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PubMed1996

Increased xylanase yield in Streptomyces lividans: dependence on number of ribosome-binding sites.

The Streptomyces lividans xylanase A (XlnA) signal peptide (sp) was replaced with the signal peptides of either mannanase A (ManA) or cellulase A (CelA), two enzymes secreted by S. lividans. Depending on the location of the ribosome binding sites (RBS) with respect to a potential initiation codon, the length of the putative sps of ManA and CelA is either 34 or 43 amino acids and 27 or 46 amino acids, respectively. The sequence encoding these sps were fused to the xylanase A gene (xlnA). Clones harboring the short sps of ManA and CelA produced as much xylanase as the clone with the control wild-type sp sequence of XlnA. In clones containing the long sps of ManA and CelA, the XlnA production was enhanced 1.5- and 2.5-fold, respectively. These XlnA yields are reduced by half and one third respectively when the internal initiation codons of the long sp sequences of ManA and CelA are mutated. Since these clones exhibited the same transcription levels, the results indicate that both RBSs are used concomitantly in S. lividans to increase the enzyme production at the translational level. However, when the short and long sps of ManA were fused to the long CelA sp sequence, giving constructs containing respectively 3 and 4 RBSs, a decrease in xylanase production was observed.

Amino Acid Sequence↗

PubMed1993

Alteration of the Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader by mitochondrial gene replacement and functional interaction with the translational activator protein PET111.

The ability to replace wild-type mitochondrial DNA sequences in yeast with in vitro-generated mutations has been exploited to study the mechanism by which the nuclearly encoded PET111 protein specifically activates translation of the mitochondrially coded COX2 mRNA. We have generated three mutations in vitro that alter the COX2 mRNA 5'-untranslated leader (UTL) and introduced them into the mitochondrial genome, replacing the wild-type sequence. None of the mutations significantly affected the steady-state level of COX2 mRNA. Deletion of a single base at position -24 (relative to the translation initiation codon) in the 5'-UTL (cox2-11) reduced COX2 mRNA translation and respiratory growth, whereas insertion of four bases in place of the deleted base (cox2-12) and deletion of bases -30 to -2 (cox2-13) completely blocked both. Six spontaneous nuclear mutations were selected as suppressors of the single-base 5'-UTL deletion, cox2-11. One of these mapped to PET111 and was shown to be a missense mutation that changed residue 652 from Ala to Thr. This suppressor, PET111-20, failed to suppress the 29-base deletion, cox2-13, but very weakly suppressed the insertion mutation, cox2-12. PET111-20 also enhanced translation of a partially functional COX2 mRNA with a wild-type 5'-UTL but a mutant initiation codon. Although overexpression of the wild-type PET111 protein caused weak suppression of the single-base deletion, cox2-11, the PET111-20 suppressor mutation did not function simply by increasing the level of the protein. These results demonstrate an intimate functional interaction between the translational activator protein and the mRNA 5'-UTL and suggest that they may interact directly.

Alleles↗

PubMed1985

The structure and expression of the murine gene encoding granulocyte-macrophage colony stimulating factor: evidence for utilisation of alternative promoters.

Two overlapping genomic clones containing the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene have been isolated. On the basis of transfection experiments, we have established that a 9-kb BamHI fragment from one of these recombinants encodes biologically active GM-CSF. As deduced from nucleotide sequence analysis, the GM-CSF gene comprises four exons encompassing 2.5 kb of genomic DNA. Primer extension analysis of GM-CSF mRNA identifies a transcriptional initiation site 35 bp upstream of a single translational initiation codon in-frame with the GM-CSF coding sequences and 28 bp downstream of a TATA promoter consensus sequence. Pre-GM-CSF molecules encoded by mRNAs originating from this promoter would include a hydrophobic leader sequence typical for a secreted protein. Intriguingly, sequences present at the 5' end of a GM-CSF cDNA clone previously isolated in our laboratory are not contained within either of the genomic clones and must therefore be transcribed from a promoter located at least 10 kb 5' of the main body of the gene. mRNAs transcribed from this alternative upstream promoter possess an additional initiating codon and potentially encode a pre-GM-CSF polypeptide with an atypical NH2-terminal leader peptide. Comparison of the nucleotide sequence of the GM-CSF gene with that of other haemopoietic growth factor genes has revealed a common decanucleotide (5'-GPuGPuTTPyCAPy-3') within their respective 5'-flanking regions which may be involved in their co-ordinate regulation.

