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PubMed2006

Characterization of recombinant Xenopus laevis type I iodothyronine deiodinase: substitution of a proline residue in the catalytic center by serine (Pro132Ser) restores sensitivity to 6-propyl-2-thiouracil.

In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T(3) in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T(4) or reverse T(3) (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (K(m)) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T(4) (K(m) 0.5 microm) and rT3 (K(m) 0.5 microm) and inner-ring deiodinase activity with T(4) (K(m) 0.4 microm). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC(50) > 1 mm) but was sensitive to gold thioglucose (IC(50) 0.1 mum) and iodoacetate (IC(50) 10 microm). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T(4) and rT3 (K(m) about 4 microm) and was highly sensitive to 6-PTU (IC(50) 2 microm). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.

3' Untranslated Regions↗
PubMed2003

Promoter activities in the baculovirus envelope glycoprotein gp64 gene.

Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4-4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13 - 22 times in Bm-N cells and over 7000 - 14,000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73 - 78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.

Animals↗
PubMed2000

Identification of a noncanonical signal for transcription of a novel subgenomic mRNA of mouse hepatitis virus: implication for the mechanism of coronavirus RNA transcription.

Subgenomic RNA transcription of coronaviruses involves the interaction between the leader (or antileader) and the intergenic (IG) sequences. However, it is not clear how these two sequences interact with each other. In this report, a previously unrecognized minor species of subgenomic mRNA, termed mRNA5-1, was identified in cells infected with mouse hepatitis virus (MHV) strains JHM2c, JHM(2), JHM(3), A59, and MHV-1. Sequence analysis revealed that the leader-body fusion site of the mRNA is located at approximately 150 nucleotides (nt) downstream of the consensus IG sequence for mRNA 5 and did not have sequence homology with any known IG consensus sequences. To determine whether this sequence functions independently as a promoter, we cloned a 140-nt sequence (from approximately 70 nt upstream to approximately 70 nt downstream of the fusion site) from viral genomic RNA and placed it in front of a reporter gene in the defective-interfering (DI) RNA-chloramphenicol acetyltransferase (CAT) reporter vector. Transfection of the reporter RNA into MHV-infected cells resulted in synthesis of a CAT-specific subgenomic mRNA detected by reverse transcription-polymerase chain reaction (RT-PCR). The strength of this promoter was similar to that of the IG7 (for mRNA 7) as measured by the CAT activity. Deletion analysis showed that the sequence as few as 13 nt was sufficient to initiate mRNA transcription, while mutations within the 13-nt abolished mRNA transcription. In vitro translation study confirmed that the envelope (E) protein was translated from mRNA5-1, which encodes the open reading frame (ORF) 5b at its 5'-end, indicating that mRNA5-1 is a functional message. Furthermore, when the ORF5b was replaced with the CAT gene and placed in the DI in the context of viral mini-genome, CAT was expressed not only from the first ORF of mRNA5-1 but also from the second and third ORF of mRNA5 and genomic DI RNA, respectively, suggesting that more than one mechanism is involved in regulation of ORF5b expression. Our findings thus support the notion that base-pairing between the leader (or antileader) and the IG is not the sole mechanism in subgenomic RNA transcription.

Animals↗
PubMed2003

A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA.

Human papillomavirus type 16 (HPV-16) has the capacity to transform human primary keratinocytes. Maintenance of the transformed phenotype requires constitutive expression of the oncoproteins E6 and E7. The low-risk HPV types express E7 from monocistronic mRNA, but for the high-risk types, no mRNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic mRNA is not very abundant, but we have shown that an E7-luciferase fusion protein can be expressed in SiHa cells from a monocistronic HPV-16 transcript initiated at nt 542. The monocistronic mRNA expresses E7-luciferase more efficiently than the most abundant in vivo-like mRNA E6*IE7, initiated by P97 and spliced from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells.

Animals↗
PubMed1989

Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.

Amino Acid Sequence↗
PubMed1996

Identification and sequences of the Treponema pallidum mglA and mglC genes.

Treponema pallidum, the agent of syphilis, cannot be continuously cultivated in vitro. To identify treponemal genes encoding exported proteins, we performed TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Clone 6D2 was chosen for further study based on partial nucleotide sequence obtained from p6D2 containing a TnphoA insertion. A complete open reading frame (orf1) and a truncated orf (orf2) were identified in the treponemal DNA of p6D2. Orf1 encodes a hydrophobic protein of 531 amino acids with a calculated M(r) of 57,882 Da. The deduced amino acid sequence of Orf1 has homology to the MglC proteins of E. coli, Haemophilus influenzae, and Salmonella typhimurium. T. pallidum Orf1 (MglC) contains a conserved motif that is found in integral cytoplasmic membrane proteins of ATP-binding cassette (ABC) transport systems. T. pallidum orf2 encodes a protein of 496 amino acids with a calculated M(r) of 55,547 Da. The deduced amino acid sequence of Orf2 has homology to the MglA proteins of S. typhimurium, E. coli, H. influenzae, and Mycoplasma genitalium. Orf2 (MglA) contains two consensus ATP-binding motifs. T. pallidum mglA and mglC are located downstream of mglB, consistent with the gene order of previously identified mgl operons. The putative T. pallidum mgl operon encodes the first high-affinity ABC transport system identified in this spirochete.

