Secondary literature sources for MA3
The following references were automatically generated.
- Weber JA, Gay CV
- Expression of translation initiation factor IF2 is regulated during osteoblast differentiation.
- J Cell Biochem. 2001; 81: 700-14
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We isolated and characterized a cDNA for the N-terminal half of the eukaryotic initiation of translation factor 2 (cIF2) during a screen of chicken osteoblast cDNAs. The apparent size of the message for this protein, approximately 5.6 kb, is slightly larger in size than that for human IF2 (hIF2). There is a high degree of sequence similarity between the human and chicken N-terminal portions of the protein that extends to the encoding nucleotide sequence. The tissue specific expression pattern for cIF2 and hIF2 are similar, being moderately abundant in brain, liver, and skeletal muscle, and detectable in kidney, chondrocytes, and freshly isolated osteoblasts. The ratio of message for cIF2 to that of beta-actin was 0.10 and 0.18 for liver and brain. Message levels peak in osteoblasts between 8 and 12 days of culture, coinciding with high levels of matrix protein synthesis. At peak expression, the ratio of cIF2:beta-actin for 8 day osteoblasts was 0.76. Treatment of osteoblast cultures with cycloheximide markedly reduces the level of cIF2 message indicating that novel protein synthesis is required for its expression. Hybridization of RNA samples from either chicken osteoblasts or a human osteoblast cell line with a probe for a subunit of human eukaryotic initiation of translation factor 2 (eIF2alpha), the housekeeping initiation factor, indicates that levels of eIF2 remain low. With hIF2, cIF2 represents the only other vertebrate homolog of IF2 for which a major portion of the coding sequence has been identified. This is the first report of regulated expression for a eukaryotic IF2 and is the first demonstration of its abundance in osteoblasts. Copyright 2001 Wiley-Liss, Inc.
- Mazumder B, Seshadri V, Imataka H, Sonenberg N, Fox PL
- Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.
- Mol Cell Biol. 2001; 21: 6440-9
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Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.
- Jenkins ZA, Haag PG, Johansson HE
- Human eIF5A2 on chromosome 3q25-q27 is a phylogenetically conserved vertebrate variant of eukaryotic translation initiation factor 5A with tissue-specific expression.
- Genomics. 2001; 71: 101-9
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Eukaryotic translation initiation factor 5A (eIF5A) is an essential protein tightly linked to cellular polyamine homeostasis. It receives the unique spermidine-derived posttranslational modification hypusine that is necessary for eIF5A's biochemical activity and cellular proliferation. The eIF5A protein stimulates ribosomal peptidyl-transferase and may be involved in nucleocytoplasmic mRNA transport. Little is known about the molecular genetics of eIF5A. Here we report on the sequence and molecular characterization of human EIF5A2, a novel phylogenetically conserved gene for eIF5A. EIF5A2 stretches over 17 kb and consists of five exons and four introns. It is localized at 3q25-q27, often noted for chromosomal instability in cancers. EIF5A2 is highly expressed in testis and colorectal adenocarcinoma and at moderate levels in the brain, in contrast to the ubiquitously expressed EIF5A1 gene. Two EIF5A2 mRNAs share a 129-nt 5' UTR and a coding sequence for the 153-amino-acid eIF5AII protein, but possess two alternative 3' UTRs of 46 and 890 nt that arise through differential polyadenylation. The protein is 84% identical and 94% similar to eIF5AI. Both EIF5A genes are conserved in vertebrates. Our findings lend further support for a specialized gene expression program of polyamine metabolic proteins and regulators that function to maintain polyamine homeostasis at elevated levels during spermatogenesis. Copyright 2001 Academic Press.
- Sasahara K et al.
- Molecular cloning and expression analysis of a putative nuclear protein, SR-25.
- Biochem Biophys Res Commun. 2000; 269: 444-50
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We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.
- Nakamura Y, Takayama N, Minamitani T, Ikuta T, Ariga H, Matsumoto K
- Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1.
- Gene. 2000; 251: 55-62
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The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.
- Raught B et al.
- Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.
- EMBO J. 2000; 19: 434-44
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The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).
- Holzmann K et al.
- A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A.
- Biochem Biophys Res Commun. 2000; 267: 339-44
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Amino acid sequencing and mass spectrometry revealed identity of a human nuclear matrix protein, termed hNMP 265, with a predicted protein of gene KIAA0111. Two-dimensional electrophoresis and Northern hybridization showed the protein to ubiquitously occur in various human cell types. Exhibiting DEAD-box motifs characteristic for RNA helicases, hNMP 265 is highly similar to the human initiation factors eIF4A-I and -II. On the other hand, hNMP 265 greatly differs from the initiation factors by a N-terminal sequence rich in charged amino acids. Sequence searches and alignments indicate proteins related to hNMP 265 in other eukaryotes. Chimeras between hNMP 265 and green fluorescence protein or hapten appeared as speckles in extranucleolar regions in the nucleus, but not in the cytoplasm. Experiments with tagged deletion mutants indicated that the N-terminal amino acid sequence is necessary for nuclear localization. A putative role of hNMP 265 in pre-mRNA processing is discussed.
- Suzuki T et al.
- Molecular cloning of a novel apoptosis-related gene, human Nap1 (NCKAP1), and its possible relation to Alzheimer disease.
- Genomics. 2000; 63: 246-54
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Expression profiles of thousands of genes (cDNAs) were analyzed in sporadic Alzheimer disease (AD)-affected brains in comparison with normal subjects by using the high-density cDNA filter method and differential display analysis. Among 31 differentially expressed genes, one gene was found to be markedly depressed in AD-affected brains. A full-length (or nearly full-length) cDNA of the gene was isolated and sequenced. The cDNA turned out to be an orthologue of rat Nap1. The gene was thus designated human Nap1 (HGMW-approved symbol NCKAP1) and was mapped to human chromosome 2q32 by fluorescence in situ hybridization. Northern blotting and in situ hybridization studies showed that in brain, the gene is predominantly expressed in neuronal cells. Antisense oligo DNA of human Nap1 transcripts was found to induce apoptosis of neuronal cells. Based on these results, the possible role of human Nap1 in AD is discussed.
