Introduction of a functional P element into the germ-line of Drosophila hawaiiensis

Brennan, Michael; Rowan, Robert G.; Dickinson, W. J.

doi:10.1016/0092-8674(84)90535-x

Cited by 52 publications

(18 citation statements)

References 18 publications

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“…Genomic DNAs were purified from Oregon-R and mod(mdg4) ul flies as described previously (5). The mod(mdg4) genomic regions were amplified with Accutaq LA DNA polymerase (Sigma), using the conditions suggested by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

The gypsy Insulator Can Act as a Promoter-Specific Transcriptional Stimulator

Wei

Brennan

2001

Molecular and Cellular Biology

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Insulators define chromosomal domains such that an enhancer in one domain cannot activate a promoter in a different domain. We show that the Drosophila gypsy insulator behaves as a cis-stimulatory element in the larval fat body. Transcriptional stimulation by the insulator is distance dependent, as expected for a promoter element as opposed to an enhancer. Stimulation of a test alcohol dehydrogenase promoter requires a binding site for a GATA transcription factor, suggesting that the insulator may be facilitating access of this DNA binding protein to the promoter. Short-range stimulation requires both the Suppressor of Hairy-wing protein and the Mod(mdg4)-62.7 protein encoded by the trithorax group gene mod(mdg4). In the absence of interaction with Mod(mdg4)-62.7, the insulator is converted into a short-range transcriptional repressor but retains some cis-stimulatory activity over longer distances. These results indicate that insulator and promoter sequences share important characteristics and are not entirely distinct. We propose that the gypsy insulator can function as a promoter element and may be analogous to promoter-proximal regulatory modules that integrate input from multiple distal enhancer sequences.The normal expression of eukaryotic genes requires stimulation of promoters by enhancers that often exert their effects over many kilobases of intervening DNA (3,54). Given this ability of enhancers to function over long distances, there must be mechanisms that prevent enhancers of one gene from activating transcription of neighboring genes. Current models propose that this is accomplished by insulators, DNA sequences that define chromosomal domains such that a promoter in one domain cannot be activated by an enhancer in a different domain (23,26). For example, the scs and scsЈ sequences that flank the Drosophila hsp70 gene prevent transcriptional stimulation by the yolk protein-1 enhancer when inserted between the enhancer and an hsp 70 target promoter (32, 56). Consistently, these same sequences insulate transgenes from the chromosomal position effects characteristic of germ line transformation in Drosophila (33). Such insulators are likely to have important roles in the correct developmental regulation of many genes as exemplified by Fab-7, which separates the iab-6 and iab-7 regulatory elements of the Drosophila Bithorax complex, and the locus control region located upstream of the chicken ␤-globin locus (9,28,38).One of the most extensively studied insulators is located in the 5Ј nontranslated region of the Drosophila gypsy retrotransposon. Many of the spontaneous mutations caused by gypsy elements are due to the blocking of enhancer-promoter interactions attributable to this insulator (11,17,24,25,40). A 430-bp fragment of gypsy, termed "the gypsy insulator," placed between an enhancer and its target promoter is sufficient to block enhancer stimulation (11,17,24,45,51,60). As is true of other insulators, this same DNA fragment effectively insulates transgenes from chromosomal position effects (48).The...

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Section: Methodsmentioning

confidence: 99%

The gypsy Insulator Can Act as a Promoter-Specific Transcriptional Stimulator

Wei

Brennan

2001

Molecular and Cellular Biology

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“…Efforts to transfer the Drosophila germ-line transformation methodology to Diptera of economic or medical interest have been unsuccessful for reasons that are unclear. The D. melanogaster P-element has been used successfully in transformation of Drosophila hawaiiensis, but attempts to use P for germ-line transformation of non-Drosophila species have been unsuccessful (7,8). One possible approach would be to try in D. melanogaster transposable elements from other species, especially those distantly related to D. melanogaster.…”

Section: Introductionmentioning

confidence: 99%

Introduction of the transposable element Minos into the germ line of Drosophila melanogaster.

