Causes of insertion sequences abundance in prokaryotic genomes

Insertion sequences (ISs) are the smallest and most frequent transposable elements in prokaryotes where they play an important evolutionary role by promoting gene inactivation and genome plasticity. Their genomic abundance varies by several orders of magnitude for reasons largely unknown and widely speculated. The current availability of hundreds of genomes renders testable many of these hypotheses, notably that IS abundance correlates positively with the frequency of horizontal gene transfer (HGT), genome size, pathogenicity, nonobligatory ecological associations, and human association. We thus reannotated ISs in 262 prokaryotic genomes and tested these hypotheses showing that when using appropriate controls, there is no empirical basis for IS family specificity, pathogenicity, or human association to influence IS abundance or density. HGT seems necessary for the presence of ISs, but cannot alone explain the absence of ISs in more than 20% of the organisms, some of which showing high rates of HGT. Gene transfer is also not a significant determinant of the abundance of IS elements in genomes, suggesting that IS abundance is controlled at the level of transposition and ensuing natural selection and not at the level of infection. Prokaryotes engaging in obligatory associations have fewer ISs when controlled for genome size, but this may be caused by some being sexually isolated. Surprisingly, genome size is the only significant predictor of IS numbers and density. Alone, it explains over 40% of the variance of IS abundance. Because we find that genome size and IS abundance correlate negatively with minimal doubling times, we conclude that selection for rapid replication cannot account for the few ISs found in small genomes. Instead, we show evidence that IS numbers are controlled by the frequency of highly deleterious insertion targets. Indeed, IS abundance increases quickly with genome size, which is the exact inverse trend found for the density of genes under strong selection such as essential genes. Hence, for ISs, the bigger the genome the better.     

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

Distant horizontal gene transfer is rare for multiple families of prokaryotic insertion sequences

Horizontal gene transfer in prokaryotes is rampant on short and intermediate evolutionary time scales. It poses a fundamental problem to our ability to reconstruct the evolutionary tree of life. Is it also frequent over long evolutionary distances? To address this question, we analyzed the evolution of 2,091 insertion sequences from all 20 major families in 438 completely sequenced prokaryotic genomes. Specifically, we mapped insertion sequence occurrence on a 16S rDNA tree of the genomes we analyzed, and we also constructed phylogenetic trees of the insertion sequence transposase coding sequences. We found only 30 cases of likely horizontal transfer among distantly related prokaryotic clades. Most of these horizontal transfer events are ancient. Only seven events are recent. Almost all of these transfer events occur between pairs of human pathogens or commensals. If true also for other, non-mobile DNA, the rarity of distant horizontal transfer increases the odds of reliable phylogenetic inference from sequence data.     

Functional characterization of the prokaryotic mobile genetic element IS26

Summary IS26L and IS26R are the 820 bp long elements found as direct repeats at both ends of the kanamycin resistance transposon Tn2680. They can mediate cointegration in E. coli K12 which contains no IS26 in its chromosome. Cointegration occurs in rec ⁺ or recA ^- strains with similar frequency. Upon cointegration mediated by either IS26R or IS26L, the element is duplicated and integrated into one of many different sites. Both IS26L and IS26R carry 14 bp perfect terminal inverted repeats and generate 8 bp direct repeats at their target sequences. Deletion formation mediated by IS26R was also observed. These functional and structural features of IS26 are characteristic of a prokaryotic mobile genetic element.     

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.     

A unique element resembling a processed pseudogene

We describe a unique DNA element with structural features of a processed pseudogene but with important differences. It is located within an 8.4-kilobase pair region of chicken DNA containing five histone genes, but it is not related to these genes. The presence of terminal repeats, an open reading frame (and stop codon), polyadenylation/processing signal, and a poly(A) rich region about 20 bases 3' to this, together with a lack of 5' promoter motifs all suggest a processed pseudogene. However, no parent gene can be detected in the genome by Southern blotting experiments and, in addition, codon boundary values and mid-base correlations are not consistent with a protein coding region of a eukaryotic gene. The element was detected in DNA from different chickens and in peafowl, but not in quail, pheasant, or turkey.     

原核增强子样序列研究进展

增强子是真核细胞中增强启动子功能的顺式作用元件。随着人们对原核生物基因表达调控的深入研究,人们发现在原核生物的调控系统和某些病毒基因组DNA片段中也具有增强子样功能。这些研究对揭示原核系统的基因表达调控和增强外源基因在原核系统中的表达水平具有重要的理论意义和实用价值。     

Detecting motifs and patterns at mobile genetic element insertion site

Mobile genetic elements (MGEs) occupy major proportion of eukaryotic genomes and are present in significant numbers inprokaryote genomes also. Here we report a new method which extracts a motif at the site of insertion of MGE using tools such asDNA SCANNER. The flanking region of the insertion site is extracted and is analyzed in DNA Scanner for physiochemicalproperties like protein-interaction measures, energy profiles as well as structural parameters. In case significant signals areobserved, the most frequently occurring n-mer (5E. histolytica, signals for EhSine1 are found ataround 5 bps upstream of insertion and most frequently occurring motif is found to be AAGGT and TCGAA. Signals for Ty3element in S. cerevisiae are found at 0-3 bps upstream of tRNA, and most frequent motif is GTTCGA (6 bps), GGTTCGA (7 bps) andGGTTCGAT (8 bps). P-element of Drosophila showed remarkable dyad peaks suggesting palindromic site of insertion.     

