNase inhibitor, 40 units SP6 RNA polymerase (Boehringer Mannheim, Germany) and 1 mg human H-ras cDNA fragment (Oncogene Science Inc., Uniondale, NY) for 2 h at 37°C. The RNA probe was used to detect PCR products which was fractionated by electrophoresis on 1% agarose gel in 0.5× TBE (45 mM Tris-borate, pH 8.2 and 1 mM EDTA), then was swamped with denaturation buffer (0.5 N NaOH and 1.5 M NaCl) and neutralization buffer (1 M Tris–HCl, pH 7.5 and 1.5 M NaCl) for 30 min, respectively. After transfer of the gel to Hybond-N+ nylon membrane, the membrane was incubated in 50% (w/ v) formamide containing 0.02% (w/v) sodium dodecyl sulfate (SDS), 0.1% (w/v) N-laurylsarcosine, 5× sodium saline citrate (SSC), 2% (w/v) blocking reagent (Boehringer Mannheim, Germany) for 2 h at 42°C. For hybridization, the membrane was further incubated in the same solution containing the denatured Dig-11-UTP labeled human ras RNA fragment probe for 17 h at the same temperature. The membrane was washed twice in 2×SSC and 0.1% (w/v) SDS for 5 min at room temperature, and then twice in 0.1 ×SSC containing 0.1% (w/v) SDS at 55°C for 15 min. The nylon membrane was then incubated in antibodies against digoxigenin, conjugated with alkaline phosphatase that had been diluted 1:10 000. Visualization of bands was achieved at room temperature in the presence of 1:100 diluted CSPD® (Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2%-(5%-chloro)tricyclo[3.3. 1.13,7]decan}-4-yl)phenyl phosphate, Boehringer, Mannheim) by exposure to Kodak BioMax-MR film at room temperature for 10 h.
2.4. Dig-11 -dUTP labelling cDNA probe of ras The most conserved sequences of the Ras were used to design degenerate oligonucleotide primers: 5%-ATG(A/C)G(A/T/G/C)GA(C/T)CA(A/G)TA(C/T)ATG-3%,5%-GT(A/G)TA(A/G)AA(A/T/G/C) GC(A/G)TC(C/T)TC(A/T/G/C)AC-3%, and a firststrand cDNA pool was synthesized from shrimp (P. monodon) hepatopancreas as the template for PCR to find the specific shrimp ras cDNA frag-
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ment. The recovered ras cDNA fragment was sequenced following the dideoxynucleotide method with modification for extended DNA sequencing. Sequence alignment of the ras sequences was performed at Intelligenetics (Genbank Online Service, Mountain View, CA), using the GENALIGN program. A 239-bp Dig11-dUTP labeled probe (primers: 5%-ATGCGAACTGGGGAAGG-3%,5%-CCCATGCGGGTCTTGGC-3%) was produced by PCR. The PCR was performed in 100 ml of 20 mM Tris – HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.16 mM dTTP, 0.04 mM Dig-11-dUTP, 2.5 units of Taq DNA polymerase (Gibco BRL; MD, USA) and 200 ng shrimp ras cDNA fragment as template. The DNA probe was amplified for 30 cycles consisting of 1 min denaturation at 94°C, 1 min renaturation at 55°C, and 1 min polymerization at 72°C.
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SDS at 55°C for 15 min. The nylon membrane was then incubated in antibodies against digoxigenin, conjugated with alkaline phosphatase that had been diluted 1:10 000. Visualization of bands was achieved at room temperature in the presence of 1:100 diluted CSPD® (Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2%-(5%-chloro)tricyclo[3.3. 1.13,7]decan}-4-yl)phenyl phosphate, Boehringer, Mannheim) by exposure to Kodak BioMax-MR film at room temperature for 10 h. For quantitative studies of the relative expression of the different transcripts, an image analysis of films was performed on a photodensitometer using the Image-Quant program (GEL-PDM ANALYZER®; Silver Spring, MD). The value corresponding to the signal of each transcript accumulation was recorded.