- van Oers MM, van Marwijk M, Kwa MS, Vlak JM, Thomas AA
- Cloning and analysis of cDNAs encoding the hypusine-containing protein eIF5A of two lepidopteran insect species.
- Insect Mol Biol. 1999; 8: 531-8
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Eukaryotic initiation factor eIF5A is essential for cell viability and contains a characteristic post-translational modification of a specific lysine residue into a hypusine. cDNAs with similarity to eIF5A sequences were derived from Spodoptera exigua and S. frugiperda cDNA libraries. The deduced amino acid sequences are identical for both species and predict a protein with a molecular mass of 17.5 kDa. The Drosophila melanogaster eIF5A cDNA sequence was retrieved from the Drosophila EST Project. The predicted protein is 80% similar to Spodoptera eIF5A. A single eIF5A gene copy is present in the S. frugiperda genome, which is transcribed into four different transcripts. Infection of S. frugiperda cells with a baculovirus resulted in a strong decline of all four transcripts already at 12 h after infection. In contrast, the eIF5A protein was fairly stable up to 48 h post infection.
- Koesters R et al.
- Human eukaryotic initiation factor EIF2C1 gene: cDNA sequence, genomic organization, localization to chromosomal bands 1p34-p35, and expression.
- Genomics. 1999; 61: 210-8
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We report the cloning and characterization of the human eukaryotic protein translation initiation factor EIF2C1 gene. The human EIF2C1 gene consists of 19 exons and 18 introns that span a region of almost 50 kb. It is located on the short arm of chromosome 1 in the region 1p34-p35. This genomic region is frequently lost in human cancers such as Wilms tumors, neuroblastoma, and carcinomas of the breast, liver, and colon. The human EIF2C1 gene is ubiquitously expressed at low to medium levels. Differential polyadenylation and splicing result in a complex transcriptional pattern. The cDNA sequence is 7478 bp long and contains an extremely large 3' untranslated region of 4799 bp with multiple, short repeated segments composed of mono-, tri-, or quattronucleotides interspersed throughout. The human EIF2C1 gene belongs to a multigene family in human. It is highly conserved during evolution, sharing about 90% identity with rabbit eIF2C and 70% identity with plant AGO1 at the amino acid level. These facts suggest that human EIF2C1 might play an important physiological role.
- Lian Z et al.
- The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis.
- Oncogene. 1999; 18: 1677-87
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The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the differences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One differentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma. Introduction of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice. Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of hepatocellular carcinoma.
- Bora RS, Kanamori A, Hirabayashi Y
- Cloning and characterization of a putative mouse acetyl-CoA transporter cDNA.
- Gene. 1999; 238: 455-62
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A mouse acetyl-CoA transporter (Acatn) cDNA was isolated by PCR cloning. Mouse Acatn exhibited 92% homology with human sequence on the basis of amino-acid sequence. The predicted gene product of Acatn is a 61 kDa hydrophobic protein with six to 10 transmembrane domains. Transfection of mouse Acatn cDNA into HeLa/GT3+ cells resulted in significant increase in the amount of 9-O-acetylated gangliosides, suggesting that Acatn does play an important role in the acetylation of gangliosides. Northern blot analysis of Acatn mRNA suggested that transcript of Acatn is widely distributed in various adult tissues. Expression of Acatn was found to be developmentally regulated, with high expression levels during early embryonic stages, and then there was a subsequent decrease in expression levels in the later embryonic stages.
- Miranda-Vizuete A, Pedrajas JR, Damdimopoulos AE, Spyrou G
- Cloning and sequencing of mouse glutaredoxin (grx) cDNA.
- DNA Seq. 1999; 10: 179-82
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Glutaredoxins are small proteins (12 kDa) with a conserved active sequence Cys-Pro-Tyr(-Phe)-Cys that catalyse GSH-disulfide oxidoreduction reactions in the presence of NADPH and glutathione reductase. Many mammalian glutaredoxins have been characterized and human and pig cDNA sequence determined. However, no mouse glutaredoxin cDNA or protein sequence has yet been reported. We have cloned a cDNA from a mouse liver library that encodes the putative mouse glutaredoxin homologue. The deduced polypeptide sequence encodes a 107 amino acid protein displaying a high degree of homology with other members of the glutaredoxin family.
- Akiyama H et al.
- Cloning of a novel gene specifically expressed in clonal mouse chondroprogenitor-like EC cells, ATDC5.
- Biochim Biophys Acta. 1999; 1444: 291-4
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We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.
- Sabelli PA, Burgess SR, Valasek L, Shewry PR
- Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3).
- Mol Gen Genet. 1999; 261: 820-30
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In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation. A meristem-specific cDNA was isolated and characterised. This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis. The ZmeIF3A protein is most similar to the yeast homologue RPG1, lacking the repeated C-terminal domain characteristic of its mammalian counterparts. However, despite this similarity, it fails to replace the RPG1 protein in complementation experiments on yeast mutants. Analysis of gene expression in situ showed that the ZmeIF3A transcript is expressed in the region of the root meristem surrounding the central stele. ZmeIF3A mRNA is also expressed in the young root, the male inflorescence, and the developing cob and seed. In maize, ZmeIF3A is encoded by one or two genomic sequences. This is the first report on the isolation and characterisation of a cDNA from higher plants that encodes a product homologous to a component of the eIF3 complex.
- Kojima S, Mayumi-Matsuda K, Suzuki H, Sakata T
- Molecular cloning of rat GADD45gamma, gene induction and its role during neuronal cell death.
