INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type ofcomputer printer. The quality ofthis reproduction is dependent upon the quality ofthe copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back ofthe book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann AIbor MI 48106-1346 USA 313n61-4700 800/521-0600 --------- MOLECUIAR CInNING AND NUCLEOTIDE SEQUENCING OF FRUCTOSE 1,6 BISPHOSPHATE AIDOlASE IN NEUROSPORA CBASSA A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNNERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCES (GENETICS) MAY 1996 By Roxanne Atsuko Yamashita Dissertation Committee: W. Dorsey Stuart, Chairperson Fred Greenwood Terrence Lyttle Ming-PiMi Neil Reimer UNI Number: 9629867 UMI Microform 9629867 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 iii © Copyright 1996 by Roxanne Atsuko Yamashita iv ACKNOWLEDGMENTS I would like to thank my committee for all of their goodwill, encouragement, and helpful suggestions during the past two years. I will always be grateful to you. Dorsey, thank you for your optimism and financial support, which allowed me to concentrate solely on my research. I have enjoyed being a part of your lab. Doug, thank you for always being someone I could trust and confide in. Neil, thank you for figuring out the parameters to sequence directly from the cosmid and for all the free DNA sequencing. Dr. Hunt, thank you for your critical reading of my thesis with special regard to the DNA and protein analysis. I would also like to thank my labmates (Robert Phillips, Stacie Schwartz, CAL Long, Steve Buczynski, Doreen Morris, and Faye Nagano) and everyone in the TWL lab (Dave Baer, Kathy Houtchins, Joan Kuh, Sue Garner, and Leyla Bayraktaroglu) for their help, expertise, and chemicals. Lastly, I would like to thank my family (Mom, Dad, Scott, Dawn, and Birdie) and friends (Val Brancher, Amelia Niemi, Noelle Brestle, Renee Sims, Christine Crosby, and Chun-Wai Chan) for always being there for me. I couldn't have made it without your support all these years. THANK YOU! :) This work is dedicated to JBA. Meow! We finally made it! v ABSTRACT Fructose 1,6-bisphosphate aldolase (FBA) catalyzes the reversible cleavage of fructose 1,6 bisphosphate into glyceraldehyde 3­ phosphate and dihydroxyacetone phosphate. There are two classes of these aldolases. Class I aldolases are usually found in plants and animals, while Class II aldolases are found in fungi and bacteria. Although aldolases have been identified in a number of organisms, the present study is the first to identify FBA in a filamentous fungi. The FBA gene was identified in the phagemid clone, 26b, in the process of screening a '}..,-ZAP cDNA library for sequences coding for extracellular media proteins in N. crassa, This partial clone was used to identify the genomic clone 10:9c in the Vollmer/Yanofsky cosmid library. The putative gene is composed of 1161 bases, contains a single 63 pb intron, and codes for 366 amino acids. The FBA gene was mapped by RFLP analysis to linkage group 5 between APSc.3 and AP13.3. The cosmid 10:9c was used to subject co-transformed progeny to RIP mutations, genetically confirming the identification of a functional aldolase gene. Comparison of the FBA protein sequence with other Class II aldolases from yeast and bacteria show that at the amino acid level, FBA in N. crassa is more similar to that of E. coli, than to s. cerevisiae or S. pombe. This was interpreted as suggesting convergence due to functional similarity. Secondary structure predictions using PSA also places N. crassa and E. coli in the same macro-class "AB8BL". This structure is seen in other cytosolic vi globular protein enzymes (e.g. triose phosphate isomerase). Additionally, all Class I aldolases are in the macro-class "AB8BL". vii TABLE OF CONTENTS Acknowledgments iv Abstract v List ofTables ix List of Figures x Chapter 1: Introduction 1 Experimental organism 1 Objectives and significance 3 Protein export 4 Methods used to characterize the mechanism of action of signal sequences in protein export in Neurospora crassa 21 Fructose 1,6 bisphosphate aldolase 23 Chapter 2: Materials and Methods 30 Rabbit anti-Neurospora crassa antibodies 30 Screening of the A.-ZAP libraries using the rabbit anti-Neurospora crassa antibodies 43 Screening of the A.