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PubMed2005

The capsid protein of satellite Panicum mosaic virus contributes to systemic invasion and interacts with its helper virus.

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5'-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5'-untranslated region (5'-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5'-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5'-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.

Capsid Proteins↗
PubMed2004

Study of translational control of eukaryotic gene expression using yeast.

Eukaryotic cells respond to starvation by decreasing the rate of general protein synthesis while inducing translation of specific mRNAs encoding transcription factors GCN4 (yeast) or ATF4 (humans). Both responses are elicited by phosphorylation of translation initiation factor 2 (eIF2) and the attendant inhibition of its nucleotide exchange factor eIF2B-decreasing the binding to 40S ribosomes of methionyl initiator tRNA in the ternary complex (TC) with eIF2 and GTP. The reduction in TC levels enables scanning ribosomes to bypass the start codons of upstream open reading frames in the GCN4 mRNA leader and initiate translation at the authentic GCN4 start codon. We exploited the fact that GCN4 translation is a sensitive reporter of defects in TC recruitment to identify the catalytic and regulatory subunits of eIF2B. More recently, we implicated the C-terminal domain of eIF1A in 40S-binding of TC in vivo. Interestingly, we found that TC resides in a multifactor complex (MFC) with eIF3, eIF1, and the GTPase-activating protein for eIF2, known as eIF5. Our biochemical and genetic analyses indicate that physical interactions between MFC components enhance TC binding to 40S subunits and are required for wild-type translational control of GCN4. MFC integrity and eIF3 function also contribute to post-assembly steps in the initiation pathway that impact GCN4 expression. Thus, apart from its critical role in the starvation response, GCN4 regulation is a valuable tool for dissecting the contributions of multiple translation factors in the eukaryotic initiation pathway.

DNA-Binding Proteins↗
PubMed1992

The parainfluenza virus type 1 P/C gene uses a very efficient GUG codon to start its C' protein.

Parainfluenza virus type 1 (PIV1) and Sendai virus (SEN) are very closely related, but the PIV1 P/C gene does not contain the ACG codon which initiates the SEN C' protein. Nevertheless, a protein corresponding to the PIV1 C' protein was observed both in vivo and in vitro. The initiation site of this protein maps upstream of the PIV1 C protein AUG in a region that does not contain an AUG codon. We have used site-directed mutagenesis to demonstrate that the PIV1 C' protein initiates from a GUG codon, four codons upstream of where the ACG is found in SEN. Remarkably, this GUG appears to initiate in vivo almost as frequently as AUG in the same context. However, whereas GUG permits downstream expression of the P and C proteins, AUG in this context does not. The conservation of an upstream non-AUG initiation codon for C' among PIV1 and SEN suggests that it is important for virus replication, even though some paramyxoviruses express only the C protein and others have no C open reading frame at all.

Base Sequence↗
PubMed1990

Scanning model for translational reinitiation in eubacteria.

Premature termination of translation in eubacteria, like Escherichia coli, often leads to reinitiation at nearby start codons. Restarts also occur in response to termination at the end of natural coding regions, where they serve to enforce translational coupling between adjacent cistrons. Here, we present a model in which the terminated but not released ribosome reaches neighboring initiation codons by lateral diffusion along the mRNA. The model is based on the finding that introduction of an additional start codon between the termination and the reinitiation site consistently obstructs ribosomes to reach the authentic restart site. Instead, the ribosome now begins protein synthesis at this newly introduced AUG codon. This ribosomal scanning-like movement is bidirectional, has a radius of action of more than 40 nucleotides in the model system used, and activates the first encountered restart site. The ribosomal reach in the upstream direction is less than in the downstream one, probably due to dislodging by elongating ribosomes. The proposed model has parallels with the scanning mechanism postulated for eukaryotic translational initiation and reinitiation.

Bacteriolysis↗
PubMed2005

Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli.

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions. Including the anti-start codon in PMOs was not required for maximal inhibition. PMOs targeted to 5' nontranslated or 3' coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site. Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C+G content of targeted regions. The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted to acpP, a gene required for viability. Because inhibition by PMOs of approximately 11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions. The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO. In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs. These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets.

Bacterial Proteins↗
PubMedDate not supplied

[Cloning, sequence analysis and heterologous expression of a beta-mannanase gene from Bacillus subtilis Z-2].

