What is your diagnosis? Bone cyst causing acute lameness in a dog.
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Animals↗
Biomedical subjects
H M Robertson
Publications and source records attributed to H M Robertson.
The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells.
Mariner transposons belong to the mariner /Tc1 superfamily of class II, DNA-mediated elements. One of these transposons, Himar1 , isolated from the horn fly, is independent of host-specific factors that would limit transfer between different species, making it an ideal candidate for gene transfer technology development. To determine the activity of Himar1 transposase in mammalian cells, we introduced the Himar1 transposase gene into an adenovirus (Ad) vector under control of the phage T7 RNA polymerase promoter. Mammalian cells infected with the Ad vector carrying the Himar1 gene efficiently expressed the Himar1 transposase in the presence of T7 polymerase. In in vitro inter-plasmid transposition reactions, Himar1 transposase expressed by the Ad vector mediated precise cut-and-paste transposition and resulted in a characteristic duplication of TA at the integration site of the target plasmid. Further studies showed that this transposase was capable of catalyzing transposition between twoplasmids co-transfected into 293T7pol cells, which express T7 RNA polymerase. Combining the integration capability of mariner transposons with the transduction efficiency of Ad vectors is expected to provide a powerful tool for introducing transgenes into the host chromosome.
Adenoviridae↗
Systematic identification of essential genes by in vitro mariner mutagenesis.
Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.
Base Sequence↗
What is your diagnosis? Nasal lymphoblastic lymphosarcoma causing epistaxis in a dog.
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Animals↗
Factors affecting transposition of the Himar1 mariner transposon in vitro.
Mariner family transposable elements are widespread in animals, but their regulation is poorly understood, partly because only two are known to be functional. These are particular copies of the Dmmar1 element from Drosophila mauritiana, for example, Mos1, and the consensus sequence of the Himar1 element from the horn fly, Haematobia irritans. An in vitro transposition system was refined to investigate several parameters that influence the transposition of Himar1. Transposition products accumulated linearly over a period of 6 hr. Transposition frequency increased with temperature and was dependent on Mg2+ concentration. Transposition frequency peaked over a narrow range of transposase concentration. The decline at higher concentrations, a phenomenon observed in vivo with Mos1, supports the suggestion that mariners may be regulated in part by "overproduction inhibition." Transposition frequency decreased exponentially with increasing transposon size and was affected by the sequence of the flanking DNA of the donor site. A noticeable bias in target site usage suggests a preference for insertion into bent or bendable DNA sequences rather than any specific nucleotide sequences beyond the TA target site.
Animals↗
Two large families of chemoreceptor genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal extensive gene duplication, diversification, movement, and intron loss.
The str family of genes encoding seven-transmembrane G-protein-coupled or serpentine receptors related to the ODR-10 diacetyl chemoreceptor is very large, with at least 197 members in the Caenorhabditis elegans genome. The closely related stl family has 43 genes, and both families are distantly related to the srd family with 55 genes. Analysis of the structures of these genes indicates that a third of them are clearly or likely pseudogenes. Preliminary surveys of other candidate chemoreceptor families indicates that as many as 800 genes and pseudogenes or 6% of the genome might encode 550 functional chemoreceptors constituting 4% of the C. elegans protein complement. Phylogenetic analyses of the str and stl families, and comparisons with a few orthologs in Caenorhabditis briggsae, reveal ongoing processes of gene duplication, diversification, and movement. The reconstructed ancestral gene structures for these two families have eight introns each, four of which are homologous. Mapping of intron distributions on the phylogenetic tree reveals that each intron has been lost many times independently. Most of these introns were lost individually, which might best be explained by precise in-frame deletions involving nonhomologous recombination between short direct repeats at their termini. [Alignment of the putatively functional proteins in the str and stl families is available from Pfam (http://genome. wustl.edu/Pfam); alignments of all translations are available at http://cshl.org/gr; alignments of the genes are available from the author at hughrobe@uiuc.edu]
Amino Acid Sequence↗
Molecular evolution of the second ancient human mariner transposon, Hsmar2, illustrates patterns of neutral evolution in the human genome lineage.
A consensus sequence for the second ancient mariner identified in the human genome, Hsmar2, was constructed by majority rule from full-length and partial sequences of 44 of the +/-1000 copies in the genome. This 1300 base pair (bp) consensus has 31 bp imperfect terminal repeats (ITRs) and encodes a 351 amino acid (aa) mariner transposase. The sequence of this transposase has allowed classification of Hsmar2 as a basal lineage of the irritans subfamily of mariners, sharing at most 38% aa identity with other members of the subfamily. The individual copies in the human genome are all highly mutated from the consensus, having suffered numerous small and some large insertions and deletions (indels), including many insertions of S and J subfamily Alu elements. The copies differ, on average, from the consensus by 11.6%, have suffered 11.8 indels per kilobase (kb), and only 3.7% of the 30 hypermutable CpG dinucleotide pairs in the consensus remain intact. This level of divergence indicates that the ancestrally active Hsmar2 element represented by the consensus was present in the human genome lineage about 80 million years (Myr) ago. Each copy has apparently evolved since then largely independently of the others, and with little constraint on its transposase coding capacity. This pattern of molecular evolution fits the current model for mariner transposon evolution. These copies provide multiple independent datasets for evaluating the pattern of neutral evolution in the human genome, for example, they confirm that most indels are very short and that deletions are twice as common as insertions.
