A transposon-related palindromic repetitive sequence from C. elegans.

A family of transposon-like sequences in the C. elegans genome is described. This family, termed the Tc6 family, consists mostly of conserved, 1.6 kb elements. Four Tc6 elements or partial elements have been cloned and the DNA sequences of three were determined. One appears to be a complete element of 1603 nucleotides, consisting of a palindrome of 765 nucleotides, with a central, non-palindromic region of 73 nucleotides. Another has an identical structure except for an internal deletion. A third is a partial element terminating at a probable internal restriction site used for cloning. A fourth clone contained portions of the Tc6 sequence juxtaposed to non-Tc6 sequences. All C. elegans strains examined contain 20-30 Tc6 elements. The ends of Tc6 elements are conserved and have sequence similarity to the ends of C. elegans transposons Tc1 and Tc3. The ends of Tc6 elements also have sequence similarity to the heptamer portion of the immunoglobulin and T-cell receptor recombination signal sequence, raising the possibility of wide phylogenetic conservation of the recombination mechanism. Tc6 elements also share sequence motifs with plant-pathogenic viroid RNA's, possibly indicative of a Tc6 RNA replicative phase.     

Continuous exchange of sequence information between dispersed Tc1 transposons in the Caenorhabditis elegans genome

In a genome-wide analysis of the active transposons in Caenorhabditis elegans we determined the localization and sequence of all copies of each of the six active transposon families. Most copies of the most active transposons, Tc1 and Tc3, are intact but individually have a unique sequence, because of unique patterns of single-nucleotide polymorphisms. The sequence of each of the 32 Tc1 elements is invariant in the C. elegans strain N2, which has no germline transposition. However, at the same 32 Tc1 loci in strains with germline transposition, Tc1 elements can acquire the sequence of Tc1 elements elsewhere in the N2 genome or a chimeric sequence derived from two dispersed Tc1 elements. We hypothesize that during double-strand-break repair after Tc1 excision, the template for repair can switch from the Tc1 element on the sister chromatid or homologous chromosome to a Tc1 copy elsewhere in the genome. Thus, the population of active transposable elements in C. elegans is highly dynamic because of a continuous exchange of sequence information between individual copies, potentially allowing a higher evolution rate than that found in endogenous genes.     

Transposition of cyanobacterium insertion element ISY100 in Escherichia coli

The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.     

Characterization of a Tc1-like transposon in the Antarctic ice-fish,Chionodraco hamatus

We report the presence of Tc1 transposon-like sequences in the Antarctic ice-fish Chionodraco hamatus, belonging to the Notothenioidei. The complete DNA sequence of these transposon-like elements is reduced in length compared to other Tc1 transposons, but it appears to share significant structural similarities with them. It contains a degenerate open reading frame, whose inferred 264 amino acid sequence shares sequence similarity with the 'aspartic acid, aspartic acid (35) glutamic acid' family of transposases, particularly those from Caenorhabditis species (sp.) and Drosophila sp. Southern blot analysis and polymerase chain reaction amplification indicate that Tc1 transposon-like sequences are present in other notothenioid species, though their amount can vary in the different lineages.     

Target site choice of the related transposable elements Tc1 and Tc3 of Caenorhabditis elegans.

We have investigated the target choice of the related transposable elements Tc1 and Tc3 of the nematode C. elegans. The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions in an 1 kbp region of the genome were determined. There was no phenotypic selection for the insertions. All insertions were into the sequence TA. Both elements have a strong preference for certain positions in the 1 kbp region. Hot sites for integration are not clustered or regularly spaced. The orientation of the integrated transposon has no effect on the distribution pattern. We tested several explanations for the target site preference. If simple structural features of the DNA (e.g. bends) would mark hot sites, we would expect the patterns of the two related transposons Tc1 and Tc3 to be similar; however we found them to be completely different. Furthermore we found that the sequence at the donor site has no effect on the choice of the new insertion site, because the insertion pattern of a transposon that jumps from a transgenic donor site is identical to the insertion pattern of transposons jumping from endogenous genomic donor sites. The most likely explanation for the target choice is therefore that the primary sequence of the target site is recognized by the transposase. However, alignment of the Tc1 and Tc3 integration sites does not reveal a strong consensus sequence for either transposon.     

Sequence identity between an inverted repeat family of transposable elements in Drosophila and Caenorhabditis.

The Tc1-like transposable elements, originally described in Caenorhabditis elegans, have a much wider phylogenetic distribution than previously thought. In this paper, we demonstrate that Tc1 shares sequence identity in its open reading frame and terminal repeats with a new transposable element Barney (also known as TCb1-Transposon Caenorhabditis briggsae 1). Barney was detected and isolated by Tc1 hybridization from the closely related nematode species, Caenorhabditis briggsae. The conserved open reading frames of Tc1 and Barney share identity with a structurally similar family of elements named HB found in Drosophila melanogaster, after the introduction of 3 small centrally located deletions in HB1. These reading frames would code for proteins with 30% amino acid identity (42% when conservative changes are included). Tc1, Barney and HB1 contain highly conserved blocks of amino acids which are likely to be in the functional domains of the putative transposase.     