Amino Acid Sequence↗
PubMed1997

The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs.

The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w and CBF5) display a remarkable sequence homology with Escherichia coli tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene YNL292w coding for one of these proteins was cloned in an E.coli expression vector downstream of a His6-tag. The resulting recombinant protein (Pus4) was expressed at high level and purified to homogeneity by metal affinity chromatography on Ni2+-NTA-agarose, followed by ion-exchange chromatography on MonoQ. The purified Pus4p catalyzes the formation of pseudouridine-55 in T7 in vitro transcripts of several yeast tRNA genes. In contrast to the known yeast pseudouridine synthase (Pus1) of broad specificity, no other uridines in tRNA molecules are modified by the cloned recombinant tRNA:Psi55 synthase. The disruption of the corresponding gene YNL292w in yeast, which has no significant effect on the growth of yeast cells, leads to the complete disappearance of the Psi55 formation activity in a cell-free extract. These results allow the formal identification of the protein encoded by the yeast ORF YNL292w as the only enzyme responsible for the formation of Psi55 which is almost universally conserved in tRNAs. The substrate specificity of the purified YNL292w-encoded recombinant protein was shown to be similar to that of the native protein present in yeast cell extract. Chemical mapping of pseudouridine residues in both cytoplasmic and mitochondrial tRNAs from the yeast strain carrying the disrupted gene reveals that the same gene product is responsible for Psi55 formation in tRNAs of both cellular compartments.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus.

The nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted M(r) of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV polyhedrins. The SeMNPV polyhedrin protein was most closely related to S. frugiperda MNPV polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV polyhedrin. The size of the mRNA was approximately 1,000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the polyhedrin mRNA were located. The 5' regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus polyhedrin genes. The SeMNPV polyhedrin mRNA does not appear to be polyadenylated.

Amino Acid Sequence↗
PubMed1991

A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frame-shifting by a mechanism indistinguishable from that of retro-viruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.

Amino Acid Sequence↗
PubMed1997

The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression.

The Bacillus subtilis L-arabinose metabolic genes araA, araB and araD, encoding L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate 4-epimerase, respectively, have been cloned previously and the products of araB and araD were shown to be functionally homologous to their Escherichia coli counterparts by complementation experiments. Here we report that araA, araB and araD, whose inactivation leads to an Ara- phenotype, are the first three ORFs of a nine cistron transcriptional unit with a total length of 11 kb. This operon, called ara, is located at about 256 degrees on the B. subtilis genetic map and contains six new genes named araL, araM, araN, araP, araQ and abfA. Expression of the ara operon is directed by a strong sigma A-like promoter identified within a 150 bp DNA fragment upstream from the translation start site of araA. Analysis of the sequence of the ara operon showed that the putative products of araN, araP and araQ are homologous to bacterial components of binding-protein-dependent transport systems and abfA most probably encodes an alpha-L-arabinofuranosidase. The functions of araL and araM are unknown. An in vitro-constructed insertion-deletion mutation in the region downstream from araD allowed us to demonstrate that araL, araM, araN, araP, araQ and abfA are not essential for L-arabinose utilization. Studies with strains bearing transcriptional fusions of the operon to the E. coli lacZ gene revealed that expression from the ara promoter is induced by L-arabinose and repressed by glucose.

Amino Acid Sequence↗
PubMed1994

The ribosomal RNA gene region in Acanthamoeba castellanii mitochondrial DNA. A case of evolutionary transfer of introns between mitochondria and plastids?

