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PubMed1994

Localized expression of a novel micropia-like element in the blastoderm of Drosophila melanogaster is dependent on the anterior morphogen bicoid.

We have identified a novel transposon-like element of Drosophila melanogaster that is present in approximately 20 copies in the genome. It codes for a polyprotein containing the diagnostic sequence motifs for a nucleic acid binding CCHC protein, a proteinase, a reverse transcriptase and an integrase as typically found in retroviruses. Owing to its early expression in the blastoderm embryo, and its close relationship to micropia, a previously identified Drosophila retrotransposon, we termed the novel element "blastopia". The spatially restricted expression of blastopia transcripts in head anlagen of the blastoderm embryo is under the direct or indirect control of the Drosophila morphogen bicoid, which is normally required to establish the anterior pattern elements in the embryo. Our results suggest that a blastopia element acts as an "enhancer trap", and thereby participates in the control of an as yet unidentified gene normally expressed in the head anlagen of the embryo.

Amino Acid Sequence↗
PubMed1994

The genome of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a member of the genus Lentivirus of the family Retroviridae. FIV can infect T lymphocytes and monocytes/macrophages in vitro and in vivo, and causes an acquired immunodeficiency syndrome-like disease in cats. Several isolates of FIV from geographically distant countries have been molecularly cloned. There is considerable heterogeneity especially in Env gene among the FIV isolates and they can be divided into two or more subgroups. Like other lentiviruses, FIV has a complex genome structure. Gag gene encodes matrix, capsid and nucleocapsid proteins, and Pol gene encodes protease, reverse transcriptase, dUTPase and integrase. The dUTPase is not present in the primate lentiviruses but present in the non-primate lentiviruses. Env gene encodes surface and transmembrane envelope glycoproteins. In addition to the structural and enzymatic proteins, at least three more genes (Vif, ORF A, Rev) are present in FIV. Vif is related to the infectivity of the cell-free viruses. Rev functions in the stability and transport of incompletely spliced viral RNAs from the nucleus to cytoplasm and is indispensable for virus replication. Although the Tat protein of the primate lentiviruses is essential for virus replication, ORF A (putative Tat gene) of FIV is not essential for virus replication in established feline T lymphoblastoid cell lines. However, the ORF A gene product is related to the efficient replication of the virus in primary peripheral blood lymphocytes. In the long terminal repeat (LTR) of FIV, there are many putative binding sites for enhancer/promoter proteins. Among these binding sites, the putative AP-1 site is important for basal promoter activity of the LTR and responsible for the T cell activation signal through protein kinase C, however the site is not required for the virus replication in established feline T lymphoblastoid cell lines. Comparative study of the molecular biology of lentiviruses revealed that the genome structure, splicing pattern and functional enhancer protein-binding sites of FIV are more similar to those of the ruminant lentiviruses than those of the primate lentiviruses.

Animals↗
PubMed1996

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↗
PubMed2004

Different molecular rearrangements in the integron of the IncP-1 beta resistance plasmid pB10 isolated from a wastewater treatment plant result in elevated beta-lactam resistance levels.

The multiresistance IncP-1 beta plasmid pB10 conferring resistance to ampicillin, streptomycin, sulfonamides, tetracycline and mercury ions was previously obtained from activated sludge bacteria by applying the exogenous isolation method with Pseudomonas sp. strain GFP2 as recipient. A pB10 derivative, designated pB10-1, occurred spontaneously and displays an extended NotI restriction fragment. From the pB10 nucleotide sequence, it is known that the corresponding NotI fragment of this plasmid contains a complete class 1 integron with an oxa2 and an orfE-like gene cassette. Sequencing of the integron-specific variable region present on pB10-1 revealed that a second copy of the oxa2 gene cassette has inserted downstream of the orfE-like cassette. Sequences flanking the second oxa2 cassette indicate that this cassette was excised from pB10 and reinserted at a new site in an integrase-catalyzed manner. Duplication of the oxa2 cassette is associated with a higher level of ampicillin resistance. Another pB10 derivative, designated pB10-2, conferring higher resistance to ampicillin, was shown to carry an IS10 insertion upstream of the oxa2 cassette. Since IS10 possesses a promoter-out activity, it can be assumed that the elevated ampicillin resistance level is due to enhanced transcription of the beta-lactamase gene.

Ampicillin↗
PubMed2001

Cloning, characterization, and possible origin of the Prevotella loescheii dnaK homolog.

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella.

Amino Acid Sequence↗
PubMed2005

Molecular characterization of a gender-linked DNA marker and a related gene in Mercurialis annua L.