Toxicity symptoms caused by high expression of Tet represser in tomato (Lycopersicon esculentum Mill. L.) are alleviated by tetracycline

As a first step towards transferring a tetracycline (Tc)-inducible gene expression system to tomato, we have transformed tomato plants with the Tn10-encoded tet repressor gene (tetR). Homozygous transformed plants with high expression of tetR mRNA show a deleterious phenotype, having reduced shoot dry weights and leaf chlorophyll content, an even more marked reduction in root dry weight and leaf size, and altered photosynthetic physiology. It appears that TetR protein exerts its toxicity only when expressed beyond a threshold level and by interacting with a process that is non-limiting under slow growth conditions. The deleterious phenotype was almost completely reversed by the application of 1 mg dm^?3 Tc to plants grown in sand. The possiblity is discussed that TetR causes these symptoms by binding to a specific DNA sequence functioning as a Tet operator. The effect of Tc on growth and physiology in wild-type plants grown in sand or rockwool is described. Tc at 0.1 mg cm^?3 had no effect. Tc at 1 mg dm^?3 caused a small reduction in root growth, while 5 and 20 mg dm^?3 Tc caused large reductions in growth and photosynthetic parameters.     

Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids.

Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.     

Trypanosoma cruzi: molecular identification and characterization of new members of the Tc13 family. Description of the interaction between the Tc13 antigen from Tulahuén strain and the second extracellular loop of the beta(1)-adrenergic receptor

Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the beta(1)-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology.     

Isolation and sequence analysis ofCaenorhabditis briggsae repetitive elements related to theCaenorhabditis elegans transposon Tc1

Summary We have identified two repetitive element families in the genome of the nematodeCaenorhabditis briggsae with extensive sequence identity to theCaenorhabditis elegans transposable element Tc1. Five members each of the TCb1 (previously known as Barney) and TCb2 families were isolated by hybridization to a Tc1 probe. Tc1-hybridizing repetitive elements were grouped into either the TCb1 or TCb2 family based on cross-hybridization intensities among theC. briggsae elements. The genomic copy number of the TCb1 family is 15 and the TCb2 family copy number is 33 in theC. briggsae strain G16. The two transposable element families show numerous genomic hybridization pattern differences between twoC. briggsae strains, suggestive of transpositional activity. Two members of the TCb1 family, TCb1#5 and TCb1#10, were sequenced. Each of these two elements had suffered an independent single large deletion. TCb1#5 had a 627-bp internal deletion and TCb1#10 had lost 316 bp of one end. The two sequenced TCb1 elements were highly conserved over the sequences they shared. A 1616-bp composite TCb1 element was constructed from TCb1#5 and TCb1#10. The composite TCb1 element has 80-bp terminal inverted repeats with three nucleotide mismatches and two open reading frames (ORFs) on opposite strands. TCb1 and the 1610-bp Tc1 share 58% overall nucleotide sequence identity, and the greatest similarity occurs in their ORF1 and inverted repeat termini.     

The effects of varying the cellular and extracellular concentrations of sodium and potassium ions on the uptake of glycine by mouse ascites-tumour cells in the presence and absence of sodium cyanide

1. Tumour cells were starved to deplete them of ATP and transferred to 0.9mm-glycine in Ringer solutions containing 2mm-sodium cyanide and various Na(+) and K(+) concentrations. The uptake of glycine then usually reached a peak by about 10min. 2. When cellular [Na(+)] and extracellular [Na(+)] were each about 30m-equiv./l., the maximum amount of glycine absorbed increased between 1.2- and 3.0-fold on lowering extracellular [K(+)] from 128 to 10m-equiv./l. 3. When extracellular [Na(+)] was 150m-equiv./l., the ratio, R, of the cellular to extracellular glycine concentrations increased progressively, from near 1 to about 9, when cellular [Na(+)] was lowered from 120 to 40m-equiv./l. 4. When cellular [Na(+)] was almost constant, either at 45 or 70m-equiv./l., R fell about 14-fold when extracellular [Na(+)] varied from 150 to 16m-equiv./l. 5. Values of R near 0.2 were found when cellular [Na(+)] was about four times as large as extracellular [Na(+)]. 6. R fell about threefold when the cells were put with 12mm- instead of 0.9mm-glycine. 7. The results were taken to imply that, under these conditions, the spontaneous movements of both Na(+) and K(+) across the cell membrane, down their respective concentration gradients, served to concentrate the glycine in the tumour cells (Christensen's hypothesis).     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.     

Immunohistochemical estimation of in-situ cell cycle time in neoplastic epithelial cells in human large intestine: a new cell proliferation index

In-situ cell cycle time (in-situ Tc) of epithelial cells could be estimated by using a formula; in-situ Tc = cell proliferation rate divided by mitosis rate, on a scale of Tm (cell cycle time in M phase) arbitrary unit (AU), In order to see the nature of in-situ Tc in the adenoma-carcinoma sequence in the human large intestine, the in-situ Tc in 27 cases of adenoma and 71 cases of adenocarcinoma with adenoma components in the human large intestine was estimated by using this formula, counting proliferating cells and mitotic cells in the immunohistochemistry of Ki-67 antigen. C12 antigen was examined as an oncogenic progression indicator in the adenoma-carcinoma sequence. The in-situ Tc tended to shorten in adenoma in accordance with the histological grading of atypia but not in adenoma component. No significant differences in the in-situ Tc was recognized as a whole among adenomas, adenoma components and adenocarcinomas in the mucosa, whereas the in-situ Tc of adenoma components with moderate to severe atypia was significantly longer than that of adenocarcinomas in the mucosa (p = 0.05). The in-situ Tc lengthened in adenocarcinomas invading the submucosa and shortened in adenocarcinomas invading the proper muscular layer. The cases expressing the C12 antigen increased in order of adenoma, adenoma component and adenocarcinoma. The cases expressing the C12 antigen indicated short in situ Tc in the adenomas and adenocarcinomas but not in the adenoma components. Thus, the estimated in-situ Tc is a useful index of the oncogenetic progression, which is different from that detected by the C12 antigen.