Lecture Notes on Transposon and Transposon Tagging | BSCI 411, Study notes of Biology

Download Lecture Notes on Transposon and Transposon Tagging | BSCI 411 and more Study notes Biology in PDF only on Docsity! Read p330-347 I. Transposon and transposon tagging II. Gene structure and regulation A. Cis-regulatory elements B. Trans-acting regulators DNA-binding type non-DNA binding type Lecture 7 Ac/Ds type Retrotransposon Transposon mutagenesis Gene cloning using transposon as a probe Similar transpoable elements: Tam1 Tam3 Snapdragon Maize Ac/Ds Spm/dSpm Fig. 7.33 Retrotransposons-viral in origin Fig. 7.34 Yeast Ty Drosophila copia Maize Mu Transposon tagging using Ac/Ds in Arabidopsis Nina Fedoroff lab (http://www.lsc.psu.edu/ptl/transtag.html) -Ac/Ds transposons were isolated in Nina Fedoroff’s lab -they are shown to be able to transpose in many plants both monocot and dicot: tobacco, tomato, Arabidopsis, flax -they are powerful tools for tagging and cloning genes Figure 1: Structure of Ac- and Ds-GUS T-DNAs a. Transposon T-DNAs Os 1,4: 325 bp 360 bp 466 bp Os 2,5: 378 bp 281 bp 466 bp Ds 3,6: 512 bp 541 bp 466 bp RB ™ LB Chiorsulfuron- CaMv Transformation resistant promoter marker b. Transposase T-DNAs BamAc Lid ee aca } — 35S- tors? Tt in RB Negative selectable ‘Transformation /B NaeAc marker marker Structure of the T-DNA segments containing the transposon (a.) and the transposase gene (b.), as well as the selectable markers associated with each. The constructs used here have been deseribed in Fedoroff and Smith (Plant J., 3: 273-289 [1993)), The amount of the Ac element in each Ds construct is represented by the filled portions at the beginning, middle and end of the transposon and the exact number of base pairs is indicated above the diagram. Dsland4 contain only the first of the Ac element's 4 transcription start sites, while the others contain all of them with (Ds3 and 6) or without (Ds? and 4) part of the element's untranslated leader. Ds constructs 4-6 contain the 358 core sequence upstream from the GUS gene, while Ds constructs 1-3 do not. Abbreviations: RB: T-DNA right border; LB: T-DNA left border; GUS: B- glucuronidase; 338 and 108: the promoters of the CaM V 3585 and 195 transeripts: the Naedic contains the 2 sequence of the tobacco mosaic ; ALS: chlorsulfuron- resistant acetolactate synthase gene (Haughn et al., MoLGen. Genet. 211: 266-271 [1988]; hygro: the bacterial api gene, confers hygromycin resistance; NPTII: bacterial neomycin phosphotransferase IT gene; tms2: the agrobacterial ims? gene, confers sensitivity to auxin amides; Bam- and Nae-Ac: deleted and promoter-substituted Ac elements, Fig. 7.40 CDeT6-19-GUS expression CDeT6-19: the promoter of a drought-responsive gene ABA: abscisic acid GUS: b-glucuronidase Certain genes are only expressed under certain conditions AG RNA expressionWT Arabidopsis flower ag mutant flower Certain genes are only expressed at specific stages and tissue 2985 bp H S X H 1 W US LF Y M AD S 1 5 9 M AD S 2 9 2 5 2 0 0 2 2 6 0 3 M AD S 2 4 8 3 2 5 5 6 8 4 6 LF Y LF Y LF Y 2 6 0 8 G-box (CCACGTGG): association with environmental stimuli Arabidopsis RBCS (light-inducible) Parsley CHS (UV-light-inducible) Bean CHS (coumaric acid- inducible) Wheat EM (ABA-inducible) 16 bp -261 — sr -228 | GATAAG TCOCACGTGGC | Accompanying G-box element element -169 ee -120 | TCCACGTGGC GTCCCTCCAACCTAACC | G-box Accompanying element element 5b ne 72 bp P _55 _€——_ EEE CCTACC \\ TQGCACGTGAT CCTACC Accompanying . G-box Accompanying element element element -152 Eee -103 GACACGTGGC CGAGCAGGC G-box Accompanying element element Some Tx factors do not have DNA-binding domain (1) RNA Pol II Fig. 7.45 The repression of a-cell-specific genes in S. cerevisiae a cells Tup1 Ssn6 a 2 MCM1 a-cell specific genes Q-rich (97-118, 181-198)Tup1 Ssn6 Binding 7 WD 7061 72 (2) Transcription co-repressors Tup1