The dioecious Mercurialis annua L. was used as a model plant to study some aspects of the molecular basis of sex determination in plants. We report in this paper the characterization of a previously identified male specific DNA marker, OPB01-1562, from diploid dioecious M. annua. The marker co-segregated with male sex in the progeny of hormonally feminized males. Sequence analysis showed the presence of approximately 0.6 kb retrotransposon-like sequence at its 3' end. Homologous sequences were isolated from diploid female, hexaploid male and monoecious plants. These sequences contained RNaseH and integrase domains of reverse transcriptase and were most similar to pineapple retrotransposon dea1, hence were named M. annua retrotransposon-like sequences (MARL-1 to MARL-5). A 771 bp fragment isolated from a diploid female, named fem771, was homologous to the 5' end of OPB01-1562. Results from DNA blot hybridization suggested OPB01-1562 and fem771 to be from the same locus and MARL-1 from a different one. RNA blot hybridization with OPB01-1562 and MARL-1 detected an approximately 2.8 kb transcript which was expressed strongly in stems and flowers of females but not males. This transcript was named M. annua female expressed (Mafex). Sex linkage of OPB01-1562 and expression of Mafex detected by OPB01-1562 strongly suggested Mafex to be a candidate gene involved in sex determination in M. annua.

Amino Acid Sequence↗
PubMed2006

PpRT1: the first complete gypsy-like retrotransposon isolated in Pinus pinaster.

We have isolated and characterized a complete retrotransposon sequence, named PpRT1, from the genome of Pinus pinaster. PpRT1 is 5,966 bp long and is closely related to IFG7 gypsy retrotransposon from Pinus radiata. The long terminal repeats (LTRs) have 333 bp each and show a 5.4% sequence divergence between them. In addition to the characteristic polypurine tract (PPT) and the primer binding site (PBS), PpRT1 carries internal regions with homology to retroviral genes gag and pol. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAseH and integrase in the same typical order known for Ty3/gypsy-like retrotransposons. PpRT1 was extended from an EST database sequence indicating that its transcription is occurring in pine tissues. Southern blot analyses indicate however, that PpRT1 is present in a unique or a low number of copies in the P. pinaster genome. The differences in nucleotide sequence found between PpRT1 and IFG7 may explain the strikingly different copy number in the two pine species genome. Based on the homologies observed when comparing LTR region among different gypsy elements we propose that the highly conserved LTR regions may be useful to amplify other retrotransposon sequences of the same or close retrotransposon family.

Amino Acid Sequence↗
PubMed2002

A group of deuterostome Ty3/ gypsy-like retrotransposons with Ty1/ copia-like pol-domain orders.

Here we report the existence of an unusual group of LTR retrotransposons, termed Gmr1-like elements. The members of this group are most similar in sequence to elements of the Ty3/ gypsy group, yet, unlike typical Ty3/ gypsy elements, they have pol genes in which the integrase domain lies upstream, rather than downstream, of the reverse transcriptase domain. Such an arrangement was formerly believed to be a characteristic peculiar to Ty1/ copia elements. The group includes the previously described retrotransposon Gmr1 from the Atlantic cod Gadus morhua, together with elements from a variety of other vertebrates, such as the zebrafish Danio rerio, the pufferfish Fugu rubripes and the clawed toad Xenopus laevis, as well as elements from non-vertebrate deuterostomes such as the sea squirt Ciona intestinalis. No Gmr1-like elements were found outside of the deuterostomes, nor were any other groups of elements with atypical pol-domain orders identified. Phylogenetic analyses show that the Gmr1-like elements form a monophyletic group within the larger group of Ty3/ gypsy elements. Some of the newly identified elements appear to be structurally intact and may still be active. The identification of this group challenges some previously held beliefs concerning the structure and evolution of LTR retrotransposons.

Amino Acid Sequence↗
PubMed2003

Transcriptional analysis of the genetic elements involved in the lysogeny/lysis switch in the temperate lactococcal bacteriophage phiLC3, and identification of the Cro-like protein ORF76.

