Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

Molecular genetic analysis of the

maize terminal ear1 gene and in

silica analysis of related genes

A Thesis Presented in Partial Fulfilment of the requirements

for the Degree of Doctor of Philosophy in Plant Biology

at

Massey University

Palmerston North, New Zealand.

Daniel Charlton Jeffares

2001

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. This thesis is dedicated to Ben, Charlie and Alex.

THESIS ABSTRACT

M utants of the maize terminal ear1 (te 1 ) gene have shortened internodes, abnorma l

phyl l otaxy, leaf pattern defects and part ia l fem inisat ion of tasse ls . The te 1 gene

encodes an R NA recogn ition motif (RRM) prote in , and is expressed i n the vegetative

shoot apex in semici rcu lar rings that lateral ly oppose the posit ions of leaf primord ia

(Veit 1 998) . Th is project a imed to further characterise the molecu lar b iology and

fun ction of the te 1 gene .

Mo lecu lar genet ic stud ies aimed to further characterise the genes structure and

express ion . Genomic c lones were sequenced revea l i ng the i ntron exon structu re. 5'

RACE was used to predict a 5' transcription start site. Competitive RT-PCR showed

that te 1 transcri pts were h ighest in vegetative shoot meristems and embryos, l ower i n

ears, roots and tassels , and u ndetectab le i n leaves. Two te 1 mutant a l le les were

c loned and the j un cti ons sequenced, a further five a l le les were characterised

i ncompletely .

The T E 1 pept ide be longs to a subclass of RRM prote ins which inc ludes the

Schizosaccharomyces pombe protein ME I2 . More than 30 putative p lant Mei2-l ike

genes were identified in Genbank , no examples have been found in metazoans.

Seven Mei2-l ike genes were predicted from the completed Arabidopsis genome.

Exon structure and amino acid sequence supported three group ings of Mei2-l ike

genes. Structural p redict ions of Mei2-l ike proteins ind icate that the th i rd RRM

contained some nove l structura l features not present i n canon ica l RRM prote ins .

Attempts to study the function of the TE 1 protein in vitro were l im ited by the inabi l ity

of both E. coli and Pichia pastoris expression systems to express the fu l l l ength

prote in , probab ly due to codo n b ias . Antibodies produced to a C-termina l port ion of

the prote in d id not specifica l ly detect the TE 1 protei n i n p lant extracts without

incurr ing n on-specific act ivity .

The te 1 cDNA was ectopical ly expressed i n Arabidopsis from a copper- induc ib le

promoter both wi th and wi thout the SV40 nuclear local isat ion s igna l ( N LS) . Although

both te 1 and NLS:te 1 transgenes were detected i n transformants n o phenotypes

cons istent ly correlated with transgene expression .

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ACKNOWLEDGEM E NTS

A l ot of people have helped me with this PhD , but I would l ike to thank David Penny

and Pau la J ameson encouraging me to stay on after Honours , encourag ing me to

start my P h D, and for support ing my appl icat ions for the doctoral scho larsh ip .

Thanks for a l l the members of my fami ly for your continued encouragement, and for

your att itude to th ings academic . I t rea l ly makes a difference.

Thanks to Bruce Veit, for teaching the essentia l techn iques of molecular b io logy l ab

work , how to crit ique experiments, a l l about meristems, and many other th ings

besides. I know we haven't a lways got on Bruce, but your attitude to science, and

why we do it , is very coo l . Thanks also to Paul Reyno lds for your help at the start of

this project.

Thanks a lso to the many other people who have taught me dur ing my time at

M assey, i n part icu lar David Penny , David Fountain , Pau la Jameson , B i l l Jones, M ike

McManus , Barry Scott , Stan Moore . Its been fun . Also to the people who he lped me

negotiate the tria l s of experimental work; Carolyn Young, Lyn Watson , Carmel

G i l lman , some of the above. Cheers a lso to the Riborg Club, Anthony Poo/e, and

David Penny for t he weird and wonderfu l world o f Riborgis eigensis.

And to a l l those fel low students and friends around the Department of Plant

B io l ogy/ lMBS/DEB Lab-Boffin Lounge and associates - Ive had a darn fine t ime:

Abby, Al ic ia , Ant, Austin , Bennett, Carmel , Caro lyn & David, Em i ly , Hua i B i , Lyn , Matt

(bane of sma l l mammals) , Mu rray, Nena , Sang Dong Yoo, Tony (Yay for the DEB

Zeppelin sessions!) , Trish McLenachan , Trish Murray, Vern .

Katy. Thanks for everything , for even edit ing what to you must be mean i ngless

jargon . © Thanks for a l l your love and affection , you he lped me to reject the

hegemony of imputed needs.

I acknowledge the assistance of the Massey Doctoral Scholarsh ip .