Insertion sequences in prokaryotic genomes

Insertion sequences (ISs) are small DNA segments that are often capable of moving neighbouring genes. Over 1500 different ISs have been identified to date. They can have large and spectacular effects in shaping and reshuffling the bacterial genome. Recent studies have provided dramatic examples of such IS activity, including massive IS expansion during the emergence of some pathogenic bacterial species and the intimate involvement of ISs in assembling genes into complex plasmid structures. However, a global understanding of their impact on bacterial genomes requires detailed knowledge of their distribution across the eubacterial and archaeal kingdoms, understanding their partition between chromosomes and extra-chromosomal elements (e.g. plasmids and viruses) and the factors which influence this, and appreciation of the different transposition mechanisms in action, the target preferences and the host factors that influence transposition. In addition, defective (non- autonomous) elements, which can be complemented by related active elements in the same cell, are often overlooked in genome annotations but also contribute to the evolution of genome organisation.     

Maize genetic diversity and association mapping using transposable element insertion polymorphisms

Transposable elements are the major component of the maize genome and presumably highly polymorphic yet they have not been used in population genetics and association analyses. Using the Transposon Display method, we isolated and converted into PCR-based markers 33 Miniature Inverted Repeat Transposable Elements (MITE) polymorphic insertions. These polymorphisms were genotyped on a population-based sample of 26 American landraces for a total of 322 plants. Genetic diversity was high and partitioned within and among landraces. The genetic groups identified using Bayesian clustering were in agreement with published data based on SNPs and SSRs, indicating that MITE polymorphisms reflect maize genetic history. To explore the contribution of MITEs to phenotypic variation, we undertook an association mapping approach in a panel of 367 maize lines phenotyped for 26 traits. We found a highly significant association between the marker ZmV1-9, on chromosome 1, and male flowering time. The variance explained by this association is consistent with a flowering delay of +123 degree-days. This MITE insertion is located at only 289 nucleotides from the 3′ end of a Cytochrome P450-like gene, a region that was never identified in previous association mapping or QTL surveys. Interestingly, we found (i) a non-synonymous mutation located in the exon 2 of the gene in strong linkage disequilibrium with the MITE polymorphism, and (ii) a perfect sequence homology between the MITE sequence and a maize siRNA that could therefore potentially interfere with the expression of the Cytochrome P450-like gene. Those two observations among others offer exciting perspectives to validate functionally the role of this region on phenotypic variation.     

Molecular and genetic characterization of an Alcaligenes eutrophus insertion element.

An insertion element, ISAE1, was discovered during the molecular analysis of mutants defective in the autotrophic growth (Aut-) of Alcaligenes eutrophus H1-4, a mitomycin C-generated derivative of strain H1. ISAE1 is 1,313 bp long, has 12-bp nearly perfect inverted terminal repeats, and contains an open reading frame that has a coding capacity of 408 amino acids. Direct repeats of 8 bp were generated by insertion of ISAE1 into chromosomes or plasmids. Most insertion were found in the AT-rich target sites. The distribution of ISAE1 is limited to A. eutrophus H1 (ATCC 17698) and H16 (ATCC 17699). Variants with newly transposed copies of ISAE1 could be isolated at an elevated frequency by changing the growth conditions.     

Gene organization and target specificity of the prokaryotic mobile genetic element IS26

Summary The 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII. The 702-bp ORFI is thus likely to code for the IS26 transposase. An 82-bp long sequence from the left end of IS26 contains a promoter-like structure in front of the start of ORFI at coordinate 64. In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene. The observation provides additional evidence for the functional relevance of ORFI. Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter. Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content.     

Nucleotide sequence of IS26, a new prokaryotic mobile genetic element.

The DNA sequence of a new IS element, the IS26, is 820 bp long and carries 14 bp perfect terminal inverted repeats. Upon integration, IS26 generates an 8 bp duplication of its target sequence. A large open reading frame within IS26 could code for a protein of 234 amino acids. On its reverse strand, IS26 also carries one large open reading frame, 591 bp long, which contains no stop codon within IS26.     

A survey of bacterial insertion sequences using IScan

Bacterial insertion sequences (ISs) are the simplest kinds of bacterial mobile DNA. Evolutionary studies need consistent IS annotation across many different genomes. We have developed an open-source software package, IScan, to identify bacterial ISs and their sequence elements—inverted and target direct repeats—in multiple genomes using multiple flexible search parameters. We applied IScan to 438 completely sequenced bacterial genomes and 20 IS families. The resulting data show that ISs within a genome are extremely similar, with a mean synonymous divergence of K_s = 0.033. Our analysis substantially extends previously available information, and suggests that most ISs have entered bacterial genomes recently. By implication, their population persistence may depend on horizontal transfer. We also used IScan's ability to analyze the statistical significance of sequence similarity among many IS inverted repeats. Although the inverted repeats of insertion sequences are evolutionarily highly flexible parts of ISs, we show that this ability can be used to enrich a dataset for ISs that are likely to be functional. Applied to the thousands of genomes that will soon be available, IScan could be used for many purposes, such as mapping the evolutionary history and horizontal transfer patterns of different ISs.     

A copia-like element in Pisum demonstrates the uses of dispersed repeated sequences in genetic analysis

A DNA sequence between two legumin genes in Pisum is a member of the copia-like class of retrotransposons and represents one member of a polymorphic and heterogeneous dispersed repeated sequence family in Pisum. This sequence can be exploited in genetic studies either by RFLP analysis where several markers can be scored together, or the segregation of individual elements can be followed after PCR amplification of specific members.     

Recurrent insertion and duplication generate networks of transposable element sequences in the Drosophila melanogaster genome

Background  

The recent availability of genome sequences has provided unparalleled insights into the broad-scale patterns of transposable element (TE) sequences in eukaryotic genomes. Nevertheless, the difficulties that TEs pose for genome assembly and annotation have prevented detailed, quantitative inferences about the contribution of TEs to genomes sequences.     

Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes

Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins. These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system. The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues. Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.