Amino Acid Sequence↗

PubMed1996

Antisense oligodeoxynucleotides against the BZLF1 transcript inhibit induction of productive Epstein-Barr virus replication.

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, in latently infected cells is sufficient to induce the viral lytic cycle. The use of oligodeoxynucleotides complementary to the BZLF1 transcript was studied to inhibit this induction of productive viral replication. For this purpose, we employed oligodeoxynucleotides complementary to the translation initiation codons and their flanking sequences. Incubation of Akata cells with the 25-mer phosphodiester (PO)- or phosphorothioate (PS)-antisense oligodeoxynucleotides for 3 h before stimulation with anti-immunoglobulin G antibodies (anti-IgG) partially inhibited the anti-IgG-mediated induction of ZEBRA synthesis. Both the PO- and PS-antisense oligodeoxynucleotide treatments also suppressed the productive EBV replication (as measured by linear DNA production) in a dose-dependent manner, with much greater efficiency than did PO and PS-oligodeoxynucleotides with sense, reverse or random sequences of the same length. Another 20-mer antisense oligodeoxynucleotide complementary to sequences downstream of the translation initiation codons showed a similar inhibitory effect on EBV replication. However, the inhibition was considerably lower when the cells were treated with oligodeoxynucleotides complementary to sequences upstream of the start codons. These results indicate that BZLF1 antisense oligodeoxynucleotides inhibit the viral activation in a sequence-specific fashion. In the virus-producer cell line P3HR-1, the same PS-antisense oligodeoxynucleotides also partially suppressed the spontaneous viral replication after 6-10 days, substantially more than the PS-random oligodeoxynucleotides. Inhibition of BZLF1 appears to be sufficient to suppress the induction of EBV replication.

Antigens, Viral↗

PubMed1983

Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.

Amino Acid Sequence↗

PubMed1995

Orphan nuclear receptor HNF-4 binds to the human coagulation factor VII promoter.

The human coagulation protease factor VII plays a pivotal role in the initiation of the coagulation cascade by both the extrinsic and the intrinsic pathway. Although the gene, encoding factor VII, is expressed predominantly in the liver, the mechanisms underlying this tissue-specific expression have not been elucidated. In this study, we have analyzed the contribution of 5 kilobases upstream of the ATG translational initiation codon upon hepatic factor VII gene transcription. Transient transfection assays of a set of nested deletions in both liver and non-liver cell lines, HepG2 and HeLa respectively, indicate that several regions are involved in liver-specific expression. A slight negative effect on factor VII promoter activity in HepG2 cells is mediated by sequences upstream of position -1212. DNase I protection experiments reveal six footprints, FPVII1 through FPVII6, within the proximal 714 base pairs but a minimal promoter of 165 base pairs containing only FPVII3-6 is sufficient to confer liver-specific expression in HepG2 cells. Interestingly, FPVII6, at position -14 to +10 on the sense strand, would indicate that an as yet unknown transcription factor covers the ATG translational initiation codon. Gel retardation experiments show that the liver-enriched transcription factor HNF-4 binds specifically to footprint FPVII4 at position -71 to -49. Furthermore, a T --> A transversion, that in the HNF-4 binding site of factor IX causes a severe bleeding disorder, was introduced into the HNF-4-binding site of factor VII and reduced promoter activity by 20-50%. Coordinate HNF-4-mediated regulation of several blood protease genes as well as genes involved in lipid metabolism might account for the positive correlation of these factors with increased risk of occlusive heart diseases.

Amino Acid Sequence↗

PubMed1995

Inhibition of hepatitis C virus replication by antisense oligonucleotide in culture cells.

