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PubMed2005

Plasmodium vivax: recent world expansion and genetic identity to Plasmodium simium.

Plasmodium vivax causes the most geographically widespread human malaria, accounting annually for 70-80 million clinical cases throughout the tropical and subtropical regions of the world's continents. We have analyzed the DNA sequences of the Csp (circumsporozoite protein) gene in 24 geographically representative strains of P. vivax and 2 of P. simium, which parasitizes several species of New World monkeys. The Csp sequences are of two types, VK210 and VK247, which differ by three diagnostic amino acid replacements, one in each of the 5' and 3' terminal regions [5' nonrepeat (NR) and 3' NR] of the gene and in an insertion sequence that precedes the 3' NR region. The central region of the gene consists of approximately 38 repetitive "motifs," which are alternatively four and five amino acids long, which also are diagnostically different between the VK210 and VK247 types. There are very few synonymous substitutions within and between the two types of strains, which we hypothesize reflects that the worldwide spread of P. vivax is very recent. The two P. simium Csp sequences belong one to each of the two VK types and are genetically indistinguishable from the corresponding P. vivax strains, suggesting that at least two host transfers have occurred between humans and New World monkeys. We exclude as unlikely the possibility that the two types of sequences could have independently arisen in humans and platyrrhines by natural selection. There are reasons favoring each of the two possible directions of host transfer between humans and monkeys.

Animals↗

PubMed1996

A replication-enhancing element with transcriptional silencer activity in autonomously replicating human chromosomal fragments.

We have identified specific nucleotide sequences involved in autonomous replication of human chromosomal fragments in human cells. Nested deletion analysis of a 10.2-kb long human chromosomal fragment showed that replication efficiency of the fragment was reduced to about 50% by loss of a short specific segment. Deletions outside the segment reduced the replication efficiency depending on their lengths. By introducing linker substitutions, we found that the distinct segment required for the efficient replication consisted of an 18-bp sequence, named REE1 (Replication Enhancing Element 1). Single or tandem copies of REE1 alone had no significant replication activity, but they stimulated replication of human chromosomal DNA fragments. We found, in addition, that the REE1 sequence inserted at a site 2.7 kb upstream of the SV40 early promoter caused repression of transcription from the promoter, suggesting that REE1 had a transcriptional silencer activity. Introduction of linker substitutions into the REE1 indicated that the nucleotide sequences required for the repression of transcription were the same as those for enhancement of replication. Thus, REE1 is responsible for both enhancement of replication and repression of transcription.

Base Sequence↗

PubMed1999

A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF).

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3'-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA(Sec) is not recognized by elongation factor EF-1alpha, but is recognized specifically by a Sec-tRNA(Sec) protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA(Sec), providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.

Animals↗

PubMed1998

Identification and characterization of IS1302, a novel insertion element from Wolinella succinogenes belonging to the IS3 family.

A new insertion sequence (IS) designated IS1302 was identified in Wolinella succinogenes. IS1302 is 1,306 bp in size with 36-bp imperfect terminal inverted repeats. It contains only one open reading frame (tnpA), which encodes a putative transposase whose sequence is similar to that of transposases of various IS elements of the IS3 family. IS1302 was identified in the genome of a W. succinogenes fumarate reductase deletion mutant in which the frd operon had been replaced by the kan gene. The insertion of IS1302 occurred when the mutant was propagated in the presence of a high concentration of kanamycin. Two different target sites of IS1302 were found immediately upstream of the kan gene, where the insertion of IS1302 resulted in a duplication of 3 bp of the target DNA. Upon insertion of IS1302, new possible promoter structures of the kan gene were created, which might lead to a stimulated transcription of the kan gene and result in a selective advantage of cells containing IS1302 at one of the two target sites. Southern blot analysis suggested the presence of at least 13 copies of IS1302 in the genome of W. succinogenes. This is the first IS element discovered in W. succinogenes.

Amino Acid Sequence↗

PubMed1994

Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition?

