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Molecular cloning and characterization of a rabbit eIF2C protein
Gene 211 (1998) 187–194
Molecular cloning and characterization of a rabbit eIF2C protein Cheng Zou a, Zhongli Zhang a, Shiyong Wu b, John C. Osterman c,* a Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA b Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA c Department of Biological Science, University of Nebraska, Lincoln, NE 68588, USA Received 1 July 1997; received in revised form 20 October 1997; accepted 27 October 1997; Received by J.A. Engler
Abstract Rabbit eIF2C (94 kDa) has been shown to play important roles in the eukaryotic peptide chain initiation process. In this study, the primary structure of rabbit eIF2C is determined by cDNA cloning. Based on the partial amino acid sequences of Endolys C cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding cDNA fragment from a rabbit liver cDNA library. This fragment was subsequently used to screen for larger cDNAs. Marathon cDNA amplification and 5∞-rapid amplification of cDNA ends were used to confirm the translation start site. Sequences from the overlapping clones were assembled into a 3599-bp composite sequence, which contains a single open reading frame that translates into a 813-deduced amino acid sequence. Northern blot analysis of rabbit liver ploy(A)+ RNA yielded a single message species at approximately 4.6 kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94 kDa eIF2C detected a higher-molecular-weight polypeptide (140 kDa). No 94 kDa polypeptide was detected. The cloned cDNA was further characterized by in-vitro transcription-coupled translation in reticulocyte lysate. The translated product was precipitated with antibodies against eIF2C. Genomic Southern blot analysis indicates that the rabbit eIF2C is a single copy gene. Sequence analysis reveals that rabbit eIF2C has strong homology with a hypothetical protein in Caenorhabditis elegans. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Recombinant DNA; Sequences; Eukaryotic peptide chain initiation; 5∞-RACE; PCR
1. Introduction The first step in protein synthesis initiation in eukaryotes involves the formation of a ternary complex among eukaryotic initiation factor 2 (eIF2), Met-tRNA and i GTP. The second step is the transfer of Met-tRNA i to the 40S ribosomal subunit, and the MettRNA · 40S · mRNA complex is formed. Reports from i different labs have indicated that the above two steps are regulated by several ancillary protein factors [reviewed in Gupta et al. (1993), Hershey (1991) and Merrick and Hershey (1996)]. Several years ago, an 80-kDa eIF2C (previous name: * Corresponding author. Tel: +1 402 472 5129; Fax: +1 402 472 2083; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, complementary DNA; eIF2C, eukaryotic initiation factor 2C; EST, expressed sequence tag; 5∞-RACE, rapid amplification of a cDNA end for 5∞-region; kb, kilobase; kDa, kilodalton; nt, nucleotide(s); ORF, open reading frame; pfu, plaque forming unit; UTR, untranslated region(s). 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 10 7 - 3
Co-eIF-2A) was isolated from rabbit reticulocyte lysate (Chakravarty et al., 1985). The size of the same polypeptide has been recalibrated as 94 kDa (Gupta et al., 1993). This protein has been shown to play two important roles in the protein synthesis initiation. First, it stimulates the ternary complex formation. Second, it stabilizes the ternary complex and the MettRNA · 40S · mRNA complex in the presence of natural i mRNAs (Roy et al., 1988). These reports suggested that eIF2C is an important component of the protein synthesis in reticulocyte lysate. Chakravarty et al. (1985) have shown that the 94-kDa eIF2C has two major protease degradation products, 50 kDa and 25 kDa. All three polypeptides of eIF2C have the ability to stimulate the ternary complex formation. eIF2C-like activity has been reported to be present in widely divergent eukaryotic organisms such as mouse ascite tumor cells (Dasgupta et al., 1978), wheat germ (Osterhout et al., 1983; Seal et al., 1983), Artemia salina ( Woodley et al., 1981), and yeast (Ahmad et al., 1985); however, we do not know the exact working mechanism of eIF2C.
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In this study, we report the primary molecular structure of the eIF2C that we have cloned as part of an effort to understand, in molecular terms, how the eIF2C works in the protein synthesis initiation complex. In addition, we report the discovery of a high-molecularweight eIF2C (140 kDa) in the rabbit reticulocyte lysate.
2. Materials and methods 2.1. Materials All primers were synthesized using the facilities of the DNA Synthesis Laboratory at the University of Nebraska, Lincoln. Two cDNA libraries were purchased from Clontech (Palo Alto, CA): Library A, Rabbit Liver cDNA Library (oligo dT primed ) in lgt11 phages, and Library B, Rabbit Liver 5∞-stretch Plus cDNA Library (oligo dT+random primed ) in lgt10 phages. 