Mannanase, an extracellular enzyme catalyzes the hydrolysis of hemicelluloses to produce oligosaccharides, has a potential to be applied in food industries. In this study, a mannanase gene from B. subtilis Z-2 was isolated through PCR screening of a genomic DNA library. The nucleotide sequence of the mannanase gene, man, contained an open reading frame of 1.080 bp, which codes for a deduced 26 amino-acid signal peptide and a mature protein with the deduced molecular mass of 38 KDa. The man gene can both be expressed heterologously into the periplasm in plasmid pET22b(+) containing intact signal peptide (pET-NdeI18) and the pelB signal peptide of the pET22b(+)vector (pET-NcoI3). The Escherichia coli BL21 (DE3) containing pET-NcoI3 secreted about twice as much mannanase as that harboring pET-NdeI18. In E. coli DH5alpha, expression of man was under the control of the lac promoter in the pRK415 vector and was much more effective when the Shine-Dalgamo (SD) sequence was changed from GGGGAG to AAGGAG and the start codon was changed from TTG to ATG, respectively. These results suggest that genetic modification of the SD sequence and start codon is practical for high-level expression of mannanase in different bacterial strains.

Bacillus subtilis↗
PubMed1997

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.

Amino Acid Sequence↗
PubMed1999

Differential expression of Myc1 and Myc2 isoforms in cells transformed by eIF4E: evidence for internal ribosome repositioning in the human c-myc 5'UTR.

eIF4E is essential for translation initiation, but its overexpression causes malignant transformation. Recent work demonstrated that eIF4E/F participates in exposing and locating alternate translation start codons during scanning. Translation initiation of several important protooncogenes and growth-regulators, such as Myc and FGF-2, can start at CUG start codon(s) upstream of the normal open reading frame (ORF). The resulting amino-terminal extension alters the properties of these proteins and their intracellular distribution. In cells overexpressing eIF4E, c-myc is overexpressed and particularly the larger, CUG-initiated form (Myc1). Recent reports suggest that synthesis of Myc2, the normally expressed AUG-initiated form, is mediated by an IRES. To determine what role eIF4E might play in c-myc expression, the c-myc 5' untranslated region (UTR) was fused in-frame to CAT reporters, and several more derivative constructs were made. In vitro translation experiments (with and without eIF4E/F); expression in CHO cells transformed with eIF4E; and deletion/mutation analysis demonstrated that Myc1 is translated by a scanning mechanism, while Myc2 is translated by Internal Ribosome Repositioning. Moreover, the existence of a true IRES in the 5'UTR was contradicted by its failure to direct translation of a circular transcript, in contrast to hsp70. The c-myc 5'UTR also failed to engage in translation in the absence of functional eIF4F, after cleavage of the eIF4G component with CVB4 protease-2A. The Internal Repositioning Element (IRPE) in c-myc 5'UTR was delimited to nucleotides (nt) 394-440 from the P1 transcription start site.

5' Untranslated Regions↗
PubMed1994

Analysis of the translational initiation region on the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase (rbcL) messenger RNA.

The chloroplast mRNAs from Euglena gracilis fall into two classes. One group of mRNs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region. To investigate the start signals for E. gracilis chloroplast mRNAs that do not carry a Shine-Dalgarno sequence, 30 S initiation complex formation has been studied using a series of transcripts carrying the wild-type translational start site of ribulose-bisphosphate carboxylase/oxygenase (rbcL) or mutated derivatives of this site. Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recognizing only the correct AUG codon. The analysis of the messages from a series of deletion mutants shows that a minimum of delta 20 residues 5' to the AUG codon is required for activity. Maximal activity requires the full 55-base leader sequence. Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is delta 60% as active as the wild-type message in promoting initiation complex formation. Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only delta 1.4-2-fold. The presence of an oligodeoxynucleotide containing a strong Shine-Dalgarno sequence does not significantly inhibit the formation of initiation complexes at the rbcL start site. Similar results are obtained when initiation complexes are formed with initiation factors from either E. gracilis chloroplasts or Escherichia coli.

Animals↗
PubMed1999

In vitro codon-reading specificities of unmodified tRNA molecules with different anticodons on the sequence background of Escherichia coli tRNASer.

The codon-reading properties of wobble-position variants of the unmodified form of Escherichia coli tRNASer1 (the UGA anticodon) were measured in a cell-free translation system. Two variants, with the AGA and CGA anticodons, each exclusively read a single codon, UCU and UCG, respectively. The only case of efficient wobbling occurred with the variant with the GGA anticodon, which reads the UCU codon in addition to the UCC codon. Surprisingly, this wobble reading is more efficient than the Watson-Crick reading by the variant with the AGA anticodon. Furthermore, we prepared tRNA variants with AA, UC, and CU, instead of GA, in the second and third positions and measured their relative efficiencies in the reading of codons starting with UU, GA, and AG, respectively. The specificity concerning the wobble position is essentially the same as that in the case of the codons starting with UC.

