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PubMed2006

Dynamic evolution at pericentromeres.

Pericentromeres are exceptional genomic regions: in animals they contain extensive segmental duplications implicated in gene creation, and in plants they sustain rearrangements and insertions uncommon in euchromatin. To examine the mechanisms and patterns of plant pericentromere evolution, we compared pericentromere sequence from four Brassicaceae species separated by <15 million years (Myr). This flowering plant family is ideal for studying relationships between genome reorganization and pericentromere evolution-its members have undergone recent polyploidization and hybridization, with close relatives changing in genome size and chromosome number. Through sequence and hybridization analyses, we examined regions from Arabidopsis arenosa, Capsella rubella, and Olimarabidopsis pumila that are homologous to Arabidopsis thaliana pericentromeres (peri-CENs) III and V, and used FISH to demonstrate they have been maintained near centromere satellite arrays in each species. Sequence analysis revealed a set of highly conserved genes, yet we discovered substantial differences in intergenic length and species-specific changes in sequence content and gene density. We discovered that A. thaliana has undergone recent, significant expansions within its pericentromeres, in some cases measuring hundreds of kilobases; these findings are in marked contrast to euchromatic segments in these species that exhibit only minor length changes. While plant pericentromeres do contain some duplications, we did not find evidence of extensive segmental duplications, as has been documented in primates. Our data support a model in which plant pericentromeres may experience selective pressures distinct from euchromatin, tolerating rapid, dynamic changes in structure and sequence content, including large insertions of mobile elements, 5S rDNA arrays and pseudogenes.

Arabidopsis↗
PubMed2006

Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

Bacterial Toxins↗
PubMed2006

Doubling genome size without polyploidization: dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice.

Retrotransposons are the main components of eukaryotic genomes, representing up to 80% of some large plant genomes. These mobile elements transpose via a "copy and paste" mechanism, thus increasing their copy number while active. Their accumulation is now accepted as the main factor of genome size increase in higher eukaryotes, besides polyploidy. However, the dynamics of this process are poorly understood. In this study, we show that Oryza australiensis, a wild relative of the Asian cultivated rice O. sativa, has undergone recent bursts of three LTR-retrotransposon families. This genome has accumulated more than 90,000 retrotransposon copies during the last three million years, leading to a rapid twofold increase of its size. In addition, phenetic analyses of these retrotransposons clearly confirm that the genomic bursts occurred posterior to the radiation of the species. This provides direct evidence of retrotransposon-mediated variation of genome size within a plant genus.

Base Sequence↗
PubMed2006

Informational structure of two closely related eukaryotic genomes.

Attempts to identify a species on the basis of its DNA sequence on purely statistical grounds have been formulated for more than a decade. The most prominent of such genome signatures relies on neighborhood correlations (i.e., dinucleotide frequencies) and, consequently, attributes species identification to mechanisms operating on the dinucleotide level (e.g., neighbor-dependent mutations). For the examples of Mus musculus and Rattus norvegicus we analyze short- and intermediate-range statistical correlations in DNA sequences. These correlation profiles are computed for all chromosomes of the two species. We find that with increasing range of correlations the capacity to distinguish between the species on the basis of this correlation profile is getting better and requires ever shorter sequence segments for obtaining a full species separation. This finding suggests that distinctive traits within the sequence are situated beyond the level of few nucleotides. The large-scale statistical patterning of DNA sequences on which such genome signatures are based is thus substantially determined by mobile elements (e.g., transposons and retrotransposons). The study and interspecies comparison of such correlation profiles can, therefore, reveal features of retrotransposition, segmental duplications, and other processes of genome evolution.

Animals↗
PubMed2002

Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome.

Previous studies demonstrated that in somatic plant cells, homologous recombination (HR) is several orders of magnitude less efficient than nonhomologous end joining and that HR is little used for genomic double-strand break (DSB) repair. Here, we provide evidence that if genomic DSBs are induced in close proximity to homologous repeats, they can be repaired in up to one-third of cases by HR in transgenic tobacco. Our findings are relevant for the evolution of plant genomes because they indicate that sequences containing direct repeats such as retroelements might be less stable in plants that harbor active mobile elements than anticipated previously. Furthermore, our experimental setup enabled us to demonstrate that transgenic sequences flanked by sites of a rare cutting restriction enzyme can be excised efficiently from the genome of a higher eukaryote by HR as well as by nonhomologous end joining. This makes DSB-induced recombination an attractive alternative to the currently applied sequence-specific recombination systems used for genome manipulations, such as marker gene excision.