2.2. Isolation of cDNA clones encoding eIF2C Degenerate primers were designed, based on partial amino acid sequences from the 94 kDa eIF2C. A 940-bp cDNA fragment of eIF2C was obtained by PCR using primer 1: 5∞- GA(C/T )ATGCGGAACAAGCAGTT(C/T )CA-3∞ (sense oligo, nt 1249–1271) and primer 2: 5∞-TA(G/A)AAGTCAAACTCTGTGG(G/A)TG-3∞ (antisense oligo, nt 2189–2167). The 940-bp PCR product was randomly labeled with DIG-dUTP using the Genius System (Boehringer Mannheim), and was used to screen library A according to the procedures described in the Genius System (Boehringer Mannheim). After screening approximately 1×106 plaques, three hybridization-positive clones were identified. The longest clone had a size of 2.3 kb. The same probe was also applied to library B, where a 2.8-kb hybridization-positive clone was identified. This clone (B1) was cloned into pGEM7zf(+) vector (Promega), and followed by nested deletion and sequencing in both orientations. DNA sequencing was performed using the dideoxynucleotide chain termination method with the Sequenase version 2 kit ( US Biochemical Corp.). A 403-bp fragment of eIF2C at the 5∞-end of the 2.8 kb clone was synthesized by PCR using primer 3: 5∞-TGGCCCAGTACTTCAAGGACAGG-3∞ (sense oligo, nt 865–888) and primer 4: 5∞-TG(A/G)AACTGCTTGTTCCGCAT(A/G)TC-3∞ (antisense oligo, nt 1271–1249). This fragment was then labeled for subsequent screening of library B. A total of 11 hybridization-positive clones (B2–B12) were identified from 1×106 plaques. The size and position of all hybridization-positive clones were identified by PCR according to the following: (1) primer 5: 5∞-CCTGCCTTGAAGTACTGGGCCA-3∞ (antisense oligo, nt 888–865) and either the forward or the back-
ward universal primer (Clontech) was used for checking the length of 5∞-end; (2) primer 3 and either the forward or backward universal primer were used for checking the length of 3∞-end. B12 and B8 were two clones that contained relatively long 5∞-ends. B12 had two internal EcoRI cutting sites. B12 was purified and cut by EcoRI to give three fragments of B12.1 (1 kb), B12.2 (0.6 kb), and B12.3 (1.6 kb), as well as the two l-arms. Three pieces of inserts were separately subcloned into pGEM7zf(+) vector and sequenced from both directions. Purified B12 l-phage DNA was sequenced by the fmol PCR Sequencing System (Promega) using primer 8: 5∞-CATCTCGAAGAAATTGGCCT-3∞ (antisense oligo, nt 84–65) to obtain the junction sequence between B12.1 and B12.2. The linking orientation between B12.2 and B12.3 was established by analyzing the DNA sequence of B12.2 and B12.3. A Marathon cDNA amplification kit was purchased from Clontech. One microgram of rabbit liver poly(A)+ RNA (Clontech) was used in the Marathon cDNA amplification according to the manufacturer’s protocol. After the cDNAs were synthesized, two 5∞-RACE reactions were performed using primer AP1 (Clontech) and primer 6: 5∞-GCCACTATGTGCAGACCTCCAGG-3∞ (antisense oligo, nt 986–963), or primer AP1 and primer 7: 5∞-CGGCAGCGTGACCTCCAGCTCCAC-3∞ (antisense oligo, nt 303–280). A step-down PCR technique ( Hecker and Roux, 1996) was applied in the 5∞-RACE reaction. The conditions for the step-down PCR were 1 min at 94°C and 2 min at 72°C for three cycles, 1 min at 94°C and 2 min at 69°C for three cycles, 1 min at 94°C and 1 min at 66°C and 1 min at 68°C for three cycles, 1 min at 94°C and 1 min at 63°C and 1 min at 68°C for three cycles, 1 min at 94°C and 1 min at 60°C and 1 min at 68°C for three cycles, then 1 min at 94°C and 2 min at 68°C for 25 cycles. 5∞-RACE products from both reactions were run on a 0.8% agarose gel and followed by Southern blotting probed with oligonucleotide 8.
2.3. Northern blot analysis of eIF2C Livers of adult rabbits were rapidly removed and quickly frozen in liquid nitrogen. Two grams of the liver were added into 20 ml of tissue guanidinium solution containing 11.8 g of guanidinium isothiocyanate, 10 mM EDTA (pH=8.0), 50 mM Tris–HCl (pH=7.5) and 5% b-mercaptoethanol. The liver was immediately ground in a polytron, and the mixture was spun for 10 min at 12 000×g at 15°C. Sarkosyl (20%, 1/10 volume) was added to the supernatant and heated at 65°C for 2 min. The mixture was extracted with 25:24:1 phenol:chloroform:isoamyl alcohol, followed by 24:1 chloroform:isoamyl alcohol. Then, a 1/10 volume of 3 M NaOAc and 2.5 volumes of ethanol were added to precipitate nucleic
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acids. The pellet was resuspended in the lysis buffer from the Fast Track 2.0 kit (Invitrogen). Poly(A)+ RNA was purified using the Fast Track kit according to the manufacturer’s protocol. Five micrograms of isolated rabbit liver poly(A)+ RNA were electrophoresed on a 1% agarose-formaldehyde denaturing gel and subsequently transferred on to Biotrace HP@ membrane (Gelman Science) by vacuum blotting using 7.5 mM NaOH. The membrane was baked at 80°C for 2 h and then incubated in a prehybridizing solution containing 1% non-fat dry milk, 1 mM EDTA (pH=8.0), 6% NaH PO , 7% SDS (pH=7.2) for 2 h at 63°C. For 2 4 hybridization, the membrane was further incubated overnight at 63°C with radiolabeled eIF2C cDNA probe. A 940-bp eIF2C cDNA probe was synthesized by PCR using primers 1 and 2, and radiolabeled using a random hexamer kit ( US Biochemical Corp.). The blot was washed successively, twice in 2×SSC and 1% SDS for 30 min at 63°C and twice with 0.1× SSC for 30 min at room temperature. The mRNA level was detected by autoradiography using Kodak X-Omat AR film at −70°C with intensifying screens.
2.4. In-vitro translation of eIF2C gene pGEM7-B1 and pGEM7-B12.1 were used in constructing a full-length eIF2C cDNA in pGEM7, which contained the single ORF, 5∞-UTR and 3∞-UTR. The newly constructed pGEM7-eIF2C was used in in-vitro translation in rabbit reticulocyte lysate using the SP6 TNT lysate-coupled transcription/translation system. [35S] Methionine (American Radiolabeled Chemicals) was used for amino acid incorporation. The pGEM7-eIF2C was purified by the Qiagen Midi-prep System (Qiagen). 0.5 mg of the purified DNA was used in each reaction. pGEM7 without the insert was used as control. The reactions were run at 30°C for 90 min. The translation product was analyzed by SDS–PAGE and also by immunoprecipitation using eIF2C antibodies. In a negative control, b-actin antibody was used in the immunoprecipitation.
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autoradiography using Kodak X-Omat AR film at −70°C with intensifying screens for 72 h.
