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PubMed1994

The dsg gene of Myxococcus xanthus encodes a protein similar to translation initiation factor IF3.

The dsg mutants of Myxococcus xanthus are defective in fruiting body development and sporulation, yet they grow normally. The deduced amino acid sequence of the dsg gene product is 50 and 51% identical to the amino acid sequence of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively. However, the Dsg protein has a carboxy-terminal extension of 66 amino acids, which are absent from its E. coli and B. stearothermophilus homologs. The Shine-Dalgarno sequence GGAGG and 5 bases further upstream are identical in M. xanthus and several enteric bacteria, despite the wide phylogenetic gap between these species. The infC gene, which encodes IF3 in enteric bacteria, starts with the atypical translation initiation codon AUU, which is known to be important for regulating the cellular level of IF3 in E. coli. Translation of the Dsg protein overexpressed from the M. xanthus dsg gene in E. coli cells initiates at an AUC codon, an atypical initiation codon in the AUU class. The dsg mutants DK429 and DK439 carry the same missense mutation that changes Gly-134 to Glu in a region of amino acid identity.

Amino Acid Sequence↗
PubMed1985

The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts.

In T4-infected cells, the gene 32 monocistronic mRNA is very stable. To study the molecular basis for this stability, we have constructed chimeric plasmids containing the monocistronic promoter and the gene 32 translation initiation sequence fused to either most of the E. coli lac operon or only a segment of the lacZ gene, followed by the gene 32 transcription terminator. The resulting hybrid transcripts are unstable in uninfected cells. In phage-infected cells, however, the hybrid mRNAs are at least as stable as gene 32 mRNA itself. Analysis of other plasmid constructs indicates that the sequences on the gene 32 mRNA from its 5' end to slightly beyond the initiation codon suffice to stabilize these hybrids. Studies with a series of deletions of the gene 32 leader sequence suggest that an RNA sequence near the gene 32 initiation codon is involved. Various models to explain this mRNA stabilization are discussed.

DNA Helicases↗
PubMed1997

Inhibition of Gal beta1, 4GlcNAc alpha2,6 sialyltransferase expression by antisense-oligodeoxynucleotides.

Treatment of human colorectal carcinoma cells HT29 with specific antisense oligodeoxynucleotides led to a decreased Gal beta1,4GlcNAc alpha2,6 sialyltransferase activity on the level of protein expression as well as on the mRNA level. Antisense treatment did not effect cell viability or cell growth. Oligodeoxynucleotides which were complementary to the region upstream of the initiation codon were particularly effective in inhibition of enzyme expression. No such inhibition was found by treatment of cells with oligodeoxynucleotides complementary to the region downstream of the initiation codon or by treatment of cells with scrambled controls or sense oligodeoxynucleotides.

Cell Division↗
PubMed1997

Cleavage of viral RNA and inhibition of viral translation by hepatitis C virus RNA-specific hammerhead ribozyme in vitro.

BACKGROUND/AIMS: A hammerhead ribozyme has been used as a new way to suppress specific gene expression. We designed hammerhead ribozymes directed against hepatitis C virus RNA, and investigated their cleavage efficiency and inhibitory effect on viral translation in vitro. METHODS: Three hammerhead ribozymes bearing different cleavage sites in the core region of hepatitis C virus RNA (genotype 1b) were designed in this study. Ribozymes and the target hepatitis C virus RNA were synthesized by in vitro transcription. The cleavage efficiency was evaluated by the ribozyme cleavage assay. The inhibitory effect of the ribozyme on viral translation was further studied by the viral translation inhibition assay. RESULTS: All ribozymes specifically cleaved the target RNA of 1217 bases at a physiological temperature in a dose-dependent manner, with the specific cleavage increasing with a longer incubation period. The target RNA was cleaved most efficiently by the ribozyme with the cleavage site located nearest to the initiation codon. In the viral translation inhibition assay, all ribozymes showed a significant inhibitory effect on viral translation. The ribozyme with the cleavage site located farthest from the initiation codon blocked viral translation most efficiently, and demonstrated almost 70 to 80% inhibition. For ribozymes with the T7 transcription terminator sequence, both the target RNA cleavage and the inhibition of viral translation tended to be achieved less efficiently by ribozymes with T7 terminator than by those without it. CONCLUSIONS: These findings suggest that ribozyme-mediated hepatitis C virus RNA cleavage may serve as a new strategy in the treatment of hepatitis C virus infection.

