Recovery of DNA Sequences Flanking P-Element Insertions in Drosophila: Inverse PCR and Plasmid Rescue

2009 ◽  
Vol 2009 (4) ◽  
pp. pdb.prot5199-pdb.prot5199 ◽  
Author(s):  
A. M. Huang ◽  
E. J. Rehm ◽  
G. M. Rubin
Keyword(s):  
Dna Sequences ◽  
P Element ◽  
Inverse Pcr ◽  
Plasmid Rescue

Identification of Chromosome Inheritance Modifiers in Drosophila melanogaster

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1623-1637 ◽  
Author(s):  
Kenneth W Dobie ◽  
Cameron D Kennedy ◽  
Vivienne M Velasco ◽  
Tory L McGrath ◽  
Juliani Weko ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Biological Activity ◽  
Cancer Progression ◽  
Birth Defects ◽  
Mitotic Chromosome ◽  
P Element ◽  
Inverse Pcr ◽  
Gtpase Activating Protein ◽  
New Genes ◽  
Genomic Analyses

Abstract Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a “sensitized” minichromosome substrate (J21A) to screen ∼3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.

Restriction of P-element insertions at the Notch locus of Drosophila melanogaster

1987 ◽  
Vol 7 (4) ◽  
pp. 1545-1548
Author(s):  
M R Kelley ◽  
S Kidd ◽  
R L Berg ◽  
M W Young
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
P Element ◽  
Induced Mutations ◽  
P Elements ◽  
Target Dna ◽  
Complex Locus ◽  
Additional Level ◽  
Derivatives Of

P elements move about the Drosophila melanogaster genome in a nonrandom fashion, preferring some chromosomal targets for insertion over others (J. C. J. Eeken, F. H. Sobels, V. Hyland, and A. P. Schalet, Mutat. Res. 150:261-275, 1985; W. R. Engels, Annu. Rev. Genet. 17:315-344, 1983; M. D. Golubovsky, Y. N. Ivanov, and M. M. Green, Proc. Natl. Acad. Sci. USA 74:2973-2975, 1977; M. J. Simmons and J. K. Lim, Proc. Natl. Acad. Sci. USA 77:6042-6046, 1980). Some of this specificity may be due to recognition of a particular DNA sequence in the target DNA; derivatives of an 8-base-pair consensus sequence are occupied by these transposable elements at many different chromosomal locations (K. O'Hare and G. M. Rubin, Cell 34:25-36, 1983). An additional level of specificity of P-element insertions is described in this paper. Of 14 mutations induced in the complex locus Notch by hybrid dysgenesis, 13 involved P-element insertions at or near the transcription start site of the gene. This clustering was not seen in other transposable element-induced mutations of Notch. DNA sequences homologous to the previously described consensus target for P-element insertion are not preferentially located in this region of the locus. The choice of a chromosomal site for integration appears to be based on more subtle variations in chromosome structure that are probably associated with activation or expression of the target gene.

scholarly journals Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

2002 ◽  
Vol 76 (11) ◽  
pp. 5540-5547 ◽  
Author(s):  
Yi Feng Jin ◽  
Toshio Ishibashi ◽  
Akio Nomoto ◽  
Michiaki Masuda
Keyword(s):  
Long Terminal Repeat ◽  
Dna Sequences ◽  
Target Selection ◽  
Terminal Repeat ◽  
Inverse Pcr ◽  
Proviral Integration ◽  
Host Chromosome ◽  
Retroviral Integration ◽  
Integration Sites ◽  
Slip Method

ABSTRACT Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.

scholarly journals Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

1986 ◽  
Vol 6 (2) ◽  
pp. 663-673 ◽  
Author(s):  
E Hoffman ◽  
V Corces
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Dna Sequences ◽  
Germ Line ◽  
Initiation Site ◽  
P Element ◽  
Heat Shock Gene ◽  
Base Pairs ◽  
Consensus Sequences ◽  
Shock Gene

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.

scholarly journals Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.

