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PubMed2001

Biotransformation of phenanthrene and 1-methoxynaphthalene with Streptomyces lividans cells expressing a marine bacterial phenanthrene dioxygenase gene cluster.

The phdABCD gene cluster in a marine bacterium Nocardioides sp. strain KP7 codes for the multicomponent enzyme phenanthrene dioxygenase. phdA encoding an iron-sulfur protein large subunit alpha, phdB encoding its small subunit beta, phdC encoding ferredoxin, and phdD encoding ferredoxin reductase, were replaced in such a way that the termination codons of the preceding open reading frames were overlapped with the initiation codons of the following genes. This manipulated phdABCD gene cluster was positioned downstream of the thiostrepton-inducible promoter PtipA in a high-copy-number vector pIJ6021, and introduced into the gram-positive, soil-inhabiting, filamentous bacterium Streptomyces lividans. The recombinant S. lividans cells converted phenanthrene into a cis-diol form, which was determined to be cis-3,4-dihydroxy-3,4-dihydrophenanthrene by its UV spectral data as well as HPLC property, using the authentic sample for comparison. This biotransformation proceeded very efficiently; 200 microM and 2 mm of phenanthrene were almost completely converted to its cis-diol form in 6 h and 32 h, respectively. In addition, the S. lividans cells carrying the phdABCD gene cluster were found to transform 1-methoxynaphthalene to two products, which were identified to be 8-methoxy-2-naphthol in addition to 8-methoxy-1,2-dihydro-1,2-naphthalenediol by their EI-MS, 1H- and 13C-NMR spectral data.

Actinomycetales↗

PubMed1998

Genetic analysis of the central untranslated genome region and the proximal coding part of the F gene of wild-type and vaccine canine distemper morbilliviruses.

Located between the open reading frames encoding the matrix (M) and the fusion (F) protein the morbillivirus genome contains an unusually large non-coding intercistronic region (M-F UTR) of up to 5.6% of the full length genome. Any function(s) of this region have largely remained obscure. Here, we analyze the M-F UTR and the proximal coding part of the downstream F gene of several recent canine distemper morbillivirus (CDV) wild-type (wt) isolates and vaccine strains. While the F gene coding part appeared to be highly conserved (about 93% homology), a considerable degree of strain-specific variation of up to 21.4% was evident when comparing the M-F UTR. Phylogenetic analysis revealed a co-circulation of several contemporary CDV genotypes within a close geographic range (central Europe). A remarkably distinct CDV wt lineage, so far detected only in mustelids, is displayed. A rather non-scattered pattern of mutations within the M-F UTR suggested superimposition of RNA sequence and/or secondary structure constraints. Extensive folding in the long (460 nt) and moderately GC-rich 5'-UTR of the F mRNA was evident, particularly around the putative F protein translation initiation codon (AUG461 of the Onderstepoort vaccine strain). The region immediately preceding the putative F initiation site also harbored the only mutation unique to both vaccine strains within the F-5'UTR (position 455: Awt vs. Cvac). The putative F protein start codon, AUG461, was found to be mutated to AUA or GUA in all wt isolates analyzed and in another vaccine strain (Rockborn). Possible consequences for F protein translation initiation in wt CDV are discussed.

Animals↗

PubMed1998

The exon structure of the human MAGP-2 gene. Similarity with the MAGP-1 gene is confined to two exons encoding a cysteine-rich region.

A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.

Amino Acid Sequence↗

PubMed1994

The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations.

Mutations in the Saccharomyces cerevisiae sua8 gene were found to be suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene. Analysis of cyc1 transcripts from sua8 mutants revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a site between the aberrant and normal ATG start codons. This effect is not cyc1 gene specific since initiation at other genes, including ADH1, CYC7, and HIS4, was similarly affected, although initiation at HIS3 and SPT15 was unaffected. The SUA8 gene was cloned and partially sequenced, revealing identity to RPB1, which encodes the largest subunit of RNA polymerase II. The sua8 suppressors are the result of single amino acid replacements of highly conserved residues. Three replacements were found either within or immediately preceding homology block D, and a fourth was found adjacent to homology block H, indicating that these regions play a role in defining start sites in vivo. Nearly identical effects on start site selection were observed for sua7 suppressors, which encode altered forms of TFIIB. Synthetic lethality was associated with double sua7 sua8 suppressor mutations, and recessive sua7 mutants failed to fully complement recessive sua8 mutants in heterozygous diploids (nonallelic noncomplementation). These data indicate that the largest subunit of RNA polymerase II and TFIIB are important determinants of transcription start site selection in S. cerevisiae and suggest that this function might be conferred by interaction between these two proteins.