Amino Acid Sequence↗
PubMed1992

Regulatory elements and transcriptional regulation by testosterone and retinoic acid of the rat nerve growth factor receptor promoter.

The low-affinity nerve growth factor receptor (LNGFR) is a membrane-associated glycoprotein which is thought to participate in some of the biological activities of nerve growth factor (NGF). Expression of the LNGFR gene is known to be regulated both during development and in response to various agents in cell culture. However, molecular mechanisms responsible for the regulation have not been described. We report here an analysis of a 4.8-kb sequence from the 5'-flanking region of the rat LNGFR gene. Several regulatory elements were identified in this region by transfection of plasmid constructs containing sequences from LNGFR fused to a bacterial cat reporter gene. The proximal part of the promoter region (0.4-kb) was shown to be sufficient to support cat expression in all cell types used. A silencer element located between -1.5 kb and -1.8 kb from the start of translation, as well as an enhancer element in more upstream regions of the promoter, were identified in the phaeochromocytoma cell line, PC12, and in the Sertoli cell line, TM4, that express the LNGFR gene. Treatment of TM4 cells with retinoic acid (RA) increases the level of LNGFR mRNA twofold, while testosterone treatment results in a tenfold decrease. Regions of the promoter responsive to testosterone and RA in TM4 cells were found at -610 to -860 bp and -1840 to -4800 bp upstream from the translation start codon, respectively. A RA-responsive element active in PC12 cells is located between bp -610 to -860 from the start codon.

Animals↗
PubMed2001

The non-structural leader protein gene of foot-and-mouth disease virus is highly variable between serotypes.

Aphthoviruses are unique among picornaviruses in that they alone encode a functional L proteinase as the first component of the viral polyprotein. The L genes of a few Indian foot-and-mouth disease viruses were sequenced and compared with those available to study the extent of variation in this gene. Besides the two in-frame start codons present in all FMDV L genes, the Asia-I vaccine virus had an additional in-frame AUG (start) codon, at codon position 3. Amino acid sequence comparison revealed that 39.8% of positions were capable of accepting replacements, yet the residues of the catalytic dyad were totally conserved. Sequence comparison at the C-terminus of the protein indicated that K/R decreasing GAGQS is sufficient for L/P1 cleavage. Phylogenetic analysis based on the L gene sequences did not reveal any serotype-specific clustering. The probable implications of the observed high variability in this non-structural gene is briefly discussed.

Amino Acid Sequence↗
PubMed1991

Transcriptional analysis of the restriction and modification genes of bacteriophage P1.

Bacteriophage P1 res and mod genes encode the restriction and modification polypeptides of the Type III restriction enzyme EcoP1. Northern blot analysis using res- and mod-specific probes revealed the presence of two separate transcripts in strains harbouring the EcoP1 restriction and modification genes. Furthermore, by constructing a series of fusions with a promoter less lacZ gene, we show that both the res and mod genes are transcribed from separate promoters. A more detailed investigation of the mod promoter region revealed two promoters located some 70 and 140bp upstream from the translational start codon. In addition, another pair of promoters and a further separate promoter are located more than 500bp upstream from this start codon. Two short open reading frames are located between these distal and proximal promoter clusters. Transcription of the res gene is initiated from within the mod open reading frame from two adjacent promoters. In addition a functional promoter is located on the antisense strand close to the res promoter region. The relationship between the transcription units of the res and mod genes is discussed.

Amino Acid Sequence↗
PubMed1986

Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators.

We present evidence that the expression of rho is regulated by rho-dependent attenuation of transcription. Gene fusion analysis with nested series of deletions of rho indicated that the transcription of rho is attenuated in a rho-dependent manner in the leader region and that neither a read-through transcription from the upstream gene, trxA, nor a modulation of transcription initiation of the rho promoter is involved in the self-control of rho. S1 mapping and Northern hybridization analyses localized at least six transcription attenuation or termination sites in the region ranging from the 3' end of the trxA structural gene to the middle of the rho structural gene. Among them, the most upstream site overlapping the rho promoter sequence was assigned to the terminator for the trxA gene, and the second and third sites, mapping about 80 and 50 nucleotides upstream from the start codon of rho, were suggested to function as the major attenuation sites for regulation of the rho expression. Further, the start points of the trxA and rho RNAs were determined in an in vitro transcription system to be located 111 nucleotides (U) and 255 nucleotides (G) upstream from their respective start codons. These results necessitate revisions of previous predictions on the sites of transcriptional signals in the trxA and rho genes (S. Brown, B. Albrechtsen, S. Pedersen, and P. Klemm, J. Mol. Biol. 162:283-298, 1982; C.-J. Lim, D. Geraghty, and J. A. Fuchs, J. Bacteriol. 163:311-316, 1985; B.J. Wallace and S.R. Kushner, Gene 32:399-408, 1984).

