The mobility of transposable elements via a cut-and-paste mechanism depends on the elaboration of a nucleoprotein complex known as the synaptic complex. We show here that the Mos1 synaptic complex consists of the two inverted terminal repeats of the element brought together by a transposase tetramer and is designated paired-end complex 2 (PEC2). The assembly of PEC2 requires the formation of a simpler complex, containing one terminal repeat and two transposase molecules and designated single-end complex 2 (SEC2). In light of the formation of SEC2 and PEC2, we demonstrate the presence of two binding sites for the transposase within a single terminal repeat. We have found that the sequence of the Mos1 inverted terminal repeats contains overlapping palindromic and mirror motifs, which could account for the binding of two transposase molecules "side by side" on the same inverted terminal repeat. We provide data indicating that the Mos1 transposase dimer is formed within a single terminal repeat through a cooperative pathway. Finally, the concept of a tetrameric synaptic complex may simply account for the inability of a single mariner transposase molecule to interact at the same time with two kinds of DNA: the inverted repeat and the target DNA.Mos1 is an autonomous mariner transposable element first isolated from Drosophila mauritiana. It is 1,286 bp long, with 28-bp imperfect inverted terminal repeats (ITR), and contains a single open reading frame encoding a 345-amino-acid transposase (Tnp). On the basis of the sequence of the Tnp and the organization of the element, mariner has been grouped together with the Tc1 and pogo transposable elements to form the Tc1/mariner superfamily (27). One of the main properties of the Tc1/mariner elements is that they do not require host factors for their mobility, because the Tnp is sufficient to promote all the transposition steps. This property accounts for the wide distribution of the Tc1/mariner superfamily among eukaryotic organisms and makes it possible to develop DNA transfer vectors based on Tc1/mariner elements (22).mariner transposes by a cut-and-paste mechanism, similar to that described for related bacterial insertion sequences (4). Briefly, the two ends of the elements are brought together by transposase oligomerization to form a synaptic complex that triggers cleavages at the transposon ends. This complex is usually designated a paired-end complex (PEC). The Tnp then promotes the integration of the excised transposon at a new target site. Assembling the highly organized synaptic complex is the key of transposition, in which the cleavages progress step by step. The structure of this synaptic complex has been elucidated for several prokaryotic transposons, such as Tn5. In this case, the molecular assembly is dimeric: each doublestranded DNA molecule is bound to both Tnp subunits (8). In other cases, where no crystallographic data are available, the exact stoichiometry of the complex has yet to be defined, as explained for IS911 (21). No complete synaptic complex has s...
In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.
BackgroundMariner-like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo.ResultsThe transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency.ConclusionThe study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.
The human kallikrein-related peptidases (KLK) are serine proteases whose concentrations are often abnormal in common human malignancies and contribute to neoplastic progression through multifaceted roles. However, little attention has been paid to their synthesis and involvement in the development and dissemination of lung cancer, the leading cause of cancer mortality worldwide. We have analysed the production of KLK6 in normal lung and tumour tissues from patients with non-small cell lung cancer (NSCLC). KLK6 immunoreactivity was restricted to epithelial cells of the normal bronchi, but most of the cancer samples were moderately or highly immunoreactive, regardless of the histological subtype. In contrast, little or no KLK6 was detected in NSCLC cells. We have developed NSCLC lines expressing wild-type KLK6 in order to investigate the role of KLK6 in lung cancer biology, and analysed its impact on proliferation. Ectopic KLK6 dramatically enhanced NSCLC cell growth and KLK6-producing NSCLC cells had accelerated cell cycles, between the G1 and S phases. This was accompanied by a marked increase in cyclin E and decrease in p21. KLK6 production was also associated with enhanced synthesis of c-Myc, which is known to promote cell-cycle progression. Finally, examination of specimens from patients with NSCLC revealed that KLK6 mRNA is overexpressed in tumour tissue, and high KLK6 concentrations were associated with lower survival rates. We conclude that a high concentration of KLK6 is an indicator of tumour proliferation and an independent predictive factor in NSCLC.
This erratum is being published as a result of author's first & last names were interchanged.
Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI-2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI-2 is frequently down-regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI-2 down-regulation in the National Cancer Institute (NCI)-H460 non-small cell lung cancer cell line using specific micro interfering micro-interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI-2 down-regulation enhanced cell adhesion to collagen IV and laminin via an increase in α1 integrin on cell surface, and increased MMP expression (mainly MMP-1 and -3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI-2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP-1, -3 and -7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.
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