Alleles↗

PubMed1988

Ribosomal initiation from an ACG codon in the Sendai virus P/C mRNA.

The Sendai virus P/C mRNA expresses the P and C proteins from alternate reading frames. The C reading frame of this mRNA, however, is responsible for three proteins, C', C and Y, none of which appear to be precursors to each other in vivo. Using site-directed and deletion mutagenesis of the P/C gene cloned in SP6 and in vitro translation of the mRNAs, we show that the 5' most proximal initiation codon of the mRNA is an ACG at position 81, responsible for C' synthesis. The succeeding initiation codons, all ATGs, are responsible for the P protein (position 104), the C protein (position 114) and the Y protein(s) (either positions 183 or 201). Examination of the relative molar amounts of the C', P and C proteins found in vivo suggests that an ACG in an otherwise favorable context is almost as efficient for ribosome initiation as an ATG in a less favored context, but only 10-20% as efficient as an ATG in a more favored context. The judicious choice of increasingly more favorable initiation codons in the P/C gene allows multiple proteins to be made from a single mRNA.

Animals↗

PubMed2003

Structure and promoter activity of the human glia maturation factor-gamma gene: a TATA-less, GC-rich and bidirectional promoter.

Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity.

Base Sequence↗

PubMedDate not supplied

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchin Strongylocentrotus purpuratus.

The general organization and primary amino acid sequences of the S. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26-27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5'-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5'-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage. The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.

Actins↗

PubMed1986

Construction of plasmid vectors for the detection of streptococcal promoters.

Plasmid vectors have been constructed for detecting DNA fragments that exhibit promoter activity in Streptococcus sanguis. The plasmids are able to replicate in both S. sanguis and Escherichia coli, and contain an erythromycin resistance marker which is expressed in both hosts. Selection for promoter activity is dependent upon the insertion of appropriate DNA fragments upstream from a promoterless chloramphenicol acetyl transferase gene (cat) from Staphylococcus aureus. To facilitate this insertion, a pair of vectors, pMU1327 and pMU1328, were constructed with the polylinker from M13mp 18 in either orientation. The to transcriptional terminator of phage lambda is present downstream from cat. Translation stop codons in all reading frames are located between the polylinker and the initiation codon of cat. These plasmids have been used to isolate DNA fragments from S. sanguis, S. lactis and S. cremoris that exhibit promoter activity in S. sanguis.

Base Sequence↗

PubMed1988

Expression of active rat DNA polymerase beta in Escherichia coli.

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

Amino Acid Sequence↗

PubMed1998

Alterations of PTEN/MMAC1, a candidate tumor suppressor gene, and its homologue, PTH2, in small cell lung cancer cell lines.

Loss of heterozygosity (LOH) at chromosome 10q23-q25 is frequent in small cell lung cancer (SCLC), indicating the presence of putative tumor suppressor genes. PTEN/ MMAC1, a newly cloned candidate tumor suppressor gene at 10q23, was mutated in multiple human cancers. We investigated whether mutations of PTEN/MMAC1 play an important role in SCLC tumorigenesis. We examined 16 SCLC cell lines for PTEN/MMAC1 mRNA expression by reverse-transcriptase polymerase chain reaction (RT-PCR) and potential mutations by sequencing analysis of the PTEN/MMAC1 coding region. No mutation was observed in PTEN/MMAC1 cDNAs in 15 cell lines expressing PTEN/MMAC1. One SCLC cell line, DMS79, did not have detectable PTEN/ MMAC1 expression. Importantly, we identified a novel homologue of PTEN/MMAC1, termed PTH2, localized to chromosome 9p21-q13 and containing only ten amino acid substitutions compared with the PTEN/MMAC1 coding region. However, because the putative initiation codon for PTEN/MMAC1 gene was changed to arginine in PTH2, the translational initiation site of PTH2 is very likely to differ from that of the PTEN/MMAC1. PTH2 was expressed in two normal lung tissues and two normal colon tissues, but in only four of 16 SCLC cell lines. A missense mutation in PTH2 was identified in a SCLC cell line that did not express PTEN/MMAC1 mRNA. Our data suggest that inactivation of PTEN/ MMAC1 is a rare event in SCLC tumorigenesis. However, the PTEN/MMAC1 homologue PTH2 may play a role in SCLC tumorigenesis.

Amino Acid Sequence↗

PubMed1989

Characterization of the human 5-lipoxygenase gene.