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TABLE OF CONTENTS

THESIS A BSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ... . . . . . . . .. . .... . . . . . . . . . . . . .. . . .. . . . .. . . . . .. . . . . . .. . . . ..... . .. . . 3

ACKN OWLEDGEMENTS . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . ... .. . . . . . . . . .. . . . . . .. . . ....... .. 4

TABLE O F CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

LIST O F TABLES . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . .. . . . . . . .. . . .. . . . . . . . ... . .. . .. . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . .. ... 11

LIST OF FIGURES ................................................................................. ...................................... . 12

LIST O F GENES ........................................................................................................................... 14

CHAPTER 1 : I NTRODUCTION . ... ... . . .. . . .. . . . . .. . ... . .. . . . .. . . . .. . ... .. . . . .. . ... ... .. . . .. .. . . . . ... .. .. . ... . 1 7

1 . 1 A BSTRACT .. . . . . . ... .. ... . . ... . ... .. . . .. . ... .... . . ....... . ..... . ..... ........... ..... ........ . .. . . . . . . . . ... . ... .. 1 7 1.2. Developmental biology ...................................... ................................ ................ ......... . .. ...... 18

1.3. T h ree important aspects of plant development ............................................................... 18

1 .3a. The control of cel l division .......... .................................................................................... 1 9 1 .3b. The role of intercel lu lar trafficking in p lant development... ............................................. 20 1.3c. Structures that give rise to the p lant body .... ., ........................ . ....................................... 2 1

1.4. Shoot meristems ......................... ....... ............... ........... .......... ........ ...... ........ ........................ 22 1 .4a. Structure of shoot meristem s ........................ ., ................................................................ 22 1 .4b. Functions of the shoot meristem ........................................................ ., ............... ., .......... 25

1.5. In itiation of lateral organs ...... ......... .... . .................. ....... ........ . . .... ... . .................. ... . .............. 27 1 .5a. Simi larity of lateral organ in it iation ..... ...... ............................. ., ...................................... 27 1 .5b. Phy l lotaxy: specification of organ posit ion ..................... ................................................. 28 i.5c. Leaf initiation and acquisition of identity ..... ........ ....... ...... .......................... ., .............. 30

1.6. An introduction to the terminal ear1 gene and homologous plant genes ................... 31 1 .6a. The terminal ear1 gene of maize ................ . ............ ...................................... . , .............. 3 1 1 .6b. Plant homologues of the terminal ear1 gene: Mei2-l ike genes ............... ........ .... '" ..... 32

1.7 RNA recogn ition motif protei ns ...................................................................... ................ .... 33 1 .7a Orig ins and structures of R NA recognition m otif p rote ins ......... . ...................... ., ............. 33 1 .7b Functions of RNA recogn it ion motif p rote ins .............................................. ., ................... 36 1 .7c Biochemical function of the S. pombe MEI2 p rotein ..................................................... . . 36

1.8. Aims of this project ..... .... ..... . .............. . . .. . .......... ............. . .................. . ......... .... . ................... 37

1.9. Data produced by oth e r research e rs .......... .... .................................................................. 37

CHAPTER 2: MOLECULA R G E N ETIC ANALYSIS O F THE TERMINAL EAR1 G E N E O F MAiZE ........ . . ... . . . . ... . . . . .. . .. . . . .. . .. . . ... . ... . . ... . . ..... ... ... . . . . . . . . . . .. . . . . . . . ... .. ... ... . . . . . .. 4 1

2.1 . ABST RACT . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . ... . . . . . .. . . . . .. . . . . . . . . . . . . .. . .. . .. . . . . . . .. . . . . .... . .. . . . . . . . ... . .. .. 41

M ETHODS (2) . . . . .. . .... . . . . . . . . .... . ..... . ... .... . ... . . . ... .. .. . . . . . . .. .. . . . . .. . .. ... . . . . .... .. . ... .. . . . . . . ... . .. . . . .. 42 2.2 Gen e ral E. coli methods ....................................................................................................... 42

2.2a. Growth of E. coli . ., .............................. ..... ........................ .... ... ............. ...................... 42 2.2b. Preparation of heat shock-competent cells ... ...... . ... . .......... . .............. .......................... 42 2.2c. Transformation of E. coli by heat shock. . ....... .. . ......................................... 42 2.2d. Preparation of electrocompetent cells .. ................ ..... ....... .......................................... 43 2.2e. Transformation of E. coli by electroporat ion ................................................................... 43 2.2f. Alkaline lysis of E. coli. . . ........ ...... ....... . .......... . ..... . . .................... 43 2.2g. Lysis of E . coli by bo i l i ng us ing STET buffer ..................... ....... ................................... 44