Oligonucleotides complementary to the sequences containing the initiator codon, AUG, of the core region of positive-stranded hepatitis C virus (HCV) were tested for their effects on viral translation in a cell-free protein synthesis system and on viral replication in a human T-lymphotropic virus type I infected cell line, MT-2C, which was cloned by the limited dilution method from MT-2 cells and showed more efficient HCV replication than an uncloned population of MT-2 cells. Treatment of HCV-infected MT-2C cells with the antisense oligonucleotide (10 microM) had a dramatic inhibitory effect on viral replication. This result suggests that the antisense oligonucleotide complementary to the sequence close to the initiation codon of the core region might be useful as an antiviral agent against HCV replication.

Antiviral Agents↗

PubMed1987

Role in translation of a triple tandemly repeated sequence in the 5'-untranslated region of human thymidylate synthase mRNA.

A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negative mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5' end. The translation efficiency was also not affected by insertion of an additional repeated element into the TTR. These results suggest that the first two repeated elements at the 5' end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.

Animals↗

PubMed1989

Molecular characterization of the Trypanosoma brucei RNA polymerase I and III largest subunit genes.

We have sequenced a 7.1-kilobase fragment of the Trypanosoma brucei RNA polymerase (pol) I largest subunit gene. The 6.9-kilobase transcript from this gene is of roughly equal abundance in bloodstream- and procyclic-form trypanosomes and has a 39-nucleotide trans-spliced leader 290 nucleotides upsteam of the putative initiation codon. The 1781-amino acid trypanosome pol I polypeptide is considerably less similar to its yeast homolog than the trypanosome pol II and pol III polypeptides are to their counterparts in yeast. The yeast and trypanosome pol I polypeptides are distinguished from pol II and pol III largest subunits by two inserts, 100-200 amino acids in length, which are found in the nonconserved portions of the polypeptides. We also report our sequence of the trypanosome pol III largest subunit gene, which agrees with that reported by Köck et al. (Köck, J., Evers, R., and Cornelissen, A.W.C.A. (1988) Nucleic Acids Res. 16, 8753-8772) except for 13 nucleotide differences. Four spliced leader addition sites between 60 and 99 nucleotides upstream of the AUG initiation codon were observed for the 5.4-kilobase transcript from this gene. Four short stretches of amino acid homology between the yeast and trypanosome pol III largest subunit polypeptides were identified which appear to be specific to this class of RNA polymerase; only one short pol I-specific sequence was identified in our comparison of the yeast and trypanosome pol I largest subunit polypeptides.

Amino Acid Sequence↗

PubMed1994

Specific inhibition of c-sis protein synthesis and cell proliferation with antisense oligodeoxynucleotides in human glioma cells.

The protein encoded by c-sis is overexpressed in human neuroglial tumors and has been hypothesized to play an important role in tumorigenesis. To address this issue, we examined the effect of an 18-base pair oligodeoxynucleotide complementary to a sequence starting of ATG initiation codon of mRNA of c-sis upon glioma cell growth. First, we investigated the expression of c-sis within cultured human glioma cell lines and also fresh glioma specimens by using polymerase chain reaction. We could detect messenger ribonucleic acid transcripts of c-sis in three of four glioma cell lines and two of five glioblastoma multiforme specimens. This finding was confirmed by dot-blot hybridization with a specific oligonucleotide probe of c-sis. The antisense oligonucleotides complementary to c-sis messenger ribonucleic acid were efficiently incorporated into A172 cells in vitro, and kinetic studies showed that maximum uptake was seen after 48 hours incubation with antisense oligomers. Exposure of human glioma cell lines to antisense oligodeoxynucleotides targeted against the first initiation codon of c-sis inhibited cell proliferation in a time- and dose-dependent fashion. From flow cytometric analysis with anti-c-sis sera, it was demonstrated that the antisense oligomers specifically block the de novo synthesis of intracellular c-sis protein by glioma cells. In contrast, the corresponding sense oligomers did not inhibit either synthesis of c-sis protein or glioma cell growth. These results clearly support a role of c-sis protein in the proliferation of these human neuroglial tumors and show that inducible protein expression can be blocked by means of synthetic oligonucleotides complementary to a coding exon.

Brain Neoplasms↗

PubMed1994

Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1).

Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2000

An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus.