3. Results and discussion 3.1. Discovery of a high-molecular-weight eIF2C protein Homogenous eIF2C (94 kDa) was purified from rabbit reticulocyte lysate. This 94-kDa polypeptide was functionally tested according to the procedure described previously ( Roy et al., 1988). For antibody production, the 94-kDa eIF2C was injected into mice. Polyclonal antibody was collected and used for Western blotting ( Fig. 1). The antibody detected the purified 94-kDa polypeptide very well; however, in rabbit reticulocyte lysate, only a high-molecular-weight, approximately 140 kDa, and a 25-kDa polypeptide were detected by our eIF2C polyclonal antibodies. This experiment was repeated two more times with different batches of reticulocyte lysates, but no 94-kDa band was detected. Preincubation of this mouse antibody with purified 94-kDa eIF2C removed the cross-reactivity of this antibody with both 140-kDa and 25-kDa polypeptides. We tried to purify this 140-kDa polypeptide, but found during the purification of the 140-kDa eIF2C that it easily degraded, producing several major degradation products, including 94-kDa, 50-kDa and 25-kDa polypeptides as detected by the above antibody (data not shown). This result indicated that the 94-kDa polypeptide purified previously was a degraded portion of the
2.5. Genomic Southern blot analysis of eIF2C Equal amounts (15 mg) of rabbit genomic DNA (Clontech) were subjected to restriction enzyme digestion and separated on a 0.8% agarose gel, transferred on to Biotrace HP@ (Gelman Sciences) and hybridized at 55°C in the presence of 1% BSA, 1 mM EDTA, 7% SDS, 6% NaH PO (pH=7.2) and 10% formamide for 36 h. The 2 4 probe for hybridization was randomly labeled cDNA PCR fragment (nt 866–1271). Filters were washed according to the manufacturer’s protocol (Gelman Sciences). The hybridization signal was detected by
Fig. 1. Western blot of eIF2C. Lane 1, 500 mg of reticulocyte lysate; lane 2, 250 mg of reticulocyte lysate; lane 3, purified eIF2C (94 kDa). Proteins were denatured and run on a 15% SDS–PAGE gel, then transferred to a Biotrace NT@ (Gelman Science). Mouse polyclonal antibodies against eIF2C were used for detection.
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Fig. 2. cDNA and deduced amino acid sequences of eIF2C. The entire cDNA coding sequence plus 5∞- and 3∞- UTR are reported (GenBank Accession No. AF005355), together with the derived amino acid sequence. Numbers to the left of the sequence represent nucleotide positions, whereas numbers to the right represent amino acid positions. Oligonucleotide primers used for PCR amplifications or for sequencing are designated as short arrows over the first four bases of the corresponding DNA, oriented in the direction of extension and numbered according to their descriptions under ‘Experimental Methods’. Asterisks represent stop codons. Rabbit eIF2C peptide (a, b and c) sequences are underlined.
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140-kDa protein, and that the original size of eIF2C appears to be 140 kDa. 3.2. cDNA cloning of elF2C
Fig. 3. Southern blotting of 5∞-RACE products. 5∞-RACE PCR products was run on a 0.8% agarose gel and subsequently transferred on to Biotrace HP@ (Gelman Science). Oligonucleotide 8 was end-labeled to detect the sizes of 5∞-RACE products. Lane 1, 5∞-RACE PCR using primers 7 and AP1; lane 2, 5∞-RACE PCR using primers 6 and AP1.
Fig. 4. Northern blotting of eIF2C poly(A)+ RNA. Five micrograms of rabbit liver poly(A)+ RNA were run on a 1% agarose-formaldehyde denaturing gel and subsequently transferred on to Biotrace HP@ (Gelman Science). A 940-bp cDNA probe was used to detect eIF2C poly(A)+ RNA.
Purified 94-kDa eIF2C was cleaved using Endolys C, and the amino acid sequences of the cleaved fragments were determined at the amino acid sequencing facilities at the University of Florida (Gainesville, FL). Three peptide sequences (a, b, c) were obtained (Fig. 2). Primer 1 was made using sequence from peptide b, whereas primer 2 used the peptide c sequence. A 940-bp fragment was isolated by PCR using degenerate primers 1 and 2. This fragment was cloned, and sequencing confirmed that it contained most of the peptide b sequence. This 940-bp fragment was then used to screen library A. After screening a total of 1×106 pfu, only three hybridization-positive plaques were found, with the longest insert size of 2.3 kb. Library B, which is both oligo-dT-primed and randomly primed, was screened with the same probe. Clone B1 (2.8 kb) was sequenced from both directions twice in each direction. Sequence analysis of B1 showed that it contained all three peptide sequences. Library B was rescreened using a 403-bp probe covering the 5∞-end region of clone B1. A total of 12 hybridization-positive plaques (including B1) were found from 1×106 pfu. Among the additional 11 clones, two had relative long 5∞-ends. B8 had ~800-bp 5∞-upstream from primer 5. B12 had about 3.3 kb 5∞-upstream from primer 5; however, only B12.1 (1 kb) corresponded to eIF2C cDNA. This fragment was flanked by the EcoRI library linker sequence, indicating that B12 was a hybrid. B12.1 had a 169-bp overlapping region with B1 from nt 851 to 1019. Combining the sequence from B1 and B12.1, we created a single ORF with the AUG located at position 82 and the stop codon UGA at position 2523 ( Fig. 2). This ORF encodes 813 amino acids. The initiator AUG lies in a strong context for translation, (CGAGAUGG), with purines at positions −3, and +4 ( Kozak, 1987). No polyadenylation sequence motif has been found in the 3∞-untranslated region to date. In order to confirm the 5∞-end of the cDNA, Marathon cDNA Amplification System (Clontech) was used to make a cDNA pool from the rabbit liver poly(A)+ RNA. Modified 5∞-RACE PCR was applied to amplify the 5∞-end of eIF2C cDNA. Two different sets of primers were used for separate reactions. Southern blotting was performed to probe the maximum 5∞-RACE products in each case. ( Fig. 3). In one reaction, a ~1000-bp discrete fragment was detected, whereas in another reaction, a ~340-bp fragment was identified. In both cases, the sizes of 5∞-RACE products corresponded well with the products anticipated. This result plus other information listed above confirmed that we indeed had the whole ORF of eIF2C cDNA.
