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Results for “Codon, Initiator”

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PubMed1986

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.

Animals↗

PubMed2000

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus.

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.

5' Untranslated Regions↗

PubMed1996

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.

Animals↗

PubMed1999

Tandem duplication polymorphism upstream of the dopamine D4 receptor gene (DRD4).

The dopamine D4 receptor (DRD4) is a member of the D2-like dopamine receptor family. Polymorphisms at the DRD4 gene have been examined for association with a wide range of neuropsychiatric disorders and normal behavioral variation. The DRD4 gene is unusual in its high amount of expressed polymorphism in humans. Here we study the identification of a polymorphic tandem duplication of 120 bp located 1.2 kb upstream of the initiation codon. The duplicated region contains consensus sequences of binding sites for several known transcription factors, suggesting that different alleles may differ in their transcriptional activity. Because chimpanzees, gorillas, and orangutans lack the duplication, the duplicated allele is inferred to be derived. The frequency of this derived duplication allele ranges from 0.40-0.81 in the 11 populations from around the world typed for this polymorphism. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:705-709, 1999.

Alleles↗

PubMed1997

Juvenile-onset spinal muscular atrophy caused by compound heterozygosity for mutations in the HEXA gene.

Progressive proximal muscle weakness is present both in spinal muscular atrophy (SMA) type III (Kugelberg-Welander disease) and in GM2 gangliosidosis, diseases that segregate in an autosomal recessive fashion. The SMN gene for SMA and the HEXA gene for GM2 gangliosidosis were investigated in a woman with progressive proximal muscle weakness, long believed to be SMA type III (Kugelberg-Welander type). She and her family underwent biochemical studies for GM2 gangliosidosis. Analysis of SMN excluded SMA. Biochemical studies on GM2 gangliosidosis showed deficiency in hexosaminidase A activity and increased GM2 ganglioside accumulation in the patient's fibroblasts. The HEXA gene was first analyzed for the Gly269-->Ser mutation characteristic for adult GM2 gangliosidosis. Since the patient was carrying the adult mutation heterozygously, all 14 exons and adjacent intron sequences were analyzed. A novel mutation in exon 1 resulting in an A-to-T change in the initiation codon (ATG to TTG) was identified. The adult patient is a compound heterozygote, with each allele containing a different mutation. Although mRNA was transcribed from the novel mutant allele, expression experiments showed no enzyme activity, suggesting that neither the TTG nor an alternative codon serve as an initiation codon in the HEXA gene.

Adult↗

PubMed1998

Reinvestigation of the functions of the hydrophobic segment of Jem1p, a yeast endoplasmic reticulum membrane protein mediating nuclear fusion.

Jem1p is a DnaJ-like protein of the yeast ER membrane, which is required for nuclear membrane fusion during mating. We have determined the position of translation initiation codon for the JEM1 gene. Translation of Jem1p starts from the second ATG codon of the previously assumed JEM1 open reading frame, leading to the synthesis of a precursor protein of 645 amino acids long. The translated Jem1p precursor contains an N-terminal hydrophobic sequence that functions as a signal sequence and is removed upon import into the ER lumen. Jem1p is peripherally associated with the ER membrane probably through protein-protein interactions.

Cell Compartmentation↗

PubMed2000

Epsilon as an initiator of translation of CAT mRNA in Escherichia coli.

Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.

Base Sequence↗

PubMed2000

Prokaryotic expression, purification, and reconstitution of biological activities (Antiprotease, antitumor, and heparin-binding) for tissue factor pathway inhibitor-2.