- FEBS Lett. 1999; 446: 313-7
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To study the molecular mechanism of neuronal cell death, we carried out the screening of genes which were induced during the neuronal cell death of neuronal PC12. We cloned the cDNA of rat GADD45gamma, the third member of the GADD45 family. Induction of GADD45gamma mRNA was observed in the neuronal cell death caused by depletion of neurotrophic factor and Ca2+ ionophore treatment. Overexpression of GADD45gamma in superior cervical ganglion neurons caused cell death. These results suggest that GADD45gamma plays an important role in neuronal cell death.
- Marissen WE, Lloyd RE
- Eukaryotic translation initiation factor 4G is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells.
- Mol Cell Biol. 1998; 18: 7565-74
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Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.
- Zou C, Zhang Z, Wu S, Osterman JC
- Molecular cloning and characterization of a rabbit eIF2C protein.
- Gene. 1998; 211: 187-94
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Rabbit eIF2C (94kDa) 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 4.6kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94kDa eIF2C detected a higher-molecular-weight polypeptide (140kDa). No 94kDa 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.
- Yamagoe S, Watanabe T, Mizuno S, Suzuki K
- The mouse Lect2 gene: cloning of cDNA and genomic DNA, structural characterization and chromosomal localization.
- Gene. 1998; 216: 171-8
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We previously purified bovine leukocyte cell-derived chemotaxin 2 (LECT2) as a 16 kDa-secreted protein with a neutrophil chemotactic activity. LECT2 protein is thought to be multifunctional, since it was recently found to be identical to chondromodulin-II, a growth stimulator of chondrocyte cells. We report here the cloning and structural analysis of mouse Lect2 cDNAs and genomic DNA, and chromosomal mapping. Two types of mouse Lect2 cDNAs were cloned: one encoded the mouse counterpart of human and bovine LECT2 proteins, and the other encoded a queer type LECT2 protein whose amino-acid sequence in the carboxy terminus was different from that of the normal type LECT2 protein. The mouse Lect2 gene spanned approx. 8 kb and consisted of five exons and four introns. The genomic organization revealed that two type transcripts arose by an alternative splicing event involving exon 4. A primer extension analysis revealed that several transcription initiation sites occurred within 60-210 nucleotides upstream from the translation initiation codon. The mouse Lect2 gene was mapped to a region adjacent to D13Mit13, D13Mit21 and Il-9 on chromosome 13 by interspecific backcross mapping.
- Biederer C, Ries S, Drobnik W, Schmitz G
- Molecular cloning of human caveolin 3.
- Biochim Biophys Acta. 1998; 1406: 5-9
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We isolated a full-length cDNA encoding human caveolin 3, which is 72% and 59% homologous to human caveolin 1 and caveolin 2, respectively. Human caveolin 3 protein contains the 'caveolin signature sequence' and the 33 amino acids spanning intramembrane domain common to all caveolins. Northern blot analysis indicates that the caveolin 3 transcript is 1.6 kb in size and exclusively detectable in muscle tissue.
- Bremaud L, Laalami S, Derijard B, Cenatiempo Y
- Translation initiation factor IF2 of the myxobacterium Stigmatella aurantiaca: presence of a single species with an unusual N-terminal sequence.
- J Bacteriol. 1997; 179: 2348-55
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The structural gene for translation initiation factor IF2 (infB) was isolated from the myxobacterium Stigmatella aurantiaca on a 5.18-kb BamHI genomic restriction fragment. The infB gene (ca. 3.16 kb) encodes a 1,054-residue polypeptide with extensive homology within its G domain and C terminus with the equivalent regions of IF2s from Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus, and Streptococcus faecium. The N-terminal region does not display any significant homology to other known proteins. The S. aurantiaca infB gene encodes a single protein which cross-reacted with antiserum to E. coli IF2 and was able to complement an E. coli infB mutant. The S. aurantiaca IF2 is distinguished from all other IF2s by a sequence of 160 residues near the N terminus that has an unusual composition, made up essentially of alanine, proline, valine, and glutamic acid. Within this sequence, the pattern PXXXAP is repeated nine times. Complete deletion of this sequence did not affect the factor's function in initiation of translation and even increased its capacity to complement the E. coli infB mutant.
- Asano K, Vornlocher HP, Richter-Cook NJ, Merrick WC, Hinnebusch AG, Hershey JW
- Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.
- J Biol Chem. 1997; 272: 27042-52
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The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.
- Johnson KR, Merrick WC, Zoll WL, Zhu Y
- Identification of cDNA clones for the large subunit of eukaryotic translation initiation factor 3. Comparison of homologues from human, Nicotiana tabacum, Caenorhabditis elegans, and Saccharomyces cerevisiae.
- J Biol Chem. 1997; 272: 7106-13
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Initiation of translation in eukaryotes is mediated by a set of initiation factors. Mammalian initiation factor 3 is composed of at least 8 subunits, with the largest being about 180 kDa in size. Here we report the cloning of the p180 subunit of human eukaryotic translation initiation factor (eIF) 3. The amino acid sequence deduced from the cDNA agrees with the sequences of CNBr fragments of eIF-3, confirming the identity of the clone. The 1382 amino acid open reading frame contains a high percentage of charged residues (48%) and an unusual repetitive domain near the carboxyl terminus composed of 25 repeats of 10 amino acids each. Data base searches identified related sequences found in members of the plant and fungal kingdoms as well as in other mammals and the nematode Caenorhabditis elegans. These sequences share significant identity with the human clone and probably represent the homologues of the p180 subunit in these organisms. This is the first report identifying the sequence of the large subunit of eIF-3.
- Aoki T et al.
- Rat TAFII31 gene is induced upon programmed cell death in differentiated PC12 cells deprived of NGF.