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2.3 General D N A m ethods .......................................................................................................... 44 2.3a. Quantification of DNA and RNA... .... .............. ...... . . . . .... . . . . ...... .. .. ..... 44 2.3b. Electrophoresis of D NA . .. . ..... .. ... ... . ..... ...... ....... ........ . ... 45 2.3 c. Stan dard PCR Reaction Protocol.. ........ . .. .... 45 2.3d. Colony PCR . ... . .. 46 2.3e. Genera l scheme for vector construction . . ... . .... ..... 46

2.4 Sequencing of te1 genom ic clone ...................................................................................... 47 2.4a. Genera l scheme of project .... . ... ..... 47 2.4b. Construction of 5' end genomic subclone.. . ... .... . . . 47 2.4c. Sequencing reactions ........... . ..... . ... ..... .. ..... . ... .... 47 2.4d. Assembly of sequence files........... .......... .. . .. .. 48

2.5 General RNA m ethodology .................................................................................................. 48 2.5a. Precautions to avoid RNase contamination ........ . .. . .................. .......... 48 2. 5b. D N ase treatment of RNA p reparations. . . .......... 49

2.6 5' Rapid amplification of cDNA e nds (5' RACE) ............................................................... 49 2.6a. S u mmary of 5' RACE method.... . . .. .. . . . ........ . '" ... 49 2.6b. Special p recautions for 5'RACE experiments " .. '" ... 50 2.6c. Extraction of polyA+ RNA d i rect from tissue Iysates.. . .. ...... ...... . . . . 52 2.6d. Suitab i l ity of RNA sam ples for 5' RACE. . ... ............... . . . ..... ... . . . ........... .. 53 2.6e. Reverse transcription reaction for 5' RACE ..... .... . . ........ . ... ........ . . 53 2.6f. Synthesis of homopolymer tai l. .......... ..... ..... ...... . . . 54 2.6g. Second strand cDNA synthesis and pr imary PCR Reaction ............ .................. .......... 54 2.6h. Secondary semi-nested PCR reaction. . ....... ...... ....... ... ..... .. .... ................ 55

2.7 Quantification of te1 transcripts by competitive RT-PCR ............................................... 55 2.7a. Summary of competitive RT-PCR procedu re. .................. .. . . ........ ............. .. ... 55 2.7b. Extraction of total RNA with Qiagen RNeasy Plant Min i Kit ... . ......... .............. .......... 56 2.7 c. Acid guadin ium thiocyanate RNA extraction m ethods ...... ............. ..... .......................... 56 2.7d . Suitab i l ity of R NA sam ples for competitive RT-PCR. ................. .................................... 58 2.7e. Synthesis of altered te1 transcript... ................................................................................ 58 2.7f. Reverse transcription reaction s for RT-PCR ................................................................... 59 2.7g. Com p etitive PCR .............................................. ............................................................... 60

2.8. N o rthern blott ing .................................................................................................................. 61 2.8a. E lectroph oresis of glyoxylated RNA .. ... .......... ...... . .................. ...... .. ......................... 6 1 2.8b.Transfer o f R N A t o memb ranes .................................. ........ .... ....................................... 62 2.8c. Synthesis of radiolabel led probes. ........ ......... ... . ....... . . .. .......... ............ ........... 62 2.8d. Hybrid isation of RNA m e mbranes ...... ............................................ ................................. 63 2.9a. Summary of te1 al lele ana lys is .... .. . . . ... . .. . . . ..... . . . .. . . . . .... .. . .... . . . .... .. . ... . . .... . . . . . .. . . .. . ... . . ..... . . 63 2.9b. Extraction of maize genomic D NA ........................ ....... .......... ...................................... 64 2.9c. peR analysis of te1 m utant a l leles ................................................... ............................. 65 2.9d. Clon ing and sequencing of PCR products . . . . .... . . . . . .. . . .. .. . .. .. . .. . . . . . . . . .. . . . ....... . . . ... ..... .. .. . ... 65 2.ge. Southern Blott ing with the D I G System ....................... ................................................... 66

RESULTS ( 2) ....... . .. . .................... . . ....... . ............ . . . .... . .... . ................ . ............ ....... ..... 68 2.11. Predictio n o f transcription start s ite with 5 ' RACE ....................................................... 75

2.1 1 a. The 5' RACE protocol was i nconsistent ............................... ........................................ 75 2. 1 1 b. RT -PC R d id not confirm that the transcription start s ite was as p redicted by 5' RACE

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 2.12. Quantificatio n of te1 transcript levels ............................................................................. 78 2.13 Analysis of te1 m utant alleles ........................................................................................... 81

2.1 3a. Analysis of m utant a l leles by PCR.. . '" . .... ..... . ................... 8 1 2 . 1 3b. Southern b lots o f te1 a l leles ............................ ......... .... .... ....................... .. . ........ ..... 8 1 2.1 3c. Analysis of combined Southern a n d PCR data .. .. . . . . . .. . ..... . . . . . .. . . . . . .. . . . . . . . ..... . . . . · . . . . . . . . . . 86

