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PubMed2006

Identification of Leishmania selenoproteins and SECIS element.

Selenoproteins result from the incorporation of selenocysteine (Sec-U) at an UGA-stop codon positioned within a gene's open reading frame and directed by selenocysteine insertion sequence (SECIS) elements. Although the selenocysteine incorporation pathway has been identified in a wide range of organisms it has not yet been reported in the Kinetoplastida Leishmania and Trypanosoma. Here we present evidence consistent with the presence of a selenocysteine biosynthetic pathway in Kinetoplastida. These include the existence of SECIS-containing coding sequences in Leishmania major and Leishmania infantum, the incorporation of (75)Se into Leishmania proteins, the occurrence of selenocysteine-tRNA (tRNA (sec) (uca)) in both Leishmania and Trypanosoma and in addition the finding of all genes necessary for selenocysteine synthesis such as SELB, SELD, PSTK and SECp43. As in other eukaryotes, the Kinetoplastids have no identifiable SELA homologue. To our knowledge this is the first report on the identification of selenocysteine insertion machinery in Kinetoplastida, more specifically in Leishmania, at the sequence level.

Amino Acid Sequence↗
PubMed2026

Systematic mapping of insertion-tolerant regions enables capsid engineering of an infectious RNA phage.

RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 non-redundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein, whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from complementary DNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an Arg-Gly-Asp motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an infectious RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications.IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an infectious RNA phage, to our knowledge. This study lays the groundwork for the rational design of live RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.

Animals↗
PubMed1989

The nucleotide sequence of the first two genes of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli.

An oligonucleotide probe, derived from the N-terminal amino acid sequence of the CFA/I fimbrial subunit protein, was used to identify the gene encoding this protein within a cloned DNA fragment encoding CFA/I fimbriae. The gene (cfa b) was found and sequenced. Flanking it upstream was a gene (cfa a) encoding a protein of 206 amino acids and downstream a gene (cfa c) probably encoding an 85 kDa protein was found. This genetic organisation of the CFA/I operon differs from that of other fimbrial operons in Escherichia coli. All three proteins have signal peptides. The nucleotide sequence was analysed for homology with other sequences, secondary structure, ribosomal binding sites and possible promoter sequences. A region of dyad symmetry probably involved in the regulation of translation of the cfa c gene was found at the 5' end of this gene. A region of dyad symmetry was also observed within the cfa b gene. In front of the CFA/I operon part of insertion sequence IS2 was found. This IS2 sequence was found in a number of CFA/I plasmids, obtained from strains isolated from various geographic locations. The insertion of the IS2 element in the CFA/I operon therefore probably happened rather early during evolution of CFA/I producing Escherichia coli strains.

Amino Acid Sequence↗
PubMed1997

A cottontail rabbit papillomavirus strain (CRPVb) with strikingly divergent E6 and E7 oncoproteins: An insight in the evolution of papillomaviruses.

We previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.5% identical amino acids and differed in size by three and two amino acids, respectively. This divergence involved (i) a great number (4.4%) of nucleotide substitutions and a high rate (83.3%) of nonsynonymous mutations; (ii) mutations changing the E6 and E7 stop codons; and (iii) in-frame sequence insertions in the E6 ORF (18 nucleotides) and downstream of the mutated E7 stop codon (6 nucleotides), both likely to result from a duplication of adjacent sequences. These extensive differences could account for distinct biological and antigenic properties. Strikingly, only four (0.8%) amino acids of the L1 major capsid protein were variable. Thus, it seems likely that sequence duplications and mutations affecting stop codons exert a strong selection pressure on the fixation of nonsynonymous mutations and that phylogenetic calculations based only on point mutations may misevaluate the time scale of the evolution of papillomaviruses.

Amino Acid Sequence↗
PubMed2003

Insights into the genetic organization of the Corynebacterium diphtheriae erythromycin resistance plasmid pNG2 deduced from its complete nucleotide sequence.

