\ø\'r1 Itt' Molecular Studies of Homologous Chromosome Pairing tn Triticum aestivum by Stephen'W'. Thomas B. Ag. Sc. (Hons.) This thesís is presented for the degree of Doctor of PhilisoPhY in The Department of Plant Science Waíte Agricultural Research Institute The (Iniversity of Adelaide March 1997 ERRATA Page 11 - last paragtaPh, line 7 coñtrast to the Holliday "model" Page 15 - line 5 replace "shown to be" with "shown to @" Page23 - line 28 "initiation" Page25 - last paragraph, line 1 'þolycomplexes" Page26 - 3rd paragraph,line2 "extranuclear" Page 31 - 3rd paragraph,line 4 rep"lace "long är- õf the chromosome" with "long arm of chromosome 58" Page 35 - 2ndparagraph, line 11 "non-homologous" Page 35 - 2ndparagraph, line 14 rep"lace "Furthêrmoie in plants tri-isosomic 5BL plants" with "Furthermore in tri-isosomic 5BL plants" Page 35 -2ndparagraph,line 16 "intrachromosomal" Page 36 - 2ndparagraph, line I "non-random" Page 43 - line 2 "di ethylpyrocarb onate" Page 43 - 2ndparagraph, line I 'þrecooled" Page 46 - 4Ihparagraph, line 3 "solutiqn" Page 47 -2ndparugraph, line 2 "manafacturer's" Page 48 -Iine2 and "was" incubated ' Page 58 - line 5 "oaraffm" Page 59 - line 10 l'identified" Page 69 - paragraPh 3, line 1 ..has utilised for cloning and replace been cloning and allows" with'was allowed" "tiiilålioi Page75 - paragraPh 2,line 14 "zygotene-PachYtene" Fisure 3.5 - line 2 thã phage "red. transferred Fisure 3.5 - line 6 clõnes which "did" not Figure 3.9 - line 4 5 minutes "Ègg" exPosed Page 84 - line 5 "advancql" Page 88 - line 5 "both ends o:f' Page 88 - paragraPh 2,line 14 "pollen had been" Page 116 - line I "Appendix 2" Fiqure 5.17 - line4 "pãl¡r-otphisms ob served" Page 129 - line 3 "chromosomeg" Pase 129 - 2ndParagtaPh, line 7 "píoduction of antibodies is required" I Declaration award of any other degree This work contains no material which has been accepted for the best of my knowledge or diploma in any university or other tefiary institution and, to the or written by another person, except and belief, contains no material previously published where due reference is made in the text' the university Library' being I give consent to this copy of my thesis, when deposited in available for loan and photocopying' Stephen W. Thomas 11 Acknowledgments Dedication (1904-1939), I hope that this thesis This thesis is dedicated to mY Grandfather F. J. Wauchope would have made him Proud. il Abbreviations Comrrion abbreviations are listed in Current Protocols on CD-ROM (1993). Additional abbreviations are as follows: pci mlcrocune pg microgram C' gram ¡r1 microlite ¡rM micromolar bp base pair kb kilobase Ítl milliliüe nm nanometre mM millimolar M molar ng nanogram A null mutation SC synaptonemal complex ptu plaque forming unit DSB double strand break SPB spindle pole body DEPC di- ethyl-pyro-carbonate AW'WM {ustralia, Waite, Wheat, Meiosis UTR untranslated region Da dalton EMS Ethyl methane sulphonate lv Abstrøct Allohexaploid bread wheat (Triticum aestivum) is composed of three closely related, homoeologous genomes, A, B and D. Despite the high degree of homology between the genomes, only bivalents form during chromosome pairing at meiosis. The diploid character of hexaploid wheat arises from the action of pairing genes (Ph) located predominantly on chromosomes 3 and 5. Although cytogenetic studies of the pairing genes in hexaploid wheat have been performed for a number of years, the understanding of the molecular action of these genes remains poorly understood. Here, two approaches to the molecular characterisation of the homologous pairing process in allohexaploid wheat have been described. The isolation and characterisation of late replicating DNA in lily has indicated that this DNA species is required for correct chromosome pairing in this organism. Attempts here to isolate late replicating DNA from hexaploid wheat have proven to be unsuccessful. It is suggested that, if late replicating DNA is present in wheat, it is of a different structure than that observed in lily. In addition to studying late replicating DNA, several meiotic genes were isolated from an earþ meiosis cDNA library screened with a probe prepared by subtractive hybridisation. One of these clones, AW-WMS, was further characterised. The genes of this family are located within the ph2a deletion on the short arm of chromosome 3D and on the short arm of chromosome 34. The ph2a deletion has been characterised using a barley consensus map and is demonstrated to be a sub-terminal deletion. The AWWM5 gene is expressed predominantly at premeiotic interphase and early meiosis in both the meiocytes and the tapetum. The protein encoded by AWWM5 has putative DNA binding and membrane binding characteristics. A speculative