DISCUSSION ( 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 2.14. Analysis o f the te1 genomic sequence ........................................................................... 92 2.15 Expression studies ............................................................................................................. 92

2.1 5a. 5' RACE...... ............................ .. ..... . .. . ... ............... 92 2. 1 Sb. Expression of te1 and meristem function . ............. . ....................................... ........... 93

2.16. M utan t allele analysis ......... ...... ......................................................................................... 96

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CHAPT E R 3. IN SILICa ANALYSIS OF MEI2-LlKE G EN ES . . . ... . . . . . . . . ...... ... . . . . . . . ..... 97

3. 1 A BSTRACT ..... ..... . . . . . . . . . . . . . . .. . . . ... ..... . . .... . ..... . ....... .... . . . .... . . . . . . ... . . .... . ........ ......... . . 97

M ETHODS (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 3.2 tBLASTn searches to identify putative Mei2- l ike genes .................................................. 98 3.3. Arabidopsis gene predictio n m ethodology . ..................... ................................................ 99 3.4. Promoter a n a lysis .............................................................................................................. 100

R E S U LTS (3) . . .... . . ... ......... .... ....... ................ ..... . . ...... .............. ............. . .... ....... .... .. 102 3.4. Seven Arabidopsis Mei2- l ike genes have been identified ............................................ 102

3.4a. Prediction of Arabidopsis Mei2-like genes.. . ........ 102 3Ab. Arabidopsis Mei2-l ike cONAs and ESTs .... . . .. ..... . . ..... 103

3.5. Three RRMs can be identified in a l l complete Mei2-l ike genes . .................................. 108 3.6. The i ntron positions of plant Mei2-l ike genes a re conserved ..................................... 116 3.7 Com parison of te1, TEU and TEL2 promoters . ............................................................. 116 3.8. A large number of Mei2-l ike genes can be identified i n p lants . .................................. 119 3.9. Mei2- l ike g e n es from taxa other than p lants .................................................................. 121 3.10. Phylogenetic a n a lysis Mei2-l ike genes ............................... .......................................... 121

3 .10a. Phylogenetic analysis of complete Mei2-like genes. . .......... ... .... . ... . . . ........ . ........... 121 3.10b. Phylogen etic analysiS of incomp lete Mei2-like gene sequences and E STs .. . . . ... . . ... 122

DISCUSSION (3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 25 3.11. Function and biochem istry of MEI2 with respect to plant Mei2-like genes .. ........... 125

3.11 a. The biological context of Mei2 and plant Mei2-like genes ................... ........... ........... 125 3 .11 b. RNA dependent localisation . . . . . .... . . ........ . . .. . . . . . . .. . ............. .. ........ . . . . .... .. . . . ....... ............ . 126 3.11 c. RNA binding activity of MEI2 and plant Mei2-like genes .. ................ . . . .... . ... .. ........... 128 3 .11 d. Phosphorylation of Mei2-like p rote ins .. . .... . . ... ... . . . . . . ..... ..................................... . . . . . ... 129 3.11 e. I nteraction with M IP1 WO repeat p rotein . ... . ...... ...... ... . . . . . . .. . ......... . . . ......... . . . . . ..... . .. . . . 130

3.12. Evolution a nd derivatio n of Mei2-l ike genes ...... .................................... . ..................... 130 3 .13. Funct ions of plant Mei2-like genes ....... ... . ............ . . ....... .... ............ . ............ . . . . . . 131

3.14. Con cluding rem arks ............................. ........................................................................... 132

CHAPTER 4. EXPRESSI O N OF THE TE1 P ROTE I N I N H ETEROLOGOUS HOSTS AND PRODUCTION OF A NTIBODIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . 1 33

4 . 1 A BSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 33

M ETHODS (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 34 4.2 Gen e ral protei n methods ..................................................... .............................................. 134

4 .2a. Bradford assay estimation of p rote in concentration . .. .. ..... ......... . . . ..... . .... . ... 134 4.2b. SOS polyacrylam ide gel electrophoreSis (SOS-PAGE) .. .... . . . ... .. .. ... ........ . ....... 134 4.2c. Western blotting. l um inescent substrate ..... . .. ..... ... .. . . ... 135 4.2d. Western blotting. colorimetric substrate . . . ...... ..... . . ...... . . . . . . ........ 136 4 .2e. E nzym e l inked immuno-sorbent assay (ELlSA) ... . .. ... .. . .............. .. 136

4.3 Production of m a ltose bindin g protei n fusion proteins ................................................ 138 4.3a. Construction of m altose binding protein fusion vectors . . ... ... .... ... . . .... . . . ... . ....... . . ........ 138 4. 3b. Small scale induction of E. coli cultures harbouring expression vectors ...... ...... .. . . . .. 138 4.3c. Optimisation of M B P:TE11•286 expression conditions .. . . . . ... ... . ........ ........... ........ .... . . . .... 139 4.3d. Large scale i nductions and affin i ty p urification of MBP:TE fusion p roteins ..... ..... . .... 139