The transposase of the bacterial insertion sequence IS1 is normally expressed by inefficient translational frameshifting between an upstream reading frame which itself specifies a transposition inhibitor, InsA, and a second consecutive reading frame located immediately downstream. A fused-frame mutant which carries an additional base pair inserted at the point of frameshifting was constructed. This mutant exhibits high transposition activity and should express the transposase, InsAB', constitutively without frameshifting. Unexpectedly, a second protein species was observed to be expressed from this mutant. We demonstrate here that this protein, InsA*, results from continued frameshifting on the modified frameshift motif. The protein retains the activities of the repressor InsA. Its elimination, by further modification of the frameshift motif, results in a further increase in various transposition activities of IS1. These results support the hypothesis that a single IS1-encoded protein, InsAB', is necessary for transposition.

Bacterial Proteins↗

PubMed1995

Structure and distribution of specific cis-elements for transcriptional regulation of PHO84 in Saccharomyces cerevisiae.

Transcription of the PHO84 gene encoding a Pi transporter in Saccharomyces cerevisiae is regulated by the Pi concentration in the medium. The promoter region of PHO84 bears five copies of the motif 5'-CACGT(G/T)-3', a candidate for the upstream activation site (UAS) that binds the transcriptional activator protein of the phosphatase regulon, Pho4p. These motifs are found at nucleotides -880 (site A), -587 (B), -436 (C), -414 (D), and -262 (E) relative to the putative ATG codon of PHO84. The Pho4p binds to all five 6-bp motifs with various affinities. Deletion analysis of the PHO84 promoter using a PHO84-lacZ fusion gene and base substitutions in the 6-bp motif revealed that two copies of the 6-bp motif, either C or D, and E, are necessary and sufficient for full regulation of the PHO84 gene. Results of expression studies with a CYC1-lacZ fusion gene with various 36-bp oligonucleotides including the 30-bp sequences around site D or E, or with modified sequences, inserted in the CYC1 promoter region indicated that the 6-bps motif flanked by a thymine nucleotide at its 5' end is much less effective as a UAS site for Pho4p in vivo than other versions. Thus, the consensus sequences for phosphatase regulation are 5'-GCACGTGGG-3' and 5'-GCACGTTTT-3' which differ from the binding sequences for the Cpflp protein required for transcription of the genes in methionine biosynthesis and for centromere function. However, Pho4p binding in vitro was unaffected by modification of the 5' or 3' flanking sites of the 6-bp motif, while modification inside the 6-bp motif affected it severely. The UAS function of the GCACGTTTT motif with respect to the Pi signal depends on its orientation in the promoter sequence.

Base Sequence↗

PubMed1982

Heterochromatin markers: a search for heterochromatin specific middle repetitive sequences in Drosophila.

We sought for cloned sequences of middle repetitive (MR) complexity that mark obligatory heterochromatic regions. Total genome probes were employed in a differential screening procedure to recover X-specific, Y-specific and autosomal specific heterochromatic sequences. X-and Y-linked sequences were recovered in the same experiment. (Y-linked clones will be described elsewhere). All nine independent, non-identical X-specific clones were found to be partially homologous to one another and to type I rDNA insertion. No other X-specific Bam HI or HindIII clones were found. In situ hybridization to normal and inverted chromosomes revealed extensive homology in the heterochromatin spanning the nucleolus organizer (NOR) and the eu-heterochromatin junction. Eleven clones which are underrepresented in polytene chromosomes were selected in another differential screening. None was autosome-specific. Five were of nucleolar origin. Among them a presumptive type II 28SrDNA insertion sequence was clearly localized within the X-chromosome proximal heterochromatin in addition to the known localization of the X and Y nucleolar organizers. We mapped three clones to major sites on the Y chromosome and to secondary autosomal sites. The results are discussed with regard to the complexity of heterochromatin organization.

Animals↗

PubMed2003

The alphavbeta5 integrin of hematopoietic and nonhematopoietic cells is a transduction receptor of RGD-4C fiber-modified adenoviruses.

