First Mariner Mos1 transposase inhibitors.

We described chemical inhibitors of Mos1 transposition. Some were already known to affect a related prokaryotic transposase (Tn5) or HIV-1 integrase, whereas the other were new compounds in this field. The new compounds were all organized around a bis-(heteroaryl)maleimides scaffold. Their mechanism of action depended on the chemical substitutions on the scaffold. The cross-activity, between HIV-1 integrase and Mos1 transposase, of the new group of inhibitors showed that Mos1 transposase could constitute an excellent surrogate HIV-1 inhibitor screen.

The wild-type conformation of the Mos-1 inverted terminal repeats is suboptimal for transposition in bacteria.

The two inverted terminal repeats (ITRs) flanking the Mos-1 mariner element differ in sequence at four positions. Gel retardation experiments indicated that each of these differences has a significant impact on the quality of the interaction between the ITR and the Mos-1 transposase. We showed that the transposase binds to the 3' ITR better than to the 5' ITR. The results of transposition assays performed in Escherichia coli indicated that these differences have an influence on the rate of transposition and the stability of the transposition products. Finally, we find that the wild-type configuration of the Mos-1 element, with one 5' ITR and one 3' ITR, is less efficient for transposition in bacteria than that of an element having two 3' ITRs.

The ITR binding domain of the Mariner Mos-1 transposase.

Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active. Little is known about the biochemistry of mariner transposition. The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element. Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141. Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements.

Features of the mammal mar1 transposons in the human, sheep, cow, and mouse genomes and implications for their evolution.

Mariner-like elements (MLE) belong to the Tc1/ mariner superfamily of class II transposons. We have analyzed the mariner related to the cecropia subfamily, and called mammal mar1, in four mammalian genomes, Bos taurus (Bovidae), Homo sapiens (Primata), Mus musculus (Rodentia), and Ovis aries (Ovidae). Three kinds of MLE sequences were found in all these species: full-length 1.3-kbp elements, shorter elements 80 bp-1.2 kbp, and single inverted terminal repeats (ITRs). All the 1.3-kbp genomic copies sequenced had an open reading frame encoding a transposase interrupted by stop codons or frame shifts. Phylogenetic analysis of the full-length elements suggested at least two distinct populations of mammal mar1 elements in each species. This was confirmed by using a statistical method that allows defining populations. Finally, the evolutionary origin of the mammal mar1 elements and the paradoxes are discussed.

Identification of mariner-like elements from the root-knot nematode Meloidogyne spp.

The Meloidogyne species are agriculturally important pests widespread in the world. These polyphagous endoparasitic nematodes possess an astonishing ability to bypass the plant resistance genes in few generations. However, the genes and mechanisms involved in this molecular determinism are not yet known. Except cytogenetic and cytotaxonomic studies, few data are available concerning their genome. There is therefore an important need of molecular tools for genetic investigation of their virulence character and other aspects of host-pathogen interactions. In that respect, the presence of mariner-like-elements (MLEs) was assessed in these endoparasitic nematodes by a polymerase chain reaction (PCR) assay using degenerate primers designed from two conserved regions of the mariner transposase open reading frame (ORF). Four Meloidogyne species of the five tested revealed the presence of MLEs in their genome. Southern blot analysis indicated that sequences hybridizing to the mariner transposase-like PCR clones occur at a moderate to low copy number in the different Meloidogyne spp. genomes. The phylogenetic analysis show that the Meloidogyne MLEs may form new subfamilies of mariner. Moreover, five PCR clones were shown to possess a continuous ORF suggesting the presence of putative transposase-like coding regions.

Evidence that the alpha-subunit influences the specificity of receptor binding of the equine gonadotrophins.

Horse LH/chorionic gonadotrophin (eLH/CG) exhibits, in addition to its normal LH activity, a high FSH activity in all other species tested. Donkey LH/CG (dkLH/CG) also exhibits FSH activity in other species, but about ten times less than the horse hormone. In order to understand the molecular basis of these dual gonadotrophic activities of eLH/CG and dkLH/CG better, we expressed, in COS-7 cells, hybrids between horse and donkey subunits, between horse or donkey alpha-subunit and human CG beta (hCG beta), and also between the porcine alpha-subunit and horse or donkey LH/CG beta. The resultant recombinant hybrid hormones were measured using specific FSH and LH in vitro bioassays which give an accurate measure of receptor binding specificity and activation. Results showed that it is the beta-subunit that determines the level of FSH activity, in agreement with the belief that it is the beta-subunit which determines the specificity of action of the gonadotrophins. However, donkey LH/CG beta combined with a porcine alpha-subunit exhibited no FSH activity although it showed full LH activity. Moreover, the hybrid between horse or donkey alpha-subunit and hCG beta also exhibited only LH activity. Thus, the low FSH activity of dkLH/CG requires an equine (donkey or horse) alpha-subunit combined with dkLH/CG beta. These results provide the first evidence that an alpha-subunit can influence the specificity of action of a gonadotrophic hormone.

