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PubMed1998

Human apolipoprotein B RNA editing deaminase gene (APOBEC1).

Genomic clones encoding the human APOBEC1 gene and its 5' flanking region have been isolated and characterized. The human gene contains five coding exons. The introns dividing these exons correspond exactly to those found in the mouse gene. The translation initiation site, ATG, is located in exon 2 at the same site as in the mouse. The 5' flanking sequence contains two Alu repeats of the Sq family. Primer extension analysis demonstrated the presence of two major transcription initiation sites. The first transcription initiation site delineates the beginning of a noncoding first exon and resides downstream of the first Alu sequence. The second transcription initiation site is within the second Alu repeat. This Alu repeat resides within the first intron, which is spliced out of the transcript from the first start site. Neither transcription initiation site has a TATA or CCAT box. Comparison with the mouse gene suggests that the Alu sequence insertion split the intestinal promoter and that subsequently the down-stream Alu sequence took on a promoter function. No evidence was found for a far upstream non-tissue-specific promoter similar to that demonstrated in the mouse gene. Rather, consideration of results from the marsupial APOBEC-1 gene suggests that this upstream mouse promoter may have had a later evolutionary origin.

APOBEC-1 Deaminase↗

PubMed1995

A transposon-like sequence with short terminal inverted repeats in the nuclear genome of Chlamydomonas reinhardtii.

A 1.2 kb DNA sequence, flanked by a potential seven base target-site duplication, was found inserted into a TOC1 transposable element from Chlamydomonas reinhardtii. The insertion sequence, named TOC2, is a member of a family of repeated DNA sequences that is present in all the C. reinhardtii strains tested. It resembles class II transposable elements: it possesses short 14 bp imperfect terminal repeats that begin AGGAGGGT, and sub-terminal direct repeats located within 250 bp of the termini. No large open reading frames were found. The terminal bases and length of target-site duplication are important in classifying transposable elements. On this basis TOC2 does not fall readily into existing families of class II transposable elements found in plants.

Animals↗

PubMed1995

Long-range interaction between two promoters: activation of the leu-500 promoter by a distant upstream promoter.

The leu-500 mutation can be suppressed in S. typhimurium topA. Previous studies have demonstrated that the plasmid-borne leu-500 minimal promoter cannot be activated in topA mutants unless adjacent (< 250 bp) transcription occurs away from the leu-500 promoter (short-range promoter interaction). To search for a potential upstream promoter responsible for activation of leu-500 in the chromosomal context, we have identified the ilvlH promoter, located 1.9 kb upstream of leu-500 (long-range promoter interaction). Different from short-range promoter interaction, which is abolished by DNA sequence insertions, the long-range promoter interaction is mediated by the intervening DNA sequence. These studies suggest that the long-range interaction between a pair of divergently arrayed promoters is probably mediated by a complex process involving relay of DNA supercoiling by the DNA sequence located between the two promoters.

Base Sequence↗

PubMed1990

Current methods for YAC clone characterization.

Yeast artificial chromosome (YAC) cloning has allowed isolation of much longer DNA fragments than was previously possible. While this technology makes feasible the isolation of large regions of complex genomes in overlapping cloned segments, it has also required altered methods for manipulation of cloned DNAs. In particular, the isolation of insert sequences from YAC clones has been especially difficult. Methods for characterization of YAC inserts are described and compared.

Chromosomes, Fungal↗

PubMed2002

Allelic variation in the highly polymorphic locus pspC of Streptococcus pneumoniae.

PspC, also called SpsA, CbpA, PbcA, and Hic, is a surface protein of Streptococcus pneumoniae studied for its antigenic properties, its capability to bind secretory IgA, C3 and complement factor H, and its activity as an adhesin. In this work we characterized the pspC locus of 43 pneumococcal strains by DNA sequencing of PCR fragments. Using PCR primers designed on two unrelated open reading frames, flanking the pspC locus, it was possible to amplify the pspC locus of each of the 43 strains of S. pneumoniae. In 37 out of 43 strains there was a single copy of the pspC gene, while two tandem copies of pspC were found in the other six strains. The sequence of the pspC locus was different in each of the 43 strains. Insertion sequences were found in the pspC locus of 11 out of 43 strains. Analysis of the deduced amino acid sequence of the PspC variants showed a common organization of the molecules: (i) a 37 amino acid leader peptide which is conserved in all proteins, (ii) an N-terminal portion which is essentially alpha-helical, and is the result of assembly of eight major sequence blocks, (iii) a proline-rich region, and (iv) a C-terminal anchor responsible for the cell surface attachment. By sequence comparison we identified 11 major groups of PspC proteins. Proteins within one group displayed only minor variations of the amino acid sequence. An unexpected finding was that PspC variants could differ in the anchor sequence. While 32 of the PspC proteins displayed the typical choline binding domain of pneumococcal surface proteins, 17 other PspCs showed the LPXTG motif, which is typical of surface proteins of other gram-positive bacteria. This major difference in the anchor region was also observed in the adjacent proline-rich regions which differed considerably in size and composition.