The complete nucleotide sequence of the erythromycin resistance plasmid pNG2 from the human pathogen Corynebacterium diphtheriae S601 was determined. The plasmid has a total size of 15,100 bp and contains at least 17 coding regions. Comparative genomics identified conserved motifs within replication initiator proteins of corynebacterial plasmids and a novel nucleotide sequence feature, termed 22-bp box, located downstream of the repA gene. The erythromycin resistance determinant erm(X) is flanked by inverted repeats of the novel insertion sequence IS3504, which may be responsible for a spontaneous deletion of the antibiotic resistance gene region. Furthermore, pNG2 encodes a putative conjugative relaxase, a membrane protein of the natural resistance-associated macrophage protein (Nramp) family and a protein with Nudix hydrolase signature. Expression of the predicted coding regions of pNG2 in Escherichia coli JM109 was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) assays. The detailed annotation of the entire pNG2 sequence provided genetic information regarding its molecular evolution and its role in dissemination of antibiotic resistance genes by horizontal gene transfer.

Amino Acid Sequence↗
PubMedDate not supplied

Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2.

The subtractive suppressive hybridization (SSH), a method that allows the identification of sequences that are present in one genome (tester) but not in the other (driver), is a promising technique for the comparison of Mycoplasma agalactiae pathogenic strains. The optimal conditions for SSH were established by subtracting the M. agalactiae type strain PG2 DNA from the M. agalactiae strain 5632 DNA. Because these two strains possess different vpma gene repertoires, 5632-specific vpma sequences (and possibly other 5632-specific sequences) were predicted to be retrieved by SSH. The subtracted tester DNA was PCR-amplified and cloned into the pGEM-T easy E. coli vector. Two independent libraries were generated and used to prepare individual probes that were tested by Southern blot with genomic DNA from various field isolates and mycoplasma reference strains. Sequence analysis of two overlapping clones showed that they potentially code for a large carboxyterminal portion of a new vpma ORF. Several DNA fragments homologous to insertion sequences were also found in 5632 and related strains. These preliminary data suggest that SSH is a powerful method to investigate differences between mycoplasma strains, and may be applied to molecular epidemiology, diagnostic, and host specificity or pathogenicity determinant discovery.

Animals↗
PubMed2000

Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region.

In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene.

Animals↗
PubMed1998

Structural and functional analysis of the gene cluster encoding the enzymes of the arginine deiminase pathway of Lactobacillus sake.

Lactobacillus sake can use arginine via the arginine deiminase (ADI) pathway. We designed degenerate primers based on an alignment of known sequences of ornithine transcarbamoylase (OTC)-encoding genes in order to amplify the L. sake counterpart sequences by PCR. Screening a genomic library of L. sake in lambdaEMBL3 allowed us to isolate a clone containing a 10-kb L. sake genomic DNA insert. Sequence analysis revealed that the genes involved in arginine catabolism were clustered and encoded ADI (arcA), OTC (arcB), carbamate kinase (arcC), and a putative carrier with high similarity to the arginine/ornithine antiporter of Pseudomonas aeruginosa (arcD). Additionally, a putative transaminase-encoding gene (arcT) was located in this region. The genes followed the order arcA arcB arcC arcT arcD, which differs from that found in other microorganisms. arcA, arcB, arcC, and arcD mutants were constructed, and the ADI pathway was impaired in all of them. Transcriptional studies indicated that arcA gene is subject to catabolite repression, and under the conditions used, several transcripts could be detected, suggesting the existence of different initiation sites or processing of a larger mRNA.

Bacterial Proteins↗
PubMed2003

Transcription of quorum-sensing system genes in clinical and environmental isolates of Pseudomonas aeruginosa.