A transcriptional analysis of the lysogeny-related genes of the temperate bacteriophage Lactococcus lactis phiLC3 was performed using Northern blot hybridization during lysogeny and lytic infection by the phage. The lysogeny-related gene cluster was found to contain four promoters (P(1), P(2), Pint and P(173)), while the P(87) promoter directed transcription of orf80 and the putative gene orf87, which are located between the integrase gene and the cell lysis genes. The start sites of the transcripts were determined by primer extension. The divergently oriented lysogenic P(1) and lytic P(2) promoters located in the genetic switch region are responsible for transcription of orf286 which encodes the phage repressor, and the genes orf63 - orf76 - orf236 - orf110 - orf82 - orf57, respectively, while orf173 is transcribed from P(173). orf76 was identified as the gene encoding the Cro-like protein of phiLC3, and it was shown that ORF76 is able to bind specifically to the genetic switch region, albeit with lower affinity than does the phage repressor ORF286. ORF76 also competed with ORF286 for binding to this region. The functionality of P(1) and P(2), and their regulation by ORF286 and ORF76, was investigated using a reporter gene. In general, P(2) was a stronger promoter than P(1), but expression from both promoters, especially P(2), was regulated and modulated by flanking sequences and the presence of orf286 and orf76. ORF286 and ORF76 were both able to repress transcription from P(1) and P(2), while ORF286 was able to stimulate its own synthesis by tenfold. This work reveals the complex interplay between the regulatory elements that control the genetic switch between lysis and lysogeny in phiLC3 and other temperate phages of Lactococcus.

Bacteriophages↗
PubMed2003

Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences.

piggyBac is a short inverted-repeat-type DNA transposable element originally isolated from the genome of the moth Trichoplusia ni. It is currently the gene vector of choice for the transformation of various insect species. A few sequences with similarity to piggyBac have previously been identified from organisms such as humans ( Looper), the pufferfish Takifugu rubripes ( Pigibaku), Xenopus ( Tx), Daphnia ( Pokey), and the Oriental fruit fly Bactrocera dorsalis. We have now identified 50 piggyBac-like sequences from publicly available genome sequences and expressed sequence tags (ESTs). This survey allows the first comparative examination of the distinctive piggyBac transposase, suggesting that it might contain a highly divergent DDD domain, comparable to the widespread DDE domain found in many DNA transposases and retroviral integrases which consists of two absolutely conserved aspartic acids separated by about 70 amino acids with a highly conserved glutamic acid about 35 amino acids further away. Many piggyBac-like sequences were found in the genomes of a phylogenetically diverse range of organisms including fungi, plants, insects, crustaceans, urochordates, amphibians, fishes and mammals. Also, several instances of "domestication" of the piggyBac transposase sequence by the host genome for cellular functions were identified. Novel members of the piggyBac family may be useful in genetic engineering of many organisms.

Animals↗
PubMed2006

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

Animals↗
PubMed2000

Identification and chromosomal localization of the monkey retrotransposon in Musa sp.

Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial fraction of their total DNA content. One major group of retroelements, which includes the gypsy and copia-like elements, is distinguished by the presence of long terminal repeats (LTRs). We have identified and partially characterized a sequence from banana (Musa acuminata cv. Grand Nain) which shows significant homology to gypsy-like LTR retroelements from other species. The element, named monkey, shows a high degree of homology to the reverse transcriptase, RNase H and integrase genes of retroelements from plants, fungi and yeast. However, several stop codons are present in the major ORF of this element, suggesting that this copy of monkey, if functional, is non-autonomous. Southern analysis indicated that monkey is present in both the A and B genomes of Musa, and that it is found in 200-500 copies per haploid genome in cv. Grand Nain. Chromosomal localization by fluorescent in-situ hybridization indicates that copies of monkey are concentrated in the nucleolar organizer regions and colocalize with rRNA genes. Other copies of monkey appear to be dispersed throughout the genome.

Chromosome Mapping↗
PubMed2001

Two novel Pao-like retrotransposons (Kamikaze and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of Pao-like elements.

To characterize the structural features common to Pao-like retrotransposons, we analyzed two lambda phage clones which contain the Pao-like elements from the silkworm species Bombyx mori and B. mandarinia, and copies of Pao itself and ninja of Drosophila simulans, amplified by PCR. We previously identified two randomly amplified polymorphic DNAs (RAPDs), W-Kamikaze and W-Yamato, from B. mori and B. mandarina, which are part of two novel Pao-like retrotransposons, Kamikaze and Yamato, respectively. Complete characterization of these and other elements of this group reported here shows that Pao-like elements have common features that distinguish them from the other groups of LTR-retrotransposons. While the elements of the Ty1-copia group encode only one cysteine and histidine (Cys) motif in their gag-like region, the Pao-like elements specify three Cys motifs. The highly conserved D(35)E motif in the integrase domain of the retrotransposon polyprotein seems to be conserved in Pao-like elements, but the number of amino acid residues between D and E varies and is greater than 35. A comparison of the deduced amino acid sequences of the reverse transcriptase domain revealed the Pao-like elements are members of neither the Ty1-copia nor the gypsy-Ty3 groups. Therefore, we confirmed that the long-terminal-repeat (LTR) retrotransposons should be divided into three major groups (or families), namely the Ty1-copia, gypsy-Ty3, and Pao-like groups.