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Fig. 5. In-vitro transcription and translation and immunoprecipitation. (A) Lane 1, translation product from pGEM7-eIF2C; lane 2, translation product from pGEM-vector lacking eIF2C cDNA. (B) Lane 1, the same translation product obtained as lane 1 (A) was treated with eIF2C polyclonal antibodies and subsequently with Protein A-agarose; lane 2, the same translation product obtained as lane 1 (A) was treated with bactin antibody and subsequently with Protein A-agarose.
3.3. Northern blot analysis of eIF2C poly(A)+ RNA The size of the eIF2C poly(A)+ RNA in rabbit liver was determined by Northern blotting. A single hybridization signal migrating at about 4.6 kb was observed (Fig. 4). 3.4. In-vitro translation of the eIF2C cloned gene and characterization of the translated product To characterize the cloned eIF2C cDNA further, the pGEM7-eIF2C, which contains the 3599-bp cDNA, was subjected to in-vitro translation in reticulocyte lysate using the SP6 TNT lysate-coupled transcription/ translation system. The translated product was analyzed by SDS–PAGE followed by autoradiography. A major polypeptide at 97 kDa was observed as the translation product (Fig. 5). This polypeptide was absent in the control experiment performed using pGEM7-vector lacking eIF2C cDNA. The in-vitro translated product as prepared above was further characterized by immunoprecipitation using eIF2C polyclonal antibody. The precipitate was subsequently analyzed using SDS–PAGE and autoradiography. A radioactive signal at 97 kDa was observed. However, when b-actin antibody was used, no signal was observed. 3.5. Genomic Southern blot analysis of eIF2C Genomic DNA from rabbit kidney was digested by BglII and PstI. BglII digestion of the cDNA produces a 1.2-kb fragment detected by the probe. PstI digestion of the cDNA produces a 350-bp fragment detected by
the probe. Restriction analysis with either BglII or PstI yielded a single band (Fig. 6). Additionally, restriction analysis with EcoRI, BamHI, KpnI or HindIII all yielded a single fragment (data not shown). This indicated that the eIF2C gene exists as a single copy gene in the rabbit genome. The 9-kb BglII fragment indicates that there is at least one large intron in a region corresponding to that of cDNA from nt 197 to 1370. The weak hybridization signal (~1 kb) seen in the PstI digestion may be due to the incomplete digestion by PstI. 3.6. cDNA sequence analysis The overall GC content of the cloned eIF2C cDNA is 60%, which is relatively high compared to some other translational factors, such as human eIF-2a (GC 40%) ( Ernst et al., 1987), human eIF-2b (42%) (Pathak et al., 1988), human eIF-2c (43%) (Gaspar et al., 1994), rat p67 (45%) ( Wu et al., 1993). Most of the hybridizationpositive clones from the two-library screening were very short ( less than 1 kb). There might be some very strong secondary structure within the eIF2C mRNA due to its high GC content. As a result, when a cDNA library was constructed, the possibility of obtaining a full-length reverse transcript was decreased. From the ORF, the calculated molecular mass is 90 kDa for the rabbit eIF2C. The difference between the calculated mass and the 140 kDa polypeptide detected in the reticulocyte lysate suggests that this protein is subjected to post-translational modification. Using the MOTIF search program within the GCG package, potential modification sites were identified, which included two N-glycosylation sites, two amidation sites, eight N-myristoylation sites, and various kinds of
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in S. cerevisiae genome database, even though yeast eIF2C activity was identified previously (Ahmad et al., 1985). One possible explanation is that yeast eIF2C may have diverged such that little sequence identity exists between the rabbit and yeast proteins. The cDNA cloning of eIF2C now enables us to understand the function and acting mechanism of this protein from the molecular level.
Acknowledgement We thank Debabrata Saha for his technical help. This work was supported in part by the National Institute of General Medical Sciences (No. 22079).
References
Fig. 6. Genomic southern blotting of eIF2C. Lane 1, pGEM7-eIF2C; lane 2, PstI digestion; lane 3, BglII digestion. A cDNA probe (nt 866–1271) was used.
phosphorylation sites. WGA binding indicated that the purified eIF2C (94 kDa) from rabbit reticulocyte lysate was a glycoprotein (data not shown). In addition, this protein has a very high calculated isoelectric point of 9.81, and contains large numbers of charged amino acids: 138 basic (Arg, Lys, His) and 77 acidic (Glu, Asp) amino acids. A GenBank EST Database search revealed that several cDNAs from other mammals (human, rat) had a large degree of homology (>95% aa identity) with the rabbit eIF2C. All of them are located within the ORF of eIF2C. A GenBank search detected a putative ORF (F48F7.1) from Caenorhalidits elegans ( Wilson et al., 1994) with a high homology to the rabbit eIF2C. This F48F7.1 exhibits 74% protein sequence identity and an additional 11% similarity. The two-protein sequence similarity is considerably high, indicating that F48F7.1 could be the eIF2C’s counterpart in C. elegans. However, no homologue at both DNA and protein level is found
Ahmad, M.F., Nasrin, N., Banerjee, A.C., Gupta, N.K., 1985. Purification and properties of eukaryotic initiation factor 2 and its ancillary protein factor (Co-eIF-2A) from yeast Saccharomyces cerevisiae. J. Biol. Chem. 260, 6955–6959. Chakravarty, I., Bagchi, M.K., Roy, R., Banerjee, A.C., Gupta, N.K., 1985. Protein synthesis in rabbit reticulocytes. J. Biol. Chem. 260, 6945–6949. Dasgupta, A., Das, A., Roy, R., Ralston, R., Majumdar, A., Gupta, N.K., 1978. Protein synthesis in rabbit reticulocytes. J. Biol. Chem. 253, 6054–6059. Ernst, H., Duncan, R.F., Hershey, J.W.B., 1987. Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2: Characterization of the protein and its messenger RNA. J. Biol. Chem. 262, 1206–1212. Gaspar, N.J., Kinzy, T.G., Scherer, B.J., Humbelin, M., Hershey, J.W.B., Merrick, W.C., 1994. Translation initiation factor eIF-2: Cloning and expression of the human cDNA encoding the c-subunit. J. Biol. Chem. 269, 3415–3422. Gupta, N.K., Datta, B., Ray, M.K., Roy, A.L., 1993. Protein synthesis initiation in animal cells: mechanism of ternary and met-tRNAf-40SmRNA complex formation and the regulatory role of an eIF-2 associated 67-kDa polypeptide. In: Ilan, J. ( Ed.), Translational Regulation in Gene Expression 2. Plenum, New York, pp. 405–431. Hecker, K.H., Roux, K.H., 1996. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques 20, 478–485. Hershey, J.W.B., 1991. Annu. Rev. Biochem. 60, 717–755. Kozak, M., 1987. An analysis of 5∞-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148. Merrick, W.C., Hershey, J.W.B., 1996. The pathway and mechanism of eukaryotic protein synthesis. In: Hershey, J.W.B., Mathews, M.B., Sonenberg, N. (Eds.), Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 31–69. Osterhout, J.J., Lax, S.R., Ravel, J.M., 1983. Factors from wheat germ that enhance the activity of eukaryotic initiation factor eIF-2. J. Biol. Chem. 258, 8285–8289. Pathak, V.K., Nielsen, P.J., Trachsel, H., Hershey, J.W.B., 1988. Structure of the beta subunit of translational initiation factor eIF-2. Cell 54, 633–639. Roy, A.L., Chakrabarti, D., Datta, B., Hileman, R.E., Gupta, N.K., Natural mRNA is required for directing Met-tRNA binding to 40S f ribosomal subunits in animal cells: Involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation. 1988. Biochemistry 27, 8203–8209.