Epithelial and endothelial cells expressing the primary Coxsackie virus B adenovirus (Ad) receptor (CAR) and integrin coreceptors are natural targets of human Ad infections. The fiber knob of species A, C, D, E and F Ad serotypes binds CAR by mimicking the CAR-homodimer interface, and the penton base containing arginine-glycine-aspartate (RGD) motifs binds with low affinity to alphav integrins inducing cell activation. Here, we generated seven different genetically modified Ad vectors with RGD sequences inserted into the HI loop of fiber knob. All mutants bound and infected CAR and alphav integrin-positive epithelial cells with equal efficiencies. However, the Ads containing two additional cysteines, both N and C terminals of the RGD sequence (RGD-4C), were uniquely capable of transducing CAR-less hematopoietic and nonhematopoietic human tumor cell lines and primary melanoma cells. Both binding and transduction of RGD-4C Ad were blocked by soluble RGD peptides. Flow cytometry of cell surface integrins and virus binding to CAR-less cells in the presence of function-blocking anti-integrin antibodies indicated that the alphavbeta5 integrin, but not alphavbeta3, alphaIIbbeta3 or beta1,alpha5 or alpha6-containing integrins served as a functional transduction receptor of the RGD-4C Ads. However, in cells with low levels of alphavbeta5 integrin, the function-blocking anti-alphavbeta5 antibodies were not effective, unlike soluble RGD peptides. Collectively, our data demonstrate that the alphavbeta5 integrin is a functional transduction receptor of RGD-4C Ads in the absence of CAR, and that additional RGD receptors are targets of these viruses. The RGD-4C vectors further extend the tropism of Ads towards potential human therapies.

Adenoviridae↗

PubMed1998

Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147.

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.

Bacteriocins↗

PubMed2003

Sequences that direct significant levels of frameshifting are frequent in coding regions of Escherichia coli.

It is generally believed that significant ribosomal frameshifting during translation does not occur without a functional purpose. The distribution of two frameshift-prone sequences, A_AAA_AAG and CCC_TGA, in coding regions of Escherichia coli has been analyzed. Although a moderate level of selection against the first sequence is evident, 68 genes contain A_AAA_AAG and 19 contain CCC_TGA. The majority of those tested in their genomic context showed >1% frameshifting. Comparative sequence analysis was employed to assess a potential biological role for frameshifting in decoding these genes. Two new candidates, in pheL and ydaY, for utilized frameshifting have been identified in addition to those previously known in dnaX and nine insertion sequence elements. For the majority of the shift-prone sequences no functional role can be attributed to them, and the frameshifting is likely erroneous. However, none of frameshift sequences is in the 306 most highly expressed genes. The unexpected conclusion is that moderate frameshifting during expression of at least some other genes is not sufficiently harmful for cells to trigger strong negative evolutionary pressure.

Base Sequence↗

PubMed1996

The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators.

The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis.

Amino Acid Sequence↗

PubMed2003

Purification and characterization of alpha-galactosidase from sunflower seeds.

From 100 g sunflower seeds, 1.2 mg purified alpha-galactosidase was obtained with an overall yield of 51%. The alpha-galactosidase acted on both terminal alpha-galactosyl residues and side-chain alpha-galactosyl residues of the galactomanno-oligosaccharides and galactomannans. The cDNA coding for sunflower alpha-galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40,263. Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence. The deduced amino acid sequences of mature enzyme and alpha-galactosidases from coffee, guar and Mortierella vinacea alpha-galactosidase II showed over 81%, 77%, and 47% homology, respectively. These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups.

Amino Acid Sequence↗

PubMed1998

Structure and function of the Bacillus hybrid enzyme GluXyn-1: native-like jellyroll fold preserved after insertion of autonomous globular domain.

