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Home > Studies on Regulation of Intronic Polyadenylation by Rna Helicase P68/Ddx5 and U1snrnp in Mcf7 Cell Line

Studies on Regulation of Intronic Polyadenylation by Rna Helicase P68/Ddx5 and U1snrnp in Mcf7 Cell Line

Thesis Info

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External Link

Author

Shaheen, Sumera

Program

PhD

Institute

University of Agriculture

City

Faisalabad

Province

Punjab

Country

Pakistan

Thesis Completing Year

2013

Thesis Completion Status

Completed

Subject

Chemistry

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/2699/1/2642S.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727314086

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In eukaryotes, pre-mRNA has to undergo different processing steps; addition of cap at 5′ end, removal of introns to join functional exons, and addition of poly(A) tail at 3′ end to become fully functional mRNA. These processing events are linked to each other; one process effects efficiency of the other. Addition of polyA tail is not only limited at 3` end but poly A sequences are also found in genes (within exons and introns). The use of one of these intronic PAS results in primary transcript with different 3`UTR. DDX5 (p68) and DDX17 (p72) are RNA helicases performing different cellular functions; mi-RNA processing, transcription, mRNA processing, cell proliferation/transformation, cellular development and cancer. Four human genes MET, BCCIP, TGM2, and SMAD2 were selected to determine the relationship between p68, U1 snRNP, and activation of intronic polyadenylation. Genes were cloned into pGL3-TK vector having Firefly luciferase reporter gene. Mutation was introduced in 5`splice site to block U1. MCF7 cells were transfected with si-RNAp68/p72. After 24 hours cells were co-transfected with WT and Mut plasmids and pRL-SV40 control vector. Expression level of short isoform was determined by Dual Luciferase Reporter assay. The results suggest the role of p68 in IPA activation. Quantitative PCR was performed on uncleaved/total mRNA that confirmed the role of p68 RNA helicase in IPA acting through U1snRNP. To exclude the possibility that IPA is activated by splicing inhibition, si-RNAs against two splicing factors were used. If competitive inhibition of splicing result in IPA activation then should get the same expression level of short isoform with both si-RNAs, but it was not the case. IPA activation was seen only after si-U1 70k treatment while no or little short isoform was observed after si-U2AF65. These results clearly prove that IPA activation is not related to splicing inhibition. Overexpression of p68 and p72 enhanced the IPA event, again confirming the role of p68 in IPA. All experimental results prove that p68 activates intronic polyadenylation by removing U1 from 5`ss. Influence of p68 on IPA is not direct but it is acting through U1.
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وقت کی پابندی

وقت کی پابندی
نحمدہ ونصلی علی رسولہ الکریم امّا بعد فاعوذ بااللہ من الشیطن الرجیم
بسم اللہ الرحمن الرحیم
معزز اساتذہ کرام اور میرے ہم مکتب ساتھیو!
آج مجھے جس موضوع پر اظہار خیال کرنا ہے وہ ہے:’’وقت کی پابندی‘‘
صدرِذی وقار!
کائنات کی رنگینیاں، کا ئنات کی رعنائیاں ، کا ئنات کی دلآویز یاں، کا ئنات کاحسن و جمال۔ یہ سب وقت کی پابندی کی مرہونِ منت ہیں، دن رات کی تبدیلی ، موسموں کا آنا جانا، گلستان ہستی میں بہار کی آمد یہ ہمہ قسم کی بوقلمونیاں نظام الاوقات سے وابستہ ہیں۔
جنابِ صدر!
ستاروں کی چمک وقت پر ہوتی ہے، بدر و ہلال کی چاندنی کے لیے وقت مقرر ہے، آفتاب کی دمک کا ایک وقت مقرر ہے، آبشاروں کی کھڑ اکھڑاہٹ کا اپنا ایک وقت ہے، گلِ نرگس کی شگفتگی وقت پرممکن ہے، لالے کی حنابندی وقت پر ہوتی ہے ، مظاہر فطرت کی مشاطگی قدرت وقت پر کرتی ہے۔
صدرِ ذی وقار!
جو وقت کی قدر کرتا ہے، وقت کو ضائع ہونے سے بچاتا ہے، وقت کی پابندی اپنی عادت ثانیہ بنالیتا ہے، وقت کی پابندی اس کی زندگی کا حصہ بن جاتی ہے، وقت کے میدان کا شاہسوار بن جاتا ہے، وقت کے گلشن میں موجودگل سرسبز سے اس کی سانسیں معطر ہوجاتی ہیں، وہ چرخِ علم ودانش پرآ فتاب ِنصف النہار کی طرح چمکتا ہے۔
جنابِ صدر!
دنیا میں جس نے بھی کمال حاصل کیا وقت کی قدر کر کے کیا ، وقت کی پابندی نے اس کے افقِ حیات پر قوس قزح بنادی، آج ہم بھی کوئی مقام، کوئی مرتبہ حاصل کرنا چاہتے ہیں تو وقت کی قدرکرنا ہوگی اور وقت کی پابندی سے ہی ہماری ترقی ممکن ہے۔
یہ طاقت ہے یہ عظمت ہے یہ فتح و کامرانی ہے
جہاں میں...