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Molecular cloning and characterization of a rabbit eIF2C protein Cheng Zou a, Zhongli Zhang a, Shiyong Wu b, John C. Osterman c,* a Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA b Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA c Department of Biological Science, University of Nebraska, Lincoln, NE 68588, USA Received 1 July 1997; received in revised form 20 October 1997; accepted 27 October 1997; Received by J.A. Engler
Abstract Rabbit eIF2C (94 kDa) has been shown to play important roles in the eukaryotic peptide chain initiation process. In this study, the primary structure of rabbit eIF2C is determined by cDNA cloning. Based on the partial amino acid sequences of Endolys C cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding cDNA fragment from a rabbit liver cDNA library. This fragment was subsequently used to screen for larger cDNAs. Marathon cDNA amplification and 5∞-rapid amplification of cDNA ends were used to confirm the translation start site. Sequences from the overlapping clones were assembled into a 3599-bp composite sequence, which contains a single open reading frame that translates into a 813-deduced amino acid sequence. Northern blot analysis of rabbit liver ploy(A)+ RNA yielded a single message species at approximately 4.6 kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94 kDa eIF2C detected a higher-molecular-weight polypeptide (140 kDa). No 94 kDa polypeptide was detected. The cloned cDNA was further characterized by in-vitro transcription-coupled translation in reticulocyte lysate. The translated product was precipitated with antibodies against eIF2C. Genomic Southern blot analysis indicates that the rabbit eIF2C is a single copy gene. Sequence analysis reveals that rabbit eIF2C has strong homology with a hypothetical protein in Caenorhabditis elegans. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Recombinant DNA; Sequences; Eukaryotic peptide chain initiation; 5∞-RACE; PCR
1. Introduction The first step in protein synthesis initiation in eukaryotes involves the formation of a ternary complex among eukaryotic initiation factor 2 (eIF2), Met-tRNA and i GTP. The second step is the transfer of Met-tRNA i to the 40S ribosomal subunit, and the MettRNA · 40S · mRNA complex is formed. Reports from i different labs have indicated that the above two steps are regulated by several ancillary protein factors [reviewed in Gupta et al. (1993), Hershey (1991) and Merrick and Hershey (1996)]. Several years ago, an 80-kDa eIF2C (previous name: * Corresponding author. Tel: +1 402 472 5129; Fax: +1 402 472 2083; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, complementary DNA; eIF2C, eukaryotic initiation factor 2C; EST, expressed sequence tag; 5∞-RACE, rapid amplification of a cDNA end for 5∞-region; kb, kilobase; kDa, kilodalton; nt, nucleotide(s); ORF, open reading frame; pfu, plaque forming unit; UTR, untranslated region(s). 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 10 7 - 3
Co-eIF-2A) was isolated from rabbit reticulocyte lysate (Chakravarty et al., 1985). The size of the same polypeptide has been recalibrated as 94 kDa (Gupta et al., 1993). This protein has been shown to play two important roles in the protein synthesis initiation. First, it stimulates the ternary complex formation. Second, it stabilizes the ternary complex and the MettRNA · 40S · mRNA complex in the presence of natural i mRNAs (Roy et al., 1988). These reports suggested that eIF2C is an important component of the protein synthesis in reticulocyte lysate. Chakravarty et al. (1985) have shown that the 94-kDa eIF2C has two major protease degradation products, 50 kDa and 25 kDa. All three polypeptides of eIF2C have the ability to stimulate the ternary complex formation. eIF2C-like activity has been reported to be present in widely divergent eukaryotic organisms such as mouse ascite tumor cells (Dasgupta et al., 1978), wheat germ (Osterhout et al., 1983; Seal et al., 1983), Artemia salina ( Woodley et al., 1981), and yeast (Ahmad et al., 1985); however, we do not know the exact working mechanism of eIF2C.
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In this study, we report the primary molecular structure of the eIF2C that we have cloned as part of an effort to understand, in molecular terms, how the eIF2C works in the protein synthesis initiation complex. In addition, we report the discovery of a high-molecularweight eIF2C (140 kDa) in the rabbit reticulocyte lysate.