Hepacivirus↗
PubMed1988

Alcohol dehydrogenase gene from Alcaligenes eutrophus: subcloning, heterologous expression in Escherichia coli, sequencing, and location of Tn5 insertions.

The nucleotide sequence of the gene that encodes the fermentative, multifunctional alcohol dehydrogenase (ADH) in Alcaligenes eutrophus, and of adjacent regions on a 1.8-kilobase-pair PstI fragment was determined. From the deduced amino acid sequence, a molecular weight of 38,549 was calculated for the ADH subunit. The amino acid sequence reveals homologies from 22.3 to 26.3% with zinc-containing alcohol dehydrogenases from eucaryotic organisms (Schizosaccharomyces pombe, Zea mays, mouse, horse liver, and human liver). Most of the 22 amino acid residues, which are strictly conserved in this group of ADHs (H. Jörnvall, B. Persson, and J. Jeffery, Eur. J. Biochem. 167:195-201, 1987), either were present in the A. eutrophus enzyme or had been substituted by related amino acids. The A. eutrophus adh gene was transcribed in Escherichia coli only under the control of the lac promoter, but was not expressed by its own promoter. A sequence resembling the E. coli consensus promoter DNA sequence did not contain the invariant T, but a G, in the potential -10 region. In the transposon-induced mutants HC1409 and HC1421, which form ADH constitutively, the insertions of Tn5::mob were localized 56 and 66 base pairs, respectively, upstream of the presumptive translation initiation codon. In contrast to the promoter, the A. eutrophus ribosome-binding site with a GGAG Shine-Dalgarno sequence 6 base pairs upstream of the translation initiation codon was accepted by the E. coli translation apparatus. A stable hairpin structure, which may provide a transcription termination signal, is predicted to occur in the mRNA, with its starting point 21 base pairs downstream from the translation termination codon.

Alcaligenes↗
PubMedDate not supplied

Partial characterization of bovine elastin gene; comparison with the gene for human elastin.

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.

Animals↗
PubMed1990

Initiation of in vivo protein synthesis with non-methionine amino acids.

Methionine is the universal amino acid for initiation of protein synthesis in all known organisms. The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase. In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759]. We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine. In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons. These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E. coli protein synthesis by valine and phenylalanine. The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.

Amino Acid Sequence↗
PubMed2002

The N-terminal truncated isoform of SOCS3 translated from an alternative initiation AUG codon under stress conditions is stable due to the lack of a major ubiquitination site, Lys-6.

The suppressor of cytokine signaling-3 (SOCS3/CIS-33/SSI-3) is an important negative regulator of cytokine signaling. Here, we show that an N-terminal truncated isoform (DeltaN-SOCS3) translated from the internal AUG codon 12 was profoundly induced by endoplasmic reticulum (ER) stress- or active double-stranded RNA-activated protein kinase PKR, as a result of induction of eukaryotic initiation factor 2alpha phosphorylation. DeltaN-SOCS3 exhibited a stronger cytokine-inhibitory activity and a higher stability than WT-SOCS3 in Ba/F3 hematopoietic cells. A potential ubiquitination residue, Lys-6, at the N terminus is evolutionary conserved among SOCS3 species. The K6Q-SOCS3 mutant showed a much longer half-life than WT-SOCS3 in Ba/F3 cells. Furthermore, inhibition of the 26 S proteasome pathway increased both ubiquitination and protein levels of WT-SOCS3 but had no effect on K6Q-SOCS3. SOCS3 mutant lacking the carboxyl-terminal SOCS-box exhibited the same stability as K6Q-SOCS3. These observations suggest that the short form of SOCS3 is a naturally occurring stabilized inhibitory protein, whereas WT-SOCS3 is a short-lived protein modulated by Lys-6 ubiquitination and proteasome-dependent degradation. Our findings provide strong evidence for the first time that translational control plays an important role in stabilization and function of SOCS3.

Animals↗
PubMed1991

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.