Amino Acid Sequence↗
PubMed1998

Tdd-3, a tRNA gene-associated poly(A) retrotransposon from Dictyostelium discoideum.

The full-length 5218-bp sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by non-redundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that element insertion is orientation specific and occurs about 100 bp downstream of tRNA genes in the D. discoideum genome. These properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned cDNAs derived from Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells.

Animals↗
PubMed1999

Identification and characterization of novel retrotransposons of the gypsy type in rice.

We found that two DNA fragments, which were obtained from Oryza sativa L cv. IR36 by PCR using degenerate primers designed for amplification of a rice gene, showed homology with the rt gene encoding reverse transcriptase of the Drosophila retrotransposon gypsy. We named the element from which they originated RIRE3 (for rice retrotransposon No. 3) and analyzed it further by isolating various clones containing segments of RIRE3. Nucleotide sequencing of the clones revealed that RIRE3 has LTRs (2316 bp) and that the internal sequence (5775 bp) includes a large ORF with gag and pol regions; the pol region includes the rt gene as well as the int gene encoding integrase in this order, as in gypsy. Interestingly, the region upstream of gag in RIRE3 contained another open reading frame, here called orf0, which does not exist in gypsy or in other retrotransposons related to it. In the course of characterizing RIRE3, we obtained a further clone, which showed less homology with the pol region of RIRE3. This clone was found to be derived from another gypsy-type retrotransposon (named RIRE8) containing the LTR sequence and orf0 both of which were only weakly homologous to that in RIRE3. Further characterization of RIRE8 revealed that there were actually two subtypes of RIRE8 (named RIRE8A and RIRE8B), which show little homology to each other in the orf0 region. Although the LTRs of RIRE3 and RIRE8 elements show very weak homology with each other, there exists a conserved sequence at their termini. We therefore carried out PCR using primers which hybridize to the rt gene of RIRE3, and total genomic DNA from various monocot and dicot plants as templates, and found that a family of RIRE3 elements was present in all plants tested.

Amino Acid Sequence↗
PubMed2001

Plasmids carrying cloned fragments of RF DNA from the filamentous phage (phi)Lf can be integrated into the host chromosome via site-specific integration and homologous recombination.

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.

Base Sequence↗
PubMed2001

The effect of mutations in the Xis-binding sites on site-specific recombination in coliphage HK022.

Excisionase (Xis) is an accessory protein that is required for the excision of the related prophages lambda and HK022. Xis binds to two tandemly arranged binding sites (X1 and X2) on the P arm of the recombination sites attP and attR. Gel-retardation analyses and site-specific recombination assays were conducted on derivatives bearing site-directed mutations in the X1 and X2 sites of phage HK022. The results confirm the cooperative binding of Xis to its sites, showing that binding to X1 stimulates further binding to X2. The results also show that mutants affected in a single site are inactive in excision, whereas mutants affected in both sites, which show a complete absence of Xis binding, display significant excision activity. This restored activity is attributed to the interaction of Xis with Integrase, the protein that catalyzes the site-specific recombination reaction.

Binding Sites↗
PubMed1999

Mutations designed to alter the stability of a putative RNA duplex in the HIV-1 pol gene.

In the HIV-1 integrase coding region there is a polypurine tract (PPT) involved in the initiation of provirus plus-strand synthesis. Upstream of this PPT there is a 15-nucleotide inverted repeat (IR) complementary to most of the PPT. We have constructed one mutant with five amino acid-neutral U to C and A to G changes in the IR and one mutant with corresponding amino acid-neutral changes in the PPT. Each set of changes abolished the complementarity and suppressed the replication of HIV-1 slightly. The combination of these ten changes restored the complementarity, and doubled the calculated free energy of the putative duplex between the IR and the PPT. This double mutant did not replicate under normal conditions, possibly because the reverse transcriptase was unable to penetrate the duplex. However, when high loads of the double mutant were added to permissive cells, replicative HIV did occasionally appear. The resurrected virus harvested from these cells replicated consistently, even though the ten nucleotide changes were left unchanged. There were no compensatory mutations in the vicinity of the IR/PPT or in the reverse transcriptase gene.

Animals↗