2. Materials and methods 2.1. Materials All primers were synthesized using the facilities of the DNA Synthesis Laboratory at the University of Nebraska, Lincoln. Two cDNA libraries were purchased from Clontech (Palo Alto, CA): Library A, Rabbit Liver cDNA Library (oligo dT primed ) in lgt11 phages, and Library B, Rabbit Liver 5∞-stretch Plus cDNA Library (oligo dT+random primed ) in lgt10 phages. 2.2. Isolation of cDNA clones encoding eIF2C Degenerate primers were designed, based on partial amino acid sequences from the 94 kDa eIF2C. A 940-bp cDNA fragment of eIF2C was obtained by PCR using primer 1: 5∞- GA(C/T )ATGCGGAACAAGCAGTT(C/T )CA-3∞ (sense oligo, nt 1249–1271) and primer 2: 5∞-TA(G/A)AAGTCAAACTCTGTGG(G/A)TG-3∞ (antisense oligo, nt 2189–2167). The 940-bp PCR product was randomly labeled with DIG-dUTP using the Genius System (Boehringer Mannheim), and was used to screen library A according to the procedures described in the Genius System (Boehringer Mannheim). After screening approximately 1×106 plaques, three hybridization-positive clones were identified. The longest clone had a size of 2.3 kb. The same probe was also applied to library B, where a 2.8-kb hybridization-positive clone was identified. This clone (B1) was cloned into pGEM7zf(+) vector (Promega), and followed by nested deletion and sequencing in both orientations. DNA sequencing was performed using the dideoxynucleotide chain termination method with the Sequenase version 2 kit ( US Biochemical Corp.). A 403-bp fragment of eIF2C at the 5∞-end of the 2.8 kb clone was synthesized by PCR using primer 3: 5∞-TGGCCCAGTACTTCAAGGACAGG-3∞ (sense oligo, nt 865–888) and primer 4: 5∞-TG(A/G)AACTGCTTGTTCCGCAT(A/G)TC-3∞ (antisense oligo, nt 1271–1249). This fragment was then labeled for subsequent screening of library B. A total of 11 hybridization-positive clones (B2–B12) were identified from 1×106 plaques. The size and position of all hybridization-positive clones were identified by PCR according to the following: (1) primer 5: 5∞-CCTGCCTTGAAGTACTGGGCCA-3∞ (antisense oligo, nt 888–865) and either the forward or the back-
ward universal primer (Clontech) was used for checking the length of 5∞-end; (2) primer 3 and either the forward or backward universal primer were used for checking the length of 3∞-end. B12 and B8 were two clones that contained relatively long 5∞-ends. B12 had two internal EcoRI cutting sites. B12 was purified and cut by EcoRI to give three fragments of B12.1 (1 kb), B12.2 (0.6 kb), and B12.3 (1.6 kb), as well as the two l-arms. Three pieces of inserts were separately subcloned into pGEM7zf(+) vector and sequenced from both directions. Purified B12 l-phage DNA was sequenced by the fmol PCR Sequencing System (Promega) using primer 8: 5∞-CATCTCGAAGAAATTGGCCT-3∞ (antisense oligo, nt 84–65) to obtain the junction sequence between B12.1 and B12.2. The linking orientation between B12.2 and B12.3 was established by analyzing the DNA sequence of B12.2 and B12.3. A Marathon cDNA amplification kit was purchased from Clontech. One microgram of rabbit liver poly(A)+ RNA (Clontech) was used in the Marathon cDNA amplification according to the manufacturer’s protocol. After the cDNAs were synthesized, two 5∞-RACE reactions were performed using primer AP1 (Clontech) and primer 6: 5∞-GCCACTATGTGCAGACCTCCAGG-3∞ (antisense oligo, nt 986–963), or primer AP1 and primer 7: 5∞-CGGCAGCGTGACCTCCAGCTCCAC-3∞ (antisense oligo, nt 303–280). A step-down PCR technique ( Hecker and Roux, 1996) was applied in the 5∞-RACE reaction. The conditions for the step-down PCR were 1 min at 94°C and 2 min at 72°C for three cycles, 1 min at 94°C and 2 min at 69°C for three cycles, 1 min at 94°C and 1 min at 66°C and 1 min at 68°C for three cycles, 1 min at 94°C and 1 min at 63°C and 1 min at 68°C for three cycles, 1 min at 94°C and 1 min at 60°C and 1 min at 68°C for three cycles, then 1 min at 94°C and 2 min at 68°C for 25 cycles. 5∞-RACE products from both reactions were run on a 0.8% agarose gel and followed by Southern blotting probed with oligonucleotide 8.
2.3. Northern blot analysis of eIF2C Livers of adult rabbits were rapidly removed and quickly frozen in liquid nitrogen. Two grams of the liver were added into 20 ml of tissue guanidinium solution containing 11.8 g of guanidinium isothiocyanate, 10 mM EDTA (pH=8.0), 50 mM Tris–HCl (pH=7.5) and 5% b-mercaptoethanol. The liver was immediately ground in a polytron, and the mixture was spun for 10 min at 12 000×g at 15°C. Sarkosyl (20%, 1/10 volume) was added to the supernatant and heated at 65°C for 2 min. The mixture was extracted with 25:24:1 phenol:chloroform:isoamyl alcohol, followed by 24:1 chloroform:isoamyl alcohol. Then, a 1/10 volume of 3 M NaOAc and 2.5 volumes of ethanol were added to precipitate nucleic
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acids. The pellet was resuspended in the lysis buffer from the Fast Track 2.0 kit (Invitrogen). Poly(A)+ RNA was purified using the Fast Track kit according to the manufacturer’s protocol. Five micrograms of isolated rabbit liver poly(A)+ RNA were electrophoresed on a 1% agarose-formaldehyde denaturing gel and subsequently transferred on to Biotrace HP@ membrane (Gelman Science) by vacuum blotting using 7.5 mM NaOH. The membrane was baked at 80°C for 2 h and then incubated in a prehybridizing solution containing 1% non-fat dry milk, 1 mM EDTA (pH=8.0), 6% NaH PO , 7% SDS (pH=7.2) for 2 h at 63°C. For 2 4 hybridization, the membrane was further incubated overnight at 63°C with radiolabeled eIF2C cDNA probe. A 940-bp eIF2C cDNA probe was synthesized by PCR using primers 1 and 2, and radiolabeled using a random hexamer kit ( US Biochemical Corp.). The blot was washed successively, twice in 2×SSC and 1% SDS for 30 min at 63°C and twice with 0.1× SSC for 30 min at room temperature. The mRNA level was detected by autoradiography using Kodak X-Omat AR film at −70°C with intensifying screens.
