Insertion Patterns Research Articles

Spineless displays or why inaccurate restorations of fossil invertebrates discredit our museums

Restorations of the morphology, ecology and function of fossil invertebrates may be inferior to those of groups more familiar to the public and artist, such as tetrapods. This is illustrated by an extreme example, a drawing of the Lower Jurassic pseudo- planktonic crinoid Pentacrinites Blumenbach on display in the Manchester Museum. It is shown, accurately, attached to the underside of a floating log. However, it is incorrectly illustrated attached by a cemented holdfast rather than by cirri. Indeed, the reconstruction lacks any cirri despite Pentacrinites having a dense- ly cirriferous stem; the column is too short, has an incorrect cross-section and an erroneous pattern of columnal insertion; the cup and proximal brachial plates are too big; the pattern of arm branching is wrong; and pinnulation is inaccurate. Yet Pentacrinites is known from multiple well-preserved specimens from the Jurassic of the British Isles and was originally described more than 200 years ago. Museum restorations of fossil tetrapods have to be accurate because they are a group that we know well, yet many invertebrate groups are alien to the public and, if they are 'edu cated' by such grossly inaccurate restorations, so they shall remain.

Genetic analyses place most Spanish isolates of Beauveria bassiana in a molecular group with word-wide distribution

BackgroundThe entomopathogenic anamorphic fungus Beauveria bassiana is currently used as a biocontrol agent (BCA) of insects. Fifty-seven Beauveria bassiana isolates -53 from Spain- were characterized, integrating group I intron insertion patterns at the 3'-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-α) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of B. bassiana.ResultsGroup I intron genotype analysis was based on the four highly conserved insertion sites of the LSU (Ec2653, Ec2449, Ec2066, Ec1921). Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1-α phylogeny served to classify most of the strains in the B. bassiana s.s. (sensu stricto) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin (host, geographic location and climatic conditions) were also examined but in general no association could be found.ConclusionMost Spanish B. bassiana isolates (77.2%) are grouped into a major phylogenetic subgroup with word-wide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish B. bassiana isolates were sampled.

Characterization of Inago1 and Inago2 retrotransposons in Magnaporthe oryzae

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.

Mechanisms for maintaining cell shape in rod‐shaped Gram‐negative bacteria

For the rod-shaped Gram-negative bacterium Escherichia coli, changes in cell shape have critical consequences for motility, immune system evasion, proliferation and adhesion. For most bacteria, the peptidoglycan cell wall is both necessary and sufficient to determine cell shape. However, how the synthesis machinery assembles a peptidoglycan network with a robustly maintained micron-scale shape has remained elusive. To explore shape maintenance, we have quantified the robustness of cell shape in three Gram-negative bacteria in different genetic backgrounds and in the presence of an antibiotic that inhibits division. Building on previous modelling suggesting a prominent role for mechanical forces in shape regulation, we introduce a biophysical model for the growth dynamics of rod-shaped cells to investigate the roles of spatial regulation of peptidoglycan synthesis, glycan-strand biochemistry and mechanical stretching during insertion. Our studies reveal that rod-shape maintenance requires insertion to be insensitive to fluctuations in cell-wall density and stress, and even a simple helical pattern of insertion is sufficient for over sixfold elongation without significant loss in shape. In addition, we demonstrate that both the length and pre-stretching of newly inserted strands regulate cell width. In sum, we show that simple physical rules can allow bacteria to achieve robust, shape-preserving cell-wall growth.

Perceptual similarity in input–output mappings: A computational/experimental study of non-native speech production

This paper takes a computational/experimental approach to investigating faithfulness in input–output phonological mappings. We seek to explain the results of a speech production experiment recently reported in Davidson (2010). In that experiment, native English speakers were asked to produce phonotactically unattested consonant clusters. We argue that modifications of the target consonant clusters are best understood by considering both unfaithful phonological mappings and imprecision in the speech production mechanism. To account for the pattern of unfaithful input–output mappings, we consider an extension of the P-map hypothesis ( Steriade, 2008) to the production of phonotactically unattested target sequences. Predictions of the P-map for this data were established by a perception experiment in which participants were asked to discriminate between unattested consonant clusters, CC, and attested modifications, including epenthesis, prothesis, C 1 change and C 1 deletion. To evaluate the effects of motor noise on consonant cluster production, we constructed a computational model that allowed us to simulate consonant cluster productions under different levels of noise. Simulations reveal that, first, a large proportion of cases involving vocoid insertion, CəC, are better accounted for by noisy implementation of the target timing than by phonological epenthesis and, second, that differences in insertion patterns between stop-initial clusters and fricative-initial clusters are due to the internal temporal properties of stops and fricatives. Factoring these cases into the analysis isolates a pattern of unfaithful input–output mappings used to evaluate the P-map hypothesis. On the basis of considerable mismatches between patterns of perceptual similarity and unfaithful input–output mappings, we argue that the P-map theory of faithfulness is too restrictive to extend to the production of non-native speech.

Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

BackgroundClostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group.ResultsThe 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. However, the sequence was very different from the human C. botulinum group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between C. botulinum group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids.ConclusionsThe completion of the genome of C. botulinum group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species C. botulinum, but as a genotypic species it should also include C. novyi and C. haemolyticum. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.

An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress

Eukaryotic genomes consist to a significant extent of retrotransposons that are suppressed by host epigenetic mechanisms, preventing their uncontrolled propagation. However, it is not clear how this is achieved. Here we show that in Arabidopsis seedlings subjected to heat stress, a copia-type retrotransposon named ONSEN (Japanese 'hot spring') not only became transcriptionally active but also synthesized extrachromosomal DNA copies. Heat-induced ONSEN accumulation was stimulated in mutants impaired in the biogenesis of small interfering RNAs (siRNAs); however, there was no evidence of transposition occurring in vegetative tissues. After stress, both ONSEN transcripts and extrachromosomal DNA gradually decayed and were no longer detected after 20-30 days. Surprisingly, a high frequency of new ONSEN insertions was observed in the progeny of stressed plants deficient in siRNAs. Insertion patterns revealed that this transgenerational retrotransposition occurred during flower development and before gametogenesis. Therefore in plants with compromised siRNA biogenesis, memory of stress was maintained throughout development, priming ONSEN to transpose during differentiation of generative organs. Retrotransposition was not observed in the progeny of wild-type plants subjected to stress or in non-stressed mutant controls, pointing to a crucial role of the siRNA pathway in restricting retrotransposition triggered by environmental stress. Finally, we found that natural and experimentally induced variants in ONSEN insertions confer heat responsiveness to nearby genes, and therefore mobility bursts may generate novel, stress-responsive regulatory gene networks.

Evolution and variation of the nifD and hupL elements in the heterocystous cyanobacteria

In heterocystous cyanobacteria, heterocyst differentiation is accompanied by developmentally regulated DNA rearrangements that occur within the nifD and hupL genes, referred to as the nifD and hupL elements. These elements are segments of DNA that are embedded within the coding region of each gene and range from 4 to 24 kb in length. The nifD and hupL elements are independently excised from the genome during the later stages of differentiation by the site-specific recombinases, XisA and XisC, respectively, which are encoded within the elements themselves. Here we examine the variation and evolution of the nifD and hupL elements by comparing full-length nifD and hupL element sequences and by phylogenetic analysis of xisA and xisC gene sequences. There is considerable variation in the size and composition of the nifD and hupL elements, however, conserved regions are also present within representatives of each element. The data suggest that the nifD and hupL elements have undergone a complex pattern of insertions, deletions, translocations and sequence divergence over the course of evolution, but that conserved regions remain.

Integration profile of retroviral vector in gene therapy treated patients is cell‐specific according to gene expression and chromatin conformation of target cell

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.

Variations in BARE-1 insertion patterns in barley callus cultures

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.

The use of retrotransposon-based molecular markers to analyze genetic diversity

Summary: Molecular markers play an essential role in all aspects of genetics, modern plant breeding, in human forensics, for map-based cloning of genes, ranging from the identification of genes responsible for the desired traits to the management of backcrossing programs. Retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their “copy and paste” life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Following the demonstration that retrotransposons are ubiquitous, active, and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of genetic diversity in plants and the way for the rapid isolation of retrotransposon termini.

Influence of surface treatment on the insertion pattern of self-drilling orthodontic mini-implants

Influence of surface treatment on the insertion pattern of self-drilling orthodontic mini-implants

Alu repeat discovery and characterization within human genomes

Human genomes are now being rapidly sequenced, but not all forms of genetic variation are routinely characterized. In this study, we focus on Alu retrotransposition events and seek to characterize differences in the pattern of mobile insertion between individuals based on the analysis of eight human genomes sequenced using next-generation sequencing. Applying a rapid read-pair analysis algorithm, we discover 4342 Alu insertions not found in the human reference genome and show that 98% of a selected subset (63/64) experimentally validate. Of these new insertions, 89% correspond to AluY elements, suggesting that they arose by retrotransposition. Eighty percent of the Alu insertions have not been previously reported and more novel events were detected in Africans when compared with non-African samples (76% vs. 69%). Using these data, we develop an experimental and computational screen to identify ancestry informative Alu retrotransposition events among different human populations.