- Biochem Biophys Res Commun. 1997; 234: 230-4
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Typical programmed cell death (PCD) requires de novo macromolecular synthesis and shares common morphological changes referred to as apoptosis. To elucidate the molecular mechanism of apoptosis, we isolated cDNA clones that are induced in differentiated PC12 cells deprived of NGF by differential display method. Among such clones, homology searches revealed that the one clone encodes the rat TATA-binding-protein-associated factor TAFII31, a component of TFIID, and a transcriptional coactivator of the p53 protein. Northern analysis of various organs in human showed one band in heart, brain, skeletal muscle and pancreas, whose size is approximately 1.1 kb which identical to that of human TAFII31 mRNA, although the size of rat human TAFII31 mRNA is approximately 2.7 kb. The deduced amino acid sequence of the rat TAFII31 was 77% identical to that of the human TAFII31. Northern analysis of various organs in adult mice showed that expression levels of TAFII31 mRNA were strong in heart but weak in spleen, although this gene is ubiquitously expressed.
- Tewari M, Yu M, Ross B, Dean C, Giordano A, Rubin R
- AAC-11, a novel cDNA that inhibits apoptosis after growth factor withdrawal.
- Cancer Res. 1997; 57: 4063-9
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Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death (apoptosis). However, the specific genes and corresponding proteins that mediate survival are poorly defined. To identify potential survival genes, a cDNA library was prepared from murine fibroblasts and screened by a functional expression cloning approach. A 1023-bp cDNA, AAC-11, was identified that encodes a protein of approximately 25 kDa. The AAC-11 gene shows strong species conservation and is ubiquitously expressed in embryonic and adult tissues with multiple transcripts, as well as in various human tumor cell lines. The predicted protein contains a leucine zipper domain but lacks a DNA-binding domain. BALB/c3T3 fibroblasts that were stably transfected with AAC-11 cDNA were viable in serum-free medium for up to 12 weeks. The protective action of AAC-11 was abolished by mutation of leucines to arginines within the leucine zipper domain. We also isolated a longer AAC-11 cDNA that codes for up to an additional 290 amino-terminal amino acids but did not protect against apoptosis. The cDNA for human AAC-11 was identified and exhibits strong homology with the murine species and retains the leucine zipper domain. Western immunoblots of BALB/c3T3 cells using rabbit anti-AAC-11 polyclonal serum revealed a major native 55-kDa AAC-11 protein and a minor 25-kDa protein corresponding to the long and short forms of AAC-11 cDNA, respectively. In summary, we report a cDNA whose expression supports cell viability after withdrawal of growth factors. The corresponding native protein may function as a novel inhibitor of apoptosis.
- Matsuyama S, Kubo K, Ohashi F, Takamori Y
- Partial cloning of prohibitin cDNA from canine, feline, bovine, equine, and rabbit liver mRNA by RT-PCR.
- J Vet Med Sci. 1997; 59: 201-3
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Prohibitin is the protein which has an inhibitory function in cell growth, and its gene is suggested to be one of putative tumor suppressor genes. In this report, we described a partial cloning of prohibitin cDNAs from canine, feline, bovine, equine, and rabbit liver mRNAs by RT-PCR, and their homology analysis. The sequences of these RT-PCR products were compared with each other as well as those reported for human and rat. The homology in this region of prohibitin cDNA was approximately 90%, and the amino acid sequence of each RT-PCR product shared more than 95% identity. Therefore, it is concluded that all the RT-PCR products are a part of prohibitin cDNA of each animal.
- Asano K, Kinzy TG, Merrick WC, Hershey JW
- Conservation and diversity of eukaryotic translation initiation factor eIF3.
- J Biol Chem. 1997; 272: 1101-9
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The largest of the mammalian translation initiation factors, eIF3, consists of at least eight subunits ranging in mass from 35 to 170 kDa. eIF3 binds to the 40 S ribosome in an early step of translation initiation and promotes the binding of methionyl-tRNAi and mRNA. We report the cloning and characterization of human cDNAs encoding two of its subunits, p110 and p36. It was found that the second slowest band during polyacrylamide gel electrophresis of eIF3 subunits in sodium dodecyl sulfate contains two proteins: p110 and p116. Analysis of the cloned cDNA encoding p110 indicates that its amino acid sequence is 31% identical to that of the yeast protein, Nip1. The p116 cDNA was cloned and characterized as a human homolog of yeast Prt1, as described elsewhere (Methot, N., Rom, E., Olsen, H., and Sonenberg, N. (1997) J. Biol. Chem. 272, 1110-1116). p36 is a WD40 repeat protein, which is 46% identical to the p39 subunit of yeast eIF3 and is identical to TRIP-1, a phosphorylation substrate of the TGF-beta type II receptor. The p116, p110, and p36 subunits localize on 40 S ribosomes in cells active in translation and co-immunoprecipitate with affinity-purified antibodies against the p170 subunit, showing that these proteins are integral components of eIF3. Although p36 and p116 have homologous protein subunits in yeast eIF3, the p110 homolog, Nip1, is not detected in yeast eIF3 preparations. The results indicate both conservation and diversity in eIF3 between yeast and humans.
- Hayashi Y, Kiyono T, Fujita M, Ishibashi M
- Isolation of a novel cDNA whose corresponding mRNA is accumulated in growth-arrested confluent but not in growing sub-confluent rat 3Y1 cells.
- Biochim Biophys Acta. 1997; 1352: 145-50
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Four cDNA fragments, whose corresponding mRNAs were accumulated in growth-arrested confluent but not in growing sub-confluent rat 3Y1 cells, were isolated by the mRNA differential display method. Sequencing of the four cDNA fragments indicated that one of them was highly homologous to 3' untranslated DNA region of mouse growth-arrest specific1 cDNA, while the other three (named confluent 3Y1 cell-associated No. 1 (cca1), cca2 and cca3) were novel. Of the three novel cDNAs, we presented some characteristics of cca2 cDNA: the cDNA consisted of 1795 nucleotides with a deduced open reading frame (ORF) encoding a protein of 338 amino acids, which showed a 35% identity with rat type IV 3beta-hydroxysteroid dehydrogenase. The CCA2 protein, with a molecular weight of 38 kDa, was accumulated in 3Y1 cells under growth-arrest conditions.