The gene for human 5-lipoxygenase has been isolated from three different bacteriophage genomic libraries and a genomic cosmid library. The gene spans greater than 82 kilobases and consists of 14 exons. The size range for the exons is 82-613 base pairs, whereas that for the introns is approximately 200 bp to greater than 26 kb. A major site of transcription initiation in leukocytes was mapped to a thymidine residue 65 base pairs upstream of the ATG initiation codon by nuclease S1 protection and primer extension experiments. Other potential minor initiation sites were found. The putative promoter region contains no TATA and CCAAT sequences in the expected positions upstream of the major transcription initiation site but contains multiple GC boxes within a (G + C)-rich region, as does the immediate 5' region of the first intron. Characteristics common to the 5' end of the human 5-lipoxygenase gene and the promoter regions of the housekeeping genes raise important questions concerning the regulation of 5-lipoxygenase gene expression.

Amino Acid Sequence↗

PubMed1989

Initiation complex formation on Euglena chloroplast 30S subunits in the presence of natural mRNAs.

An in vitro system has been developed that allows the formation of translation initiation complexes with Euglena chloroplast 30S ribosomal subunits and natural mRNAs. For these experiments two regions of the Euglena chloroplast genome have been cloned behind the T7 transcriptional promoter and the corresponding RNAs synthesized in vitro. These mRNAs are capable of forming initiation complexes with chloroplast 30S subunits in the presence of fMet-tRNA and E. coli initiation factors. Deletion of the normal translation start site results in a message that is no longer recognized by the chloroplast subunits suggesting that the correct AUG initiation codon on the mRNA is being selected by the small ribosomal subunit. Initiation complex formation with the chloroplast 30S subunits is specific for chloroplast mRNAs and mRNA from the phage MS2 is not active in this system.

Animals↗

PubMed1997

Cap-independent leaky scanning as the mechanism of translation initiation of a plant viral genomic RNA.

The genome of plum pox virus contains a single open reading frame that is translated into a large polyprotein. Although the open reading frame starts at nucleotide 36 (36AUG), it is translated from the second, 147AUG, which is in a more favourable context for translation initiation. We have carried out in vitro translation and transient expression analysis in protoplasts of a nested set of substitution and deletion mutants, and the results show that no internal structure in the 5' noncoding region of plum pox virus is necessary for efficient translation initiation. On the other hand, when the cryptic 36AUG was placed in a favourable context, it turned into an efficient initiation codon in vitro. Furthermore, AUGs that were placed in a favourable context, initiating short intraleader open reading frames, repressed translation initiation from the 147AUG in vitro and in vivo. These results point to leaky scanning as the mechanism of translation initiation of plum pox virus RNA. Nevertheless, it is a peculiar leaky scanning where the initiation of translation does not require a cap structure at the 5' end. This fact is congruent with the experimentally predicted absence of a stable secondary structure at the 5' noncoding region.

Animals↗

PubMed1995

Promoter region of the transcriptional unit for human alpha 1-chimaerin, a neuron-specific GTPase-activating protein for p21rac.

alpha 1-chimaerin is a neuron-specific GTPase-activating protein for p21rac, a protein involved in morphological events. The mRNA is highly expressed in certain brain regions. It is also detected in cultured neuronal, but not in non-neuronal cells. As a first step towards understanding the mechanisms underlying this regulation, genomic clones containing the 5'-flanking region of the human alpha 1-chimaerin transcriptional unit were isolated and characterised. A cluster of multiple transcription start sites of alpha 1-chimaerin mRNAs was detected by primer-extension and S1-mapping analyses. The cluster was mapped to nucleotides -464 to -434 (relative to nucleotide A in the initiation codon) in genomic DNA. The 5'-proximal region contained no TATA box, initiator motif and Sp1-binding site. A 210-bp fragment with approximately 110 bp 5'-flanking sequence could function as a minimal promoter upon analysis using hybrid chloramphenicol acetyltransferase reporter constructs and transient transfection. Internal deletion and point-mutation experiments revealed that a GGCCAATC sequence located at nucleotides -519 to -512 was essential for alpha 1-chimaerin promoter activity. Mobility-shift assay showed the specific binding of nuclear factor(s) to this region, which was competed by the oligonucleotides corresponding to wild-type but not mutant forms. The data also suggest the existence of possible novel CCAAT-binding factor(s) interacting with the alpha 1-chimaerin CCAAT box binding site. A cell-type-preferred suppressor located in the 5'-distal region was found which may play a role in controlling neuron-specific expression of alpha 1-chimaerin mRNA. These findings of a specific promoter for alpha 1-chimaerin transcription will facilitate further studies on its neuronal-specific expression and function.

Amino Acid Sequence↗

PubMed1986

Determination of the proteolytic processing sites in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus.