Hepatitis C virus translation is initiated on a approximately 330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG(266-268) in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.

Animals↗

PubMed1990

Location of a P1 plasmid replication inhibitor determinant within the initiator gene.

The P1 plasmid replication initiator protein, RepA, binds to its own promoter and represses transcription efficiently. There are only about 20 RepA dimers present per repA gene. A possible reason for this highly restrained expression became evident when repA expression was increased by using foreign promoters: with fivefold overexpression, the replication rate was diminished, and with 40-fold overexpression, replication was not detectable. The inhibition was P1 specific: growth of Escherichia coli and replication of pSC101, R6K, and mini-F plasmids were not affected. The activity is apparently not from RepA itself. Excess purified RepA did not inhibit replication in vitro. Mutations of the repA translation initiation codon reduced synthesis of the initiator but not the inhibitory activity. Deletion from either the N- or C-terminal ends of repA (28 and 69 codons, respectively, out of the 286-codon open reading frame) affected the initiator but not the inhibitory activity. Further deletions affected both the activities. These results demonstrate that the integrity of the initiator is not required for inhibition, but involvement of an unstable initiator fragment or of initiator mRNA cannot be ruled out.

Amino Acid Sequence↗

PubMed1985

Hybridization arrest of globin synthesis in rabbit reticulocyte lysates and cells by oligodeoxyribonucleoside methylphosphonates.

Oligodeoxyribonucleoside methylphosphonates which are complementary to the 5' end, the initiation codon regions, or the coding regions of rabbit globin mRNA were synthesized. These oligomers were shown to interact with their complementary mRNA binding sites by their ability to serve as primers for reverse transcriptase. In several cases, the priming efficiency of the oligomers was enhanced when the oligomer was preannealed with the mRNA. This behavior correlates with the predicted secondary structure of the mRNA and suggests that some oligomer binding sites occur in hydrogen-bonded stem regions of the mRNA. Methylphosphonate oligomers inhibit translation of globin mRNA in reticulocyte lysates. Inhibition is due to the interaction of the oligomers with mRNA. The extent of inhibition is affected by the sequence and chain length of the oligomer, the location of the oligomer binding site on the mRNA, and the secondary structure of the binding site. Oligomers which bind to the 5' end and initiation codon regions of beta-globin mRNA inhibit both alpha- and beta-globin synthesis whereas oligomers which bind to the coding region of alpha-globin mRNA or the coding region of beta-globin mRNA inhibit translation of their target mRNA in a specific manner. Oligodeoxyribonucleoside methylphosphonates inhibit globin synthesis in rabbit reticulocytes. The effects of various oligomers on cellular globin synthesis are similar to those in the lysate system and suggest that the conformation of globin mRNA is the same in both systems during translation.

Animals↗

PubMed2003

The simian immunodeficiency virus 5' untranslated leader sequence plays a role in intracellular viral protein accumulation and in RNA packaging.

We investigated the role of 5' untranslated leader sequences of simian immunodeficiency virus (SIV(mac239)) in RNA encapsidation and protein expression. A series of progressively longer deletion mutants was constructed with a common endpoint six nucleotides upstream of the gag initiation codon and another endpoint at the 3' end of the primer binding site (PBS). We found that efficient intracellular Gag-Pol protein accumulation required the region between the PBS and splice donor (SD) site. Marked reduction of genomic RNA packaging was observed with all the deletion mutants that involved sequences at both the 5' and at the 3' ends of the major SD site, and increased nonspecific RNA incorporation could be detected in these mutants. RNA encapsidation was affected only modestly by a deletion of 54 nucleotides at the 3' end of the SD site when the mutant construct pDelta54 was transfected alone. In contrast, the amount of pDelta54 genomic RNA incorporated into particles was reduced more than 10-fold when this mutant was cotransfected with a construct specifying an RNA molecule with a wild-type packaging signal. Therefore, we conclude that the 175 nucleotides located 5' of the gag initiation codon are critical for efficient and selective incorporation of genomic RNA into virions. This location of the SIV Psi element provides the means for efficient discrimination between viral genomic and spliced RNAs.