الحديث الضعيف وما يتعلق به من الأحكام

Legitimation among scholars, since they fall to category of hadith dho’if (weak). Therefrom, several scholars argued that we might use them for hujjah mutlaq (absolute argumentation), while some others said it might be wiser not to use them at all. Yet there is also another opinion which said it could be used under special conditions. Based on this, this study aims to uncover and shed light the disagreements above scientifically, as well as to find he differences and the influence of the jurisprudence of law-making (fiqh). Then, the researchers sought to raise a strong opinion based on the arguments presented in the thesis, so which the researchers and or anyone who wants to practice the Hadith may find helpful.

Evaluation of Synthetic Epsps Gene in Genetically Transformed Plants

Codon optimized synthetic epsps gene was used to develop transgenic tobacco and sugarcane lines using Agrobacterium and biolistic approach, respectively. Putative transgenic tobacco lines analyzed by qRT-PCR showed variable mRNA transcript levels. Southern hybridization performed for determining gene integration patterns showed multiple copy inserts (1-4) of transgene in various tested lines. Immunoblot strips confirmed the translation of cp4-epsps mRNA transcripts in T1 line. Malachite green assay showed variable EPSPS activity levels in various lines in response to glyphosate application. Transgenic tobacco lines exposed to 1% (v/v) glyphosate at 4 to 5 leaf stage showed variable resistance levels ranging from moderate level tolerance to high tolerance. The cp4-epsps mRNA transcript levels varied from genotype to genotype in tested transgenic sugarcane lines regardless of the integration of multiple copy number (3-6) of transgene. Genetic transformation with pyramided constructs results in the integration of multiple genes at a single locus, which is desirable in terms of expression efficiency of transgenes. The cp4-epsps gene was pyramided with insect resistant cry1Ac and cry2Ab genes and cloned in pSb187 vector was used to genetically transform tobacco using Agrobacterium mediated approach. Transgenic lines were confirmed through PCR using gene specific primers of respective genes. The mRNA transcript level of cp4-epsps gene detected through qRT-PCR using GAPDH gene as an internal control was found to be variable among different transgenic lines. Transgenic lines were tested for checking the translational efficiency of all three genes using immunoblot strips. Transgenic tobacco lines found expressing all three proteins were subjected to detached leaf insect bioassays to evaluate the effectiveness of Bt genes in putative transgenic tobacco against insect attack using first instar larvae from Spodoptera littura and Helicoverpa armigera. Transgenic tobacco found to be resistant against these insects showed that cry1Ac and cry2Ab genes were efficient enough in pyramided form. When these transgenic tobacco lines were exposed to glyphosate at 10 to 12 leaf stage, chlorotic symptoms were observed which hinted that herbicide tolerance potential of transgene declined with increasing age of the plants. In order to overcome the spatio temporal gene expression issues, a dual copy epsps expression cassette synthesized under different regulatory elements, cloned in pGreen0029 binary vector was used to generate transgenic tobacco. These tobacco lines exhibited excellent resistance against 19% (v/v) glyphosate at 10-12 leaf stage. This construct has shown tremendous potential regarding transgene expression levels which is required in the field crops. This dual cassette cp4-epsps gene has been recommended for generation of glyphosate resistant cotton and other commercially important cultivars.