David J. Finnegan

Eukaryotic transposable elements and genome evolution

The changes in DNA sequence that have taken place during the evolution of eukaryotic genomes cannot be accounted for simply by base substitutions; some more complex mutations must have taken place as well. Transposable elements can affect gene structure and expression in several ways that suggest that they may have contributed to these evolutionary events.

The molecular basis of I-R hybrid Dysgenesis in drosophila melanogaster: Identification, cloning, and properties of the I factor

We have analyzed two mutations of the white-eye gene, which arose in flies subject to I-R hybrid dysgenesis. These mutations are associated with insertions of apparently identical 5.4 kb sequences, which we have cloned. We believe that these insertions are copies of the I factor controlling I-R hybrid dysgenesis. The I factor is not a member of the copia-like or fold-back classes of transposable elements and has no sequence homology with the P factor that controls P-M dysgenesis. All strains of D. melanogaster contain I-factor sequences. Those present in reactive strains must represent inactive I elements. I elements have a remarkably similar sequence organization in all reactive strains and are located in peri-centromeric regions. Inducer strains appear to contain both I elements, located in peri-centromeric regions, and 10-15 copies of the complete I factor at sites on the chromosome arms.

Transposition of the Drosophila element mariner into the chicken germ line

The ability of the Drosophila transposable element mariner to transpose in the chicken was tested using a plasmid carrying an active mariner element injected into chick zygotes. Surviving embryos and chicks were analyzed for presence of mariner. Analysis of embryos that survived for at least 12 days of development indicated that mariner had transposed at high frequency into the chicken genome. Germline transmission of mariner from one of three surviving birds confirmed transposition. Analysis of the first-generation (G1) chicks showed that they each contained between one and three copies of mariner. Six different transposition events were represented in the G1 birds, and the transposition was catalyzed by expression of the mariner elements transposase gene. Transmission from G1 to G2 occurred at a 1:1 ratio. Mariner therefore has potential for development as a vector for transgenesis in avian species.

Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.

Genome evolution: Sex and the transposable element

Mating systems are thought to play an important role in determining the fate of genomic parasites such as transposable elements. This is supported by recent studies which indicate that asexual genomes may be structured very differently to those of sexual species.

Mechanism of Mos1 transposition: insights from structural analysis.

We present the crystal structure of the catalytic domain of Mos1 transposase, a member of the Tc1/mariner family of transposases. The structure comprises an RNase H‐like core, bringing together an aspartic acid triad to form the active site, capped by N‐ and C‐terminal α‐helices. We have solved structures with either one Mg2+ or two Mn2+ ions in the active site, consistent with a two‐metal mechanism for catalysis. The lack of hairpin‐stabilizing structural motifs is consistent with the absence of a hairpin intermediate in Mos1 excision. We have built a model for the DNA‐binding domain of Mos1 transposase, based on the structure of the bipartite DNA‐binding domain of Tc3 transposase. Combining this with the crystal structure of the catalytic domain provides a model for the paired‐end complex formed between a dimer of Mos1 transposase and inverted repeat DNA. The implications for the mechanisms of first and second strand cleavage are discussed.

Excision of the Drosophila mariner transposon Mos1: Comparison with bacterial transposition and V(D)J recombination

It has been proposed that the modern immune system has evolved from a transposon in an ancient vertebrate. While much is known about the mechanism by which bacterial transposable elements catalyze double-strand breaks at their ends, less is known about how eukaryotic transposable elements carry out these reactions. We have examined the mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage. We show that the nontransferred strand is cleaved initially, unlike prokaryotic transposons which cleave the transferred strand first. First strand cleavage is not tightly coupled to second strand cleavage and can occur independently of synapsis, as happens in V(D)J recombination but not in transposition of prokaryotic transposons. Unlike V(D)J recombination, however, second strand cleavage of mariner does not occur via a hairpin intermediate.

Two classes of Flac mutants insensitive to transfer inhibition by an F-like R factor

SummaryMutants of Flac episomes whose transfer is no longer inhibited by the fi+ R factor R100 are shown to be of at least two types. One is recessive in transient heterozygotes containing Fhis and R100 in addition to the Flac mutant. The other is dominant. The occurrence of recessive mutants suggests that inhibition of F transfer by R100 requires an F-specified gene product in addition to that produced by the R factor. Synthesis of the F product or its interaction with the R100 product to give the true inhibitor seems to be a slow process. Since the inhibitor and mutations of the transfer gene traJ both affect a plasmid-specific transfer product, F-pilus formation, and surface exclusion, we propose that the inhibitor prevents the synthesis or function of the traJ product.

Identification of a potential RNA intermediate for transposition of the LINE-like element I factor in Drosophila melanogaster.

The I factor, a transposable element related to mammalian LINEs, controls the I‐R system of hybrid dysgenesis in Drosophila melanogaster. It transposes at high frequency in the germ‐line of the female progeny of crosses between females of the reactive class of strains and males of the inducer class. The structure and DNA sequence of the I factor suggest that it transposes by reverse transcription of an RNA intermediate. Northern blot and S1 mapping experiments show that a full‐length RNA of the I factor is synthesized specifically in the conditions of which I factors transpose. This RNA has all characteristics of a transposition intermediate. It is only found in the ovaries of dysgenic females suggesting that I factor activity is restricted to this tissue because of regulation at the level of the initiation of transcription or RNA stability.

Transposable elements: How non-LTR retrotransposons do it

The source of the enzyme activity responsible for the transposition of retrotransposons of the type that lack terminal repeats has at last been identified: in L1Hs elements, it is encoded by the second open reading frame and is a nuclease related to the apurinic repair endonucleases.