5' Untranslated Regions↗

PubMed1997

[Regulation of translation of the distal lacZ gene in polycistronic mRNA by the ribosome stream from the proximal gene].

Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed. These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence. The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII. Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation. The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids. We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.

Genes↗

PubMed1998

Experimental study on pathogenicity of precore mutants in Hepadnaviridae.

OBJECTIVE: To study the replicative competency and pathogenicity of precore gene mutants of duck hepatitis B virus (DHBV) in the duck model. METHODS: Three site-directed point mutations in the precore region of cloned DHBV were constructed. Head-to-tail dimers were formed. The three plasmids were named: pEDM1-2 (initiation codon ATG mutated to TTG), pEDM2-2 (an "A" was inserted down stream of codon 12, leading to frame shift in the distal end of precore region), pEDM3-2 (codon 38 was changed from TAT to TAA, leading to a stop codon at the 3'-end). Mutants and wild-type cloned DNA dimers were first separately used to transfect LMH cells (a chicken hepatoma cell line) and viruses were collected from supernatant and used to infect 6 one-day-old ducklings per group. Serum duck hepatitis B surface antigen (DHBsAg) and DHBV DNA were assayed. Six weeks after infection, ducks were killed and liver tissues were studied for histopathological changes. RESULTS: After transfection, pEDM1-2, pEDM2-2 and pEDM3-2 expressed similar level of DHBsAg. Replication of pEDM1-2 and pEDM3-2 was similar to that of the wild type clone, while pEDM2-2 replicated at a significantly decreased level. Infection study employing the supernatant of transfected cells was as follows: pEDM1-2 infected 5/6 ducklings, pEDM2-2 non infected, pEDM3-2 infected 2/6 ducklings, wild type virus infected 6/6 ducklings. Positive serum samples from both pEDM1-2 and pEDM3-2 were at a lower serum DHBV level compared to that of the wild type virus. Pathological changes were more significant in pEDM3-2 infected duck livers, with numerous inflammatory cells in portal tract and infiltration into parenchyma. CONCLUSIONS: Mutations in the initiating codon or generation of a stop codon at the 3'-end of the precore region resulted in decreased replication competency of DHBV, while frame-shift mutation of the precore region, covering the epsilon encapsidation signal abolished the replication of DHBV. When the mutants replicated in hosts, more severe pathological changes were observed in ducks infected with mutant harboring a stop codon at the 3'-end. Data suggest that replicative-competent DHBV precore mutant can be more pathogenic than wild-type DHBV.

Animals↗

PubMed2004

Transcriptional and translational regulation of the marRAB multiple antibiotic resistance operon in Escherichia coli.

The marRAB multiple antibiotic resistance operon of Escherichia coli is autorepressed by MarR. MarR binds to two palindromic sequences in vitro: site I lies between and overlaps the -35 and -10 hexamers for RNA polymerase binding; site II lies between the transcription start site and the GTG initiation codon of marR. To assess the importance of these sites in vivo, the effects of mutant sites on transcription were analysed using fusions to lacZ in the presence and absence of wild-type MarR. When both sites were wild type, transcription in the derepressed marR-deleted strain was 19-fold that of the wild-type strain; when only site I or site II was wild type, this ratio was reduced to 4.3- and 2.6-fold, respectively, showing that full repression requires both sites, but some repression can occur at one site independently of the other. Translational fusions of the wild-type promoter to lacZ demonstrated that marR translation proceeds at only 4.5% of the transcription rate. Analysis of translational fusions with mutant leader sequences demonstrated that the principal reason for inefficient translation is a weak Shine-Dalgarno (SD) sequence, AGG(G). Although the SD sequence is located within the potential stem-loop structure of site II, no evidence for occlusion of the SD sequence was found in the wild-type strain. However, a single basepair mutation that strengthens the stem-loop structure drastically reduced the translational efficiency. Substitution of ATG for GTG as the initiation codon increased translational efficiency by 50%. Increasing the 5 bp spacing between the SD sequence and the GTG codon by one to four bases reduced the translational efficiency by 50-75%. Inefficient translation of marR may help to sensitize the cell to environmental signals.

Bacterial Proteins↗