4.4. Productio n of anti MBP:TEjl.286.antibody ................ ...................................................... 140

4.4a I noculation of rabbits and collection of sera . .. ....... .... .. ........ ... ... .. . ....... .... .. . . . . .. ... . . . . . 140 4Ab. Affin ity p u rification of polyclonal antibody .... . ... . .... .... . . . ...... ...... . ... . . . . . ......... . . . . . . .. .. . . 141

4.5 H istidine-tagged construct with ArgU tran sfer RNA gene ........................................... . 142 4.5a. Construct ion of h istidin e tagging vector.... . ...... .......... . . .. .... .. ........ .. . . ... .......... · ..... . . ... 142

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4.5b. Subcloning the ArgU tRNA gene by peR..... . .... 142 4.5c. Expression trials with histidine tagging vector.... . 143 4.5d. Purification of histidine-tagged TE1 p rotein.......... ............. . . .... 143

4.6 Transformation of Pichia pastoris with integratin g expression constructs .............. 144 4.6a. Overview of Pichia expression system '. . . .. 144 4.6b. Production of integrating expression vector in E coli.... . ..... 145 4.6c . Preparation of electro-competant Pichia cells. . .. 145 4.6d. Electroporation of Pici?ia cells.. 145 4.6e. Selection of transform ants with increasing numbers of integrations.. . 146 4.6f. Extraction of Pici?ia genomic DNA .. . 147 4.6g. Southern blotting.... .. ...... . .. . ..... 147 4 .6h. Induction trials with transformed Pichia pastoris . . ....... ..... . . .. ........... 148

R ES U LTS (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50 4.7 Only the a m ino-terminal half of the TE1 protei n was produced as MBP fusions ...... 153 4.8 Expression of f u l l length TE1 protein from the ArgU tRNA construct a nd the A rgU tRNA-su pplemented pSBET vector ................ ........................................................................ 155 4.9 The Pichia pastoris expression system did not produce s i g n ifican t quantities of the TE1 protei n ................................................................................................................................. 159

4.9a. AnalysiS of p rotein expression by transformed Pici?ia pastoris lines ......... ............ ...... 159 4.9b. AnalysiS of the integrations of transformed Pichia pastoris lines ................... ............ 161

4.10. Characterisatio n of the activity of polyclonal sera .............. ....................................... 163 4.10a. I mm une sera from both rabbits had activity against the MBP:TE11'286 p rotein ........ 163 4.10b. Pre-immune sera B had activity against the M B P p rotein ..... ......... ..... ..... .. .... .. ..... 164 4.10c. Antibody activity against MBP and TE11•186 portions of the fusion protein ............... 165 4 .10d. The affinity purified antibody has activity against a l l maize tissues .......................... 167

DISCUSS ION (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 69 4.11. Why was the TE1 protei n not produced i n E. coli? ..................................................... 169 4.12. Why was the TE1 protei n not produced i n Pichia pastoris? ..................................... 172 4.13. The 'non specific' activity of the polyc lona l a ntibodies ............................................. 172 4.14. Concluding rem a rks and future prospects .................................................................. 173

CHAPTER 5: ECTOPIC EXPRESSION OF TEL G EN ES I N ARABIDOPS/S . . . . . . . . . . 1 74

5 . 1 ABSTRACT .... . . . . . . . . . . . . . . . . . .. . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . .. . . . . . . . . . . .. . . . . . . 1 74

M ETHODS (5) . . . . .. . . . . . . . . . . . . . . . . . ........................... .. .. . .. .. . . . . . . . . . . . . . . . . .... . . . . . . . . . . .. . . . . . . . . .. . . . 1 75 5.2 Construction of vectors for transformation of Arabidopsis . ........................................ 175

5.2a. Construction of copper-inducible transformation vectors . .. .. . ............. 175 5.2b. Construction of glucocorticoid transformation vectors ............ . . .................................. 177

5.3 Tra nsformation of Agrobacterium .................................................................................... 178 5.4 Growth of Arabidopsis ....... ................................................................................................ 178 5.5 Transformation of Arabidopsis by vacuum i nfi ltratio n ................................................. 179

5.5a. Overview of the procedure ................ ......... ........ .... .... ........... . .. ........ 179 5.5b. Vacuum infiltration ............................... ....... . ... ... . .... ..... ... . ........... ....... ................ 179 5.5c. Selection of transformants. .. . ...,.. ..... . ........ 180