3T3 Cells↗
PubMed1993

Regulation of c-myb oncogene expression in immature and mature murine T cells.

The c-myb oncogene encodes a nuclear binding protein which may play a major role in differentiation during early T cell development. However, the functionally important transcription regions in the GC promoter site have not been defined and the significance of the regulation of this promoter site in T cell differentiation has not been determined. Therefore, the promoter strength was determined by measurement of the CAT activity in cell extracts of EL-4 cells that were transfected with a CAT expression vector that contained cloned segments of the 5' myb gene. Stepwise removal of DNA sequences between -2300 bp and -346 bp upstream from the ATG initiation codon resulted in a gradual loss of 50% of CAT activity, whereas deletion of DNA sequences from -346 to -295 and -232 to -155 bp upstream from the ATG initiation codon eliminated promoter activity. On analysis of the CAT activity after transfection of various cell lines with these same constructs, it was found that the same two promoter regions were required for high CAT activity in all the cell lines, including murine cell lines which express the alpha/beta TCR and high levels of c-myb (BW5147), the alpha/beta TCR and low levels of c-myb (Yac-1), or the gamma/delta TCR (KN 12.1 and KN 2.4 T), a murine fibroblast T cell line (NIH-3T3), and a human epithelial cell line (HeLa). However, the CAT activity did not correlate with steady state levels of expression of the c-myb gene in the murine cell lines. Our data indicate that the c-myb oncogene promoter is constitutively expressed is highly dependent on a limited region of the 5' myb gene, requires two DNA elements for optimal activity, and is functional in diverse T cell lines.

3T3 Cells↗
PubMed2005

Construction of a bispecific antisense oligonucleotide containing multiple binding sites for the treatment of hormone insensitive prostate tumors.

Antisense oligonucleotides (oligos) have demonstrated efficacy for the treatment of various cancers, infectious diseases and metabolic disorders. While most studies have utilized single oligos either administered alone, or more recently in combination therapy with other drugs, some investigators have administered more than one oligo in a combined administration or have designed oligos which target multiple proteins (those which share mRNA sequence homology). Antisense oligos inhibit mRNA translation through complementary base pair binding, often about the AUG initiation codon. This inhibition is further enhanced through destruction of the mRNA:oligo hybrid by RNAse H. Construction of an oligo with multiple binding sites located about the respective mRNA initiation codons could simultaneously block translation of more than one protein, even those unrelated in sequence. Such binding could produce a complex mRNA:oligo hybrid more prone to degradation and clearance. Furthermore, such a formulation would increase oligo specific activity and cellular uptake, reduce toxicity, and stabilize at 1:1 the ratio between multiple oligo active sites which otherwise must be comparably delivered (in amount) and individually targeted. The activity of these newly constructed oligos could be tested in both in vitro and in vivo prostate tumor models, utilizing the hormone sensitive LNCaP and the hormone insensitive PC-3 lines. In vitro testing would evaluate oligos administered either alone or in combination with other chemotherapeutics. In vivo testing would administer the oligos to tumors carried in athymic nude mice by either intratumoral inoculation or by using a diffusion pump. Antisense oligos which target proteins associated with growth factors or their receptors, could have a role in the treatment of human prostate cancers when administered with hormone deprivation therapy, or against tumors which have already become hormone insensitive. In the later case, such treatment could form the basis of a second tier of therapy based upon growth factor deprivation.

Androgens↗
PubMed2002

Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes.

To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.

Amino Acids↗
PubMed1993

Characterization of the gene for the human high affinity IgE receptor (Fc epsilon RI) alpha-chain.