We report the expression of tissue factor pathway inhibitor-2 (TFPI-2) (also known as PP-5, placental protein-5; MSPI, matrix-associated serine protease inhibitor) in E. coli as a 25-kDa nonglycosylated protein with a glycine substituted for aspartic acid at the amino terminus. High-level expression of TFPI-2 was obtained with pRE1 expression vector under the transcriptional and translational controls of the lambdaP(L) promoter and lambdacII ribosome-binding site, respectively, with ATG initiation codon. TFPI-2 was produced as inclusion bodies and accounted for 25-30% of the total E. coli proteins. The inclusion bodies containing TFPI-2 were solubilized with urea, sulfitolyzed, purified, and refolded through a disulfide interchange reaction. The refolded E. coli TFPI-2 inhibited plasmin with an inhibition constant (K(i)) of 5 nM that is similar with the TFPI-2 expressed in a mammalian system. The refolded E. coli TFPI-2 bound heparin and also inhibited plasmin, regardless of whether the enzyme was in the fluid phase or was bound to the membranes of HT-1080 fibrosarcoma cells. In addition, refolded E. coli TFPI-2 inhibited radiolabeled matrix degradation and Matrigel matrix invasion by HT-1080 fibrosarcoma cells and B16-F10 melanoma cells. Together, our results suggest that glycosylation is not essential for antiprotease, antitumor, and matrix-binding activities of TFPI-2. Based on these collective data, we conclude that a biologically active nonglycosylated TFPI-2 can be produced in E. coli and that the protein can be produced in high-enough quantities to conduct in vivo studies for determination of the role of this inhibitor in tumor invasion and metastasis.

Amino Acid Substitution↗

PubMed1997

Analysis of the 5' flanking region of the human galactocerebrosidase (GALC) gene.

Galactocerebrosidase (GALC) is the lysosomal enzyme deficient in human and certain animal species with globoid cell leukodystrophy (GLD) or Krabbe disease. It catalyzes the hydrolysis of specific galactolipids including galactosylceramide and psychosine. The GALC protein is found in very low amounts in all tissues, which delayed its purification and the subsequent cloning of its cDNA and gene. We previously published the exon-intron organization of the human gene, but did not functionally analyze the 5' flanking region. We now provide a description of this GC-rich region which includes one potential YY1 element and one potential SP1 binding site. There are 13 GGC trinucleotides within the first 150 bp preceding the initiation codon. The 5' end of intron 1 contains six potential Sp1 binding sites, one AP1 binding site, and eight AP2 binding sites. A construct containing nucleotides -176 to -24 had the strongest promoter activity using a vector containing the chloramphenicol acetyltransferase reporter gene. We also provide evidence for the presence of inhibitory sequences located immediately upstream of the promoter region, and within the first 234 nucleotides of intron 1. These elements together with a suboptimal nucleotide at position +4 may explain the low level of GALC protein in all cell types.

Cloning, Molecular↗

PubMed1998

Structural organization and sequence of CLN2, the defective gene in classical late infantile neuronal ceroid lipofuscinosis.

Mutations in the CLN2 gene result in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disease. In this report, we present the complete sequence of the human CLN2 gene and define its physical relationship with two other genes that have been previously mapped to chromosome 11p15. The CLN2 gene consists of 13 exons and 12 introns and spans 6.65 kb. By S1 mapping and primer extension, the 5'-terminus of the CLN2 mRNA was mapped to 32 nucleotides upstream of the proposed initiation codon. A number of other elements were found to be located in close proximity to CLN2, including the gene encoding transcription factor TAFII30, the gene encoding intregrin-linked kinase, and an approximately 914-bp fragment that is 82% identical to antithrombin III. In addition, an EST cDNA clone that is transcribed on the strand opposite to CLN2 and that overlaps a portion of the CLN2 gene was identified. Finally, a set of primer pairs are presented for the amplification of the coding sequences, putative promoter, and splice junctions of the CLN2 gene. Taken together, this information will facilitate the molecular analysis of and genetic testing for classical LINCL.

Amino Acid Sequence↗

PubMed1998

The mouse tumor necrosis factor receptor 2 gene: genomic structure and characterization of the two transcripts.