Acanthamoeba castellanii, an amoeboid protozoan, occupies an intriguing position in phylogenetic trees based on nuclear rRNA sequences, branching together with or near (as an outgroup to) green algae and land plants. To gain insight into the organization, expression and evolutionary affiliations of the mtDNA of this non-photosynthetic protist, we determined the sequence of a 7778 base-pair region containing the single-copy large subunit (LSU) and small subunit (SSU) rRNA genes (rnl and rns, respectively) of the approximately 40 kilobase-pair A. castellanii mitochondrial genome. We also sequenced the 5'- and 3'-terminal portions of the corresponding LSU and SSU rRNAs. In A. castellanii mtDNA, rnl is flanked both upstream and downstream by a cluster of five tRNA genes, with rns and then cox1 (the cytochrome oxidase subunit 1 gene) following immediately further downstream. These genes are all in the same transcriptional orientation and are separated by only short non-coding spacers. Although rnl and rns are organized in a novel way in A. castellanii mtDNA, their SSU and LSU rRNA products are strikingly similar to their eubacterial homologs in primary sequence, secondary structure and post-transcriptional modification. In these characteristics, the A. castellanii mitochondrial rRNAs much more closely resemble their counterparts in land plants than do the corresponding mitochondrial rRNAs in the green alga, Chlamydomonas reinhardtii. Although no intervening sequences have so far been found in the mitochondrial rnl of angiosperms (flowering plants), A. castellanii mitochondrial rnl contains three group I introns, all located within highly conserved regions in the 3'-half of the gene and each possessing a free-standing open reading frame (ORF). The insertion site of one of these introns is identical to that of the single group I intron in the chloroplast rnl of C. reinhardtii, and sequence comparison reveals that these two introns (one mitochondrial, the other chloroplast) are structurally homologous both within the core region and within the ORFs they encode. These observations are indicative of intron movement between mitochondria and chloroplasts, either intracellularly in a photosynthetic, remote common ancestor of A. castellanii and C. reinhardtii or, more recently, as a result of an intercellular exchange of genetic information.

Acanthamoeba↗
PubMed1995

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis.

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. The gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).

Amino Acid Sequence↗
PubMed1990

Sequence and expression analysis of potential nonstructural proteins of 4.9, 4.8, 12.7, and 9.5 kDa encoded between the spike and membrane protein genes of the bovine coronavirus.

The nucleotide sequence between the spike and membrane protein genes in the bovine coronavirus (BCV) genome was determined by sequencing cDNA clones of the genome, and open reading frames potentially encoding proteins of 4.9, 4.8, 12.7, and 9.5 kDa, in that order, were identified. The 4.9- and 4.8-kDa proteins appear to be vestiges of an 11-kDa protein for which a single nucleotide deletion event in the central part of the gene gave rise to a stop codon. The consensus CYAAAC sequence precedes the 4.9-, 12.7-, and 9.5-kDa ORFs and predicts that transcription will start from each of these sites. Northern analyses using sequence-specific probes and oligo(dT)-selected RNA demonstrated that the predicted transcripts are made, and that these correspond to mRNAs 4, 5, and 5-1. BCV mRNA 4 appears to be a counterpart to mouse hepatitis virus (MHV) mRNA 4 which, in the MHV JHM strain, encodes the putative 15.2-kDa nonstructural protein. BCV mRNAs 5 and 5-1 appear to be used for the synthesis of the 12.7- and 9.5-kDa proteins, respectively, which demonstrates a pattern of expression strikingly different from that utilized by MHV. MHV makes its homologs of the 12.7- and 9.5-kDa proteins from the single mRNA 5. In vitro translation analyses demonstrated that the BCV 9.5-kDa protein, unlike its MHV counterpart, is poorly made from downstream initiation of translation. Thus, from a comparison between BCV and MHV we find evolutionary evidence for the importance of the CYAAAC sequence in regulating coronavirus transcription.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of a full-length rat sucrase-isomaltase-encoding cDNA.

Sucrase-isomaltase (SI) has been widely used as a marker enzyme to study cellular differentiation in the small intestine. We isolated a 6.1-kb SI cDNA clone (GC1.4) from a size-fractionated cDNA library from rat intestine. Sequencing of this cDNA clone showed 6066 nucleotides (nt) with an open reading frame (ORF) of 1841 amino acids (aa). The nt sequence correctly predicts several known aa stretches in the protein. The deduced as sequence showed 78 and 75% overall identity with the rabbit and human SI, respectively. At the active sites of both S and I, the rat nt sequence encodes stretches of 14 and 16 aa, respectively, which show 100% identity to rabbit and human SI. In the region immediately beyond the transmembrane domain, the rat sequence encodes an extra 10 aa, as compared to rabbit and human. This 10-aa insertion consists almost entirely of Pro, Ser and Thr, and may be responsible for additional O-glycosylations of rat SI. The cDNA contains a 3'-UTR (untranslated region) of 499 nt with polyadenylation signal sequence and a poly(A) tract. The ATG start codon was found 41 nt downstream from the 5' end of the cDNA. Primer extension experiments showed the cap site to be 61 nt upstream from the start codon. The results indicate that our cDNA clone lacks only 20 nt in the 5'-UTR. Given that this cDNA encodes the entire coding region of SI, it should be useful in elucidating the regulatory mechanisms of SI biosynthesis, localization and targeting during rat intestinal development and differentiation.

Amino Acid Sequence↗
PubMed2004

A novel lipase/chaperone pair from Ralstonia sp. M1: analysis of the folding interaction and evidence for gene loss in R. solanacearum.