Amino Acid Sequence↗
Molecular evolution of an ancient mariner transposon, Hsmar1, in the human genome.
A confident consensus sequence for Hsmar1, the first mariner transposon recognized in the human genome, was generated using three genomic and 15 cDNA sequences. It is thought to represent the ancestrally active copy that invaded an early primate genome. The consensus is 1287 base pairs (bp) long, has 30 bp perfect inverted terminal repeats (ITRs), and encodes a 343 amino acid (aa) mariner transposase. Each copy has diverged from the consensus largely independently of the others and mostly neutrally, and most are now defective. They differ from the consensus by an average of 7.8% in DNA sequence and 7.5 indels per kilobase, both of which values indicate that the copies were formed about 50 Myr ago. On average, only 20% of the 73 surmised CpG hypermutable sites in the consensus remain. A remarkable exception to this loss of functionality is revealed by a set of ten cDNA clones derived from a particular genomic copy that has diverged only 2.4% from the consensus, retained 54% of its hypermutable CpG pairs, and which has a full-length transposase open reading frame. The complete sequence of one of these cDNAs (NIB1543) indicates that the transposase gene of this copy may have been conserved because it is spliced to a human cellular gene encoding a SET domain protein. A specific PCR assay was used to reveal the presence of Hsmar1 copies in all primates examined representing all major lineages, but not in close relatives of primates. PCR fragments cloned and sequenced from a representative sample of primates confirmed that Hsmar1 copies are present in all major lineages, and also revealed another cecropia subfamily mariner in prosimians only, and a third highly divergent mariner present in the greater slow loris Nycticebus coucang. There are about 200 copies of Hsmar1 in the human genome, as well as +/-2400 copies of a derived 80 bp paired ITR structure and +/-4600 copies of solo ITRs. Thus, this transposon had a considerable insertional mutagenic effect on past primate genomes.
Amino Acid Sequence↗
The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets.
A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.
Animals↗
Neuroglian is expressed on cells destined to form the prothoracic glands of Manduca embryos as they segregate from surrounding cells and rearrange during morphogenesis.
A cell surface protein (3B11) is differentially expressed in the embryonic labial segment of Manduca as two circular monolayers of epithelial cells invaginate and segregate from surrounding epithelial cells. The cells that invaginate and preferentially express 3B11 represent the presumptive prothoracic glands. These cells continue to express protein 3B11 as they rearrange to form first a three-dimensional aggregate and later anastomosing filaments of cells. In the differentiated prothoracic gland, expression of 3B11 is restricted to sites of cell-cell contact. Cloning and sequencing of the cDNA for protein 3B11 revealed that this protein is the Manduca counterpart of Drosophila neuroglian and mouse L1. These surface proteins are known to function as adhesion/recognition molecules during development. Manduca neuroglian shares 58 and 31% identity respectively with the Drosophila and mouse proteins and has a cytoplasmic domain of over 100 amino acids.
Amino Acid Sequence↗
Ancient DNA from amber fossil bees?
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Amber↗
Members of the pogo superfamily of DNA-mediated transposons in the human genome.
A new superfamily of transposons from fungi, nematodes, and flies related to the pogo element of Drosophila melanogaster was recognized that represents a branch of the extended superfamily of transposase and integrase proteins sharing a common D.D35E catalytic domain. Searches of human sequences in the public databases for similarity to this domain revealed at least two members of this new superfamily, with many highly mutated copies, in the human genome. A full-length consensus was constructed for one of them, which includes the MER37 medium reiteration frequency sequence recognized previously, from 343 human sequence accessions (261 of which are unique). Most of these were Expressed Sequence Tags, some were Sequence-Tagged Sites, and a few are from long genomic sequences. The 2417 bp consensus has the hallmarks of a pogo superfamily transposon, including 12 bp inverted terminal repeats, and encodes two long open reading frames. The first ORF encodes a polypeptide with 42% amino acid sequence identity to pogo in the D.D35E region. The second element shows 49% amino acid sequence identity with the first, and 40% with pogo in this region. These elements coincide with those described recently as Tigger1 and Tigger2, respectively. These transposons appear to have been active 80-90 Myr ago in the genome of an early primate or primate ancestor.