The mouse TNFR2 gene has been cloned, sequenced, and characterized as a gene spanning >44 kb of the genome. By alignment of five genomic clones we have established that TNFR2 consists of 10 exons and 9 introns with exons ranging in size from 35 bp to 2.6 kb and introns ranging from 322 bp to >16 kb. All splice acceptor and donor sites conform to the canonical AG/GT rule. The translation initiation and termination sites are located in exon 1 and 10, respectively. Although TNFR2 lacks a canonical TATA box, the gene is transcribed from a unique start site located 70 bp upstream of the ATG initiation codon that conforms to the consensus Inr motif. Several cis-elements for transcription factors were identified in the 5' flanking region, including NF-1, Sp-1, AP2, gamma-IRE, and NF-kappaBeta motifs. Functional analysis indicates that the region -705/-412 contains a negative cis-acting element and that the minimal promoter contains motifs that confer LPS inducibility. Two mouse TNFR2 mRNAs of 3.2 and 4.1 kb are detected by Northern blot analysis, but until now their origin has not been explained. No evidence of alternative splicing of the coding exons was found. However, hybridization studies and amplification of cDNA ends suggest the use of a noncanonical polyadenylation signal in the untranslated region of exon 10. A comparative analysis of the 3' untranslated regions of the human and mouse TNFR2 genes shows highly divergent 3' ends. The possibility of an ancestral mouse TNFR2 mRNA similar to the short transcript is discussed.

Amino Acid Sequence↗

PubMed1998

The truncated form of the Epstein-Barr virus LMP-1 is dispensable or complimentable by the full-length form in virus infection and replication.

The Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1) gene of the Akata virus strain was cloned, and its nucleotide sequence was determined. Compared with the B95-8 strain, the translation initiation codon for the truncated LMP-1 gene, which is expressed in the lytic cycle, was lost. Immunoblotting showed that Akata EBV produces no truncated LMP-1 protein in any state and that the full-length LMP-1 protein is expressed at a significant level during lytic infection. The results suggest that the truncated LMP-1 protein is dispensable for EBV infection and replication or that the full-length form can "functionally" complement the truncated form if the truncated form has a function.

Base Sequence↗

PubMed1999

Internal initiation of translation of bovine viral diarrhea virus RNA.

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon.

Animals↗

PubMed2002

The product of the UL12.5 gene of herpes simplex virus type 1 is not essential for lytic viral growth and is not specifically associated with capsids.

The herpes simplex virus type 1 UL12 gene encodes a pH-dependent deoxyribonuclease termed alkaline nuclease. An N-terminally truncated version of the UL12 gene, called UL12.5, was shown to be translated independently from a subgenic mRNA which shares its 3' terminus with the full-length UL12 mRNA. We showed previously that the UL12.5 gene product cannot compensate for the absence of the full-length UL12 gene product (R. Martinez, L. Shao, J. C. Bronstein, P. C. Weber, and S. K. Weller, 1996, Virology 215, 152-164); however, it was not known whether UL12.5 itself performs an essential function during lytic viral growth. In this article the initiation codon for the UL12.5 gene product was mapped and altered to create a gene no longer capable of producing UL12.5. This mutation was introduced into the viral genome to create a virus which was capable of producing full-length UL12 but not UL12.5. The growth properties of this virus indicate that UL12.5 is not essential for viral growth in culture. UL12.5 was previously reported to represent a capsid-associated form of alkaline nuclease (J. C. Bronstein, S. K. Weller, and P. C. Weber, 1997, J. Virol. 71, 3039-3047). Sucrose sedimentation analysis of capsids from cells infected with wild-type or mutant viruses indicates that both UL12 and UL12.5 are found in fractions from across the sucrose gradient which do not always correlate with the presence of viral capsids. Furthermore, UL12.5 is found in fractions across the gradient even in cells infected under conditions in which no capsids are formed. These results indicate that UL12.5 does not specifically associate with viral capsids. Taken together, these data indicate that UL12.5 is not likely to play an important role in lytic viral infection.

Animals↗

PubMed1996

Cloning and analysis of the gene for cystathionine gamma-synthase from Arabidopsis thaliana.