-ZAP libraries using degenerate oligonucleotides based on N-terminal sequencinR S2 Purification and isolation of the phagemids S6 Isolation of the genomic sequence using a eDNA probe 63 Automated sequencing of the phagemid and cosmid clones 73 Sequencing 7S Sl Nuclease detection of introns 78 Restriction Fragment Length Polymorphism (RFLP) rtiappinR.82 viii Repeat Induced Point Mutations (RIP) : 86 DNA and protein sequence analysis 92 Secondary structure analysis ofthe aldolase protein 96 Chapter 3: Results 97 Identification of secreted proteins in Neurospora 97 Analysis of the aldolase gene 99 Sl Nuclease detection of Introns 115 RFLP mappfug of the aldolase gene 117 Repeat Induced Point (RIP) mutations 118 DNA and protein sequence analysis 131 Secondary structure analysis ofthe aldolase protein 148 Chapter 4: Discussion 163 Fructose 1,6-bisphosphate aldolase 163 A.-ZAP library screeninR 164 Identification of fructose 1,6 bisphosphate aldolase 165 Repeat Induced Point mutations 166 Alternatives to using RIP to detect a functional aldolase protein 170 Analysis of the aldolase gene 171 DNA and protein sequence analysis 174 Secondary structure analysis ofthe aldolase protein 168 Chapter 5: Conclusion 181 Appendix: N-terminal protein sequences for glucose bands 1 and 2 and sucrose bands 1 and 2 183 References 185 ----- - .._._--------.-- ix LIST OF TABLES Table fggg 1. Class I and Class II fructose 1,6 bisphosphate aldolases 27 2. Clones identified from the A-ZAP library ·.. 103 3. Consensus table for introns 112 4. Codon usage for the fructose 1,6 bisphosphate aldolase gene in N. crassa 116 5. DNA and protein similarity between Class II aldolases .132 6. DNA and protein distance ma~ 142 7. DNA matrix ofnon-synonymous VS. synonymous changes 145 x LIST OF FIGURES Figt;re, Pa..K~ 1. Life cycle of Neurospora 2 2. Protein export in prokaryotes and eukaryotes 5 3. Consensus model for the SPD mechanism model 8 4. SRP model 9 5. ABC transporters in prokaryotes and eukaryotes 14 6. The glycolytic pathway 25 7. Sequencing strategy for aldolase gene in Neurospora crassa 29 8. SDS-PAGE ofmedia proteins isolated for N-terminal sequencinR 100 9. Amino acid sequence for sucrose band 3 101 10. Design ofdegenerate oligonucleotide probe for sucrose band 3 102 11. Plaque lifts from A-ZAP library screeninK 104 12. Phagemid sequence from eDNA, 26b 105 13. Cosmid sequence from 10:9c in the Vollmer/Yanofsky genomic library 106 14. Proposed sequence offructose 1,6 bisphosphate aldolase l07 15. Mechanism for Sl Nuclease detection ofintrons 119 16. Sl Nuclease detection blots 120 17. RFLP check on parental strains 121 18. RFLP blot for aldolase 122 19. RFLP mapping of aldolase 123 xi 20. Map of linkage group 5 124 21. G/C to NT mutations 125 22. Proposed mechanism of Repeat Induced Point (RIP) mutations 128 23. Three phenotypes from RIP FBA strains 129 24. RIP of the FBA gene 130 25. Aligned DNA sequences of Class I and Class II aldolases 133 26. Aligned protein sequences ofClass I and Class II aldolases 139 27. Unrooted Neighbor-joining tree based on DNA distances for 5 Class II aldolases 143 28. Unrooted Neighbor-joining tree based on protein distances for 5 Class II aldolases ·.. 144 29. Unrooted Neighbor-joining tree for non-synonymous DNA changes 146 30. Bootstrap showing a protein consensus tree .147 31. PSA analysis results for N. crassa 153 32. PSA analysis results for S. pombe 154 33. PSA analysis results for S. cerevisiae .155 34. PSA analysis results for E. coli 156 35. PSA analysis results for C. glutamicum 157 36. Secondary structure probabilities for N. crassa 158 37. Secondary structure probabilities for S. pombe 159 38. Secondary structure probabilities for S. cerevisiae 160 39. Secondary structure probabilities for E. coli 161 40. Secondary structure probabilities for C. glutamicum 162 xii 41. Similarity between the oligonucleotide probe and the putative aldolase sequence 167 1 CHAPTER 1 INTRODUCTION Experimental organism Neurospora crassa is a eukaryotic fungi belonging to the class Ascomycetes. It is commonly known as the orange-bread mold. Neurospora crassa has a great deal of genetic history, produces no afflatoxins, can be propagated vegetatively in both liquid and solid media asexually, such that strains with a particular genotype can be produced indefinitely, and is able to secrete proteins into liquid media.