- Lopez Ribera I, Ruiz-Avila L, Puigdomenech P
- The eukaryotic translation initiation factor 5, eIF-5, a protein from Zea mays, containing a zinc-finger structure, binds nucleic acids in a zinc-dependent manner.
- Biochem Biophys Res Commun. 1997; 236: 510-6
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A maize cDNA encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from an 8-day-old seedling cDNA library. The 1975 bp cDNA encodes a protein of 451 amino acids, with a predicted molecular weight of 49.04 kDa, and hybridizes to a single sequence in the maize genome. The deduced sequence contains motifs characteristic of proteins belonging to the GPTase superfamily, a zinc finger well conserved in all the protein sequences for eIF-5 reported so far, and a fragment also present in prokaryotic and chloroplast L11 ribosomal protein. Polymer-binding assays have been used to assess the predicted RNA binding property of the protein and to characterize its function. It is shown that the eIF-5-encoded protein binds to single-stranded DNA and to polyuridylic acid and that the binding is dependent on the presence of Zn2+ ions. These results suggest that the zinc-finger structure is involved in the binding of the eIF-5 protein to RNA.
- Mirbod F, Nakashima S, Kitajima Y, Ghannoum MA, Cannon RD, Nozawa Y
- Molecular cloning of a gene encoding translation initiation factor (TIF) from Candida albicans.
- J Med Vet Mycol. 1996; 34: 393-400
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The differential display technique was applied to compare mRNAs from two clinical isolates of Candida albicans with different virulence; high (potent strain, 16240) and low (weak strain, 18084) extracellular phospholipase activities. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. A complementary DNA fragment seen distinctly in the potent phospholipase producing strain was highly homologous to the yeast translation initiation factor (TIF). The selected DNA fragment was then used as a probe to isolate its corresponding complementary DNA clone from a library of C. albicans genomic DNA. The sequence of isolated gene revealed an open reading frame of 1194 nucleotides with the potential to encode a protein of 397 amino acids with a predicted molecular weight of 43 kDa. Over its entire length, the amino acid sequence showed strong homology (78-89%) to Saccharomyces cerevisiae TIF and (63-80%) to mouse eIF-4A proteins. Therefore, our C. albicans gene was identified to be TIF (Ca TIF). Northern blot analysis in the two strains of C. albicans revealed that Ca TIF expression is 1.5-fold higher in the potent phospholipase producing strain. The restriction endonuclease digestion of genomic DNA from this potent strain revealed at least two hybridized bands in Southern blot analysis, suggesting two or more closely related sequences in the C. albicans genome.
- Si K, Das K, Maitra U
- Characterization of multiple mRNAs that encode mammalian translation initiation factor 5 (eIF-5).
- J Biol Chem. 1996; 271: 16934-8
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Eukaryotic translation initiation factor 5 (eIF-5) interacts with the 40 S initiation complex (40S.mRNA.MettRNAf.eIF-2.GTP) to promote the hydrolysis of bound GTP with the concomitant joining of the 60 S ribosomal subunit to the 40 S initiation complex to form a functional 80 S initiation complex. In this paper, the multiple mRNAs that encode mammalian eIF-5 have been characterized. In rat tissues, three major eIF-5 mRNAs of 3.5, 2.8, and 2.2 kilobases in length are detected. All major eIF-5 mRNAs are initiated from a single transcription initiation site, contain identical 5'-untranslated and coding regions, but differ from one another only in the length of their 3'-untranslated regions. The different lengths of the 3'-untranslated region of eIF-5 mRNAs are generated by the use of alternative polyadenylation signals. Additionally, we demonstrate tissue-specific variations in eIF-5 mRNA expression as well as preference for polyadenylation sites. These results should lead to increased understanding of the regulation of eIF-5 gene expression.
- Chang AC, Dunham MA, Jeffrey KJ, Reddel RR
- Molecular cloning and characterization of mouse stanniocalcin cDNA.
- Mol Cell Endocrinol. 1996; 124: 185-7
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In bony fish, stanniocalcin is a glycoprotein hormone thought to be an important regulator of calcium uptake from the aquatic environment. Although stanniocalcin was previously thought to be unique to fish, recent evidence has indicated the existence of a human homologue. To facilitate studies of the function of stanniocalcin in mammals, we have now isolated the mouse stanniocalcin cDNA. This cDNA encodes a predicted protein of the same length as its human counterpart and with a high level of similarity (238/247) amino acids are identical, and five represent conservative changes). As in human, the mRNA is expressed in many mouse tissues, suggesting that mammalian stanniocalcin has a paracrine rather than endocrine role.
- Tenhunen K, Laan M, Manninen T, Palotie A, Peltonen L, Jalanko A
- Molecular cloning, chromosomal assignment, and expression of the mouse aspartylglucosaminidase gene.
- Genomics. 1995; 30: 244-50
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Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to human lysosomal storage disease aspartylglucosaminuria. Here, we describe isolation, chromosomal location, genomic structure, and tissue-specific expression of the mouse Aga gene as well as the intracellular processing of the mouse Aga polypeptide and compare these characteristics to human AGA. The mouse Aga gene was localized to the central area of the B region of chromosome 8, which represents the synteny group in the human chromosome 4q telomeric region where the human AGA gene is located. The mouse gene spans an 11-kb genomic region and contains nine exons and eight introns, which is analogous to the human gene. Furthermore, the exon-intron boundaries of the mouse and human genes are identically positioned. The nucleotide sequence identity of the cDNA and deduced amino acid sequence identity of the protein are 84.4 and 82.4%, respectively. However, the mouse Aga cDNA contains untranslated regions that are shorter than those in the human cDNA, and only one 1.2-kb mRNA transcript is produced in mouse versus two transcripts in human. Expression of the mouse Aga cDNA in COS-1 cells showed that the mouse Aga polypeptide was processed similarly to the human counterpart.
