Characterization of the sbr gene in Neumspora crassa Yanhua Yan A thesis submitted to the Faculty of Graduated Shidies and Research In partial fuifillment of the requinments for the degree of Master of Science Ottawa-Carleton Institute of Biology Carleton University Ottawa, Ontario Canada National Library Bibliothèque nationale ($1 of Canada du Canada Acquisitions and Acquisitions et Bibliog aphic Sew ices services bibliographiques 395 Wellington Street 395. rue WuNinglwi Ottawa ON KIA ON4 OUawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Librw of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriiute or seU reproduire, prêter, distribuer ou copies of ths thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract Insertional mutation of the Neurospora crassa (N. crassa) pal1 kown (sbr) gene results in a stnking, colonial morphology. This gene was cloned previously, but only one 5' truncated cDNA sequence had been recovered by conventional screening of several libraries (Carnpsell, 1998). The cloned sbr gene was proposed as a regulatory gene because it encodes a helix-tum-helix (HTH), a putative zinc finger, and a glutamine-rich region. This study completed the sbr gene sequence and mapped the gene to linkage group III. The sbr gene has one intron and encodes a protein of 612 amino acids with two glutamine-rich regions at the N-terminal. Complementation with a sbr genomic clone recovered the wild type phenotype. SBR protein was expressed and purified under native condition. In a protein-DNA binding assay in which total genomic DNA digested by restriction enzymes was allowed to bind with SBR protein, three genomic fragments fiom size 0.3kb to 0.8kb were isolated fiom protein-DNA complexes. Electrophoretic mobility shifi assay indicated SBR-DNA binding was specific. Protein from an expression clone with deletion of the putative zinc finger lost its DNA binding ability. Searching the N. crassa genome database retrieved a sequence containhg the 0.3kb fragment. The possibility that the isolated fragments contain the SBR responsive regulatory elements has been suggested with bioinfomatic programs in prediction of genes associated with these fragments and in searching transcription factor binding sites. Some expression plasmids with deletion at different part of the sbr gene were cloned for funher çtudy in detennining functional domains. III To my wife Za ju Acknowledgments First of dl, 1 wish to express my deep gratitude to my excellent supervisor Dr. P. John Vierula for his guidance and assistance during the development of this thesis. I thank my cornmittee memben, Dr. Myron Smith and Dr. Doug Johnson, for their involvement and suggestions. Thanks also go to grad students Weiping Chen and Alex Valencia who shared the lab and gave me help whenever available. Table of content TITLE ................................................... .... .... .......... .........*......... ........ .... ....... ..... ...... ............ 1 ACCEPTANCE PAGE ......... ................................................. ...m...e... e.o.............. II ACKNOWLEDCMENTS ...................................................................... ..... ......... V TABLE OF CONTENT ...................................... ............. ....... ........................................ VI LIST OF TABLES...................................... ... .. .. .............e... ........ ................ ....................... X LIST OF ABBREVlATIONS ............................................... ...................... ............ ......... XI .-in Expression and purijication of histidine-tagged regtriatory protein .................. .. ... .. .. ..... .... ........... 9 B. Electrophoretic mobiliiy shiji arsay ................+..................~............,,............,..............---....-...-.--....II C.Foorprinring ........ .................................................................................................................. 12 D. Idenrtfiing the DNA - binding region of a DNA binding protein .. ... .. .. 13 E. Ident~fYingthe DATA setpence bound to a putative transcription factor .. .. .. ... ... ........ .. ....... 14 OBJECTIVES OF THIS STUDY ~o~e~aa~aa~eaa~ae~eoo~~ae~a~~aaa~~~~~a~moaaaeoaaaaoaaaeaooo16 A. Extrûcting ENA Jiom phage clone isoiatedfiom genomic librq................................. ................. 18 B. Subcloning sbr genomic DM.... .. ... .. ..... ... ... -..a-. -- .... .. 19 TRANSFORMATIONOF E. COLI CELLS.......~~....~...~....~~...~~~.~~..~~~~~...~~~.~~~~~~..~...~~~~~~~.~~.~..~~~~.~.~....19 A. Chernical transformation... ..... .. -.. ... .... -... --- -.. - -- -- - - -- ----.-.--. .... .. .. .. .. .. .. .. .. ... 1 9 B. Electroporation. ................................... .............................................................................. 20 EXTRACTIONOF PLASMID DNA FROM E. COUa.aaooeo....aa~a.~~aaaa~~ee~a~oa~aaaooaaaoaaaaoa2 1 DNA SEQUENCING..aa.aa.e.om..a..a.aooaaoaaaoe~emoaom*aeoaooaooaoaao*o.a~aoaoaoaaoooao~~~aaa~aoaaomoaaa~oo~aoa~oa*aaoooaamoaaa~aoaoa 22 COMPLEMENTATIONSTUDY: ~%.ANSFORMINGsbr MUTANT eo.m....em.~~oa~o..a.momm.~ooaoa.aaaoaaoaaoaae 23 A . Preparation of sbr spheroplasts...................................................................................................... 23 B . Electroporation of sbr:..................................................................................................................... 24 GENEMAPPING .........m..~=***~**~**~********m*~****~~~*~o~**~*o~~~~~~~*a~~***e*~o~~~.~*~..~~~~*o~*am*****mo*~~****~~~~~m****~*~~*~~..25 A . Growing map strains. ...................................................................................................................... 25 B . Extraction ofgenornic DM................................ .., ....................................................................... 25 C. Hybridization ..................................................................................................................................26 SOUTHERNBLOT .........*.........~~~~~~~~~~~~~~~~mm~~~~~~m~~~~~o~~~~~a~~~~~~~~~~~~~~~~~~~~~~~~a~~~~~~~~~~~~~~~~~e~~e26 RADIO LABELINC DNA PROBE ................................................................................................27 HYBRIDIUTIONAND FILM DEVELOPMENT .............................................................................28 A . Extraction of total RNA ................................................................................................................... 29 B . Reverse transcription....................................................................................................................... 30 PCR PROTOCOL ....................................................................................o............................3 f .-i . Transjormution of bacteria.............................................................................................................. 31 B . Expression of SBR protein ................................................................................................................32 C. Protein purijication ..................................... ............................................................................... 32 SDS-POLYACRYLAMIDEGELELECTROPHORESIS (PAGE) ..........................*.............. 33 WESTERNBLOT AND IMMUNOSTAINING .................................................................................34 ISOLATINCSBR BINDINC TARCET DNA ELEMENTS .............................................................35 ELECTROPHORESISMOBtLITY SHIFT ASSAY WMSA) ............................................................37 4 . preparation of probe DNA: ........................................................................................................ 37 B . Preparation of non-spectfic cornpetitor......................................................................................... 38 C. Prorein-DNA binding ...................................................................................................................... 38 D . Electrophoresis ............................................................................................................................... 38 DNA ANALYSIS WITH BIOlNFORMATIC PROGRAMS ...................*.*..................................38 RESULTS ................................................................................................................................................. 41 GENOMICCLONE AND SEQUENCE............................................................................................ 41 sbr cDNA SEQ~NCE................................................................................................................ 44 A . Amplijication of sbr cDNA S'end ............................................................................................... 44 B . Sequence of the RT-PCR product and the tramlation srart site .....................................................