2.6. Assay of protein geranylgeranyltransferase I acti6ity
2.5. RNA extractions and Northern Blotting Total RNA extractions were made following the guanidine thiocyanate method (Chirgwin et al., 1979) using the rapid RNA extraction kit from TEL-TEST (Friendswood, TX) after disruption of organs in liquid nitrogen. Total RNA was isolated from 1 g fresh weight individual organs and quantified by spectrophotometry. A 239 bp Dig-11-dUTP labeled fragment of the shrimp (P. monodon) ras cDNA was used to probe the shrimp (P. monodon) total RNA which was fractionated by denaturing electrophoresis on 1.2% agarose-formaldehyde gel in 20 mM MOPS, pH 7.0, 8 mM sodium acetate, and 1 mM EDTA. The agarose gel was treated consecutively with denaturation buffer (0.05 N NaOH and 150 mM NaCl) and neutralization buffer (100 mM Tris – HCl, pH 7.5 and 150 mM NaCl) for 30 min, respectively. Transfer to nylon membrane (Hybond N+, Amersham), the membrane was incubated in 50% (w/v) formamide containing 0.02% (w/v) sodium dodecyl sulfate (SDS), 0.1% (w/v) N-laurylsarcosine, 5 × sodium saline citrate (SSC), 2% (w/v) blocking reagent (Boehringer Mannheim, Germany) for 2 h at 42°C. For hybridization, the membrane was further incubated in the same solution containing the denatured Dig-11-dUTP labeled shrimp ras cDNA probe for 17 h at the same temperature. The membrane was washed twice in 2× SSC and 0.1% (w/v) SDS for 5 min at room temperature, and then twice in 0.1× SSC containing 0.1% (w/v)
For the assay of protein geranylgeranyltransferase I activity, peptides were synthesized by the solid-phase method (Merrifield, 1986). The standard assay as described previously (Lin and Chuang, 1998) involved monitoring the transfer of [3H]-geranylgeranyl moieties from all-trans[3H]-geranylgeranyl pyrophosphate to KRKCIVF. Each reaction mixture contained 50 mM Tris–HCl, pH 8.0, 1 mM DTT, 250 mM KRKCIVF, 0.4 mM [3H]-geranylgeranyl pyrophosphate (15 000 dpm/pmol), and 10–100 mg of protein geranylgeranyltransferase I in a total volume of 50 ml. After incubation for 30 min at 37°C, samples (40 ml) were applied to paper strips (1 ×2 cm2; p81 phosphocellulose paper; Whatman, Clifton, NJ) that had been numbered in pencil. After the liquid had permeated the paper, the strips were immersed in 10 ml/strip of a mixture of ethanol and phosphoric acid (prepared by mixing equal volumes of 95% ethanol and 75 mM phosphoric acid) four times (10 min/time) at room temperature. Background values were obtained by using the reaction mixture with buffer in place of the enzyme, and these samples were the last to be added to the washing solution. The means of triplicate results were used for determination of each activity, background value, and specific radioactivity. One unit of activity was defined as the amount that incorporated 1 pmol of [3H]-geranylgeranyl group per minute into the synthetic peptide under the conditions of the reaction.
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Fig. 1. Ras probe from the hepatopancreas of shrimp P. monodon. The PCR products with degenerate oligonucleotide primers, 5%-ATG(A/C)G(A/T/G/C)GA(C/T)CA(A/G)TA(C/T)ATG-3%,5%-GT(A/G)TA(A/G)AA(A/T/G/C)GC(A/G)TC(C/T)TC(A/T/G/C)AC3%, were synthesized from hepatopancreas of P. monodon (Pm) and Penaeus japonicus (Pj) and fractionated by electrophoresis on 1% agarose gel in 0.5 ×TBE (45 mM Tris–borate, pH 8.2 and 1 mM EDTA) and stained with ethidium bromide (A). The gel was incubated consecutively with denaturation buffer (0.5 N NaOH and 1.5 M NaCl) and neutralization buffer (1 M Tris – HCl, pH 7.5 and 1.5 M NaCl) for 30 min, respectively before blotting to Hybond-N+ nylon membrane and reacting with the Dig-11-UTP labeled human H-ras RNA fragment (B). Nucleotide sequence (C) of the probe isolated from the hepatopancreas cDNA pool of P. monodon was compared with that of P. japonicus. The difference in bases was marked with asterisks. Alignment of the amino acid sequence (D) of Ras probe protein from P. monodon, P. japonicus, and KB-Ras of mammals. The conserved regions involved in GTP metabolism are boxed. Identical positions are marked by dots.
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2.7. Quantitation of protein Amounts of protein were determined by Lowry’s method (Lowry et al., 1951) or by the Micro BCA* Protein Assay (Pierce, Rockford, IL), using bovine serum albumin as the reference substance.