Alleles↗

PubMed1995

A multiplex polymerase chain reaction for distinguishing Mycobacterium tuberculosis from Mycobacterium tuberculosis complex.

A multiplex polymerase chain reaction has been developed which is able to distinguish Mycobacterium tuberculosis from other members of the M. tuberculosis complex. The assay is based on the simultaneous amplification of two different targets: a 396bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 245bp fragment from the M. tuberculosis complex insertion sequence IS986. Results have been obtained for 54 mycobacterial strains including five non-M. tuberculosis complex isolates. All 49 strains of the M. tuberculosis complex were positive for IS986 but only the 27 M. tuberculosis isolates were positive for both IS986 and mtp40.

Base Sequence↗

PubMed1989

The occurrence of long ribosomal transcripts homologous to type I insertions in bobbed mutants of Drosophila melanogaster.

In Drosophila melanogaster up to two thirds of the rDNA genes contain insertion sequences of two types in the 28S coding region. Comparison of the ribosomal insertion transcripts in the wild type and in two bobbed mutants reared at two temperatures showed that the level of type I transcripts is dependent on both the number of genes with type I insertions in the bobbed loci and the intensity of bobbed phenotype. Importantly, a long transcript of 8.7 kb hybridized to the ribosomal probe, the INS I probe and also to the restriction fragment of the rDNA downstream of the point of insertion was found in one bobbed mutant. This result and also those from sandwich hybridization indicate that some interrupted ribosomal genes are functional.

Animals↗

PubMed2004

Shuffling of Sulfolobus genomes by autonomous and non-autonomous mobile elements.

Each of the sequenced Sulfolobus genomes contains large numbers of putatively mobile elements, both IS elements (insertion sequence elements) and MITEs (miniature inverted-repeat transposable elements). There are 344 in the 3.0 Mb genome of Sulfolobus solfataricus P2 and 95 in the 2.7 Mb genome of Sulfolobus tokodaii. In the former they constitute more than 10% of the genome. Experimental data suggest that transposition of IS elements occurs frequently. Moreover, the gene order between the two organisms differs greatly, indicating that multiple rearrangements have occurred. This has also led to considerable speculation as to how the cells are viable. Recently, a third Sulfolobus genome was completed which contains no IS elements or MITEs. This enabled us to compare the gene orders of the three genomes and provide evidence for mobile element-induced rearrangements of sections of the genomes.

DNA Transposable Elements↗

PubMed1990

Spinach cytosolic fructose-1,6-bisphosphatase. Purification, enzyme properties and structural comparisons.

Cytosolic fructose-1,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6-bisphosphate and Mg2+ are 1.5 microM and 260 mM, respectively, and its Vmax is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 microM and 120 microM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-1,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-1,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-1,6-bisphosphatases from other species reveal similarity ranging over 47-54%, which is higher than the 40-45% similarity between the chloroplast enzyme and gluconeogenic fructose-1,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-1,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-1,6-bisphosphatases.

Amino Acid Sequence↗

PubMed1992

Functional analysis of alternatively spliced transcripts of the human histidine decarboxylase gene and its expression in human tissues and basophilic leukemia cells.