- Brander KA, Kuhlemeier C
- A pollen-specific DEAD-box protein related to translation initiation factor eIF-4A from tobacco.
- Plant Mol Biol. 1995; 27: 637-49
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A pollen-specific sequence, NeIF-4A8, has been isolated from a cDNA library from mature pollen of Nicotiana tabacum cv. Samsun. NeIF-4A8 is a full-length cDNA whose deduced amino acid sequence exhibits high homology to the eucaryotic translation initiation factor eIF-4A from mouse, Drosophila and tobacco. eIF-4A is an RNA helicase which belongs to the supergene family of DEAD-box proteins. Northern blot analysis with a gene-specific probe showed strict anther-specific expression of NeIF-4A8 starting at microspore mitosis. With antibodies raised against tobacco eIF-4A the presence of abundant eIF-4A-related proteins in developing anthers and pollen grains was demonstrated. The genomic analysis shows that the coding region is split by three introns whereas a large, fourth intron is situated in the 5'-untranslated region. A promoter construct with 2137 bp of upstream sequence fused to the GUS reporter gene was used to confirm that the expression is confined to the haploid cells within the anther. NeIF-4A8 is a prime candidate formediating translational control in the developing male gametophyte.
- Miyagi Y et al.
- Abundant expression of translation initiation factor EIF-4E in post-meiotic germ cells of the rat testis.
- Lab Invest. 1995; 73: 890-8
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BACKGROUND: Eukaryotic translation initiation factor EIF-4E is a key component in the regulation of translational efficiency of MRNAs and its increased expression may accelerate cell growth and division. EIF-4E has the potential to transform rat embryo fibroblast cells by cooperation with v-Myc or adenovirus E1A. Our previous study showed that a variety of tumor cells examined exhibited elevated levels of eIF-4E gene expression. Thus it is thought that overexpression of EIF-4E can result in aberrant growth and cell transformation. EXPERIMENTAL DESIGN: To characterize rat EIF-4E, a clone containing rat eIF-4E cDNA was isolated from a rat testis cDNA library by screening with a synthetic probe prepared by the reverse transcription-polymerase chain reaction (RT-PCR) method. Based on the conserved nucleotide sequences between mouse and human eIF-4E cDNA, two oligonucleotide primers for RT-PCR were synthesized chemically. The cloned cDNA was sequenced and used as a probe for analysis of the expression level of eIF-4E mRNA in normal, differentiated rat tissues by Northern blot analysis. In situ hybridization analysis with digoxigenin-labeled antisense eIF-4E RNA as a probe was carried out to identify the eIF-4E-expressing sites in rat tissues. In addition, to analyse the phosphorylation level of EIF-4E, the proteins were fractionated from rat tissues by affinity column chromatography followed by isoelectric focusing (IEF)-gel electrophoresis. RESULTS: The nucleotide sequence of the eIF-4E cDNA is highly conserved in human, rat, and mouse. Extraordinarily elevated expression, more than 50-fold compared with that in the adult rat prostate, of eIF-4E was observed in testicular germ cells of rats of reproductive age, which was much greater than that in any tumor cell lines examined so far. The amount of EIF-4E fractionated from the adult rat testis was approximately 10 times higher than that from the adult rat liver. At least half of the purified testicular EIF-4E proteins were phosphorylated, a ratio similar to that in other rat tissues such as liver. In situ hybridization analysis demonstrated that elevated expression of eIF-4E mRNA was mainly observed in post-meiotic germ cells. CONCLUSIONS: Full activity of EIF-4E in translation requires the phosphorylation of the protein on a specific serine residue. Thus the elevated level of EIF-4E observed in the adult rat testis should be reflected in increase of the functional activity of EIF-4E. Based on the results of in situ hybridization analysis and characterization of EIF-4E, it was concluded that abundant EIF-4E in the testis may play an important role in spermatogenesis through translational regulation of stage-specific mRNAs during germ cell development.
- Wakiyama M, Saigoh M, Shiokawa K, Miura K
- mRNA encoding the translation initiation factor eIF-4E is expressed early in Xenopus embryogenesis.
- FEBS Lett. 1995; 360: 191-3
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The translation initiation factor eIF-4E plays an important role in regulating the overall rate of translation in eukaryotic cells. To investigate the expression of eIF-4E itself, we characterized the eIF-4E mRNA expressed in Xenopus embryos. 5'-RACE was performed to determine the 5'-end of the mRNA and the result predicts isoforms differing at the amino-terminal end. Expression of the eIF-4E mRNA in Xenopus oocytes and embryos was examined by RT-PCR. Xenopus eIF-4E mRNA is produced during oogenesis and persists during the early stages of embryogenesis as a maternal mRNA.
- Ma L, Spremulli LL
- Cloning and sequence analysis of the human mitochondrial translational initiation factor 2 cDNA.
- J Biol Chem. 1995; 270: 1859-65
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Complete cDNAs encoding human mitochondrial translational initiation factor 2 (IF-2mt) have been obtained from liver, heart, and fetal brain cDNA libraries. These cDNAs have a long open reading frame 2181 residues in length encoding a protein of 727 amino acids. Overall, human IF-2mt has 30-40% identity to the corresponding prokaryotic factors. Surprisingly, it is no more homologous to yeast IF-2mt than to the IF-2s from bacterial sources. The greatest region of conservation lies in the G-domain of this factor with less conservation in the COOH-terminal half of the protein and very little homology near the amino terminus. The 5'-untranslated leaders of the liver and heart cDNAs contain a number of short open reading frames. These sequences may play a role in the translational activity of the IF-2mt mRNA. Northern analysis indicates that the IF-2mt gene is expressed in all tissues but that the level of expression varies over a wide range.