3. Results and discussions
3.1. Preparation of a Ras cDNA probe of shrimp P. monodon The most conserved amino acid sequences of the Ras were used to design degenerate oligonucleotide primers, and a first-strand cDNA pool from hepatopancreas of P. monodon and P. japonicus was the template for polymerase chain amplification (Fig. 1(A)). The Southern blot revealed that P. monodon and P. japonicus express similarly sized ras cDNA fragment, 275 bp, to react with Dig-11-UTP labeled RNA ras probe of human (Fig. 1(B)). A detailed comparison of the recovered ras probe, 239-bp product, in P. monodon and P. japonicus demonstrated two bases differences (Fig. 1(C)) and identical amino acids sequences (Fig. 1(D)).
3.2. Tissue distribution of Ras mRNA in the mature shrimp P. monodon
Fig. 2. Comparison of ras expression in hepatopancreas, gill, muscle and heart of the mature shrimp P. monodon. Total RNA (20 mg) from hepatopancreas, gill, muscle and heart were electrophoresed on a 1.2% agarose-formaldehyde gel and transferred to a nylon membrane. The blot was hybridized with the shrimp (P. monodon) ras 239bp Dig-11-dUTP labeled probe and exposed for 10 h (A). The gel stained with ethidium bromide before transfer to the nylon filter is presented in the bottom panel (B). The position of the 28S and 18S rRNA is indicated.
The Ras gene expresses more than one transcript in tissues of P. monodon, in agreement with the previous findings of P. japonicus (Huang and Chuang, 1999). Total RNA from shrimp hepatopancreas showed three prominent bands to react with the shrimp (P. monodon) ras probe, 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) with the transcript of R1 showing a lower expression on the northern blot. Analyses of RNA from gill and heart were similar (Fig. 2(A)). Some intermediate transcripts corresponding to 5.0 kb, 3.5 kb, and 1.6 kb were observed in the preparation of gill and heart. In contrast, negligible signal was detected with the Ras probe when comparable amounts of RNA from muscle were analyzed. Differences in the level of ras expression among the organs examined are evident. The highest expression of Ras
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Fig. 3. Ras expression during shrimp embryonic development. Stages were electrophoresed on a 1.2% agarose-formaldehyde gel transferred to nylon membrane. The blot was hybridized with 239bp Dig-11-dUTP labeled shrimp (P. monodon) ras probe (A). ethidium bromide stained RNA prior to transfer is presented in (B). Radiolabelled bands on autoradiogram were quantified analyzed with Phosphor-Imager Analyzer (Molecular Dynamics, Sunnyvale, CA) (C). N: nauplius; Z: protozoea; M: mysis; post-larvae stages.
was observed in gill. Within a particular organ, an equal ratio of the two smallest transcripts, R2 and R3, is always seen and the amount of the largest transcript, R1, is usually less than that of either of the smaller transcripts, R2 and R3.
and The and PL:
3.3. Ras expression in the early embryonic de6elopment of shrimp P. monodon Ras has been shown to be developmentally regulated in mammals (Tanaka et al., 1986). A
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Northern blot was prepared with these RNA samples and hybridized with a ras probe. In embryonic stages, a change in the relative proportion of the three transcripts, R1, R2 and R3, was found (Fig. 3(A)). Messenger RNA amounts of R1 and R2 are increased largely from a minimum in the nauplius stage to a maximum in the post-larvae stage (Fig. 3(C)). However, R3 peaked during the protozoea stage. These results suggest differential Ras synthesis is required during the premolt.
3.4. Prominent geranylgeranylation in early de6elopmental stages Protein geranylgeranylation is the major controlling event in the cell cycle progression through G1 phase, whereas protein farnesylation is not involved (Vogt et al., 1996). In our previous studies, protein geranylgeranyltransferase I of the shrimp
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P. japonicus effectively catalyzed the transfer of [3H]-geranylgeranyl pyrophosphate to Ras carboxylterminal CAAX (Lin and Chuang, 1998; Huang and Chuang, 1999). Radioactive experiments demonstrated that the prenylation by protein geranylgeranyltransferase I in the early nauplius stage exceeded that of the protozoea by 3-fold, mysis stage by 7-fold, and post-larvae stage by 2.5-fold, respectively (Fig. 4). Thus, Ras is not the major substrate for protein geranylgeranyltransferase I in the nauplius stage as evidenced by the low expression of its mRNA (Fig. 3). However, we can not exclude the possibility that the substrate for protein geranylgeranyltransferase I at the nauplius stage derives from a reservoir of ras mRNA transcripts or Ras proteins. Further studies comparing the regulation of Ras at the nauplius stage by prenylation, transcription and translation are under way. In the meanwhile, we are interested in probing the regulation of the ERK mitogenic signaling cascade (Coso et al., 1995; Minden et al., 1995) by the other geranylgeranylated GTP binding proteins, such as Rac (Kinsella et al., 1991) and Rho (Adamson et al., 1992; Katayama et al., 1999), during embryogenesis of shrimp, which is without the control of mos (Sagata et al., 1988, 1989).