model on the action of the pairing genes in allohexaploid wheat, and the putative function of the AW.WM5 gene is discussed. Table of Contents Declaration Acknowledgements u Abbreviations üi Abstract iv Chapter.l - Review of Literature 1.1 Introduction 1 2 1.2 The Classical View of Meiosis and Mitosis 3 1.3 Model Organisms for the Study of Meiosis 1.3.1 The Lower Eukaryotes-Yeasts and Other Fungi J 1.3.2 Plants 4 1.3.3 Animals 5 7 1.4 The Synaptonemal ComPlex 1.5 Genetic Recombination at Meiosis 10 1.5.1 The Recombination Process 10 1.5.1.1 The Holliday Model l1 1.5.1 .2 The Meselson-Radding Model t2 1.5.1.3 The Double Strand Break Repair Model 1.5.2 Genes Required for Recombination 1.6 Chromosome Pairing 1.6.1 Premeiotic or Somatic Association of Homologous Chromosomes 1.6.2 Specific Interactions of Homologous Chromosomes at Prophase 1.6.3 Long Range Interaction of Chromosomes I.6.4 Chance Contacts between Homologous Chromosomes in Random Motion 1.6.5 Interaction of Chromosomes with Intranuclear Structures 1.6.6 The Mechanism of Homology Recognition 1.6.7 The Presence of Specific Pairing Sites 1.6.8 The Initimate Assessment of Homology at The DNA Level 1.6.9 A Model for Homologous Chromosome Alignment and Synapsis 1.7 Chromosome Pairin g in Triticum aestivum 1.7.1 The Genetic Control of Chromosome Pairing I.7.2The Mechanism of Ph Action 1.7.3 The Biochemical Mode of Ph Action 1.7.4 Ph Mutants 1.8 Research Aims 41 Chapter 2'Materials and Methods 4l 2.1 Plants and Growth Conditions 4l 2.2 Collection and Staging Of Meiocytes 42 2.3 Isolation of Nucleic Acids from Plant Material 42 2.3.1Extraction Of DNA from Plant Tissue 42 2.3.1J Miniprep Isolation of DNA for Small scale Applications 42 2.3.l.zMidiPreP Extraction of DNA 43 2.3.2 RNA Extraction from Plant Material 43 2.4 Punfication and Precipiøtion of Nucleic Acids 43 2.4.1 Phenol:Chloroform Extraction of Nucleic Acids 44 2.4.2 Precipitation of Nucleic Acids 44 2.5 E. coli Recombinant Techniques 44 2.5.1L\gation of DNA into Plasmids 44 2.S.2Transformation of E' coli 44 2.5.2.I Preparation of competent cells for Transformation 45 2.5.2.2 Transformationof E. coli 45 2.5.3 Isolation of Plasmids from E. coli 45 2.5.3.1Small Scale Isolation of Plasmids 46 2.5.3.2large Scale Isolation of Plasmids 46 2.6 Bactenophage Recombinant Techniques 46 2.6.I Preparation of E coliHoststrains for Infection with Phage 46 2.6.2Preparation of DNA from Lambda Phage 47 2.7 Preparation of a oDNA Library 47 2.7.1 Isolation of Poly(A) RNA from Total RNA Samples 47 2.7.2Preparation of the cDNA Library 47 2.7.2.I Production of Double Stranded cDNA and Ligation into ),9110 48 2.7.2.2Packaging and Transfection of the Phage Library 48 2.8 Southern Transfer and DNA Hybridisation 48 2.g.1 DNA Gel Electrophoresis and transfer to Membranes 48 2.8.2 Transfer of Phage Plaques to Membranes 49 2. 8.3 Prehybridisation of Membranes 49 2.8.4 Hybridisation of Membranes 2.8.4.I Preparation of Radio-Labelled Probe 49 49 2.8.4.2 Hybridisation Conditions 50 2. 8.5 V/ashing Hybridisation Membranes 50 2.9 Northern Transfer and DNAiRNA Hybridisation 50 2.9.I ElectroPhoresis of RNA 50 2.9.1.1 Gel PreParation 50 2.9 .l .2 SamPle PreParation 51 2.9.2 Northern Transfer and Hybridisation 51 2.9.2.1Transfer of RNA to Membranes 51 2.9 .2.2 Prehybridisation Conditions 51 2.9 .2.3 Hybridisation Conditions 5l 2.10 Isolation of Late Replicating DNA from wheat Meiocytes 5l 2.10.1 DNA PreParations 52 2.1 0.2 Isopycnic Centrifu gation 52 2.10.3 Digestion of DNA with Mung Bean Nuclease I 53 z.l}. Fractionation of DNA on a Continuous Caesium Chloride Gradient 53 2.10.5 End Labelling of DNA with cr-32P-dCTP wheat 54 2.l0.6Preparation of a Lambda Library of High Density DNA from Meiocytes 55 2.11 Preparation of an Early Meiosis Subtractive Probe from Wheat 55 2.ll.l Production of mRNA Populations with Subtractive oligolinkers 56 z.ll.zPolymerase Chain Reaction Amplification of cDNA Populations 2.1I.3 Amplification of Immature Pollen cDNA and Photobiotin Labelling 56 57 2.ll.4Detection of Biotin Incorporation into oDNA 57 2.1 I .5 Subtractive Hybridisation 58 2.I2 Characterisation of WM27 58 2.!2.|Subcloning the Meiosis Specific Region of pWM27 59 z.l2.2Northern Hybridisation with pWM2Tms 60 2.12.3 Preliminary Mapping of The pWM2Tms Clone 6l 2.12.4 Re-Screen of the cDNA Library of Genomic and Full 61 2.lz.sProduction of a wheat Genomic Library and Isolation length oDNA Clones 6l 2.12.5.1 Partial Digestion with EcoRl 6T 2.12.5.2Fractionation on a Sucrose Gradient 62 2.12.5.3 Ligation to Vector and Packaging 62 2.12.5.4 Plating and Titre of Phage 62 2.12.5.5 Screening of the Genomic Library 62 2.12.6 Subcloning the Genomic Insert 63 2.12.7 Construction of a Nested Deletion Library 64 2.