THE ISOLATION AND CHARACTERIZATION OF A P. ANGULOSUS HOMEOBOX. Peter Lance Pfeffer Thesis submitted in fulfillment of the requirements for the degree of DOCTOR of PHILOSOPHY in the Department of Biochemistry,University Faculty ofof Science, Cape Town University of Cape Town. Cape Town, January 1991 . • The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town i CERTIFICATE OF SUPERVISOR In terms of paragraph eight of "General regulations for the degree of Ph.D.", I, as supervisor of the candidate P.L.Pfeffer, certify that I approve of the incorporation into this thesis of material that has already been published or submitted for publication. Professor C. von Holt Professor in the Department of Biochemistry and Director of the UCT-FRD Research Center for Molecular Biology. ii ACKNOWLEDGEMENTS I wish to thank Professor Claus von Holt, my supervisor, for his excellent guidance, support and encouragement during the course of this thesis. Rob Warren for his helpful suggestions and many fruitful discussions. My colleagues in lab 402 for their moral support and joviality, making the lab a second home. Special thanks to Sylva Schwager and Madhu Chauhan. Dr. W.McGinnis for the kind donation of the Antennapedia homeobox-containing plasmid p903G. The Foundation for Research Development and the University of Cape Town Research Committee for their generous financial support. My wife Cynthia, my mother and my brother for their continual enthusiasm and interest in my work. iii INDEX THE ISOLATION AND CHARACTERIZATION OF A SEA URCHIN HOMEOBOX CONTENTS Certificate of supervisor .................................. i Acknowledgements ......................................... :~~ Index . ................................................... 11..1 Summary . .................................................. vi Abbreviations ............................................ vii CHAPTER 1: LITERATURE REVIEW THE STRUCTURE AND FUNCTION OF HOMEOBOXES 1.1 Historical perspective ................................. 1 1. 2 Classes of Homeoboxes .................................. 2 1.3 Structure of the homeodomain ........................... 5 1. 4 Binding studies ........................................ 6 1.5 Structural features of Homeodomain-proteins ........... 11 1.6 The function of homeodomain proteins .................. 13 1.6.1 Experimental approaches ........................ 13 1.6.2 Transcription factors .......................... 14 1. 6. 3 Conclusions .................................... 21 1.7 Homeobox genes and Development ........................ 24 1. 7 .1 Drosophila ..................................... 25 1.7.1.1 Early embryogenesis ..................... 25 1.7.1.2 The bicoid protein gradient ............. 25 1.7.1.3 The segmented subdivision of the embryo.27 1.7.1.4 The homeotic genes ...................... 30 1.7.1.5 Interpretations ......................... 32 1.7.1.6 Later embryogenesis ..................... 33 1. 7. 2 Vertebrates .................................... 34 1 . 7 . 3 Echinoderms .................................... 3 9 1. 7. 4 Nematodes ...................................... 40 CHAPTER 2: THE ISOLATION OF A SEA URCHIN HOMEOBOX 2.1 Library construction ................................. 42 2.2 Ligation ............................................. 44 2. 3 Library evaluation ................................... 45 2. 4 Screening . ........................................... 4 6 2. 5 Characterization . .................................... 4 7 2. 6 Subcloning ........................................... 5 O iv CHAPTER 3: SEQUENCE CHARACTERIZATION 3 • 1 Method ............................................... 51 3.1.1. Sequencing strategy ........................... 52 3.2 Results and Discussion ................................ 53 CHAPTER 4: RESTRICTION FRAGMENT LENGTH POLYMORPHISM 4.1. Restriction fragments ............................... 57 4. 2. Copy number ......................................... 58 4.3. Degree of polymorphism .............................. 59 4.3.1 Statistical method for predicting the frequency of restriction fragments ............ 60 4.3.2 Comparing predicted and observed polymorphism.62 CHAPTER 5: EXPRESSION 5. 1 RNA dot blots ........................................ 64 5. 2 RNAase ti tra tions .................................... 6 5 5 . 3 RNAase mapping . ...................................... 6 7 5 . 4 Northern blots .................................... : .. 7 0 5. 5 Discussion ........................................... 71 CHAPTER 6: CONCLUSION ............................... .... 7 2 CHAPTER 7: METHODS AND MATERIALS 7.1 Sea urchins 7 .1. 