Computer analyses reveal a hobo-like element in the nematode Caenorhabditis elegans, which presents a conserved transposase domain common with the Tc1-Mariner transposon family.

The present report describes the use of computer analyses to reveal a hobo-like element in the genome of Caenorhabditis elegans. This hobo-like sequence is 3039 bp long, contains two inverted terminal repeats of 25-27 bp and probably does not encoded a functional transposase. Sequence comparisons suggest that each transposase of hobo elements probably has a D(D/S)E motif. Thus the transposases of the hAT superfamily of transposons appear to be close to the other transposases and intregrases.

Human and other mammalian genomes contain transposons of the mariner family.

Internal fragments of the putative transposase gene of mariner-like elements (MLEs) were amplified from human, mouse, rat, chinese hamster, sheep and bovine genomic DNAs by polymerase chain reaction (PCR). The sequences identified in human, ovine and bovine genomes correspond to ancient degenerate transposons. Screening mammalian sequence libraries identified a truncated element in the human ABL gene and the sequence of its 5'-ITR was determined. This ITR sequences were used in PCR experiments with DNA from six mammalian species and detected full-sized and deleted MLEs. The presence of MLE in mammalian genomes demonstrates that they are ubiquitous mobile elements found from fungi to man. This observation strongly raises the possibility that MLE could constitute tools for the modification of eucaryotic genomes.

Liver-enriched HNF-3 alpha and ubiquitous factors interact with the human transferrin gene enhancer.

The human transferrin gene enhancer is organized in two domains. Domain A contains a single enhanson designated Ia. Domain B contains four enhansons named Ib, II, III and IV. We demonstrate here that the liver-enriched transcription factor HNF-3 alpha interacts with enhanson Ia and that enhansons Ib and IV are binding sites for members of the NF1 family. In addition, enhansons II and III seem to be respectively the targets for the AP4 protein and for EIII, a factor not yet completely identified. Analysis of mutated enhancer regions establishes that each enhanson is required for full enhancer activity and that the proteins binding to enhansons II, III and IV may interact within a multiprotein complex. This enhancer region presents no activity in the Sertoli cells of testis, where transferrin is also synthesized. We demonstrate that in Sertoli cells, the members of the HNF-3 family are not expressed; this fact may account for the inactivity of the enhancer in these cells.

Characterization of the active part of the human transferrin gene enhancer and purification of two liver nuclear factors interacting with the TGTTTGC motif present in this region.

The human transferrin gene enhancer is composed of two functional domains (A and B). We have previously shown that domain A is able to mediate enhancer activity in transient expression experiments. Here, we show that multimers of the domain A single enhanson coupled to a canonical TATA box are sufficient to promote transcription in vitro with liver nuclear extracts. Gel mobility shift assays reveal the binding of two liver nuclear factors to this enhanson, and methylation interference experiments show that the motif 5'-TGTTTGCTTT-3' is the target site for these factors. This was confirmed by the use of mutants in gel retardation assays and transient expression experiments. The two proteins interacting with the decanucleotide have been purified from rat liver nuclear extracts by DNA affinity chromatography. The purified proteins named enhancer-binding protein (EBP)-45 and EBP-40 appear as single polypeptide bands with respective molecular masses of 45 and 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The TGTTTGC motif was found to be important for the hepatocyte-specific expression of several genes; and in some cases, it was demonstrated that the transcription factor CCAAT/enhancer-binding protein (C/EBP) is able to bind to this sequence. In vitro experiments show that EBP-45 and EBP-40 are different from C/EBP; they also show that the two proteins interact with the TGTTTGC motif present in control regions of other hepatic genes, such as the mouse albumin enhancer eH and hepatitis B virus enhancer E elements.

The enhancer of the human transferrin gene is organized in two structural and functional domains.

We previously identified a 300-base pair long enhancer, located 3.6 kilobases upstream of the cap site of the human transferrin gene. A 5' deletion up to position 86 of the enhancer resulted in complete loss of the enhancer activity. Here we show by competition footprint analysis, gel retardation assays, and transient expression studies in hepatoma and HeLa cells that the enhancer is composed of two distinct structural and functional domains, A (nucleotides 1-86) and B (nucleotides 87-291). Each domain is a proto-enhancer of a different type. Domain A is a proto-enhancer that, when multimerized, is able by itself to stimulate transcription from the heterologous SV40 promoter, both in Hep3B and HeLa cells. It contains the octanucleotide TGTTTGCT sequence and is the binding site of two liver-specific nuclear factors and of a different HeLa nuclear factor. Domain B contains four binding sites interacting with several liver nuclear proteins. In order to bind, any of these proteins requires the presence of all the others. This domain is able to block the activity of a downstream negative element, but it has no enhancer activity by itself. In the presence of the transferrin promoter, full enhancer activity requires the association of the two domains A and B.