Rescue of Drosophila engrailed mutants with a highly divergent mosquito engrailed cDNA using a homing, enhancer-trapping transposon

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1531-1541
Author(s):  
M. Whiteley ◽  
J.A. Kassis
Keyword(s):  
Homologous Recombination ◽  
Transposable Elements ◽  
Anopheles Gambiae ◽  
Genomic Dna ◽  
P Element ◽  
Null Mutation ◽  
Coding Region ◽  
High Frequencies ◽  
Enhancer Trapping ◽  
Regulatory Dna

Specific fragments of Drosophila regulatory DNA can alter the insertional specificity of transposable elements causing them to ‘home’ to their parent gene. We used this property to insert a transposon-encoded functional coding region near a defective one and rescue a null mutation. This approach differs from homologous recombination in that the endogenous defective coding region is left in place and the genomic DNA is altered by the addition of the therapeutic transposon. We constructed a P-element-based transposon in which an engrailed cDNA from Anopheles gambiae (a mosquito) is expressed from a Drosophila engrailed minimal promoter. The promoter fragment used includes 2.6 kb of regulatory DNA that causes transposons to home to the endogenous Drosophila engrailed gene at high frequencies. We inserted this transposon onto a Drosophila chromosome that produces no functional engrailed proteins. When this transposon integrated near the engrailed promoter, adult viability was restored to engrailed mutant flies showing that the highly divergent mosquito engrailed protein can replace the Drosophila engrailed protein at all stages of development. Insertion of this transposon into the adjacent invected gene, which is transcribed in a pattern similar to engrailed, led to only embryonic rescue, suggesting an important difference in the regulation of these two genes.

scholarly journals hobo enhancer trapping mutagenesis in Drosophila reveals an insertion specificity different from P elements.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 1063-1076 ◽  
Author(s):  
D Smith ◽  
J Wohlgemuth ◽  
B R Calvi ◽  
I Franklin ◽  
W M Gelbart
Keyword(s):  
Drosophila Melanogaster ◽  
Transposable Element ◽  
Enhancer Trap ◽  
P Element ◽  
Present Evidence ◽  
P Elements ◽  
Element Insertion ◽  
Enhancer Trapping ◽  
Indispensable Tool

Abstract P element enhancer trapping has become an indispensable tool in the analysis of the Drosophila melanogaster genome. However, there is great variation in the mutability of loci by these elements such that some loci are relatively refractory to insertion. We have developed the hobo transposable element for use in enhancer trapping and we describe the results of a hobo enhancer trap screen. In addition, we present evidence that a hobo enhancer trap element has a pattern of insertion into the genome that is different from the distribution of P elements in the available database. Hence, hobo insertion may facilitate access to genes resistant to P element insertion.

Evaluation of the tshr gene reveals polymorphisms associated with typical symptoms in primary congenital hypothyroidism

2016 ◽  
Vol 29 (1) ◽  
Author(s):  
Erik Artur Cortinhas Alves ◽  
Raissa Coelho Andrade ◽  
Carlos Eduardo de Melo Amaral ◽  
Milena Coelho Fernandes Caldato ◽  
Adriana Maria Rocha Bastos ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Peripheral Blood ◽  
Genomic Dna ◽  
Gene Mutations ◽  
Molecular Heterogeneity ◽  
Coding Region ◽  
Blood Samples ◽  
Live Births ◽  
Tshr Gene ◽  
Inactivating Mutations

AbstractPrimary congenital hypothyroidism (PCH) has an incidence of approximately 1 in each 3000–4000 live births. In the last two decades, nearly 50 types of the distinct inactivating mutations have already been described in the coding region of the tshr gene. The aim of present study was to investigate tshr gene mutations in patients with primary congenital hypothyroidism, analyzing a sample of 106 patients that were diagnosed with PCH. Genomic DNA was isolated from peripheral blood samples, and 10 exons from the TSH receptor were automatically sequenced. Five nucleotide alterations (P52T, N187N, A459A, L645L, and D727E. N187N and D727E polymorphisms) were associated with positive medical history. In view of the clinical, biochemical and molecular heterogeneity of the etiology of the PCH, the study of polymorphisms is critical for investigating the possible associations with prevailing symptoms of this disorder.