- Deiss LP, Feinstein E, Berissi H, Cohen O, Kimchi A
- Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death.
- Genes Dev. 1995; 9: 15-30
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Programmed cell death is often triggered by the interaction of some cytokines with their cell surface receptors. Here, we report that gamma interferon (IFN-gamma) induced in HeLa cells a type of cell death that had cytological characteristics of programmed cell death. In this system we have identified two novel genes whose expression was indispensable for the execution of this type of cell death. The rescue was based on positive growth selection of cells after transfection with antisense cDNA expression libraries. The antisense RNA-mediated inactivation of the two novel genes protected the cells from the IFN-gamma-induced cell death but not from the cytostatic effects of the cytokine or from a necrotic type of cell death. One of those genes (DAP-1) is expressed as a single 2.4-kb mRNA that codes for a basic, proline-rich, 15-kD protein. The second is transcribed into a single 6.3-kb mRNA and codes for a unique 160-kD calmodulin-dependent serine/threonine kinase (DAP kinase) that carries eight ankyrin repeats. The expression levels of the two DAP proteins were selectively reduced by the corresponding antisense RNAs. Altogether, it is suggested that these two novel genes are candidates for positive mediators of programmed cell death that is induced by IFN-gamma.
- Ma J, Farwell MA, Burkhart WA, Spremulli LL
- Cloning and sequence analysis of the cDNA for bovine mitochondrial translational initiation factor 2.
- Biochim Biophys Acta. 1995; 1261: 321-4
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The complete sequence of the cDNA encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained by library screening followed by 3'-RACE PCR. The open reading frame for bovine IF-2mt encodes a protein of 727 amino acids. The sequence of bovine IF-2mt exhibits 85% identity to human IF-2mt, but only 38% identity to yeast IF-2mt and 39% identity to Escherichia coli IF-2 alpha.
- Kasperaitis MA, Voorma HO, Thomas AA
- The amino acid sequence of eukaryotic translation initiation factor 1 and its similarity to yeast initiation factor SUI1.
- FEBS Lett. 1995; 365: 47-50
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Eukaryotic initiation factor eIF-1 was purified from rabbit reticulocytes. Amino acid sequence analysis revealed that the protein contained a blocked amino-terminus. After cleavage with the endoproteinase Asp-N, three peptides were sequenced. The obtained partial sequences were identical to sequences of SUI1ISO1, the human homologue of the yeast translation initiation factor SUI1. The SUI1 gene product was identified as a protein involved in the recognition of the protein synthesis initiation codon. A similar mode of action has been suggested for eIF-1.
- Kissil JL, Deiss LP, Bayewitch M, Raveh T, Khaspekov G, Kimchi A
- Isolation of DAP3, a novel mediator of interferon-gamma-induced cell death.
- J Biol Chem. 1995; 270: 27932-6
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Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.
- Edery I, Chu LL, Sonenberg N, Pelletier J
- An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture).
- Mol Cell Biol. 1995; 15: 3363-71
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The ability to generate cDNA libraries is one of the most fundamental procedures in contemporary molecular biology. One of the major drawbacks of current methods is that most cDNAs present in any given library are incomplete, rendering the characterization of genes an inefficient and time-consuming task. We have developed an affinity selection procedure using a fusion protein containing the murine cap-binding protein (eukaryotic initiation factor 4E), coupled to a solid support matrix, that allows for the purification of mRNAs via the 5' cap structure. When combined with a single-strand-specific RNase digestion step, specific retention of complete cDNA-RNA duplexes following first-strand synthesis is achieved. This method can be used to generate cDNA libraries in which polyadenylated and nonpolyadenylated mRNAs are equally represented and to enrich for full-length or 5'-end clones, thus facilitating cDNA cloning and promoter mapping.
- Koettnitz K, Kappel B, Baumruker T, Hauber J, Bevec D
- The genomic structure encoding human initiation factor eIF-5A.
- Gene. 1994; 144: 249-52
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A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additional eIF-5A-related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A. The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.
- Owttrim GW, Mandel T, Trachsel H, Thomas AA, Kuhlemeier C
- Characterization of the tobacco eIF-4A gene family.
- Plant Mol Biol. 1994; 26: 1747-57
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Characterization of cDNAs encoding eukaryotic translation initiation factor 4A (eIF-4A) indicates the expression of a minimum of ten related genes in tobacco leaf cells. The ten groups fall into two gene families, NeIF-4A2 and NeIF-4A3. The majority of the cDNAs exhibit significant sequence similarity to the NeIF-4A2 family at both the DNA and deduced amino acid levels. Northern analysis using specific probes indicates variable expression of four family members in various tobacco organs. Western analysis, using an anti-tobacco eIF-4A polyclonal antibody, reveals a complex pattern of immunologically related polypeptides of approximately 46 kDa. Subcellular fractionation suggests that at least one eIF-4A-related polypeptide is located in the chloroplast where it is ribosome-associated.
- Dever TE, Wei CL, Benkowski LA, Browning K, Merrick WC, Hershey JW
- Determination of the amino acid sequence of rabbit, human, and wheat germ protein synthesis factor eIF-4C by cloning and chemical sequencing.