Agrobacterium tumefaciens↗
PubMed1996

Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera.

Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short-inverted-repeat elements are characterized by their extreme specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggyBac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN-368 cells or IPLB-SF21 AE cells. The precise excision of these elements in infected IPLB-SF21 AE cells occurs in the absence of either tagalong or piggyBac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion in a single family of unique transposons.

Animals↗
PubMed2004

High similarity between flanking regions of different microsatellites detected within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia.

Microsatellite flanking regions have been compared in two butterfly species. Several microsatellite flanking regions showed high similarity to one another among different microsatellites within a same species, but very few similarities were found between species. This can be the consequence of either duplication/multiplication events involving large regions containing microsatellites or of microsatellites imbedded in minisatellite regions. The multiplication of microsatellites might also be linked to mobile elements. Furthermore, crossing over between nonhomologous microsatellites can lead to the exchange of the flanking regions between microsatellites. The same phenomenon was observed in both studied butterfly species but not in Aphis fabae (Hemiptera), which was screened at the same time using the same protocol. These findings might explain, at least partially, why microsatellite isolation in Lepidoptera has been relatively unsuccessful so far.

Animals↗
PubMed1993

Identification and characterization of IS1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family.

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS1138, and the target site into which it inserted were determined. IS1138 consists of 1288 bp with 18 bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3 bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS1138 are present in most, if not all, strains of M. pulmonis, but IS1138-like sequences were not detected in other mycoplasmal species.

Amino Acid Sequence↗
PubMed1998

A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences.

A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

Cloning, Molecular↗
PubMed1992

Is there a connection between genomic changes and wide hybridization?

The structural organization of a set of highly repetitive DNA sequences (HRS) of barley (Hordeum vulgare) was studied by blot-hybridization in the genomes of seven Hordeum L. species and several Hordeum x Secale hybrids. The copy numbers of the sequences, and length and intensity of the hybridization fragments varied among barley species; so, this set appeared to be useful as molecular markers for barley species. Structural rearrangements of some HRS were observed in hybrids. It was noteworthy that the genomic changes in the hybrids partially coincided with those that take place during species divergence; so, chromosomal rearrangements are likely to proceed according to certain rules. The possibility of cryptic mobile elements participating in the genomic rearrangements under stress factors of the remote hybridization, is discussed, the primary structure of a Bam HI fragment (999 bp long) of Hordeum vulgare DNA being the example.

Base Sequence↗
PubMed2005

Adaptation of intronic homing endonuclease for successful horizontal transmission.

Group I introns are thought to be self-propagating mobile elements, and are distributed over a wide range of organisms through horizontal transmission. Intron invasion is initiated through cleavage of a target DNA by a homing endonuclease encoded in an open reading frame (ORF) found within the intron. The intron is likely of no benefit to the host cell and is not maintained over time, leading to the accumulation of mutations after intron invasion. Therefore, regular invasional transmission of the intron to a new species at least once before its degeneration is likely essential for its evolutionary long-term existence. In many cases, the target is in a protein-coding region which is well conserved among organisms, but contains ambiguity at the third nucleotide position of the codon. Consequently, the homing endonuclease might be adapted to overcome sequence polymorphisms at the target site. To address whether codon degeneracy affects horizontal transmission, we investigated the recognition properties of a homing enzyme, I-CsmI, that is encoded in the intronic ORF of a group I intron located in the mitochondrial COB gene of the unicellular green alga Chlamydomonas smithii. We successfully expressed and purified three types of N-terminally truncated I-CsmI polypeptides, and assayed the efficiency of cleavage for 81 substrates containing single nucleotide substitutions. We found a slight but significant tendency that I-CsmI cleaves substrates containing a silent or tolerated amino acid change more efficiently than nonsilent or nontolerated ones. The published recognition properties of I-SpomI, I-ScaI, and I-SceII were reconsidered from this point of view, and we detected proficient adaptation of I-SpomI, I-ScaI, and I-SceII for target site sequence degeneracy. Based on the results described above, we propose that intronic homing enzymes are adapted to cleave sequences that might appear at the target region in various species, however, such adaptation becomes less prominent in proportion to the time elapsed after intron invasion into a new host.