Acknowledgements
Fig. 4. Analysis of Ras acylation by protein geranylgeranyltransferase I in shrimp at different developmental stages. Synthesized Ras-carboxylterminal peptide KRKCIVF (250 mM) was prenylated by the protein geranylgeranyltransferase I from different developmental stages in 0.4 mM [3H]-geranylgeranyl pyrophosphate (15 000 dpm/pmol), 50 mM Tris–HCl, pH 8.0 and 1 mM DTT at 37°C for 30 min. Samples (40 ml) were applied to paper strips (1× 2 cm2; p81 phosphocellulose paper; Whatman, Clifton, NJ) that had been numbered in pencil. After the liquid had permeated the paper, the strips were immersed in 10 ml/strip of a mixture of ethanol and phosphoric acid (prepared by mixing equal volumes of 95% ethanol and 75 mM phosphoric acid) four times (10 min/time) at room temperature. Background values were obtained by using the reaction mixture with buffer in place of the enzyme, and these samples were the last to be added to the washing solution. The means of triplicate results were used for determination of each activity, background value, and specific radioactivity. One unit of activity was defined as the amount that incorporated 1 pmol of [3H]-geranylgeranyl group per minute into the synthetic peptide under the conditions of the reaction.
We are grateful to the National Science Council and the Agriculture Council for financial support. We also appreciate the help of Mr. Kuan-Fu Liu at the supply of embryos from Tung Kang Marine Laboratory, Taiwan Fisheries Research Institute, Tung Kang, Ping Tung, Taiwan. C.-F. H. is a recipient of a National Science Council Graduate Fellowship.
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Differential expression of ras in organs and embryos of shrimp Penaeus monodon (Crustacea: Decapoda) Chein-Fuang Huang a, Wen-Yi Chen a, Nin-Nin Chuang b,* a
Department of Zoology, National Taiwan Uni6ersity, Nankang 11529, Taipei, Taiwan b Institute of Zoology, Academia Sinica, Nankang 11529, Taipei, Taiwan
Received 12 May 1999; received in revised form 30 November 1999; accepted 6 December 1999
Abstract Total RNA from shrimp hepatopancreas of Penaeus monodon showed three prominent bands that react with the shrimp ras probe, a 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) on the northern blot. The R1 is the least abundant. Analyses of total RNA from gill and heart were similar to each other. The highest expression of Ras was observed in the gill, while a negligible signal was detected with the Ras probe in muscle. Ras expression is developmentally regulated in embryonic stages of shrimp. Messenger RNA levels of ras were increased from a minimum in the nauplius stage to a maximum in the post-larvae stage for R1 and R2. R3 showed a maximum at the protozoea stage. On the other hand, the activity of protein geranylgeranyltransferase I was increased significantly in the early nauplius stage. No correlative increase of prenylation activity by protein geranylgeranyltransferase I was observed with the transcription activity of ras. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ras; Protein geranylgeranyltransferase I; Shrimp; Embryo; Hepatopancreas; Gill; Heart; Mitogen; Aquaculture
1. Introduction Ras proteins are membrane-associated small guanine nucleotides binding proteins and are required at different stages of the cell cycle (Moodie and Wolfman, 1994). In particular, it has been shown that Ras is required for cell cycle progression from G0 to G1 (Dobrowolski et al., 1994) and in the G1/S transition (Stacey et al., 1991; Lu and Campisi, 1992; Howe et al., 1993). In mammals, four isoforms of Ras exist: H-Ras, N-Ras, KARas, and KB-Ras (Lowy and Willumsen, 1993). They are products of three genes, with KA-Ras and KB-Ras being splice variants of the same gene. Among them, the protein sequences are 80% * Corresponding author. Tel./fax: + 886-2-27899531. E-mail address: [email protected] (N.-N. Chuang)