- J Biol Chem. 1994; 269: 3212-8
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The small eukaryotic initiation factor (eIF)-4C is implicated in the initiation pathway, where it enhances ribosome dissociation into subunits and stabilizes the binding of the initiator Met-tRNA(i) to 40 S ribosomal subunits. In order to elucidate the function of eIF-4C, its structure has been further characterized. The amino acid sequence of many peptides from rabbit reticulocyte and wheat germ eIF-4C have been determined chemically. From the chemical sequencing of the rabbit protein, it was noted that at least two different eIF-4C molecules were present which differed by conservative substitutions at three positions (2 aspartic acid for glutamic acid switches and 1 valine for isoleucine switch). By the use of unique sequences with low codon degeneracy, primers were used to obtain a polymerase chain reaction product of appropriate size and sequence. This product was then used to isolate full-length coding sequence cDNA clones for human eIF-4C. A similar strategy was used to design PCR primers and then isolate a wheat cDNA clone which lacked the coding region for the first 23 amino acids, but contained a complete 3'-untranslated region. The protein amino acid sequence of wheat germ eIF-4C is 68% identical with the mammalian protein, and, allowing for the most conservative substitutions, the proteins are 76% similar. Both the mammalian and wheat germ proteins are 143 amino acids in length and have molecular weights of about 16,400. A unique feature of eIF-4C is its apparent "polarity" as 9 of the first 15 amino acids are basic while 13 of the last 20 amino acids are acidic. This dipole nature may enable the protein to interact with both the ribosome (perhaps via the rRNA) and other translation initiation factors.
- Nakashima T et al.
- Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells.
- Mol Cell Biol. 1993; 13: 6367-74
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The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.
- Das K, Chevesich J, Maitra U
- Molecular cloning and expression of cDNA for mammalian translation initiation factor 5.
- Proc Natl Acad Sci U S A. 1993; 90: 3058-62
- Display abstract
Eukaryotic translation initiation factor 5 (eIF-5) catalyzes the hydrolysis of GTP bound to the 40S ribosomal initiation complex (40S.AUG.Met-tRNAf-eIF-2.GTP) with the subsequent joining of a 60S ribosomal subunit resulting in the formation of a functional 80S initiation complex. A rat cDNA that encodes eIF-5 has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. The 3.55-kb cDNA encodes a protein of 429 amino acids (calculated M(r) 48,926) with properties that are similar to eIF-5 isolated from rabbit reticulocyte lysates. The deduced amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.
- Metz AM, Browning KS
- Sequence of a cDNA encoding wheat eukaryotic protein synthesis initiation factor 4A.
- Gene. 1993; 131: 299-300
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A cDNA encoding wheat translation initiation factor 4A (eIF-4A) was isolated from a wheat cDNA library and sequenced. The deduced wheat eIF-4A amino acid sequence from the cDNA is compared to eIF-4A from Arabidopsis thaliana, tobacco, mouse and Saccharomyces cerevisiae. Putative RNA helicase motifs, and putative ATP-binding and hydrolysis sites are identified.
- Chakravarti D, Maitra U
- Eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae. Cloning, characterization, and expression of the gene encoding the 45,346-Da protein.
- J Biol Chem. 1993; 268: 10524-33
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Eukaryotic translation initiation factor 5 (eIF-5) catalyzes hydrolysis of GTP bound to a 40 S ribosomal initiation complex with the subsequent joining of a 60 S ribosomal subunit to form an 80 S initiation complex. The yeast gene that encodes eIF-5, designated TIF5, has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. TIF5 is a single-copy gene that maps on yeast chromosome XVI and is essential for cell viability. The gene contains an intron-free open reading frame that encodes a protein of calculated M(r) 45,346 in close agreement with the apparent molecular weight of eIF-5 isolated from yeast cells. Sequence analysis of the gene reveals several interesting features. First, the presence of two in-frame translational start sites located 51 base pairs apart suggests the possibility that two proteins, differing by an amino-terminal extension of 17 amino acids, could be generated from the TIF5 gene via differential translational starts. This would explain the presence, in yeast cell lysates, of two forms of eIF-5 differing in molecular weight by about 2,000. Second, the predicted amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.
- Jaramillo M, Pelletier J, Edery I, Nielsen PJ, Sonenberg N
- Multiple mRNAs encode the murine translation initiation factor eIF-4E.
- J Biol Chem. 1991; 266: 10446-51
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All eukaryotic cellular mRNAs (except organellar) possess at their 5' end the structure m7GpppX (where X is any nucleotide) termed the "cap." The cap structure facilitates the melting of mRNA 5' secondary structure through the action of initiation factor-4F (eIF-4F) in conjunction with eIF-4B. eIF-4F consists of three subunits of which one, eIF-4E (eIF-4E has recently been designated eIF-4 alpha according to the Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (Safer, B. (1989) Eur. J. Biochem. 186, 1-3)), contains the cap binding site. Several lines of evidence suggest that eIF-4E regulates the rate of translation initiation. Consequently, changes in cellular eIF-4E levels could control growth and differentiation. To investigate the possibility that eIF-4E expression is regulated, we studied the pattern of eIF-4E expression in several cell lines. Here, we show the existence of multiple mRNAs for eIF-4E that are generated by differential polyadenylation. In addition, we show tissue-specific differences in eIF-4E mRNA expression and utilization of polyadenylation sites.
- Milburn SC, Hershey JW, Davies MV, Kelleher K, Kaufman RJ
- Cloning and expression of eukaryotic initiation factor 4B cDNA: sequence determination identifies a common RNA recognition motif.
- EMBO J. 1990; 9: 2783-90
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Eukaryotic protein synthesis initiation factor 4B (eIF-4B) is an 80,000 dalton polypeptide which is essential for the binding of mRNA to ribosomes. A highly purified preparation of eIF-4B from HeLa cells was subjected to enzymatic cleavage and amino-terminal amino acid sequence analysis. Degenerate oligonucleotide probes were used to isolate a 3851 bp cDNA encoding eIF-4B from a human cDNA library. The DNA encodes a protein comprising 611 residues with a mass of 69,843 daltons. The amino-terminal domain of eIF-4B contains a consensus RNA binding domain present in a number of other RNA binding proteins. Expression of eIF-4B in transfected COS-1 cells yielded a polypeptide which reacted with anti-eIF-4B antiserum and comigrated with purified eIF-4B. Expression of eIF-4B in COS-1 cells resulted in a general inhibition of translation, possibly due to a 50-fold eIF-4B overproduction.