Chromosome Deletion↗
PubMed1994

Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF.

The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M(r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products. In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells.

Animals↗
PubMed2000

Characterization of a DNA region containing 5'-(CAAT)(n)-3' DNA sequences involved in lipooligosaccharide biosynthesis in Haemophilus somnus.

Repetitive tetranucleotide sequences of 5'-(CAAT)(n)-3' have been identified at the 5' end of an open reading frame (ORF) named lob1 from Haemophilus somnus strain 738. Based on sequence analysis, lob1 has 59% DNA homology to lex2B, which is involved in lipooligosaccharide (LOS) biosynthesis in H. influenzae. We now report that the number of 5'-CAAT-3' repeats in lob1 varied from 31-35, but that 94% of colonies contained 33 repeats of 5'-CAAT-3' downstream of two potential start codons, as determined by DNA sequence analysis of the 5'-CAAT-3' region from individual colonies. If transcription began with the start codon closest to the 5'-CAAT-3' repeats, a protein of 34.5 kDa would be encoded when 33 repeats were present. However, we could not establish a correlation between the number of 5'-CAAT-3' repeats in lob1 with a specific LOS electrophoretic profile or reactivity with two LOS monoclonal antibodies, indicating multiple genes control LOS phase variation in H. somnus. Complementation of strain 129Pt with lob1 containing 33 5 '-CAAT-3' repeats in shuttle vector pLS88 resulted in transformants 129Pt(pLSlob1-33A) and 129Pt(pLSlob1-33B), both of which demonstrated the same altered LOS electrophoretic profile. Unlike strain 129Pt, both transformants underwent limited LOS phase variation, which correlated with variation in the number of 5'-CAAT-3' repeats in pLSlob1-33. Nanoelectrospray-mass spectrometry of O-deacylated LOS indicated that transformant 129Pt(pLSlob1-33A) LOS was composed of a different distribution of glycoforms than LOS of the parent strain. The ratio of glucose to galactose changed from 1:2 in strain 129Pt LOS to 2:1 in transformant 129Pt(pLSlob1-33A) LOS, as determined by gas chromatography-mass spectrometry. Nuclear magnetic resonance spectroscopy confirmed and extended these observations. Transformant 129Pt(pLSlob1-33A) was constitutively more reactive in colony immunoblotting to polyclonal antiserum made to purified strain 738 LOS, and was more susceptible to complement-mediated killing in the presence of anti-738 LOS serum than parent strain 129Pt. Based on these results, Lob1 appears to be a phase variable galactosyl transferase involved in LOS biosynthesis in H. somnus.

Animals↗
PubMed2000

Naturally occurring woodchuck hepatitis virus (WHV) deletion mutants in chronically WHV-infected woodchucks.

Deletion mutants of hepatitis B virus (HBV) are often found in chronically HBV-infected patients. It has not been possible to study the significance of such deletion mutants on liver diseases in a suitable animal model. In this study, we characterized naturally occurring deletion mutants of woodchuck hepatitis virus (WHV) in 11 chronically WHV-infected woodchucks. Deletions within the WHV preS region (nt 2992-338) had a length of 72 or 84 bp and were located in the amino terminal part of preS1. Internal deletions within the core gene (CID) had variable lengths (103 to 312 bp) and were identified within the center of this gene (nt 2021-2587). Four of seven CIDs were in-frame deletions, whereas the remaining three CIDs were out-of-frame deletions and led to the interruption of the reading frame. Sequence analysis of cloned PCR products of CIDs showed that heterogeneous WHV deletion mutants coexisted in single woodchucks. In addition, WHV genomes with double deletions in the preS1 and the core region could be found. We were unable to detect the expression of truncated core proteins in transfection experiments. The CID mutations led to a marked increase of the expression of the luciferase gene which was fused to the start codon of WHV polymerase, probably due to the shortening of the untranslated region or the removal of AUGs preceding the polymerase start codon. The characterization of naturally occurring WHV deletion mutants will allow us to study their biological and pathogenic properties in the woodchuck model in the future.

Animals↗