Chromosomes 2q, 4q and 13q21 Are Preferential Targets of Epstein-Barr Virus (EBV) Integration In Burkitt's Lymphoma Cell Lines

AbstractAbstract 4452The Epstein-Barr virus (EBV) was first described in 1964 as a possible cause of Burkitt's lymphoma (BL), the most common childhood cancer in equatorial Africa. EBV is an oncogenic virus found in about 95% of the endemic BL. In latently infected cells, the EBV DNA can be maintained in episomal form, but integrated EBV could also be present. Data on the exact integration loci of EBV in BL are very rare, however, integration of EBV-DNA into the human genome has been pointed as an important mechanism in malignant cellular transformation. We have investigated the EBV integration loci (EBV-IL) in 20 BL cell lines using fluorescence in situ hybridization (FISH).FISH was performed using, as EBV-DNA probe, a biotinylated 3,000 bp fragment amplified by long distance-PCR from DNA of the EBV-positive BL cell line Raji. FISH on metaphase spreads of the BL cell line Namalwa was performed to validate the quality of the EBV-FISH probe. Negative controls were performed on EBV-negative BL cell lines CA-46, CW698, and Tanoue. Integration of EBV was defined by the presence of symmetrical doublet hybridization signals at the same chromosomal loci in both sister chromatids. A minimum of 15 well-conserved and complete metaphases was evaluated in each case.As expected, Namalwa showed symmetrical doublet hybridization signals of our EBV-FISH probe on chromosome 1p35. EBV-negative cell lines did not show hybridization signals of the probe. We detected 632 EBV-IL in the total of metaphases analyzed in 20 BL cell lines. Integration of EBV was seen in all chromosomal arms except Yp, 8p, 14p, 20q, 21p, and 22p. An analysis of the chromosomal distribution of all EBV-IL revealed a pattern of preferential insertion for EBV-IL on chromosomes 13q (14% of the metaphases), 2q and 4q (12%, respectively), 7q (10,5%), 3q (9%), and 17q (6,5%).Regarding the recurrency of EBV-IL, the BL cell lines were divided in 4 groups. The first group consisted of 8 cell lines with high-recurrent EBV-IL at a specific chromosomal band (75%-100% of the metaphases analyzed). This first group showed specific EBV-IL at a precise chromosomal location on homologous chromatids on 2p23 (Seraphine), 2q21~31 (Switzer), 22q12 (PA-682), 7p22 and 13q21 (AG876), 7q11 and 17q25 (Naliaka), and 7q21 and 13q21 (LY-67). Interestingly, in BL60 and Maku, the unique EBV-IL visualized was localized near the translocation breakpoint junction of a der(19)t(17;19) and a der(13)t(3;13), respectively. The second group contained 5 cell lines with medium-recurrent EBV-IL (27%-50%). In this group, we observed metaphases with 1 to 3 recurrent EBV-IL localized on 2p12 (Rael), 15q11~14 (JI), 15q13~15 (JBL2), 4q21 and 4q32 (BL16), and 3q21~26, 4p12, and 13q21 (Akuba). The third and fourth groups comprised 4 cell lines with low-recurrent EBV-IL (13%-20%) and 3 cell lines with absence of recurrent EBV-IL, respectively. Non-recurrently integrated EBV-DNA signals on additional chromosomes were observed in cell lines of all 4 groups, except in 4 of the high-recurrent group. We further investigated the distribution of the EBV-IL of groups 3 and 4, of those in groups 1 and 2 with a recurrency less than 27% of the metaphases, and of those that were non-recurrently integrated. We observed again that the viral integration targeted preferentially chromosomes 2q (21%), 4q (14%), and 3q (11%).Our results identify for the first time the integration loci of EBV in BL cell lines and that this integration succeed preferentially on chromosomes 2q, 3q, and 4q. Moreover, in 12 cell lines, this integration ocurred non-randomly at a specific chromosomal sites, targeting 13q21 recurrently. Whether the integration of EBV in these loci affects the expression of genes important for the pathogenesis of BL or whether the EBV integration contributes to an enhanced chromosomal instability remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Determinants That Specify the Integration Pattern of Retrotransposon Tf1 in the fbp1 Promoter of Schizosaccharomyces pombe

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses and, as such, are important models for the study of viral integration and target site selection. The transposon Tf1 of Schizosaccharomyces pombe integrates with a strong preference for the promoters of polymerase II (Pol II)-transcribed genes. Previous work in vivo with plasmid-based targets revealed that the patterns of insertion were promoter specific and highly reproducible. To determine which features of promoters are recognized by Tf1, we studied integration in a promoter that has been characterized. The promoter of fbp1 has two upstream activating sequences, UAS1 and UAS2. We found that integration was targeted to two windows, one 180 nucleotides (nt) upstream and the other 30 to 40 nt downstream of UAS1. A series of deletions in the promoter showed that the integration activities of these two regions functioned autonomously. Integration assays of UAS2 and of a synthetic promoter demonstrated that strong promoter activity alone was not sufficient to direct integration. The factors that modulate the transcription activities of UAS1 and UAS2 include the activators Atf1p, Pcr1p, and Rst2p as well as the repressors Tup11p, Tup12p, and Pka1p. Strains lacking each of these proteins revealed that Atf1p alone mediated the sites of integration. These data indicate that Atf1p plays a direct and specific role in targeting integration in the promoter of fbp1.

