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D J Finnegan

Publications and source records attributed to D J Finnegan.

At least 19 recordsLinked to original sources
PubMed2001

DNA-binding activity and subunit interaction of the mariner transposase.

Mos1 is a member of the mariner/Tc1 family of transposable elements originally identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase-transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino acids. This requires a putative helix-turn-helix motif between residues 88 and 108. Binding is preferentially to the right hand end, which differs at four positions from the repeat at the left end. Cleavage of Mos1 by transposase is also preferentially at the right hand end. Wild-type transposase monomers interact with each other in a yeast two-hybrid assay and we have used this to isolate mutations resulting in reduced interaction. These mutations lie along the length of the protein, indicating that transposase-transposase interactions are not due to a single interaction domain. One such mutation which retains both DNA-binding and catalytic activity has greatly reduced ability to excise Mos1 from plasmid DNA through coordinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase-transposase interaction is required to form a synaptic complex necessary for coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequences with GC base pairs. This is the first example of a mariner/Tc1 transposase with altered target specificity.

Amino Acid Sequence↗
PubMed2001

Drosophila homolog of the myotonic dystrophy-associated gene, SIX5, is required for muscle and gonad development.

SIX5 belongs to a family of highly conserved homeodomain transcription factors implicated in development and disease. The mammalian SIX5/SIX4 gene pair is likely to be involved in the development of mesodermal structures. Moreover, a variety of data have implicated human SIX5 dysfunction as a contributor to myotonic dystrophy type 1 (DM1), a condition characterized by a number of pathologies including muscle defects and testicular atrophy. However, this link remains controversial. Here, we investigate the Drosophila gene, D-Six4, which is the closest homolog to SIX5 of the three Drosophila Six family members. We show by mutant analysis that D-Six4 is required for the normal development of muscle and the mesodermal component of the gonad. Moreover, adult males with defective D-Six4 genes exhibit testicular reduction. We propose that D-Six4 directly or indirectly regulates genes involved in the cell recognition events required for myoblast fusion and the germline:soma interaction. While the exact phenotypic relationship between D-Six4 and SIX4/5 remains to be elucidated, the defects in D-Six4 mutant flies suggest that human SIX5 should be more strongly considered as being responsible for the muscle wasting and testicular atrophy phenotypes in DM1.

Animals↗
PubMed1999

Pogo transposase contains a putative helix-turn-helix DNA binding domain that recognises a 12 bp sequence within the terminal inverted repeats.

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.

Amino Acid Sequence↗
PubMed1998

Copy number control of a transposable element, the I factor, a LINE-like element in Drosophila.

The I factor is a LINE-like transposable element in Drosophila. Most strains of Drosophila melanogaster, inducer strains, contain 10-15 copies of the I factor per haploid genome located in the euchromatic regions of the chromosome arms. These are not present in a few strains known as reactive strains. I factors transpose at low frequency in inducer strains but at high frequency in the female progeny of crosses between reactive and inducer flies. We have found that the activity of the I factor promoter is sensitive to the number of copies of the first 186 nucleotides of the I factor sequence, which constitutes the 5'-untranslated region. The activity of the I factor decreases as the copy number of this sequence increases.

Animals↗
PubMed1997

A LINE-like transposable element in Drosophila, the I factor, encodes a protein with properties similar to those of retroviral nucleocapsids.

I factors are members of the LINE-like family of transposable elements and move by reverse transcription of an RNA intermediate. Complete I factors contain two open reading frames. The amino acid sequence encoded by the first of these, ORF1, includes the motif CX2CX4HX4C that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences CX2CX4HX6C and CX2CX9HX6C. The function of this protein is unknown. We have expressed this protein in Escherichia coli and Spodoptera frugiperda cells and have shown that it binds both DNA and RNA but without any evidence for sequence specificity. The properties of deletion derivatives of the protein indicate that more than one region is responsible for DNA binding and that the CCHC motif is not essential for this. The ORF1 protein expressed in either E. coli or Spodoptera cells forms high molecular weight structures that require the region of the protein including the CCHC motif for their formation. This protein can also accelerate the annealing of complementary single-stranded oligonucleotides. These results suggest that this protein may associate with the RNA transposition intermediates of the I factor to form particles that enter the nucleus during transposition and that it may stimulate both the priming of reverse transcription and integration. This may be generally true for the product of the first open reading frame of LINE-like elements.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Control of expression of the I factor, a LINE-like transposable element in Drosophila melanogaster.