5.6 Induct ion conditions for plants with copper-inducible tra nsgenes ............................ 181 5.6a. I nduction in synthetic media.. ................... .. ... ......... . . ....... 181 5.6b. I nduction in soil . .... ......... .... .... ... ........ . . .... .. . ... .......... ..... ................ ............... 181

5.7. Dexamethasone applicatio n to TEL2:glucocorticoid receptor plants ........................ 181 5.8. Assays of transgene expression ..................................................................................... 182

5.8a. N orthern blotting .... .. . . . . . . ...... . . . .......... . . . . ........... 182 5.8b. Western blotting ........ ... . .. .. .... ... .. .. 182

RESULTS (5 ) . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . 1 84

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5.10. Initial analysis of copper-inducible transform ants ..................................................... 184 5.1 0a. Transgenic p lants transformed with copper inducible constructs contained one or two integrations ofT -DNA . .. . . .. . . . . . . . . . 184 5.1 0b The te1 transgene was expressed in Arabidopsis from the copper inducible p romoter . . . ...... . .... .. .. ... . . . . . . ... ...... .... .. .. . . . 186

5.11. Expression of the te1 transgene did not correlate with any observable phenotypes ...................................................................................................................................................... 188

5.1 1 a. Neither vegetative nor floral morphology was affected by fe 1 transgene expression .............. ...... . . . ....... 1 88

5 1 1 b The wnnkled leaf phenotype did not correlate with transgene expression .. ... . .. 1 89 5 1 1 c The antl_MB P·TEi1•286 antibody did not d etect the TEi protein In transgenic p lants ................. ..... ... .. . .............. . ........ 1% 5. 1 1 d. Aerial rosettes formed, but they were not due to expression of the te 1 transgene . . 1 96

5.12. Initial observations of TEL2 glucocorticoid fusion plants showed n o clear phenotype .............. . ... . ................... ........................... ............................................. ..................... 198

DISCUSSION (5) ... . . ... . . . . . ... . . . . . ... ................ ......... .. ....... . .. . . . ... .... . . . . . . . .......... . . . . . . . .... . 199 5.12 The aerial rosette phenotype is the result of g rowth conditions ............................... 199 5.13. Expression of the m aize te1 gene or the Arabidopsis TEL2 gene in Arabidopsis did not produce any consistent phenotype ........... .. .............................................................. 200

5. 1 3a. Technical issues with te1 transgene expression.... . .... 200 5i3b. Technical issues with TEL2:GR transgene expression . . . .. .. . . ..... . . 201 5.1 3c. Biological aspects of TEL genes .. . ........... . ........... 201 5.1 4d. A reinterpretation of overexpression phenotypes based on recent data .. . . . .. . . . 203

5.15. Con cluding remarks . .............................. ................................................ ......................... 203

CHAPTER 6. SYNOPSIS ....... ...... ...... .. . . .... . .. . . . . .. . .. . . ... ....... . ... ... ... .... . . .... . ... .. . . ...... . . 206 6.1. Recapitulation of the aims of the project ... . . .................................................................. 206 6.2. Homology, o rthologs and genome duplications ................... ....... . . ...................... ......... 207 6.3. Models of TEL gene function ............................................................................................ 208 6.4. A fin al summary a nd some new questions ...... . ...................... .......... ............. ................ 209

REFERENCES . .. . . . .... . . . . ..... ...... ... . .. ... ... ... ... . .. .. . .. . . .... . .. . . ... . .... . . . ........... .. . . ... . .. . . . ..... . 2 1 1

A P P E NDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Appendix 1. Olig o n ucleotide P rimers ....................... .................................... ................. ........ 227 Appendix 2. PCR P rograms ... ..... ............................................................................................. 229 Appendix 3. List of Suppliers ............................. . ..................... ................................... ............ 232 Appendix 4. Assembly of te1 gene sequencing reactions . ................................................. 233 Appendix 5. Subclones of the 12.4 kb Kpnl te1 genomic clon e . ..... . .................... . ............. 234 Appendix 7. Pedigrees of m aize stocks . ... . ............................................................................ 235 Appendix 8. PCR produ cts from te1 m utant alleles ..................................... ..... .................. . 235 Appen dix 9. Southern Blot D ata .......... . ................................ ................................................... 236

Appendix 9. 1 . Southern blot one . . . .... . . . .. . ... . . . . . . .. . ... . .. . ... . . .. .. .... ... . ... . . .... . .. . . . .. ... . ... . . . . . . . . . . .. . . . .. . . 2 36 Appendix 9.2. Southern blot two. . . ...... 2 37 Appendix 9.3. Southern blot three . . .. . ... ...... .. .. . .... .. . .... . . ..... . . . .. . . .. .... . ...... . .... . ... . ... . .. . .... . . , .... .... . 2 38