The bottom-component RNA (B-RNA) of cowpea mosaic virus is expressed by the production of a approximately 200,000-dalton polyprotein (200K polyprotein), from which the functional proteins are formed by specific proteolytic cleavages. Partial amino-terminal sequences of the various B-RNA-encoded proteins have now been determined. Comparison of the information obtained with the B-RNA sequence allowed the localization of the coding regions for these proteins on B-RNA, the calculation of their precise molecular weights, and the determination of the cleavage sites at which they are released from the polyprotein precursor. Sequence analysis of the 32K protein, which is derived from the amino-terminal end of the 200K polyprotein, indicated that the AUG codon at nucleotide position 207 of the RNA sequence is the translation initiation codon. Sequence analysis of the 170K, 110K, 87K, 84K, 60K, and 58K proteins revealed the existence of three types of cleavage site in the 200K polyprotein: glutamine-serine (two sites), glutamine-methionine (one site), and glutamine-glycine (one site) amino acid pairs. The nature of these cleavage sites suggested that two different viral proteases are involved in the processing of the B-RNA-encoded polyprotein.

Journal Article↗

PubMed2005

Initiation of protein synthesis in bacteria.

Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process. Subsequent sections describe the structure, function, and interactions of the mRNA, the initiator tRNA, and the initiation factors IF1, IF2, and IF3. Finally, we provide an overview of mechanisms of regulation of the translation initiation event. Translation occurs on ribonucleoprotein complexes called ribosomes. The ribosome is composed of a large subunit and a small subunit that hold the activities of peptidyltransfer and decode the triplet code of the mRNA, respectively. Translation initiation is promoted by IF1, IF2, and IF3, which mediate base pairing of the initiator tRNA anticodon to the mRNA initiation codon located in the ribosomal P-site. The mechanism of translation initiation differs for canonical and leaderless mRNAs, since the latter is dependent on the relative level of the initiation factors. Regulation of translation occurs primarily in the initiation phase. Secondary structures at the mRNA ribosomal binding site (RBS) inhibit translation initiation. The accessibility of the RBS is regulated by temperature and binding of small metabolites, proteins, or antisense RNAs. The future challenge is to obtain atomic-resolution structures of complete initiation complexes in order to understand the mechanism of translation initiation in molecular detail.

Bacteria↗

PubMed2004

L-Myc protein synthesis is initiated by internal ribosome entry.

An internal ribosome entry segment (IRES) has been identified in the 5' untranslated region (5' UTR) of two members of the myc family of proto-oncogenes, c-myc and N-myc. Hence, the synthesis of c-Myc and N-Myc polypeptides can involve the alternative mechanism of internal initiation. Here, we show that the 5' UTR of L-myc, another myc family member, also contains an IRES. Previous studies have shown that the translation of mRNAs containing the c-myc and N-myc IRESs can involve both cap-dependent initiation and internal initiation. In contrast, the data presented here suggest that internal initiation can account for all of the translation initiation that occurs on an mRNA with the L-myc IRES in its 5' UTR. Like many other cellular IRESs, the L-myc IRES appears to be modular in nature and the entire 5' UTR is required for maximum IRES efficiency. The ribosome entry window within the L-myc IRES is located some distance upstream of the initiation codon, and thus, this IRES uses a "land and scan" mechanism to initiate translation. Finally, we have derived a secondary structural model for the IRES. The model confirms that the L-myc IRES is highly structured and predicts that a pseudoknot may form near the 5' end of the mRNA.

5' Untranslated Regions↗

PubMed1991

Expression in bacteria of a polypeptide encoded by a transforming fragment of herpes simplex virus type 2.

We have constructed a plasmid (pMD2) containing the 38,000 MW polypeptide (38K polypeptide) gene from the transforming Bg1II-N fragment of HSV-2 fused to the amino-terminal portion of the beta-galactosidase gene in plasmid pUC8. Nucleotide sequence determination around the fusion-junction confirmed that the viral gene sequences starting at its second codon is in the correct reading frame in relation to the translation initiation codon of beta-galactosidase. The lac control sequences direct the synthesis of a 39K protein. This protein was shown to be structurally related to the 38K protein from HSV-2-infected cells by partial proteolytic cleavage analysis. Furthermore, antiserum directed against HSV-2-infected cells, as well as a monoclonal antibody against the 38K viral polypeptide and antibodies raised against a synthetic peptide corresponding to the nine C-terminal amino acid residues of the 38K viral protein, detected the fusion protein in bacteria containing the recombinant plasmid pMD2 but not in Escherichia coli containing a related plasmid or no plasmid.

Animals↗