Quorum sensing (QS)-based transcriptional responses in Pseudomonas aeruginosa have been defined on the basis of increases in transcript levels of QS-controlled genes such as lasB and aprA following the hierarchical transcriptional increases of central controllers such as the lasR gene. These increases occur at high bacterial concentrations such as early-stationary-phase growth in vitro. However, the extent to which the increases occur in a variety of clinical and environmental isolates has not been determined nor is there extensive information on allelic variation in lasR genes. An analysis of the sequences of the lasR gene among 66 clinical and environmental isolates showed that 81% have a sequence either identical to that of strain PAO1 or with a silent mutation, 15% have nucleotide changes resulting in amino acid changes, and 5% have an insertion sequence in the lasR gene. Using real-time PCR to quantify transcript levels of lasR, lasB, and aprA in the early log and early stationary phases among 35 isolates from bacteremia and pneumonia cases and the environment, we found most (33 of 35) strains had increases in lasR transcripts in early stationary phase but with a very wide range of final transcript levels per cell. There was a strong correlation (r(2) = 0.84) between early-log- and early-stationary-phase transcript levels in all strains, but this finding remained true only for the 50% of strains above the median level of lasR found in early log phase. There were significant (P < 0.05) but weak-to-modest correlations of lasR transcript levels with aprA (r(2) = 0.2) and lasB (r(2) = 0.5) transcript levels, but again this correlation occurred only in the 50% of P. aeruginosa strains with the highest levels of lasR transcripts in early stationary phase. There were no differences in distribution of lasR alleles among the bacteremia, pneumonia, or environmental isolates. Overall, only about 50% of P. aeruginosa strains from clinical and environmental sources show a lasR-dependent increase in the transcription of aprA and lasB genes, indicating that for about 50% of clinical isolates this regulatory system may not play a significant role in pathogenesis.

Bacterial Proteins↗
PubMed1995

Rapid methods for population-scale analysis for gene polymorphisms: the ACE gene as an example.

OBJECTIVE: To obtain rapid, high throughput genotyping of the angiotensin converting enzyme (ACE) gene intron 16 insertion/deletion polymorphism. METHODS: DNA was obtained from whole blood samples by a simple liquid phase methanol extraction procedure. The ACE gene was amplified by the polymerase chain reaction (PCR) using two oligonucleotide primers (ACE1 and ACE3) outside the insertion sequence and one primer (ACE2) inside the sequence. Microtitre array diagonal gel electrophoresis (MADGE) was used to determine genotypes. RESULTS: 84 and 65 bp PCR products indicating the presence of deletion (D) and insertion (I) alleles, respectively, were clearly resolved after electrophoresis on a 7.5% polyacrylamide gel. Up to 480 DNA samples on 5 gels could be genotyped in a single electrophoresis run, or up to 1000 samples in a working day. CONCLUSIONS: A simplified DNA extraction protocol coupled to the high throughput capability of the MADGE electrophoretic system for genotyping enables analysis of large populations for association studies of ACE genotype with cardiac disease events.

Alleles↗
PubMed1982

Transposon Tn10: genetic organization, regulation, and insertion specificity.

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.

Base Sequence↗
PubMed2003

Cowpea mosaic virus-based chimaeras. Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses.

Expression of foreign peptides on the surface of cowpea mosaic virus particles leads to the creation of chimaeras with a variety of phenotypes and yields. Two factors were shown to be particularly significant in determining the properties of a given chimaera: the length of the inserted sequence and its isoelectric point. The deleterious effect of high isoelectric point on the ability of chimeras to produce a systemic infection occurs irrespective of the site of insertion of the peptide. Ultrastructural analysis of tissue infected with chimaeras with different phenotypes showed that all produced particles with a tendency to aggregate, irrespective of the size or isoelectric point of the insert. Host range and transmission studies revealed that the expression of a foreign peptide did not (1) alter the virus host range, (2) increase the rate of transmission by beetles or through seed, or (3) change the insect vector specificity. These findings have implications for both the utility and the biosafety of Cowpea mosaic virus-based chimaeras.

Amino Acid Sequence↗
PubMed1993

Glycophorin B and glycophorin E genes arose from the glycophorin A ancestral gene via two duplications during primate evolution.