Appendix 10. Supplementary file disk ....... ... . ....... . . . .................................................. ...... ....... 239 Gene p rediction s . . ... ... . ... . . .. . .. . . . . . . . . . . . . ... . . .. . . . . . . .. .. . .... . . . . ... . . ... . . .. .. . .. . . . . .. .... . . . . . . . .... . . . . ..... ... . . . . . . ... 2 39

Appendix 11. The concurrent RNA world project.. .............. . ................ .... . ..... ......... ... ....... ... 240

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1 1

LIST OF TABLES

Table 2 . 1 . te 1 mutant a l le les 56

Tab le 2 . 2 . S pl i ce s i tes i n te1 gene 60

Tab le 3 . 1 . Arabidopsis Mei2� l i ke genes 95

Tab le 3 .2 . EST h its from Arabidopsis BLASTn searches of genomic sequences 96

Tab le 3 . 3 . Mei2- l i ke genes from p lants 1 09

Tab le 4 . 1 . Expression vectors 1 39

Tab le 4 . 2 . Resistance of transgenic Pichia l i nes G41 8 1 5 1

Tab le 5 . 1 . Tra nsgenic Arabidopsis constructs 1 69

Tab le 5 . 2 . Leaf number frequencies i n te 1 -expressing Arabidopsis 1 78

Tab le 5 . 3 . Copper- inducib le tran sforma nt l ines ; transgene expression and the

wrink led phenotype

1 79

12

LIST OF F IGU RES

Figure 1 . 1 . Symplast ic fie lds i n the SAM 23

F igure 1 . 2 . Overlay o f five R R M domains 35

F igure 2 . 1 . Overview of the 5' RACE protocol 5 1

F igu re 2 .2 . The structure of the te 1 gene 69

F igure 2 .3 . Genomic sequence o f t he te 1 gene 70

F igu re 2 .4 . 5 ' RAC E ge ls 77

F igure 2 .5 . Competitive RT -PCR quantificat ion o f te 1 transcript 79

F igu re 2 . 6 . RNA samples used for competitive RT-PCR 80

F igure 2 .7 . te1 a l le le Southern b lot 1 83

F igu re 2 . 8 . te 1 a l le le Southern b lot 2 84

F igure 2 .9 . te 1 a l le le Southern b lo t 3 85

F igure 2 . 1 0. Summary of te1-1 a lle le PCR and Southern ana lysis 87

F igure 2 . 1 1 . Summary of te1-ems a l le le PCR and Southern ana lysis 88

Figu re 2.1 2 . Summary of te1-mum4 a l le le PCR and Southern ana lysis 89

F igure 2 . 1 3 . Summary of te1-mum6 a l le le PCR a n d Southern ana lys is 90

Figu re 2 . 1 4 . Summary of te1-mum7 al le le PCR and Southern ana lysis 9 1

F igu re 2 . 1 5 . Expression level s of t e 1 and internode lengths 95

F igure 3 . 1 . tBLASTn search for Mei2-l i ke gene 99

F igure 3 .2 . Netgene2 graph ica l output for promoter ana lysis 1 03

F igu re 3 . 3. Al ignment of fu l l l ength Mei2-l i ke prote ins 1 09

Figure 3 .4 . Structure-based a l i gnment of RRM3 1 1 4

F igure 3 . 5 . U nique C-term ina l ahel ix o f RRM3 1 1 5

F igure 3 .6 . I ntron posit ions i n Mei2- l i ke genes 1 1 7

F igure 3 .7 . Promoter e lements i n te 1 , TEL 1 and TEL2 1 1 8

F igure 3 .8 . Maximum pars imony tree of a l l fu l l length Mei2- l ike genes 1 23

F igure 3 . 9. Maximum pars imony tree inc lud ing part ia l Mei2- l ike

genes and ESTs 1 24

F igure 4 . 1 . Vectors constructed for TE1 protei n express ion 1 5 1

F igure 4 . 2 . SOS-PAGE ge l of affin ity purified M B P:TE 1 prote ins 1 54

F igure 4 .3 . Western b lot of 6H :TE1 i nduction from pOJ06 vector 1 56

Figu re 4 .4 . Coomassie stai ned SOS-PAGE gel of affin ity purified

6H :TE1 protei n 1 57

F igure 4 . 5 .

F igure 4 . 6 .

F igure 4 . 7 .

F igure 4 . 8.

TE1 expression from the pSBET vector

SOS-PAGE gel of Pichia i nductions

Southern b lot of transgenic Pichia l i nes

EL lSA data from raw sera

1 58

1 60

1 62

1 64

F igure 4 . 9 . Western b lot , pre- immune sera B activity aga inst MBP 1 66

F igure 4 . 1 0 . Western b lot showing Sera A activity aga inst

both port ions of MBP :TE1 1 -286 1 66

F igure 4 . 1 1 . Western b lot showing n on-specific activity of affin ity

purified a nt ibody 1 68

F igure 4 . 1 2 . The d istribut ion of rare codons i n the fe 1 read ing frame 1 7 1

F igu re 5 . 1 . Vectors constructed for expression of fe 1 and

TEL2 i n Arabidopsis 1 76

F igu re 5.2 .