identical with major differences residing in their carboxyl termini, including CAAX motif of prenylation (Del Villar et al., 1996). Lipid modification of Ras results in an increase in its intrinsic affinity for the plasma membrane where Ras protein can participate in signal transduction (Zhang et al., 1997). The addition to Ras of isoprenoid residues, such as geranylgeranyl (20carbon) and farnesyl residues (15-carbon), is determined by the X residue of the carboxyl terminal CAAX (C, cysteine; A, an aliphatic amino acid) of proteins. If X is leucine or phenylalanine, the protein is geranylgeranylated (Casey et al., 1991; Moores et al., 1991; Yokoyama et al., 1991); if X is methionine, serine, alanine or glutamine, the protein is farnesylated (Hancock et al., 1989; Schaber et al., 1990; Reiss et al., 1991). Ras is preferentially farnesylated in mammals
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(James et al., 1995), but geranylgeranylated in shrimp (Lin and Chuang, 1998; Huang and Chuang, 1999). Abolishing prenylation disrupts the association of Ras with membranes, thereby disrupting its function (Der and Cox, 1991; Kato et al., 1992). Inhibitors of prenylation are effective at suppressing the growth of tumor cells possessing oncogenic Ras (Hancock et al., 1989; Seabra et al., 1991; Sun et al., 1995; Lerner et al., 1997). Ras is a specific and interesting regulation target for the applications in aquaculture, as suggested by the fact that microinjection of oncogenic mammalian Ras proteins into Xenopus lae6is oocytes would induce cellular divisions (Pomerance et al., 1992). Since the cultivation of penaeid shrimp is a worldwide economically important industry. In this report, we undertake the characterization of Ras at early developmental stages in the tropical shrimp Penaeus monodon, and probe the correlation of prenylation activity with the Ras expression. We attempt in the future the transfer of Ras into developing embryos to probe its effect on growth. In addition, we plan to use Ras for further investigations of the precise mechanism by which the mitogenic signal induces maturation and releases the M-phase arrest in oocytes of shrimp, as suggested by the results of vertebrates (Rhodes et al., 1994, 1997).
2. Materials and methods
2.1. Materials All reagents used were of the highest grade available commercially.
2.2. Experimental animals Shrimp (P. monodon), collected off the coast of Taiwan, were kept at 18°C for less than 3 days before experiments in a recirculating seawater system. Organs were dissected out immediately after shrimp had been killed and frozen in liquid nitrogen and stored at −80°C.
2.3. Southern blotting of shrimp ras PCR products The Dig-11-UTP labeled human H-ras RNA fragment was synthesized by 1× transcription
buffer (40 mM Tris–HCl, pH 8.0, 6 mM MgCl2, 10 mM DTT, 2 mM spermidine), 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.65 mM UTP, 0.35 mM Dig-11-UTP, 30 units RNase inhibitor, 40 units SP6 RNA polymerase (Boehringer Mannheim, Germany) and 1 mg human H-ras cDNA fragment (Oncogene Science Inc., Uniondale, NY) for 2 h at 37°C. The RNA probe was used to detect PCR products which was fractionated by electrophoresis on 1% agarose gel in 0.5× TBE (45 mM Tris-borate, pH 8.2 and 1 mM EDTA), then was swamped with denaturation buffer (0.5 N NaOH and 1.5 M NaCl) and neutralization buffer (1 M Tris–HCl, pH 7.5 and 1.5 M NaCl) for 30 min, respectively. After transfer of the gel to Hybond-N+ nylon membrane, the membrane was incubated in 50% (w/ v) formamide containing 0.02% (w/v) sodium dodecyl sulfate (SDS), 0.1% (w/v) N-laurylsarcosine, 5× sodium saline citrate (SSC), 2% (w/v) blocking reagent (Boehringer Mannheim, Germany) for 2 h at 42°C. For hybridization, the membrane was further incubated in the same solution containing the denatured Dig-11-UTP labeled human ras RNA fragment probe for 17 h at the same temperature. The membrane was washed twice in 2×SSC and 0.1% (w/v) SDS for 5 min at room temperature, and then twice in 0.1 ×SSC containing 0.1% (w/v) SDS at 55°C for 15 min. The nylon membrane was then incubated in antibodies against digoxigenin, conjugated with alkaline phosphatase that had been diluted 1:10 000. Visualization of bands was achieved at room temperature in the presence of 1:100 diluted CSPD® (Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2%-(5%-chloro)tricyclo[3.3. 1.13,7]decan}-4-yl)phenyl phosphate, Boehringer, Mannheim) by exposure to Kodak BioMax-MR film at room temperature for 10 h.