Human glycophorin A, B, and E genes are homologous from the 5'-flanking region to 1 kilobase downstream from the exon encoding the transmembrane region. Analysis of human Alu sequences at the transition site from the homologous to nonhomologous region suggested that the GPA gene most closely resembles the ancestral gene, whereas GPB and GPE genes arose by homologous recombination within the Alu repetitive sequence, and acquired 3' sequences from an unrelated gene (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623; Kudo, S., and Fukuda, M. (1990) J. Biol. Chem. 265, 1102-1110). To understand glycophorin gene evolution in primate phylogeny, transmembrane and Alu regions of several primate genomes were amplified by the polymerase chain reaction and their sequences were analyzed. These studies revealed that the GPA gene was present in all primates studied, and the GPB gene was present in pygmy chimpanzee, chimpanzee, and gorilla, but absent from orangutan and gibbon. GPE gene was present in all species with a GPB gene, but was detected in only 7 out of 16 gorillas. The 24-base pair insertion sequence found in the transmembrane exon of the human GPE gene was shown to be derived from the ancestral GPB gene and was inserted into the ancestral GPE gene prior to gorilla divergence. The recombination site in the GPA gene was confirmed to be within an Alu repetitive sequence. We conclude that GPB and GPE genes arose from an ancestral GPA gene via two gene duplications occurring during primate evolution, prior to gorilla divergence.

Amino Acid Sequence↗
PubMed1995

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.

Base Sequence↗
PubMed2001

Type II iodothyronine deiodinase is preferentially expressed in rainbow trout (Oncorhynchus mykiss) liver and gonads.

It is well admitted that thyroid hormones (TH) play a role in the development of vertebrates. The major secretory product of the thyroid is a pro-hormone, T(4), which is activated in peripheral tissues by outer ring deiodination to T(3). We have isolated from rainbow trout testis, a full length cDNA encoding type II iodothyronine deiodinase (rtD2). The cDNA was 2410 nucleotides long and coded for a polypeptide of 264 amino acids including a selenocysteine residue. The predicted molecular weight of rtD2 was 29.3 kDa and the isoelectric point 8.71. The deduced amino acids sequence showed 80% identity with Fundulus heteroclitus D2 (fhD2) but only 68-69% identity with rat, mouse, and human D2. The 3' UTR contained a putative selenocysteine insertion sequence (SECIS) similar to that described in human cDNA. The rtD2 gene was isolated and the gene structure was similar to that described in human with two exons separated by a large intron. We studied rtD2 gene expression by Northern blot analysis using total RNA extracted from testis, ovary, and other tissues. We found a high expression of a 3 kb transcript in liver and in gonads. A lower expression was also detected in posterior kidney. In testis, rtD2 mRNA expression was dependent on spermatogenic stages: it increased at the onset of spermatogenesis. Our results show that the structural characteristics of the D2 protein and gene have been highly conserved during evolution. The rtD2 mRNA expression in the gonads suggests that rtD2 may be a key factor regulating local supply of active T(3) during rainbow trout gametogenesis.

3' Untranslated Regions↗
PubMed1985

Isomerization of herpes simplex virus 1 genome: identification of the cis-acting and recombination sites within the domain of the a sequence.

Previous studies have shown that the a sequence located at the termini and at the junction between the L and S components is the site-specific, cis-acting sequence mediating the inversions of herpes simplex virus 1 DNA. We constructed mutated a sequences, inserted them into the thymidine kinase gene, and recombined them into the L component of the viral genome. Deletion of Uc or Ub domains of the a sequence did not affect inversions, whereas the deletion of direct repeat #4 (DR4) drastically reduced their frequency. Deletion of both direct repeat #2 (DR2) and DR4 abolished inversions. Recombinational events leading to inversions appear to occur through DR2, and possibly DR4. These results complement previous studies showing that most of one DR1 sequence can also be dispensed with and are consistent with the hypothesis that DR4 and possibly DR2 are the cis-acting sites for the inversions mediated by the a sequence.

Base Sequence↗
PubMed2000

Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.

3' Untranslated Regions↗