Loukeris

Arcà

Livadaras

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

104

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A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Orosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp7O-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp7O-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three ofthe insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp7O-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.Transposable elements have been used as vectors for stable germ-line transformation in Drosophila melanogaster. The mobile element P, present in some D. melanogaster populations, was the first element shown to insert into germ-line chromosomes in embryos (1). Typically, P-element-mediated transformation is achieved by coinjecting into preblastoderm embryos a mixture of two plasmids: one expressing transposase but unable to transpose and one carrying a gene (or genes) of interest flanked by the inverted terminal repeats of the element. Transformants are detected among the progeny of the injected individuals through the expression of dominant phenotypes. Germ-line transformation has revolutionized Drosophila research through the introduction of powerful methodologies such as analysis of in vitro mutagenized genes, gene cloning by transposon tagging, and enhancer trapping.Two other elements unrelated to P, hobo and mariner, have been shown to be able to transpose in the genome of D.melanogaster. hobo is found in some but not all D. melanogaster populations and causes hybrid dysgenesis (2, 3). Defective hobo elements containing foreign DNA can transpose into germline chromosomes from plasmids if coinjected into preblastoderm embryos along with a full-length element that can provide transposase (4). mariner, an element found in Drosophila mauritiana but not in D. melanogaster, has been introduced into D. melanogaster and shown to induce in trans mobilization of a nonautonomous mariner element (5, 6). Efforts to transfer the Drosophila germ-line transformation me...

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“…In undertaking such tests, it is encouraging that the P elements derived from D. melanogaster are active in distantly related Hawaiian Drosophilid species (36). Perhaps the PAT-1 element could be introduced into C. elegans by germ-line microinjection (37,38) and subsequently screened for progeny containing integrated PAT-1 sequences.…”

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confidence: 99%

Isolation and characterization of a nematode transposable element from Panagrellus redivivus.

Link¹,

Graf-Whitsel²,

Wood³

1987

Proc. Natl. Acad. Sci. U.S.A.

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We have isolated a transposable element, designated PAT-1, from the free-living nematode Panagrellus redivivus. P. redivivus strain C15 was found to have a high spontaneous mutation frequency compared to the standard Caenorhabditis elegans laboratory strain N2. To characterize the genetic lesions occurring in spontaneous C15 mutants, we molecularly cloned the homolog of the C. elegans unc-22 gene from wild-type P. redivivus and two strains carrying spontaneous mutations in this gene. One of these mutations resulted from the insertion of a 4.8-kilobase segment of repetitive DNA. This repetitive element (PAT-I) varies in copy number (10-50 copies) and location in different P. redivivus strains and is absent from C. elegans. The element could be useful as a transformation vector for C. elegans. Our approach is a general one that could be used to isolate additional nematode transposons from other species.The nematode Caenorhabditis elegans has been intensively studied as a model developmental system, and a few of its developmentally important genes have been molecularly cloned (1, 2). Characterization of these genes has been hampered by the lack of an efficient DNA transformation system. In Drosophila inelanogaster, P elements have proven to be extremely useful transformation vectors for stable reintroduction of cloned genes (3,4 MATERIALS AND METHODS Maintenance of Strains. P. redivivi's strains were routinely maintained at 20'C and handled essentially as described for C. elegans (18). The C15 strain (19) was obtained from M. R.Samoiloff(University of Manitoba); the PS strain from P. W. Sternberg (Massachusetts Institute of Technology); the SC strain from the Caenorhabditis Genetics Center (Division of Biological Sciences, University of Missouri); the W strain from J. Pasternak (University of Waterloo); and the G strain from E. Geraert (State University of Ghent, Belgium). The C15 strain is the product of nine sibling matings (19); the remaining strains are not inbred. P. rediviv'its unc-22 ("twitcher") mutants were maintained by mating individual female siblings of twitcher males to wild-type males; half of these matings produced twitcher males.Construction of Genomic Libraries. Genomic DNA from wild-type P. redivivits was prepared from mixed-stage populations of animals harvested from NG plates (18) 5325The publication costs of this article were defrayed in part by page charge paytnent. This article must therefore be hereby marked 'advertiisemzent in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Introduction of a functional P element into the germ-line of Drosophila hawaiiensis

Cited by 52 publications

References 18 publications

The gypsy Insulator Can Act as a Promoter-Specific Transcriptional Stimulator

The gypsy Insulator Can Act as a Promoter-Specific Transcriptional Stimulator

Introduction of the transposable element Minos into the germ line of Drosophila melanogaster.

Isolation and characterization of a nematode transposable element from Panagrellus redivivus.

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