2.4. Dig-11 -dUTP labelling cDNA probe of ras The most conserved sequences of the Ras were used to design degenerate oligonucleotide primers: 5%-ATG(A/C)G(A/T/G/C)GA(C/T)CA(A/G)TA(C/T)ATG-3%,5%-GT(A/G)TA(A/G)AA(A/T/G/C) GC(A/G)TC(C/T)TC(A/T/G/C)AC-3%, and a firststrand cDNA pool was synthesized from shrimp (P. monodon) hepatopancreas as the template for PCR to find the specific shrimp ras cDNA frag-
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ment. The recovered ras cDNA fragment was sequenced following the dideoxynucleotide method with modification for extended DNA sequencing. Sequence alignment of the ras sequences was performed at Intelligenetics (Genbank Online Service, Mountain View, CA), using the GENALIGN program. A 239-bp Dig11-dUTP labeled probe (primers: 5%-ATGCGAACTGGGGAAGG-3%,5%-CCCATGCGGGTCTTGGC-3%) was produced by PCR. The PCR was performed in 100 ml of 20 mM Tris – HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.16 mM dTTP, 0.04 mM Dig-11-dUTP, 2.5 units of Taq DNA polymerase (Gibco BRL; MD, USA) and 200 ng shrimp ras cDNA fragment as template. The DNA probe was amplified for 30 cycles consisting of 1 min denaturation at 94°C, 1 min renaturation at 55°C, and 1 min polymerization at 72°C.
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SDS at 55°C for 15 min. The nylon membrane was then incubated in antibodies against digoxigenin, conjugated with alkaline phosphatase that had been diluted 1:10 000. Visualization of bands was achieved at room temperature in the presence of 1:100 diluted CSPD® (Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2%-(5%-chloro)tricyclo[3.3. 1.13,7]decan}-4-yl)phenyl phosphate, Boehringer, Mannheim) by exposure to Kodak BioMax-MR film at room temperature for 10 h. For quantitative studies of the relative expression of the different transcripts, an image analysis of films was performed on a photodensitometer using the Image-Quant program (GEL-PDM ANALYZER®; Silver Spring, MD). The value corresponding to the signal of each transcript accumulation was recorded.
2.6. Assay of protein geranylgeranyltransferase I acti6ity
2.5. RNA extractions and Northern Blotting Total RNA extractions were made following the guanidine thiocyanate method (Chirgwin et al., 1979) using the rapid RNA extraction kit from TEL-TEST (Friendswood, TX) after disruption of organs in liquid nitrogen. Total RNA was isolated from 1 g fresh weight individual organs and quantified by spectrophotometry. A 239 bp Dig-11-dUTP labeled fragment of the shrimp (P. monodon) ras cDNA was used to probe the shrimp (P. monodon) total RNA which was fractionated by denaturing electrophoresis on 1.2% agarose-formaldehyde gel in 20 mM MOPS, pH 7.0, 8 mM sodium acetate, and 1 mM EDTA. The agarose gel was treated consecutively with denaturation buffer (0.05 N NaOH and 150 mM NaCl) and neutralization buffer (100 mM Tris – HCl, pH 7.5 and 150 mM NaCl) for 30 min, respectively. Transfer to nylon membrane (Hybond N+, Amersham), the membrane was incubated in 50% (w/v) formamide containing 0.02% (w/v) sodium dodecyl sulfate (SDS), 0.1% (w/v) N-laurylsarcosine, 5 × sodium saline citrate (SSC), 2% (w/v) blocking reagent (Boehringer Mannheim, Germany) for 2 h at 42°C. For hybridization, the membrane was further incubated in the same solution containing the denatured Dig-11-dUTP labeled shrimp ras cDNA probe for 17 h at the same temperature. The membrane was washed twice in 2× SSC and 0.1% (w/v) SDS for 5 min at room temperature, and then twice in 0.1× SSC containing 0.1% (w/v)
For the assay of protein geranylgeranyltransferase I activity, peptides were synthesized by the solid-phase method (Merrifield, 1986). The standard assay as described previously (Lin and Chuang, 1998) involved monitoring the transfer of [3H]-geranylgeranyl moieties from all-trans[3H]-geranylgeranyl pyrophosphate to KRKCIVF. Each reaction mixture contained 50 mM Tris–HCl, pH 8.0, 1 mM DTT, 250 mM KRKCIVF, 0.4 mM [3H]-geranylgeranyl pyrophosphate (15 000 dpm/pmol), and 10–100 mg of protein geranylgeranyltransferase I in a total volume of 50 ml. After incubation for 30 min at 37°C, samples (40 ml) were applied to paper strips (1 ×2 cm2; p81 phosphocellulose paper; Whatman, Clifton, NJ) that had been numbered in pencil. After the liquid had permeated the paper, the strips were immersed in 10 ml/strip of a mixture of ethanol and phosphoric acid (prepared by mixing equal volumes of 95% ethanol and 75 mM phosphoric acid) four times (10 min/time) at room temperature. Background values were obtained by using the reaction mixture with buffer in place of the enzyme, and these samples were the last to be added to the washing solution. The means of triplicate results were used for determination of each activity, background value, and specific radioactivity. One unit of activity was defined as the amount that incorporated 1 pmol of [3H]-geranylgeranyl group per minute into the synthetic peptide under the conditions of the reaction.