The 1,3-1,4-beta-glucanase from Bacillus macerans (wtGLU) and the 1, 4-beta-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 A resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

Bacillus↗

PubMed1995

A novel cis-acting element in a liver cytochrome P450 3A gene confers synergistic induction by glucocorticoids plus antiglucocorticoids.

The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16 alpha-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5'-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA.

Animals↗

PubMed1993

DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco.

DST elements are highly conserved sequences located in the 3' untranslated regions (UTRs) of a set of unstable soybean transcripts known as the small auxin-up RNAs (SAURs). To test whether DST sequences could function as mRNA instability determinants in plants, a model system was developed to facilitate the direct measurement of mRNA decay rates in stably transformed cells of tobacco. Initial experiments established that the chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) transcripts degraded with similar half-lives in this system. In addition, their decay kinetics mirrored the apparent decay kinetics of the corresponding transcripts produced in transgenic plants under the control of a regulated promoter (Cab-1). The model system was then used to measure the decay rates of GUS reporter transcripts containing copies of the DST sequence inserted into the 3'UTR. An unmodified CAT gene introduced on the same vector served as the internal reference. These experiments and a parallel set utilizing a beta-globin reporter gene demonstrated that a synthetic dimer of the DST sequence was sufficient to destabilize both reporter transcripts in stably transformed tobacco cells. The decrease in transcript stability caused by the DST sequences in cultured cells was paralleled by a coordinate decrease in transcript abundance in transgenic tobacco plants. The implications of these results for the potential function of DST sequences within the SAUR transcripts are discussed.

Base Sequence↗

PubMed1995

High-level expression of recombinant pea chloroplast fructose-1,6-bisphosphatase and mutagenesis of its regulatory site.

The cDNA fragment coding for mature chloroplast pea fructose-1,6-bisphosphatase [Fru(1,6)P2ase] was introduced by PCR into the expression vector pET-3d resulting in the construction pET-FBP. After transformation of BL21 (DE3) Escherichia coli cells by the pET-FBP plasmid and induction with isopropyl thio-beta-D-galactoside, high-level expression of the recombinant enzyme was achieved. The protein could be purified in three days by a simple procedure which includes heat treatment, ammonium sulfate fractionation, DEAE Sephacel and ACA 44 chromatographies with a yield of 20 mg/l culture. In every respect, the recombinant enzyme was similar to plant chloroplast Fru(1,6)P2ase and, in particular, its reactivity with Mg2+ and redox regulatory properties were conserved. In a second series of experiments based on three-dimensional modeling of the chloroplast protein and sequence alignments, two cysteine residues of the recombinant enzyme (Cys173 and Cys178) were mutated into serine residues. An active enzyme, which did not respond to thiol reagents and to light activation, was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplast enzyme.

Amino Acid Sequence↗

PubMed2006

Internal promoter characterization and expression of the Deinococcus radiodurans pprI-folP gene cluster.

PprI is a general gene switch responsible for the extraordinary radioresistance of Deinococcus radiodurans. From NCBI DNA sequence analysis, it was predicted that the translation start codon of the downstream folP (DR0168) gene overlaps the pprI (DR0167) stop codon, suggesting that these genes may form an operon. In this study, we show that a mutant containing an inserted sequence in folP does not grow unless folate is added to the medium, but is not affected in extreme radioresistance, whereas a pprI disruptant strain could grow in the absence of folate. It was found that expression of a pprI-lacZ fusion is constitutive and unaltered following ionizing radiation as is the production of the PprI protein. PprI protein is not expressed if its promoter is deleted and the transcription from the entire pprI promoter is essential for radioresistance of D. radiodurans. However, the deletion of pprI promoter has no effect on the expression of the folP-lacZ fusion. Primer extension analysis of the folP promoter region shows that folP is transcribed from its own promoter located within the pprI structural gene. All these results do support neither the existence of a pprI-folP operon nor a regulatory role of FolP in pprI expression.

Bacterial Proteins↗

PubMed1999

Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the Shigella flexneri chromosome.

The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.

Chromosomes, Bacterial↗