Algal Proteins↗
PubMed2004

Ca2+ dependency of 'Ca2+-independent' exocytosis in SPOC1 airway goblet cells.

SPOC1 airway goblet cells secrete mucin in response to P2Y2 receptor agonists and to secretagogues, phorbol 12-myristate 13-acetate (PMA) and ionomycin, which mobilize elements of the phospholipase C pathway, PKC and Ca2+, respectively. Previous studies demonstrated that mucin secretion from SLO-permeabilized, EGTA-buffered SPOC1 cells was stimulated by PMA at low Ca2+ levels (< 0.1 microm), consistent with the notion that regulated exocytosis may occur by Ca2+-independent pathways. We tested the alternative hypothesis that PMA-induced mucin secretion is, in fact, a Ca2+-dependent process under the conditions of low bulk Ca2+, one that is permitted in the typical SLO-permeabilized cell model by the slow binding kinetics of EGTA. Both IP3 and elevated bulk Ca2+ activated mucin secretion in SPOC1 cells buffered by EGTA, suggesting that IP3 generates a local Ca2+ gradient in the vicinity of the secretory granules to the degree necessary to trigger exocytosis. BAPTA, which binds Ca2+ approximately 100-fold faster than EGTA, diminished IP3-induced mucin release over a range of concentrations by > or = 69%, yet maintained an essentially normal mucin secretory response to elevated bulk Ca2+ in permeabilized SPOC1 cells. BAPTA also diminished the mucin secretory response of permeabilized cells to PMA, relative to the EGTA-buffered control: at PMA below 30 nm, BAPTA abolished the secretory response, and at higher concentrations it was reduced significantly relative to the EGTA-buffered controls. PMA-induced secretion in EGTA was insensitive to heparin. These results suggest that Ca2+ is released locally during PMA-induced exocytosis, by an IP3-independent mechanism.

Animals↗
PubMed1991

Possible horizontal transfer of Drosophila genes by the mite Proctolaelaps regalis.

There is strong inferential evidence for recent horizontal gene transfer of the P (mobile) element to Drosophila melanogaster from a species of the Drosophila willistoni group. One potential vector of this transfer is a semiparasitic mite, Proctolaelaps regalis DeLeon, whose morphology, behavior, and co-occurrence with Drosophila are consistent with the properties necessary for such a vector. Southern blot hybridization, polymerase chain reaction (PCR) amplification, and DNA sequencing showed that samples of P. regalis associated with a P strain of D. melanogaster carried P element sequences. Similarly, Drosophila ribosomal DNA sequences were identified in P. regalis samples that had been associated with Drosophila cultures. These results have potentially important evolutionary implications, not only for understanding the mechanisms by which genes may be transferred between reproductively isolated species, but also for improved detection of some host-parasite and predator-prey relationships.

Animals↗
PubMed2000

Selfish DNA in protein-coding genes of Rickettsia.

Rickettsia conorii, the aetiological agent of Mediterranean spotted fever, is an intracellular bacterium transmitted by ticks. Preliminary analyses of the nearly complete genome sequence of R. conorii have revealed 44 occurrences of a previously undescribed palindromic repeat (150 base pairs long) throughout the genome. Unexpectedly, this repeat was found inserted in-frame within 19 different R. conorii open reading frames likely to encode functional proteins. We found the same repeat in proteins of other Rickettsia species. The finding of a mobile element inserted in many unrelated genes suggests the potential role of selfish DNA in the creation of new protein sequences.

Bacterial Proteins↗
PubMed1991

Characterization of the gentamicin resistance transposon Tn5281 from Enterococcus faecalis and comparison to staphylococcal transposons Tn4001 and Tn4031.