A cDNA clone, CGS1, encoding cystathionine gamma-synthase (CGS) from Arabidopsis thaliana was selected by complementation of CGS mutant strain of Escherichia coli (metB). Cells expressing CGS1 can grow on medium lacking Met and contain CGS enzyme activity. Genomic DNA blot analysis of A. thaliana revealed that there is a single gene homologous with CGS1. A genomic fragment carrying CGS1 was cloned and sequenced. Through combined analysis of the cDNA and genomic clone it was determined that the CGS1 coding sequence is 1692 bp, encodes a 563 amino acid, 60 kDa protein, and is interrupted by ten introns. A transcriptional initiation site was detected 260 bp 5' of the initiator codon. The predicted amino acid sequence of CGS1 contains a consensus pyridoxal phosphate-binding site and is similar to MetB of E. coli, with which it is 35 percent identical. The CGS1 product has a sequence at the amino terminus that resembles a transit peptide for localization to plastids. At least 160 amino acids from the amino terminus of the CGS1 enzyme are not essential for enzymatic activity.

Amino Acid Sequence↗

PubMed1997

Polyketide synthase gene pksM from Aspergillus terreus expressed during growth phase.

The polyketide synthase gene pksM was detected in the genomic DNA library of Aspergillus terreus by hybridization with the 6-methylsalicylic acid synthase (6-MSAS) gene of Penicillium patulum as a probe. 9524 bp of the cloned DNA were sequenced and a 5.5 kb open reading frame was revealed. A single intron (62 bp) was identified in the conserved position. Two transcription start points were determined within the 5'-flanking region at 50 and 72 (major) bp upstream from the putative translation initiation codon ATG. The conserved active site motifs for ketosynthase, acyltransferase, dehydratase, ketoreductase and acyl carrier protein were found within the predicted polypeptide consisting of 1803 amino acids. Unlike the P. patulum 6-MSAS gene, the transcription of pksM from A. terreus was observed in the middle of the vegetative growth phase.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed2001

Sialyl Lewis(x)-liposomes as vehicles for site-directed, E-selectin-mediated drug transfer into activated endothelial cells.

E-selectin, exclusively expressed on activated endothelial cells, is a potential target for site-directed delivery of agents. We and others have shown that sialyl LewisX-liposomes (sLe(x)-liposomes) are recognized by E-selectin. We now report an approach employing sLe(x)-liposomes to deliver antisense oligonucleotides (AS-ODNs) directed against the adhesion molecule ICAM-1 to activated vascular endothelial cells. ICAM-1 expression was analyzed at the protein level by immunofluorescence and a cell surface ELISA, and at the RNA level by RT-PCR. We have investigated two different AS-ODNs complementary to the 3' untranslated region and the AUG translation initiation codon of ICAM-1 mRNA. Both inhibited protein expression, but did not influence the mRNA level, pointing to a hybridization of AS-ODNs with the mRNA in the cytoplasm. Our results demonstrate the feasibility of a novel approach for the delivery of agents to activated endothelial cells by glycoliposomes targeted to E-selectin.

Cells, Cultured↗

PubMed2000

The broad bean nodulin VfENOD18 is a member of a novel family of plant proteins with homologies to the bacterial MJ0577 superfamily.

Full-length transcript sequences were isolated from broad bean root nodules, which encode a novel nodulin designated VfENOD18. The corresponding transcripts were detected in early and in late stages of nodule development and were localized exclusively in the nitrogen-fixing zone III. The VfENOD18 sequence is not only homologous to a number of ESTs from various mono- and dicotyledonous plants, but also to the ATP-binding protein MJ0577 from Methanococcus jannaschii and to a range of bacterial proteins that belong to the MJ0577 superfamily. Hence, VfENOD18 is a member of a ubiquitous family of plant proteins that might function as ATP-binding proteins or ATPases. On the genomic level, VfENOD18 genes can be divided into two groups on the basis of differences in their 5' UTRs. One group lacks the 5' UTR region including the ATG initiation codon, whereas the second group contained the complete 5' UTR region. Further upstream of this VfENOD18 gene, a retrotransposon sequence was identified. The -14/-964 VfENOD18 promoter fragment was devoid of complete organ-specific elements known from other nodulin gene promoters. Nevertheless, this region was able to mediate full promoter activity in the central region of transgenic Vicia hirsuta root nodules.

5' Untranslated Regions↗