L-Histidine decarboxylase (HisDC) is the enzyme catalyzing the formation of histamine from L-histidine. HisDC activity is expressed specifically in mast cells/basophils, endocrine cells in stomach, and histaminergic neurons in brain. As a first step in the analysis of the regulation of HisDC gene expression, we have cloned the cDNA coding for HisDC from a cDNA library of a human basophilic leukemia cell line, KU-812-F. We identified two types of HisDC cDNA, representing the 2.4-kb and 3.4-kb HisDC mRNA constitutively expressed in these cells. Sequence analysis of these cDNA revealed that the 3.4-kb mRNA contains the insert sequence of 824 bases and suggests that both 2.4-kb and 3.4-kb mRNA may represent the alternatively spliced transcripts of the HisDC gene. Using expression plasmids containing a cDNA for each HisDC mRNA, we analyzed the function of possible HisDC isoforms. We show that only the 2.4-kb mRNA encodes functional HisDC and is expressed in human brain and lung. However, we were unable to detect the 3.4-kb mRNA in these tissues. Thus, the 3.4-kb mRNA may be generated by KU-812-F cell-specific splicing of the HisDC gene transcripts. Furthermore, we demonstrated the increase in the level of 2.4-kb HisDC mRNA and HisDC activity in KU-812-F cells following treatment with phorbol 12-myristate 13-acetate.

Alternative Splicing↗

PubMed1997

Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST.

The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Base Sequence↗

PubMed2000

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence.

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.

Animals↗

PubMed2005

Evolutionary changes of the flhDC flagellar master operon in Shigella strains.

Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC, was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of non-flagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli, with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene clusters.

Base Sequence↗

PubMed2005

Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica.

Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed.

Journal Article↗

PubMed2006

Distribution of a transposon-like element carrying bla(CMY-2) among Salmonella and other Enterobacteriaceae.

OBJECTIVES: The dissemination of cephamycin resistance in Enterobacteriaceae and its correlation with a transposon-like DNA element consisting of a specific tnpA-bla(CMY-2)-blc-sugE structure were investigated. METHODS: A total of 140 enterobacterial isolates belonging to 17 species (10 genera) of Enterobacteriaceae phenotypically characterized as putative AmpC-producers were evaluated. The isolates were examined by PCR analysis, DNA-DNA hybridization and nucleotide sequencing. RESULTS: The bla(CMY-2)-carrying element was detected in 34 isolates from 10 species (9 genera), including all 14 Salmonella and 4 Shigella isolates as well as 7 of the 10 Escherichia coli isolates tested. The remaining 9 isolates were from 112 isolates of the other 14 species tested. The genetic structure of the bla(CMY-2)-carrying element was identical in 29 isolates, while in 3 E. coli and 2 Citrobacter isolates an additional insertion sequence IS1 was found inserted at various nucleotide positions close to the 3' end, either within or downstream, of tnpA. In 12 of the 14 representative isolates examined, the bla(CMY-2)-carrying element was found inserted in the finQ gene of various-sized plasmids with highly conserved 8 bp direct repeats flanking the junction regions. Among the other 106 non-CMY-2-producing isolates, plasmid-mediated ampC genes were found only in one isolate of Enterobacter aerogenes which carried a bla(DHA-1)-like gene. CONCLUSIONS: bla(CMY-2) is the most prevalent plasmid-mediated ampC gene among Enterobacteriaceae. All the bla(CMY-2) genes identified in the present study were associated with a specific transposon-like element that may be responsible for the spread of bla(CMY-2) among Enterobacteriaceae.

Cephalosporin Resistance↗

PubMed1991

The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5.

A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.

Amino Acid Sequence↗

PubMed1999

IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans.

Thiobacillus ferroxidans ATCC 19859 undergoes rapid phenotypic switching between a wild-type state characterized by the ability to oxidize ferrous iron (FeII) and reduced sulfur compounds and a mutant state where it has lost the capacity to oxidize FeII but retains the ability to oxidize sulfur. The mutant has also gained the capacity to swarm. It is proposed that loss of FeII oxidation is due to the reversible transposition of the insertion sequence IST1 into resB encoding a putative cytochrome c-type biogenesis protein. Downstream from resB and co-transcribed with it is resC, encoding another putative cytochrome biogenesis protein. IST1 insertional inactivation of resB could result in the loss of activity of its target c-type cytochrome(s). This putative target cytochrome(s) is proposed to be essential for FeII oxidation but not for sulfur oxidation. Curiously, resB and resC pertain to the proposed system II cytochrome biogenesis pathway whereas gamma Proteobacteria, of which T. ferrooxidans is a member, normally use system I. This could represent an example of lateral gene transfer.

Bacterial Proteins↗

PubMedDate not supplied

Molecular characterization of reticuloendotheliosis virus insertions in the genome of field and vaccine strains of fowl poxvirus.

Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent "variant" FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5' long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3' LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3' LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5' LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3' LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5' LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5' LTR sequences, positive PCR results with primers that amplify the 5' LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3' LTR PCR.

Animals↗