1. Embryos ....................................... 7 5 7 .1. 2 Adult tissues .................................. 75 7.2 Nucleic acid isolation 7. 2 .1 Genomic DNA .................................... 76 7.2.2 Plasmid DNA .................................... 76 7 . 2 . 3 Lambda DNA . 7 6 7 . 2 . 4 Total RNA . ..................................... 7 6 7.2.5 Poly A+ RNA .................................... 77 7. 2. 6 RNA content of embryo .......................... 7 7 7.3 Sea urchin genomic library 7. 3. 1 Vector and Host ................................ 7 8 7.3.2 Ligation ....................................... 78 7. 3. 3 Packaging ...................................... 78 7. 3. 4 Titration ...................................... 79 7.3.5 Amplification .................................. 79 7. 3. 6 Screening . ..................................... 80 7.4 Microbial techniques 7. 4 .1 Competent cells ................................ 80 7. 4. 2 Transformation ................................. 81 Index V 7.5 Nucleic acid manipulation 7.5.1 DNA Enzymatic manipulation ..................... 81 7.5.1.1 Restriction enzymes ..................... 81 7.5.1.2 Klenow fragment ......................... 82 7.5.1.3 Nick translations ....................... 82 7. 5. 1. 4 Ligations ............................... 83 7.5.1.5 Dephosphorylations ...................... 83 7.5.2 RNA Manipulations .............................. 83 7. 5. 2 .1 RNAases ................................. 83 7. 5. 2. 2 Transcriptions .......................... 84 7.5.2.3 Glyoxal denaturation .................... 85 7.5.3 General nucleic acid manipulations ............. 85 7.5.3.1 Ethanol precipitation ................... 85 7. 5. 3. 2 Spin columns ............................ 86 7.5.3.3 Concentration determinations ............ 86 7.5.3.4 Gel electrophoretic purification . ........................... 86 7.5.3.5 TCA precipitation ....................... 86 7.5.3.6 Sucrose gradient size fractionation ..... 87 7.6 Electrophoresis 7.6.1 Agarose ........................................ 87 7.6.2 Non-denaturing acrylamide ...................... 88 7.6.3 Denaturing acrylamide RNA gels ................. 88 7. 6. 4 Sequencing gels ................................ 88 7.7 Nucleic acid transfer and hybridization 7. 7 .1 Southern blots ................................. 89 7 • 7 • 2 Northern blots ................................. 8 9 7 . 7 . 3 RNA Dot blots .................................. 9 O 7. 7. 4 Hybridization .................................. 90 7.8 RN.Aase titration and mapping .......................... 91 7.9 Photographic techniques 7. 9. 1 Photography . ................................... 9 2 7 . 9. 2 Autoradiography ................................ 9 2 7.9.3 Microdensitometer scanning ..................... 92 CHAPTER 8 : REFERENCES ................................... 9 3 Index vi SUMMARY THE ISOLATION AND CHARACTERIZATION OF A P.ANGULOSUS HOMEOBOX The aim of this thesis was to isolate and characterize a homeobox-containing gene of the South African sea urchin Parechinus angulosus. This was achieved by constructing a genomic library of several individuals and screening this library using a probe containing the Antennapedia homeobox. Eight clones were isolated and shown to represent different alleles of the same gene. One clone was sequenced, revealing a homeobox which was termed PaHboxl. This homeobox was compared to published homeobox sequences and shown to be a member of the Antp (Hoxl.l) subclass (table 1.1). A splice donor site was identified 23 bp upstream of the homeobox and the observation confirmed by RNAase mapping. PaHboxl is situated in a genomic area showing a significantly higher degree of restriction fragment polymorphism than expected. This was shown by a statistical analysis which should be of general value in the interpretation of such polymorphisms. The expression of PaHboxl was examined by RNAase protection assays and Northern blotting. Two distinct phases of expression were observed - during embryogenesis PaHboxl is expressed transiently at low levels in 11,5 hr mesenchyme blastula stage embryos (44 ± 8 transcripts per embryo) with levels 3-5 fold lower 2,5 hr before and after this stage. Expression is observed again at up to 160 fold higher levels in the adult with maximal expression in testis (11 transcripts per 10 pg total RNA), and increasingly lower levels in intestines, ovary and Aristotle's lantern. Two transcripts of size 5,2 and 5,7 kbp were observed. Expression in Aristotle's lantern and embryonic stages could not be detected by Northern analysis. vii ABBREVIATIONS