The Fc epsilon RI couples the mast cell-surface binding of IgE and Ag to a complex series of intracellular events culminating in cell activation and degranulation. The alpha-chain of Fc epsilon RI constitutes the Ig-binding subunit of this heterotetrameric receptor, and is itself a member of the Ig gene superfamily. We have isolated a human genomic DNA clone containing the entire Fc epsilon RI alpha gene, and completely sequenced a region from 1257 bp 5' of the transcription start site, to 513 bp 3' of the last exon of the gene. As with the previously characterized rat and mouse genes, human Fc epsilon RI alpha consists of five exons and four introns, and spans 5889 bp of genomic DNA. The splice donor and acceptor sites deduced by comparison with the cDNA sequence corresponded exactly to the locations found in analogous rodent genes. By mapping the 5' end of Fc epsilon RI alpha transcripts we found three major transcription initiation sites 24, 27, and 29 bp upstream of the ATG translation initiation codon. As well, several longer minor transcripts were seen, with a maximum of 60 nt of 5'-untranslated sequence. About 650 bp of DNA upstream of the ATG translation initiation codon were compared among human, rat, and mouse Fc epsilon RI alpha sequences in search of common motifs that might mediate conserved regulatory interactions with DNA binding proteins. A 172-bp region of the human Fc epsilon RI alpha 5'-flanking sequence was highly conserved in both rodent species. Further studies will be required to determine whether these or other sequences are involved in Fc epsilon RI alpha gene regulation.

Base Sequence↗
PubMed2001

Dual targeting of phage-type RNA polymerase to both mitochondria and plastids is due to alternative translation initiation in single transcripts.

We isolated and sequenced a nuclear gene and cDNA encoding a bacteriophage T7-type RNA polymerase, NsRpoT-B, from Nicotiana sylvestris. The gene, NsRpoT-B, consists of 19 exons and 18 introns and encodes a polypeptide of 1020 amino acid residues. The predicted NsRpoT-B protein shows 71% amino acid identity with NsRpoT-A which is a mitochondrial protein. Quantitative RT-PCR revealed that steady-state NsRpoT-B mRNA accumulation is highest in the mature leaves and lowest in the cotyledons. Transient expression assays in protoplasts from N. sylvestris leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-B encodes dual targeting signals directing the protein into mitochondria and plastids. This strongly suggests that NsRpoT-B functions as an RNA polymerase transcribing genes from two different plant organelle genomes. NsRpoT-B transcripts have two potential translation initiation codons. An in vitro translation assay indicated that a chimeric mRNA encoding the N-terminal NsRpoT-B fused to an sGFP produced two polypeptides translated from the first and second initiation codons. This implies that the dual targeting of NsRpoT-B protein is regulated, in part, at the level of translation. We have designated this protein NsRpoTpm.

Alternative Splicing↗
PubMed1982

Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus.

Several functions of the retrovirus genome involve structural features in the vicinity of its 5' terminus. In an effort to further elucidate the relationship between structure and function in retrovirus RNA, we have determined the sequence of the first 1,010 nucleotides at the 5' end of the genome of Rous sarcoma virus by using the Maxam-Gilbert method to sequence suitable domains in cloned Rous sarcoma virus DNA. The results (i) locate the initiation codon for the gag gene of Rous sarcoma virus 372 nucleotides from the 5' end of viral RNA; (ii) demonstrate that this codon is preceded by three methionine codons that are apparently not used in translation; (iii) sustain previous conclusions that the principal site to which ribosomes bind on the Rous sarcoma virus genome in vitro does not contain the initiation codon for gag; (iv) permit deduction of the amino acid sequence of a viral structural protein, p19; (v) confirm the amino-terminal sequence of Pr76gag; and (vi) substantiate the identification of a splice donor site described in the accompanying manuscript (Hackett et al., J. Virol., 41:527-534, 1982).

Avian Sarcoma Viruses↗
PubMed1996

Structure of the pcbC gene encoding 2,3-dihydroxybiphenyl dioxygenase of Pseudomonas sp. P20.

Pseudomonas sp. P20 is a soil bacterium growing in biphenyl or 4-chlorobiphenyl as the sole carbon and energy source, where the initial catabolism of biphenyl to form benzoate is catalyzed by four enzymes encoded in the pcbABCD gene cluster. The nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene corresponding to the pcbC gene was determined. The pcbC gene was composed of 921 base pairs with an ATG initiation codon and a TGA termination codon, which can encode a polypeptide of molecular mass 34 kDa containing 306 amino acids. A promoter-like sequence and ribosome-binding sequence were identified upstream of the initiation codon. The deduced amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase exhibited 47% identity with that of corresponding enzyme of Pseudomonas sp. DJ-12, and less than 35% identity with those of other extradiol-type dioxygenases.

Amino Acid Sequence↗
PubMed1986

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

Base Sequence↗