Splicing of the Sinorhizobium meliloti RmInt1 group II intron provides evidence of retroelement behavior

Group II introns act as both large catalytic RNAs and mobile retroelements. They are found in organelle and bacterial genomes and are spliced via a lariat intermediate, in a mechanism similar to that of spliceosomal introns. However, their distribution and insertion patterns, particularly for bacterial group II introns, suggest that they function and behave more like retroelements than organelle introns. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. This group II intron is excised, in vivo and in vitro, as intron lariats. However, the complete splicing reaction in vivo remains to be elucidated. A lacZ reporter gene system, northern blotting and real-time reverse transcription were carried out to investigate RmInt1 splicing activity. Splicing efficiency of 0.07 ± 0.02% was recorded. These findings suggest that bacterial group II introns function more like retroelements than spliceosomal introns. Their location is consistent with a role for these introns in preventing the spread of other potentially harmful mobile elements in bacteria.

Do symplasmic networks in cambial zones correspond with secondary growth patterns?

The plasmodesmal (PD) network in the cambial zone of Arabidopsis thaliana hypocotyls was analysed using electron microscopy and dye-coupling studies and compared to those of internodes of Populus nigra and Solanum lycopersicum. In all species, PD densities and frequencies undergo alterations in topologically successive cambial walls reflecting species-specific patterns of PD degradation and PD insertion during cell development. Longitudinal PD fission is responsible for an abrupt increment of PD numbers in specific walls of the youngest derivatives at the xylem and/or phloem side. Here, PDs seem to mediate positional signalling to control tissue fate and early cell determination. PD numbers at all cambial interfaces of A. thaliana correspond to those of the herbaceous tomato, but are higher with the woody poplar. This suggests a positive correlation between PD frequencies and the rapidity of cell division activity. Photoactivated green fluorescent protein (26 kDa) did not diffuse through cambial PDs of A. thaliana. This is in keeping with the common size exclusion limit (SEL) of 8-10 kDa observed for PDs at the youngest interfaces of tomato and poplar which may mediate diffusive exchange of developmental signals of equal molecular size. The regular growth patterns in internodal cambial zones of poplar and tomato result from synchronized cell division activity of neighbouring initials. A. thaliana hypocotyls have an irregular mode of secondary growth. Here, signalling through PDs in misaligned radial walls between non-homologous derivatives may control tissue development. The observed organizational differences between the cambia cast doubts on the suitability of A. thaliana as a model plant for cambial research.

Ring Transformations of Pyrazolones Via Azo-Olefins

Instead of substitution, 4-halo-pyrazolones undergo ring opening to azo-olefins (2, 12, 16) on reaction with nucleophiles. Attempted Hofmann degradation of N-Haloamides 4 has to compete with either ring closure, involving NHCl-shift to 7, or formation of mesoionic compounds 10, whose pyrazolone ring shows a different pattern of nitrogen insertion. Substitution of cyclic amines for chlorine in azo-olefin 12 is followed by rearrangement to the bicycle 15. Access to 1,2,3-thiadiazolium salts (18, 22) is possible by electrophilic attack of sulfur reagents to azo-olefins.

Analysis of plant diversity with retrotransposon-based molecular markers

Retrotransposons are both major generators of genetic diversity and tools for detecting the genomic changes associated with their activity because they create large and stable insertions in the genome. After the demonstration that retrotransposons are ubiquitous, active and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. These have found applications ranging from the mapping of genes responsible for particular traits and the management of backcrossing programs to analysis of population structure and diversity of wild species. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of population diversity in plants.

A new deep-water cardinalfish (Neamia: Apogonidae) from Kiritimati Atoll, Kiribati

A single specimen of Neamia xenica, new species, was collected at 93 m utilizing mixed-gas rebreathers. This cardinalfish has pored lateral-line scales (extending from the posttemporal to caudal fin) at a smaller length than in Neamia articycla, a quasi-ovoid cluster of melanophores on the opercle without associated narrow dark marks (a round or oval dark opercular spot with pale outer region outlined with narrow dark marks in N. articycla and N. notula, respectively), and 14 pectoral fin-rays (14 fin rays in N. articycla and N. notula and 17–21 fin rays in N. octospina). Dorsal and anal insertion patterns of supraneurals, pterygiophores, and vertebral spines are reported for all species of Neamia. Species of Apogonichthys with 14 pectoral-fin rays may be confused with species of Neamia but do not have an opercular spot and have five free hypurals.