I factors are LINE-like transposable elements in the genome of Drosophila melanogaster. They normally transpose infrequently but are activated in the germline of female progeny of crosses between males of a strain that contains complete elements, an I or inducer strain and females of a strain that does not, an R or reactive strain. This causes a phenomenon known as I-R hybrid dysgenesis. We have previously shown that the I factor promoter lies between nucleotides 1 and 30. Here we demonstrate that expression of this promoter is regulated by nucleotides 41-186 of the I factor. This sequence can act as an enhancer as it stimulates expression of the hsp7O promoter in ovaries in the absence of heat-shock. Within this region there is a site that is required for promoter activity and that is recognized by a sequence-specific binding protein. We propose that this protein contributes to the enhancer activity of nucleotides 41-186 and that reduced I factor expression in inducer strains is due to titration of this protein or others that interact with it.

Animals↗
PubMed1995

BS a novel LINE-like element in Drosophila melanogaster.

Transposable elements with long terminal inverted repeats are rare and only one family of elements of this sort has been identified in the genome of Drosophila melanogaster. An insertion associated with the HSBS mutation of the achaete-scute complex has been reported to be a second element of this type. We have determined the complete sequence of this insertion and have shown that it is in fact two copies of a new LINE-like transposable element, that we have called BS, inserted in opposite orientation 337 bp apart. Like other elements of this type, BS has two open reading frames that appear to encode a gag-like polypeptide and a reverse transcriptase. There are few complete BS elements in the five strains of D.melanogaster that we have tested and they appear to transpose infrequently. The events that may have lead to the double BS insertion are discussed in terms of the supposed mechanism of transposition of LINE-like elements.

Amino Acid Sequence↗
PubMed1993

The 5' untranslated region of the I factor, a long interspersed nuclear element-like retrotransposon of Drosophila melanogaster, contains an internal promoter and sequences that regulate expression.

The I-R system of hybrid dysgenesis in Drosophila melanogaster is controlled by a long interspersed nuclear element-like retroposon, the I factor. Transposition of the I factor occurs at a high frequency only in the ovaries of females produced by crossing males of inducer strains that contain functional I factors with females of reactive strains that lack them. In this study, the 5' untranslated region of the I factor was joined to the chloramphenicol acetyltransferase gene, and activity was assayed in transfected D. melanogaster tissue culture cells and transformed flies. The results have identified a promoter that lies within the first 186 pb of the I factor. Deletion analysis shows that nucleotides +1 to +40 are sufficient for high promoter activity and accurate transcription initiation. This region contains sequences that are found in a class of RNA polymerase II promoters that lack both a TATA box and CpG-rich motifs. In transformed flies, high levels of expression from nucleotides +1 to +186 are confined to the ovaries of reactive females, suggesting that the promoter is involved in the tissue and cytotype specificity of transposition.

Animals↗
PubMed1992

Transposable elements.

Transposable elements comprise a major fraction of eukaryotic genomes. They are studied both because of their intrinsic biological interest and because they can be exploited as valuable research tools. Many interesting papers dealing with various aspects of the biology of these elements have been published during the past year and a number of new elements have been reported. Four areas in which particularly valuable contributions have been made are the mechanisms of transposition, the regulation of transposition, the use of transposable elements as research tools, and the biological function of transposable elements.

Animals↗
PubMed1991

Polypeptide components of Drosophila small nuclear ribonucleoprotein particles.

In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.

Animals↗
PubMed1991

Evidence for retrotransposition of the I factor, a LINE element of Drosophila melanogaster.

LINEs are transposable elements found in various eukaryotes such as plants, protists, insects, and mammals. Their transposition is usually difficult to study, particularly in humans, where some diseases have been shown to result from LINE insertion mutations. This is due to the fact that most copies of any particular family of elements are defective and that their transposition frequency is low. By contrast, the I factor of Drosophila melanogaster transposes at high frequency during I-R hybrid dysgenesis and is a good model for studying the LINE element superfamily. LINEs encode putative polypeptides showing similarities with viral reverse transcriptases but, unlike viral retrotransposons, they do not have terminal repeats and their ability to transpose by reverse transcription has previously only been inferred from structural analysis. Here we present direct evidence for LINE retrotransposition. Transposition of an I factor marked by an intron resulted in accurate removal of the intron.

Animals↗
PubMed1990

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.

Animals↗
PubMed1989

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.

Base Sequence↗