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

scholarly journals Genotypic effects, maternal effects and grand-maternal effects of immobilized derivatives of the transposable element mariner.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 183-192
Author(s):  
A R Lohe ◽  
D A Lidholm ◽  
D L Hartl
Keyword(s):  
Maternal Effects ◽  
Maternal Effect ◽  
P Element ◽  
Transformation Rate ◽  
Coding Region ◽  
Genotypic Effect ◽  
Exogenous Dna ◽  
Germline Expression ◽  
Helper Plasmids ◽  
Female Germline

Abstract The baseline rate of spontaneous integration of the autonomous mariner element Mos1 into the germline of Drosophila melanogaster is estimated as 16 +/- 5% (mean +/- SE) among fertile G0 flies. However, the transformation rate is reduced approximately 20-fold in Mos1 constructs with exogenous DNA in the size range 5-12 kb inserted into the SacI site. To provide alternative Mos1 helper plasmids for transformation experiments, two types of Mos1-promoter fusions were constructed: hsp-70:Mos1 and hsp26-Sgs3:Mos1. The former has the Mos1 coding region driven by the hsp70 heat-shock promoter; the latter has it driven by the basal Sgs3 promoter under the control of the hsp26 female-germline specific transcriptional regulator. When introduced into D. melanogaster by P-element-mediated germline transformation, these elements are unable to transpose or excise in the presence of autonomous Mos1-related elements (they are "marooned") because the 5' inverted repeat of Mos1 is missing. As expected, the hsp26-Sgs3:Mos1 fusions exhibit a significantly greater rate of germline excision of a target mariner element than do the hsp70:Mos1 fusions. Unexpectedly, the rate of excision of target mariner elements induced by hsp26-Sgs3:Mos1 is the same in the male germline as in the female germline. Both hsp:Mos1 fusions show strong germline expression and a maternal effect of the mariner transposase. A significant grand-maternal effect of the hsp:Mos1 fusions was also detected as a result of a maternal effect on the germline of the F1 progeny. Among flies carrying the promoter fusions inherited maternally, about three-quarters of the overall rate of germline excision derives from the direct genotypic effect and about one-quarter results from the grand-maternal effect. Despite the strong somatic expression of the hsp:Mos1 fusions, mariner transformants carrying a white+ reporter gene at the SacI site remained stable in the soma.

scholarly journals Genetic study of motor functions in Drosophila melanogaster

2012 ◽  
Vol 10 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Sergey A Fedotov ◽  
Julia V Bragina ◽  
Nataliya G Besedina ◽  
Larisa V Danilenkova ◽  
Elena A Kamysheva ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Motor Activity ◽  
Genomic Dna ◽  
Molecular Mechanisms ◽  
Central Pattern Generators ◽  
Courtship Song ◽  
P Element ◽  
Rhythmic Motor ◽  
Pattern Generators ◽  
First Time

To investigate molecular mechanisms of central pattern generators (CPG s) functioning, we carried out a screening of collection of Drosophila P-insertional mutants for strong deviations in locomotion and courtship song. In 21 mutants, the site of the P-insertion was localized by sequencing of the fragments of genomic DNA flanking the P-element. Bioinformational analysis revealed a list of candidate genes, potential players in development and functioning of CPG s. Possible involvement of certain identified genes in rhythmic motor activity is suggested for the first time (CG15630, Map205).

scholarly journals The Two Drosophila Telomeric Transposable Elements Have Very Different Patterns of Transcription

1999 ◽  
Vol 19 (1) ◽  
pp. 873-881 ◽  
Author(s):  
O. N. Danilevskaya ◽  
K. L. Traverse ◽  
N. C. Hogan ◽  
P. G. DeBaryshe ◽  
M. L. Pardue
Keyword(s):  
Transposable Elements ◽  
Sequence Similarity ◽  
Sequence Divergence ◽  
Full Length ◽  
Coding Region ◽  
Free Standing ◽  
Reading Frame ◽  
Sequence Organization ◽  
Sense Strand ◽  
Cell Fractions