2.4. In-vitro translation of eIF2C gene pGEM7-B1 and pGEM7-B12.1 were used in constructing a full-length eIF2C cDNA in pGEM7, which contained the single ORF, 5∞-UTR and 3∞-UTR. The newly constructed pGEM7-eIF2C was used in in-vitro translation in rabbit reticulocyte lysate using the SP6 TNT lysate-coupled transcription/translation system. [35S] Methionine (American Radiolabeled Chemicals) was used for amino acid incorporation. The pGEM7-eIF2C was purified by the Qiagen Midi-prep System (Qiagen). 0.5 mg of the purified DNA was used in each reaction. pGEM7 without the insert was used as control. The reactions were run at 30°C for 90 min. The translation product was analyzed by SDS–PAGE and also by immunoprecipitation using eIF2C antibodies. In a negative control, b-actin antibody was used in the immunoprecipitation.
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autoradiography using Kodak X-Omat AR film at −70°C with intensifying screens for 72 h.
3. Results and discussion 3.1. Discovery of a high-molecular-weight eIF2C protein Homogenous eIF2C (94 kDa) was purified from rabbit reticulocyte lysate. This 94-kDa polypeptide was functionally tested according to the procedure described previously ( Roy et al., 1988). For antibody production, the 94-kDa eIF2C was injected into mice. Polyclonal antibody was collected and used for Western blotting ( Fig. 1). The antibody detected the purified 94-kDa polypeptide very well; however, in rabbit reticulocyte lysate, only a high-molecular-weight, approximately 140 kDa, and a 25-kDa polypeptide were detected by our eIF2C polyclonal antibodies. This experiment was repeated two more times with different batches of reticulocyte lysates, but no 94-kDa band was detected. Preincubation of this mouse antibody with purified 94-kDa eIF2C removed the cross-reactivity of this antibody with both 140-kDa and 25-kDa polypeptides. We tried to purify this 140-kDa polypeptide, but found during the purification of the 140-kDa eIF2C that it easily degraded, producing several major degradation products, including 94-kDa, 50-kDa and 25-kDa polypeptides as detected by the above antibody (data not shown). This result indicated that the 94-kDa polypeptide purified previously was a degraded portion of the
2.5. Genomic Southern blot analysis of eIF2C Equal amounts (15 mg) of rabbit genomic DNA (Clontech) were subjected to restriction enzyme digestion and separated on a 0.8% agarose gel, transferred on to Biotrace HP@ (Gelman Sciences) and hybridized at 55°C in the presence of 1% BSA, 1 mM EDTA, 7% SDS, 6% NaH PO (pH=7.2) and 10% formamide for 36 h. The 2 4 probe for hybridization was randomly labeled cDNA PCR fragment (nt 866–1271). Filters were washed according to the manufacturer’s protocol (Gelman Sciences). The hybridization signal was detected by
Fig. 1. Western blot of eIF2C. Lane 1, 500 mg of reticulocyte lysate; lane 2, 250 mg of reticulocyte lysate; lane 3, purified eIF2C (94 kDa). Proteins were denatured and run on a 15% SDS–PAGE gel, then transferred to a Biotrace NT@ (Gelman Science). Mouse polyclonal antibodies against eIF2C were used for detection.
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Fig. 2. cDNA and deduced amino acid sequences of eIF2C. The entire cDNA coding sequence plus 5∞- and 3∞- UTR are reported (GenBank Accession No. AF005355), together with the derived amino acid sequence. Numbers to the left of the sequence represent nucleotide positions, whereas numbers to the right represent amino acid positions. Oligonucleotide primers used for PCR amplifications or for sequencing are designated as short arrows over the first four bases of the corresponding DNA, oriented in the direction of extension and numbered according to their descriptions under ‘Experimental Methods’. Asterisks represent stop codons. Rabbit eIF2C peptide (a, b and c) sequences are underlined.
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140-kDa protein, and that the original size of eIF2C appears to be 140 kDa. 3.2. cDNA cloning of elF2C
Fig. 3. Southern blotting of 5∞-RACE products. 5∞-RACE PCR products was run on a 0.8% agarose gel and subsequently transferred on to Biotrace HP@ (Gelman Science). Oligonucleotide 8 was end-labeled to detect the sizes of 5∞-RACE products. Lane 1, 5∞-RACE PCR using primers 7 and AP1; lane 2, 5∞-RACE PCR using primers 6 and AP1.
Fig. 4. Northern blotting of eIF2C poly(A)+ RNA. Five micrograms of rabbit liver poly(A)+ RNA were run on a 1% agarose-formaldehyde denaturing gel and subsequently transferred on to Biotrace HP@ (Gelman Science). A 940-bp cDNA probe was used to detect eIF2C poly(A)+ RNA.
Purified 94-kDa eIF2C was cleaved using Endolys C, and the amino acid sequences of the cleaved fragments were determined at the amino acid sequencing facilities at the University of Florida (Gainesville, FL). Three peptide sequences (a, b, c) were obtained (Fig. 2). Primer 1 was made using sequence from peptide b, whereas primer 2 used the peptide c sequence. A 940-bp fragment was isolated by PCR using degenerate primers 1 and 2. This fragment was cloned, and sequencing confirmed that it contained most of the peptide b sequence. This 940-bp fragment was then used to screen library A. After screening a total of 1×106 pfu, only three hybridization-positive plaques were found, with the longest insert size of 2.3 kb. Library B, which is both oligo-dT-primed and randomly primed, was screened with the same probe. Clone B1 (2.8 kb) was sequenced from both directions twice in each direction. Sequence analysis of B1 showed that it contained all three peptide sequences. Library B was rescreened using a 403-bp probe covering the 5∞-end region of clone B1. A total of 12 hybridization-positive plaques (including B1) were found from 1×106 pfu. Among the additional 11 clones, two had relative long 5∞-ends. B8 had ~800-bp 5∞-upstream from primer 5. B12 had about 3.3 kb 5∞-upstream from primer 5; however, only B12.1 (1 kb) corresponded to eIF2C cDNA. This fragment was flanked by the EcoRI library linker sequence, indicating that B12 was a hybrid. B12.1 had a 169-bp overlapping region with B1 from nt 851 to 1019. Combining the sequence from B1 and B12.1, we created a single ORF with the AUG located at position 82 and the stop codon UGA at position 2523 ( Fig. 2). This ORF encodes 813 amino acids. The initiator AUG lies in a strong context for translation, (CGAGAUGG), with purines at positions −3, and +4 ( Kozak, 1987). No polyadenylation sequence motif has been found in the 3∞-untranslated region to date. In order to confirm the 5∞-end of the cDNA, Marathon cDNA Amplification System (Clontech) was used to make a cDNA pool from the rabbit liver poly(A)+ RNA. Modified 5∞-RACE PCR was applied to amplify the 5∞-end of eIF2C cDNA. Two different sets of primers were used for separate reactions. Southern blotting was performed to probe the maximum 5∞-RACE products in each case. ( Fig. 3). In one reaction, a ~1000-bp discrete fragment was detected, whereas in another reaction, a ~340-bp fragment was identified. In both cases, the sizes of 5∞-RACE products corresponded well with the products anticipated. This result plus other information listed above confirmed that we indeed had the whole ORF of eIF2C cDNA.