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Fig. 1. Ras probe from the hepatopancreas of shrimp P. monodon. The PCR products with degenerate oligonucleotide primers, 5%-ATG(A/C)G(A/T/G/C)GA(C/T)CA(A/G)TA(C/T)ATG-3%,5%-GT(A/G)TA(A/G)AA(A/T/G/C)GC(A/G)TC(C/T)TC(A/T/G/C)AC3%, were synthesized from hepatopancreas of P. monodon (Pm) and Penaeus japonicus (Pj) and fractionated by electrophoresis on 1% agarose gel in 0.5 ×TBE (45 mM Tris–borate, pH 8.2 and 1 mM EDTA) and stained with ethidium bromide (A). The gel was incubated consecutively with denaturation buffer (0.5 N NaOH and 1.5 M NaCl) and neutralization buffer (1 M Tris – HCl, pH 7.5 and 1.5 M NaCl) for 30 min, respectively before blotting to Hybond-N+ nylon membrane and reacting with the Dig-11-UTP labeled human H-ras RNA fragment (B). Nucleotide sequence (C) of the probe isolated from the hepatopancreas cDNA pool of P. monodon was compared with that of P. japonicus. The difference in bases was marked with asterisks. Alignment of the amino acid sequence (D) of Ras probe protein from P. monodon, P. japonicus, and KB-Ras of mammals. The conserved regions involved in GTP metabolism are boxed. Identical positions are marked by dots.
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2.7. Quantitation of protein Amounts of protein were determined by Lowry’s method (Lowry et al., 1951) or by the Micro BCA* Protein Assay (Pierce, Rockford, IL), using bovine serum albumin as the reference substance.
3. Results and discussions
3.1. Preparation of a Ras cDNA probe of shrimp P. monodon The most conserved amino acid sequences of the Ras were used to design degenerate oligonucleotide primers, and a first-strand cDNA pool from hepatopancreas of P. monodon and P. japonicus was the template for polymerase chain amplification (Fig. 1(A)). The Southern blot revealed that P. monodon and P. japonicus express similarly sized ras cDNA fragment, 275 bp, to react with Dig-11-UTP labeled RNA ras probe of human (Fig. 1(B)). A detailed comparison of the recovered ras probe, 239-bp product, in P. monodon and P. japonicus demonstrated two bases differences (Fig. 1(C)) and identical amino acids sequences (Fig. 1(D)).
3.2. Tissue distribution of Ras mRNA in the mature shrimp P. monodon
Fig. 2. Comparison of ras expression in hepatopancreas, gill, muscle and heart of the mature shrimp P. monodon. Total RNA (20 mg) from hepatopancreas, gill, muscle and heart were electrophoresed on a 1.2% agarose-formaldehyde gel and transferred to a nylon membrane. The blot was hybridized with the shrimp (P. monodon) ras 239bp Dig-11-dUTP labeled probe and exposed for 10 h (A). The gel stained with ethidium bromide before transfer to the nylon filter is presented in the bottom panel (B). The position of the 28S and 18S rRNA is indicated.
The Ras gene expresses more than one transcript in tissues of P. monodon, in agreement with the previous findings of P. japonicus (Huang and Chuang, 1999). Total RNA from shrimp hepatopancreas showed three prominent bands to react with the shrimp (P. monodon) ras probe, 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) with the transcript of R1 showing a lower expression on the northern blot. Analyses of RNA from gill and heart were similar (Fig. 2(A)). Some intermediate transcripts corresponding to 5.0 kb, 3.5 kb, and 1.6 kb were observed in the preparation of gill and heart. In contrast, negligible signal was detected with the Ras probe when comparable amounts of RNA from muscle were analyzed. Differences in the level of ras expression among the organs examined are evident. The highest expression of Ras
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Fig. 3. Ras expression during shrimp embryonic development. Stages were electrophoresed on a 1.2% agarose-formaldehyde gel transferred to nylon membrane. The blot was hybridized with 239bp Dig-11-dUTP labeled shrimp (P. monodon) ras probe (A). ethidium bromide stained RNA prior to transfer is presented in (B). Radiolabelled bands on autoradiogram were quantified analyzed with Phosphor-Imager Analyzer (Molecular Dynamics, Sunnyvale, CA) (C). N: nauplius; Z: protozoea; M: mysis; post-larvae stages.