Amino Acid Sequence↗
A purified mariner transposase is sufficient to mediate transposition in vitro.
Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.
Animals↗
The genomes of most animals have multiple members of the Tc1 family of transposable elements.
A PCR assay was employed to detect sequence homologous to the transposase gene of the Tc1 family of transposable elements in a wide variety of animals. Amplification products of the appropriate size were obtained from most insects (92 of 108 examined; 85%), most other invertebrates (33 of 43; 77%), and many vertebrates (18 of 36; 50%). Sequencing a sample of cloned PCR products from eight insects, one hydra, and two frogs revealed that each had multiple distinct members of the family in their genomes. In the most extreme case, the horn fly Haematobia irritans yielded evidence of seventeen distinct types of Tc1 family elements. Most of the sequences obtained indicate that the elements are within the range of variation already known from fungi, nematodes, flies, fish and frogs. Some, however, had novel length variants or divergent sequences, indicating that they represent new subfamilies of these transposons. These results indicate that this family of transposons is extremely common in animal genomes, with multiple representatives in most genomes.
Amino Acid Sequence↗
Reconstructing the ancient mariners of humans.
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Amino Acid Sequence↗
Wolbachia infections and the expression of cytoplasmic incompatibility in Drosophila sechellia and D. mauritiana.
Various stocks of Drosophila mauritiana and D. sechellia were found to be infected with Wolbachia, a Rickettsia-like bacterium that is known to cause cytoplasmic incompatibility and other reproductive abnormalities in arthropods. Testing for the expression of cytoplasmic incompatibility in these two species showed partial incompatibility in D. sechellia but no expression of incompatibility in D. mauritiana. To determine whether absence of cytoplasmic incompatibility in D. mauritiana was due to either the bacterial or host genome, we transferred bacteria from D. mauritiana into an uninfected strain of D. simulans, a host species known to express high levels of incompatibility with endogenous Wolbachia. We also performed the reciprocal transfer of the natural D. simulans Riverside infection into a tetracycline-treated stock of D. mauritiana. In each case, the ability to express incompatibility was unaltered by the different host genetic background. These experiments indicate that in D. simulans and D. mauritiana expression of the cytoplasmic incompatibility phenotype is determined by the bacterial strain and that D. mauritiana harbors a neutral strain of Wolbachia.
Animals↗
Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera.
Transposable elements of the mariner family are widespread among insects and other invertebrates, and initial analyses of their relationships indicated frequent occurrence of horizontal transfers between hosts. A specific PCR assay was used to screen for additional members of the irritans subfamily of mariners in more than 400 arthropod species. Phylogenetic analysis of cloned PCR fragments indicated that relatively recent horizontal transfers had occurred into the lineages of a fruit fly Drosophila ananassae, the horn fly Haematobia irritans, the African malaria vector mosquito Anopheles gambiae, and a green lacewing Chrysoperla plorabunda. Genomic dot-blot analysis revealed that the copy number in these species varies widely, from about 17,000 copies in the horn fly to three copies in D. ananassae. Multiple copies were sequenced from genomic clones from each of these species and four others with related elements. These sequences confirmed the PCR results, revealing extremely similar elements in each of these four species (greater than 88% DNA and 95% amino acid identity). In particular, the consensus sequence of the transposase gene of the horn fly elements differs by just two base pairs out of 1,044 from that of the lacewing elements. The mosquito lineage has diverged from the other Diptera for over 200 Myr, and the neuropteran last shared a common ancestor with them more than 265 Myr ago, so this high similarity implies that these transposons recently transferred horizontally into each lineage. Their presence in only the closest relatives in at least the lacewing lineage supports this hypothesis. Such horizontal transfers provide an explanation for the evolutionary persistence and widespread distribution of mariner transposons. We propose that the ability to transfer horizontally to new hosts before extinction by mutation in the current host constitutes the primary selective constraint maintaining the sequence conservation of mariners and perhaps other DNA-mediated elements.
Amino Acid Sequence↗
Distribution of transposable elements in arthropods.
Transposable elements of the DNA-mediated and RNA-mediated classes found in arthropods are briefly described and their distribution reviewed. The distribution patterns of DNA-mediated elements are extremely patchy and the principal cause appears to be the horizontal transfer of elements between host lineages. In the best documented case of mariner elements, these hosts can be in different orders of insects, classes of arthropods, and even other phyla of animals. RNA-mediated elements appear to undergo much longer periods of vertical evolution within host lineages, and evidence for their horizontal transfer remains scant. The evolutionary relationships of many of these transposons have recently been illuminated by phylogenetic analyses of the reverse-transcriptase enzymes of the RNA-mediated elements, and the recognition that the transposases of some of the DNA-mediated elements are distantly related to in the integrases of some of the RNA-mediated elements.
Animals↗