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Fig. 5. In-vitro transcription and translation and immunoprecipitation. (A) Lane 1, translation product from pGEM7-eIF2C; lane 2, translation product from pGEM-vector lacking eIF2C cDNA. (B) Lane 1, the same translation product obtained as lane 1 (A) was treated with eIF2C polyclonal antibodies and subsequently with Protein A-agarose; lane 2, the same translation product obtained as lane 1 (A) was treated with bactin antibody and subsequently with Protein A-agarose.
3.3. Northern blot analysis of eIF2C poly(A)+ RNA The size of the eIF2C poly(A)+ RNA in rabbit liver was determined by Northern blotting. A single hybridization signal migrating at about 4.6 kb was observed (Fig. 4). 3.4. In-vitro translation of the eIF2C cloned gene and characterization of the translated product To characterize the cloned eIF2C cDNA further, the pGEM7-eIF2C, which contains the 3599-bp cDNA, was subjected to in-vitro translation in reticulocyte lysate using the SP6 TNT lysate-coupled transcription/ translation system. The translated product was analyzed by SDS–PAGE followed by autoradiography. A major polypeptide at 97 kDa was observed as the translation product (Fig. 5). This polypeptide was absent in the control experiment performed using pGEM7-vector lacking eIF2C cDNA. The in-vitro translated product as prepared above was further characterized by immunoprecipitation using eIF2C polyclonal antibody. The precipitate was subsequently analyzed using SDS–PAGE and autoradiography. A radioactive signal at 97 kDa was observed. However, when b-actin antibody was used, no signal was observed. 3.5. Genomic Southern blot analysis of eIF2C Genomic DNA from rabbit kidney was digested by BglII and PstI. BglII digestion of the cDNA produces a 1.2-kb fragment detected by the probe. PstI digestion of the cDNA produces a 350-bp fragment detected by
the probe. Restriction analysis with either BglII or PstI yielded a single band (Fig. 6). Additionally, restriction analysis with EcoRI, BamHI, KpnI or HindIII all yielded a single fragment (data not shown). This indicated that the eIF2C gene exists as a single copy gene in the rabbit genome. The 9-kb BglII fragment indicates that there is at least one large intron in a region corresponding to that of cDNA from nt 197 to 1370. The weak hybridization signal (~1 kb) seen in the PstI digestion may be due to the incomplete digestion by PstI. 3.6. cDNA sequence analysis The overall GC content of the cloned eIF2C cDNA is 60%, which is relatively high compared to some other translational factors, such as human eIF-2a (GC 40%) ( Ernst et al., 1987), human eIF-2b (42%) (Pathak et al., 1988), human eIF-2c (43%) (Gaspar et al., 1994), rat p67 (45%) ( Wu et al., 1993). Most of the hybridizationpositive clones from the two-library screening were very short ( less than 1 kb). There might be some very strong secondary structure within the eIF2C mRNA due to its high GC content. As a result, when a cDNA library was constructed, the possibility of obtaining a full-length reverse transcript was decreased. From the ORF, the calculated molecular mass is 90 kDa for the rabbit eIF2C. The difference between the calculated mass and the 140 kDa polypeptide detected in the reticulocyte lysate suggests that this protein is subjected to post-translational modification. Using the MOTIF search program within the GCG package, potential modification sites were identified, which included two N-glycosylation sites, two amidation sites, eight N-myristoylation sites, and various kinds of
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in S. cerevisiae genome database, even though yeast eIF2C activity was identified previously (Ahmad et al., 1985). One possible explanation is that yeast eIF2C may have diverged such that little sequence identity exists between the rabbit and yeast proteins. The cDNA cloning of eIF2C now enables us to understand the function and acting mechanism of this protein from the molecular level.
Acknowledgement We thank Debabrata Saha for his technical help. This work was supported in part by the National Institute of General Medical Sciences (No. 22079).
References
Fig. 6. Genomic southern blotting of eIF2C. Lane 1, pGEM7-eIF2C; lane 2, PstI digestion; lane 3, BglII digestion. A cDNA probe (nt 866–1271) was used.
phosphorylation sites. WGA binding indicated that the purified eIF2C (94 kDa) from rabbit reticulocyte lysate was a glycoprotein (data not shown). In addition, this protein has a very high calculated isoelectric point of 9.81, and contains large numbers of charged amino acids: 138 basic (Arg, Lys, His) and 77 acidic (Glu, Asp) amino acids. A GenBank EST Database search revealed that several cDNAs from other mammals (human, rat) had a large degree of homology (>95% aa identity) with the rabbit eIF2C. All of them are located within the ORF of eIF2C. A GenBank search detected a putative ORF (F48F7.1) from Caenorhalidits elegans ( Wilson et al., 1994) with a high homology to the rabbit eIF2C. This F48F7.1 exhibits 74% protein sequence identity and an additional 11% similarity. The two-protein sequence similarity is considerably high, indicating that F48F7.1 could be the eIF2C’s counterpart in C. elegans. However, no homologue at both DNA and protein level is found
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