In Enterococcus faecalis, the genetic determinant encoding gentamicin resistance (Gmr) on the conjugative plasmid pBEM10 previously has been shown to be on a mobile element. In the current study, this element, termed Tn5281, was shown to relocate in the absence of homologous recombination in E. faecalis UV202. On the basis of restriction endonuclease analysis and DNA-DNA hybridization studies, Tn5281 was shown to be similar, if not identical, to the Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in U.S. isolates of Staphylococcus epidermidis, since all three of these transposons have symmetrically located HindIII (2.5 kb apart), ClaI (slightly more than 2.5 kb apart), and HaeIII (3.9 kb apart) sites. Restriction endonuclease digestion patterns of Tn5281 generated with HincII, ScaI, and AluI were also consistent with Tn4001 and Tn4031. By using a probe specific for the external portion of the terminal inverted repeat of Tn4031, it was determined that each terminus of Tn5281 contained a 0.35-kb HaeIII fragment and a 0.7-kb HindIII-HaeIII fragment. The sizes of these fragments are identical to those found in the staphylococcal transposons, which is a further indication that inverted repeats like IS256 are present in Tn5281. A 1-kb HaeIII fragment in pBEM10 also hybridized with this probe, which indicates that Tn5281 in pBEM10 contains a double copy of the inverted repeat at one end.

Conjugation, Genetic↗
PubMed1994

Dissemination among staphylococci of DNA sequences associated with methicillin resistance.

DNA probes consisting of pUC19 containing cloned Staphylococcus aureus chromosomal fragments were constructed from two methicillin-resistant S. aureus strains with different DNA sequences 5' to mecA, the gene that mediates methicillin resistance. The probe from one strain, BMS1, contained a portion of the regulatory sequences (the terminal 641 bp of mecR1 and all of mecI) associated with the induction and repression of mecA transcription (pGO195). The second probe, from strain COL (pGO198), contained DNA not found in strain BMS1. This DNA was within the sequences added at the site of a mecR1 deletion. Genomic digests of 14 S. aureus isolates recovered between 1961 and 1969 all hybridized with pGO198. In contrast, 78% (36 of 46) of the S. aureus organisms isolated since 1988 hybridized with pGO195 but not with pGO198; the remainder hybridized with pGO198. No S. aureus isolates hybridized with both probes. Staphylococcus epidermidis digests hybridized with pGO198 (46%), pGO195 (14%), or both probes (35%); all 20 Staphylococcus haemolyticus isolates hybridized with pGO198. The restriction fragment length polymorphism patterns of all pGO198-hybridizing regions in S. aureus were identical to those in strain COL. In addition, the mecR1 deletion junction nucleotide sequences of eight S. aureus and six S. epidermidis isolates were identical. However, 21 of 23 S. epidermidis and all 20 S. haemolyticus isolates had from 5 to more than 20 additional chromosomal bands that hybridized with pGO198; none of 21 S. aureus isolates had additional hybridizing bands. These data suggest that the additional DNA responsible for the mecR1 deletion was part of a repetitive, and possibly mobile, element resident in coagulase-negative staphylococci but not in S. aureus. These data also support a hypothesis that the deletion event occurred in a coagulase-negative staphylococcus with subsequent acquisition of the interrupted sequences by S. aureus.

Base Sequence↗
PubMed1995

PCR mapping of integrons reveals several novel combinations of resistance genes.

The integron is a new type of mobile element which has evolved by a site-specific recombinational mechanism. Integrons consist of two conserved segments of DNA separated by a variable region containing one or more genes integrated as cassettes. Oligonucleotide probes specific for the conserved segments have revealed that integrons are widespread in recently isolated clinical bacteria. Also, by using oligonucleotide probes for several antibiotic resistance genes, we have found novel combinations of resistance genes in these strains. By using PCR, we have determined the content and order of the resistance genes inserted between the conserved segments in the integrons of these clinical isolates. PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons and dissemination of antibiotic resistance genes.

Base Sequence↗
PubMed1997

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.

Amino Acid Sequence↗