A microbial strain (referred to as M1) that produces an extracellular lipase was isolated from a soil sample in Vietnam, and identified as a Ralstonia species by partial sequencing of its 16S rDNA. A genomic library was constructed from Pst I fragments, and a colony showing lipase activity was selected for further analysis. Sequencing of the 4.7-kb insert in this clone (named M1-72) revealed one incomplete and three complete ORFs, predicted to encode a partial hypothetical glutaminyl tRNA synthetase (304 aa), a hypothetical transmembrane protein (500 aa), a lipase (328 aa) and a lipase chaperone (352 aa), respectively. Alignment of the insert sequence with the corresponding region of the genome of R. solanacearum GMI1000 (GenBank Accession No. AL646081) confirmed the presence in the latter of the genes for the hypothetical transmembrane protein and glutaminyl tRNA synthetase, which exhibited 89-91% identity to their counterparts in M1. However, R. solanacearum GMI1000 lacks the complete lipase-encoding gene and the major part of the chaperone-encoding gene, creating a so-called "black hole". The deduced amino acid sequences of the products of the lipase gene lipA and chaperone gene lipB from strain M1 shared 49.3-60.3% and 23.9-32.7% identity, respectively, with those of the Burkholderia lipase/chaperone subfamily I.2. lipB is located downstream of lipA, and separated from it by only 9 bp, and each gene has a putative ribosome binding site. The mature lipase LipA, a His-tagged derivative (LipAhis), the tagged full-length chaperone LipBhis and a truncated form (DeltaLipBhis) lacking the 56 N-terminal residues were expressed in Escherichia coli BL21. LipA, LipAhis and DeltaLipBhis could be expressed at high levels (70, 15 and 12 mg/g wet cells, respectively) and were easily purified. However, LipBhis was expressed at a much lower level which precluded purification. The specific activity of purified LipAhis, expressed on its own, was very low (<52 U/mg). However, after co-incubation with the purified DeltaLipBhis in vitro, the specific activity of the enzyme was markedly enhanced, indicating that the chaperone facilitated correct folding of the enzyme. A lipase:chaperone ratio of 1:10 was found to be optimal, yielding an enzyme preparation with a specific activity of 650 U/mg.

Amino Acid Sequence↗
PubMed1993

Structural requirements for efficient translational frameshifting in the synthesis of the putative viral RNA-dependent RNA polymerase of potato leafroll virus.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).

Base Sequence↗
PubMed2000

Enhanced expression of a calcium-dependent protein kinase from the moss Funaria hygrometrica under nutritional starvation.

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss Funaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA of Funaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in the Funaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene from Funaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of the Funaria gene shows extensive homology with CDPKs from higher plants, 73% identity with the Fragaria CDPK and 71% identity with CDPK isoform 7 of Arabidopsis. Phylogenetic analysis revealed that the Funaria CDPK is closer to the CDPKs from higher plants like strawberry and Arabidopsis as compared to those from lower plants such as the liverwort Marchantia, the green alga Chlamydomonas or another moss Tortula. Northern analysis shows enhanced expression of the CDPK transcript within 24-48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.

Amino Acid Sequence↗
PubMed1993

The nac (nitrogen assimilation control) gene from Klebsiella aerogenes.

The Klebsiella aerogenes nac gene, whose product is necessary for nitrogen regulation of a number of operons, was identified and its DNA sequence determined. The nac sequence predicted a protein a 305 amino acids with a strong similarity to members of the LysR family of regulatory proteins, especially OxyR from Escherichia coli. Analysis of proteins expressed in minicells showed that nac is a single-gene operon whose product has an apparent molecular weight of about 32 kDa as measured in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immediately downstream from nac is a two-gene operon, the first gene of which encodes another member of the LysR family. Upstream from nac is a tRNAAsn gene transcribed divergently from nac. About 60 bp upstream from the nac open reading frame lies a sequence nearly identical to the consensus for sigma 54-dependent promoters, with the conserved GG and GC nucleotides at -26 and -14 relative to the start of transcription. About 130 bp farther upstream (at -153 relative to the start of transcription) is a sequence nearly identical to the transcriptional activator NTRC-responsive enhancer consensus. Another weaker NTRC-binding site is located adjacent to this site (at -133 relative to the start of transcription). Thus, we propose that nac is transcribed by RNA polymerase carrying sigma 54 in response to the nitrogen regulatory (NTR) system. A transposon located between the promoter and the nac ORF prevented NTR-mediated expression of nac, supporting this identification of the promoter sequence. The insertion of over 5 kb of transposon DNA between the enhancer and its target promoter had only a weak effect on enhancer-mediated regulation, suggesting that enhancers may be able to act at a considerable distance on the bacterial chromosome.

Amino Acid Sequence↗