ABSTRACT The transposable elements HeT-A and TARTconstitute the telomeres of Drosophila chromosomes. Both are non-long terminal repeat (LTR) retrotransposons, sharing the remarkable property of transposing only to chromosome ends. In addition, strong sequence similarity of their gag proteins indicates that these coding regions share a common ancestor. These findings led to the assumption that HeT-A andTART are closely related. However, we now find that these elements produce quite different sets of transcripts. HeT-Aproduces only sense-strand transcripts of the full-length element, whereas TART produces both sense and antisense full-length RNAs, with antisense transcripts in more than 10-fold excess over sense RNA. In addition, features of TART sequence organization resemble those of a subclass of non-LTR elements characterized by unequal terminal repeats. Thus, the ancestral gag sequence appears to have become incorporated in two different types of elements, possibly with different functions in the telomere. HeT-Atranscripts are found in both nuclear and cytoplasmic cell fractions, consistent with roles as both mRNA and transposition template. In contrast, both sense and antisense TART transcripts are almost entirely concentrated in nuclear fractions. Also,TART open reading frame 2 probes detect a cytoplasmic mRNA for reverse transcriptase (RT), with no similarity to TARTsequence 5′ or 3′ of the RT coding region. This RNA could be a processed TART transcript or the product of a “free-standing” RT gene. Either origin would be novel. The distinctive transcription patterns of both HeT-A andTART are conserved in Drosophila yakuba, despite significant sequence divergence. The conservation argues that these sets of transcripts are important to the function(s) ofHeT-A and TART.

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

Characterization and proposed spontaneous deletion mechanism of AVR-Pik locus in Pyricularia oryzae

2021 ◽  
Author(s):  
Phyo Han Thwin ◽  
Mai Funabiki ◽  
Yuki Tomita ◽  
Takehiko Yamazaki ◽  
Ayumi Abe ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Transposable Elements ◽  
Host Plant ◽  
Phytopathogenic Fungi ◽  
Nucleotide Sequences ◽  
Pyricularia Oryzae ◽  
Avirulence Gene ◽  
Spontaneous Mutant

Abstract In phytopathogenic fungi, a mutation in the avirulence gene can lead to the breakdown of resistance in the host plant. The nucleotide sequences of the AVR-Pik locus in the strain Ina168 and its spontaneous mutant Ina168m95-5 of Pyricularia oryzae were determined. An AVR-Pik spontaneous deletion mechanism of Ina168m95-5, including multiple homologous recombination events involving repetitive transposable elements, is proposed.

Nkx5-1 controls semicircular canal formation in the mouse inner ear

Development ◽  
1998 ◽  
Vol 125 (1) ◽  
pp. 33-39 ◽  
Author(s):  
T. Hadrys ◽  
T. Braun ◽  
S. Rinkwitz-Brandt ◽  
H.H. Arnold ◽  
E. Bober
Keyword(s):  
Homologous Recombination ◽  
Inner Ear ◽  
Homeobox Gene ◽  
Semicircular Canals ◽  
Otic Vesicle ◽  
Null Mutation ◽  
Complex Structures ◽  
Vestibular Organ ◽  
Inner Ear Development ◽  
Vestibular Organs

The inner ear develops from the otic vesicle, a one-cell-thick epithelium, which eventually transforms into highly complex structures including the sensory organs for balance (vestibulum) and hearing (cochlea). Several mouse inner ear mutations with hearing and balance defects have been described but for most the underlying genes have not been identified, for example, the genes controlling the development of the vestibular organs. Here, we report the inactivation of the homeobox gene, Nkx5-1, by homologous recombination in mice. This gene is expressed in vestibular structures throughout inner ear development. Mice carrying the Nkx5-1 null mutation exhibit behavioural abnormalities that resemble the typical hyperactivity and circling movements of the shaker/waltzer type mutants. The balance defect correlates with severe malformations of the vestibular organ in Nkx5-1(−/−) mutants, which fail to develop the semicircular canals. Nkx5-1 is the first ear-specific molecule identified to play a crucial role in the formation of the mammalian vestibular system.

scholarly journals Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 219-223 ◽  
Author(s):  
M Poncz ◽  
S Surrey ◽  
P LaRocco ◽  
MJ Weiss ◽  
EF Rappaport ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Genomic Dna ◽  
Cdna Probe ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Coding Region ◽  
Platelet Factor ◽  
Amino Acid Homology

Abstract We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

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