1986 ◽  
Vol 6 (5) ◽  
pp. 1520-1528 ◽  
Author(s):  
D Y Chang ◽  
B Wisely ◽  
S M Huang ◽  
R A Voelker
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Insertion Site ◽  
Hybrid Dysgenesis ◽  
P Element ◽  
Dna Transformation ◽  
Wild Type ◽  
Induced Mutations ◽  
X Ray ◽  
Element Insertion

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

scholarly journals The Drosophila ACE3 chorion element autonomously induces amplification.

1992 ◽  
Vol 12 (5) ◽  
pp. 2444-2453 ◽  
Author(s):  
J L Carminati ◽  
C G Johnston ◽  
T L Orr-Weaver
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Regulatory Elements ◽  
P Element ◽  
Follicle Cells ◽  
Control Element ◽  
The Third ◽  
Transposon Insertion ◽  
Multiple Copies ◽  
Flanking Sequences

The Drosophila chorion genes amplify in the follicle cells by repeated rounds of reinitiation of DNA replication. ACE3 (amplification control element from the third chromosome) has been identified by a series of deletion experiments as an important control element for amplification of the third-chromosome chorion cluster. Several elements that quantitatively enhance amplification also have been defined. We show that a single 440-bp ACE3 sequence is sufficient to regulate amplification with proper developmental specificity autonomously from other chorion DNA sequences and regulatory elements. Although ACE3 is sufficient for amplification, the levels of amplification are low even when ACE3 is present in multiple copies. When controlled solely by ACE3, amplification initiates either at ACE3 or within closely linked sequences. Amplification of an ACE3 transposon insertion produces a gradient of amplified DNA that extends into flanking sequences approximately the same distance as does the amplification gradient at the endogenous chorion locus. The profile and extent of the amplified gradient imply that the low levels of amplification observed are the result of limited rounds of initiation of DNA replication. Transposon inserts containing multiple copies of ACE3 in a tandem, head-to-tail array are maintained stably in the chromosome. However, mobilization of the P-element transposons containing ACE3 multimers results in deletions within the array at a high frequency.

T-DNA tagging and characterisation of a novel meristem-specific promoter from tobacco

1998 ◽  
Vol 25 (6) ◽  
pp. 637 ◽  
Author(s):  
Stephen R. Mudge ◽  
Robert G. Birch
Keyword(s):  
Dna Sequences ◽  
Promoter Sequence ◽  
Single Copy ◽  
Firefly Luciferase ◽  
Inverse Pcr ◽  
Tobacco Plants ◽  
Apical Meristems ◽  
Expression Of Genes ◽  
Tobacco Genome ◽  
Plant Promoters

We have evaluated T-DNA mediated plant promoter tagging, with a left-border-linked promoterless firefly luciferase (luc) construct, as a strategy for the isolation of novel plant promoters. In a population of approximately 300 transformed tobacco plants, 10 lines showed LUC activity, including novel tissue-specific and developmental patterns of expression. One line, showing LUC activity only in the shoot and root apical meristems, was further characterised. Inverse PCR was used to amplify a 1.5 kb fragment of plant DNA flanking the single-copy T-DNA insertion in this line. With the exception of a 249 bp highly repetitive element, this sequence is present as a single copy in the tobacco genome, and is not homologous to any previously characterised DNA sequences. Sequence analysis revealed the presence of several motifs that may be involved in transcriptional regulation. Transgenic tobacco plants transformed with a transcriptional fusion of this putative promoter sequence to the β-glucuronidase (uidA) reporter gene, showed GUS activity confined to the shoot tip and mature pollen. This promoter may be useful to direct the expression of genes controlling the transition to flowering, or genes to reduce losses due to pests and stresses damaging plant apical meristems.

Drosophila P element transposase recognizes internal P element DNA sequences

Cell ◽  
1989 ◽  
Vol 59 (2) ◽  
pp. 359-371 ◽  
Author(s):  
Paul D. Kaufman ◽  
Rhonda F. Doll ◽  
Donald C. Rio
Keyword(s):  
Dna Sequences ◽  
P Element
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