was observed in gill. Within a particular organ, an equal ratio of the two smallest transcripts, R2 and R3, is always seen and the amount of the largest transcript, R1, is usually less than that of either of the smaller transcripts, R2 and R3.
and The and PL:
3.3. Ras expression in the early embryonic de6elopment of shrimp P. monodon Ras has been shown to be developmentally regulated in mammals (Tanaka et al., 1986). A
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Northern blot was prepared with these RNA samples and hybridized with a ras probe. In embryonic stages, a change in the relative proportion of the three transcripts, R1, R2 and R3, was found (Fig. 3(A)). Messenger RNA amounts of R1 and R2 are increased largely from a minimum in the nauplius stage to a maximum in the post-larvae stage (Fig. 3(C)). However, R3 peaked during the protozoea stage. These results suggest differential Ras synthesis is required during the premolt.
3.4. Prominent geranylgeranylation in early de6elopmental stages Protein geranylgeranylation is the major controlling event in the cell cycle progression through G1 phase, whereas protein farnesylation is not involved (Vogt et al., 1996). In our previous studies, protein geranylgeranyltransferase I of the shrimp
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P. japonicus effectively catalyzed the transfer of [3H]-geranylgeranyl pyrophosphate to Ras carboxylterminal CAAX (Lin and Chuang, 1998; Huang and Chuang, 1999). Radioactive experiments demonstrated that the prenylation by protein geranylgeranyltransferase I in the early nauplius stage exceeded that of the protozoea by 3-fold, mysis stage by 7-fold, and post-larvae stage by 2.5-fold, respectively (Fig. 4). Thus, Ras is not the major substrate for protein geranylgeranyltransferase I in the nauplius stage as evidenced by the low expression of its mRNA (Fig. 3). However, we can not exclude the possibility that the substrate for protein geranylgeranyltransferase I at the nauplius stage derives from a reservoir of ras mRNA transcripts or Ras proteins. Further studies comparing the regulation of Ras at the nauplius stage by prenylation, transcription and translation are under way. In the meanwhile, we are interested in probing the regulation of the ERK mitogenic signaling cascade (Coso et al., 1995; Minden et al., 1995) by the other geranylgeranylated GTP binding proteins, such as Rac (Kinsella et al., 1991) and Rho (Adamson et al., 1992; Katayama et al., 1999), during embryogenesis of shrimp, which is without the control of mos (Sagata et al., 1988, 1989).
Acknowledgements
Fig. 4. Analysis of Ras acylation by protein geranylgeranyltransferase I in shrimp at different developmental stages. Synthesized Ras-carboxylterminal peptide KRKCIVF (250 mM) was prenylated by the protein geranylgeranyltransferase I from different developmental stages in 0.4 mM [3H]-geranylgeranyl pyrophosphate (15 000 dpm/pmol), 50 mM Tris–HCl, pH 8.0 and 1 mM DTT at 37°C for 30 min. Samples (40 ml) were applied to paper strips (1× 2 cm2; p81 phosphocellulose paper; Whatman, Clifton, NJ) that had been numbered in pencil. After the liquid had permeated the paper, the strips were immersed in 10 ml/strip of a mixture of ethanol and phosphoric acid (prepared by mixing equal volumes of 95% ethanol and 75 mM phosphoric acid) four times (10 min/time) at room temperature. Background values were obtained by using the reaction mixture with buffer in place of the enzyme, and these samples were the last to be added to the washing solution. The means of triplicate results were used for determination of each activity, background value, and specific radioactivity. One unit of activity was defined as the amount that incorporated 1 pmol of [3H]-geranylgeranyl group per minute into the synthetic peptide under the conditions of the reaction.
We are grateful to the National Science Council and the Agriculture Council for financial support. We also appreciate the help of Mr. Kuan-Fu Liu at the supply of embryos from Tung Kang Marine Laboratory, Taiwan Fisheries Research Institute, Tung Kang, Ping Tung, Taiwan. C.-F. H. is a recipient of a National Science Council Graduate Fellowship.
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