F igure 5 .3 .

S .4a .

S .4b .

S .4c .

F igure 5 . 5 .

Southern b lot of tra nsgeneic Arabidopsis l i nes

Northern b lot of transgeneic Arabidopsis l i nes

Wrin kled and normal Arabidopsis s ib l ings i n pot

Wrin kled l eaf phenotype in te 1 transgenic l i ne

Wrin kled l eaf phenotype i n NLS:fe 1 transgen ic l i ne

Northern b lot showing fe 1 expression i n i nd iv idua l wri n kled

1 85

1 87

1 9 1

1 92

1 93

vs. normal s ib l ings 1 94

F igure 5 . 6 . Northern b lot showing te 1 expression i n poo ls of wrin kled

vs. normal s i b l i ngs 1 95

F igure 5 . 7 . The aer ia l rosette phenotype 1 98

13

ABBREVIATIONS

2-ME Amp BC I P Cefo OEPC D IG OMPC

OMSO dNTPs OTT E OTA EMS

GA3 Gent I PTG Kan LB NaOAc N BT PBS PCR PCR p N P P polyA+ rpm RRM RT-PCR SAM SOS S OS-PAGE Spec SSC

Tris xg

2-mercaptoethanol (�-mercaptoethanol ) ampici l l i n (superscript, concentration i n �lg/ml ) x-phosphate/5-bromo-ch loro- indoly l-phosphate Cefotaxime (superscript, concentrat ion in �g/ml ) d iethylpyrocarbonate O igoxigen i n d imethylpyrocarbonate

d imethy lsu lphoxide deoxyribonucleotide triphosphates d ith iothreitol d isod ium ethy lened iaminetetra-acetate ethylmethane su lfonate

G ibere l l i c acid 3 gentamycin (superscript, concentratio n i n �g/ml) I sopro pylthio-/3-0-ga lactas ide kanamycin (superscript, concentrat ion i n �lg/ml) Lur ia Bertann i med ium sod i u m acetate 4 N itrob lue tetrazo l i um ch lo ride phosphate buffered sa l ine Polymerase cha in reactio n polymerase cha in reactio n p-Nitrophenyl phosphate d isod i um Polyadenylated RNA revo lut ions per m in ute RNA recogn it ion motif Reverse transcri ption-po lymerase chain react ion shoot a pical m eristem s sod i um dodecyl su lfate SOS po lyacry laminde ge l e lectrophoresis spect inomycin (superscript, conce ntrat ion i n �g/ml ) sod i u m chlor ide, sod i um citrate 20 x SSC is 3 M NaCI , 0 .3M sod i um citrate pH 7 .0 Tri s (hydroxymethyl )aminomethane m u lt ip les of gravitationa l force

14

TABLE OF GEN ES

A consistent nomenclature is used throughout this thesis to describe genes and p roteins. Genes names are a lways ita l i cised, protei ns a lways l isted i n block capitals with suffixes describ ing the amino acids incl uded for part ia l peptides. e .g . the port io n of the te 1 gene was expressed to produce the peptide TE1 1 -286

Gene symbol Gene Species

15

A CE 1 meta l loth ionein regu l atory prote i n Saccharomyces cerevisiae A ML 1, A ML2, AML3, Arabidopsis Mei2-l i ke Arabidopsis thaliana A ML4, A ML5 A N T AINTEGUMENTA Arabidopsis thaliana CDC2a cel l d ivisio n cycle 2a Arabidopsis thaliana CLV 1, CLV2, CLV3 CLAVA TA Arabidopsis thaliana CUC 1, CUC2 CUPSHAPED COTYLEDONS 1, 2 Arabidopsis tha/iana FIL FILAMENTOUS FLOWER Arabidopsis tha/iana gn 1 Gnarly Zea mays GR g lucocortico id receptor mammalian kn 1 knotted 1 Zea mays M B P maltose b ind ing prote i n E. coli Mei2 Meiosis deficient 2 Schizosaccharomyces

pombe MG0 1, MG02 MGOUN Arabidopsis thaliana nptll neomycin phoshotransferase synthetic OSH 1 Oryza sativa homeobox1 Oryza sativa S TM SHOOTMERIS TEMLESS Arabidopsis thaliana T7 10 phage T7 protein 1 0 phage T7 te1 teminal ear 1 Zea mays TEL 1 , TEL2 terminal ear1-like Arabidopsis tha/iana UFO UNUSUAL FLORAL ORGANS Arabidopsis thaliana WUS WUSCHEL Arabidopsis thaliana YAB3 YABBY3 Arabidopsis thaliana