Université d'Ottawa University of Ottawa

163 Pages·2001·8.73 MB·English

Université d'Ottawa University of Ottawa

IDENTDFTCATION AND MOLECULAR CHA.lUCTERISATION
OF A PUTATIVE GENE FOR CELL DETACHZNG FACTOR
FROM lRTCHOMONAS VAGI1VALIS
A Thesis Submitted to the
School of Graduate Studies
University of Ottawa
In Partial Fulfilment of the Requirernents for the
Degree of Master of Science
Department of B iochemistry, Microbiology, and Immunology
Faculty of Medicine
BY
Kiera L. Delgaty
@GeraL . Delgaty, Ottawa, Canada, 2000.
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Trichomonas vaginah (TV)i s the etiologic agent of trichornoniasis, a sexually
transmitted disease of significant socio-economic importance. Cell-fiee filtrate fiom TV
growth media causes rounding and detachment of adherent monolayers of human
epithelial cells in culture. Cell detaching factor (CDF), a 200 kDa glycoprotein in the
punfied preparation of the cell-free filtrate, is believed to play an important role in the
pathogenesis of TV. The identification of the gene for CDF would therefore allow further
characterisation of the protein and its expression. A cDNA clone (CDF-2) was identified
in a TV cDNA library by immunological screen with rabbit anti-sem prepared against a
punfied preparation of CDF. This clone contained an open reading fiame (ORF) that was
believed to represent a partial coding sequence for CDF. A genome walk initiated fkom
CDF-2 using Rapid Amplification of Genomic DNA Ends (RAGE)i dentified a contiguous
6.5 kb ORF. Northern analysis, which identified a 6.5-7 kb transcnpt, supported the size
of the ORF, while Rapid Amplification of cDNA Ends (RACE) identified a putative 5' end
of the ORF and identified a 3 1 bp 3' untranslated region. The ORF is predicted to encode
a 232 kDa polypeptide containing 42 EEKPKL repeats, but has no sequence homology
with any previously reparted protein- The full-length ORF was amplified from TV
genomic DNA by PCR and cloned into the plasmid vector pCR2.1-TOPO. The ORF was
transcribed in vitro using T7 RNA polymerase and translated in rabbit reticulocyte lysate
in the presence of 35~-methionine. Several high moiecular weight proteins (>200, 165,
150, 130, 118, 80, 47 kDa) were produced. Two polypeptides of 205 and 220 kDa were
immunoprecipitated fiom the in vitro translation mix by the anti-CDF serum. These data
suggest that the gene encoding TV CDF has been idensede
I would like to t h dD r. Gary Garber for allowing me the opportunity to work in
his laboratory, and for hÎs hancial support throughout the project. He has provided
many opportunities that have rounded out my training, and through my experiences in his
lab, 1 have grown both personally and professionally. Most of dl, 1 wish to thank him for
believing in me.
Special thanlcs to Dr. Ken Dirnock, who has supe~sedan d guided my research
through to success. He has always been enthusiastic, but patient, and he has given me
support, reaswance, encouragement, and gentle nudges when 1 needed them.
I gratefully acknowledge the support of the other rnembers of my Thesis Advisory
Cornmittee: Dr. J. R Dillon who taught me the value of critical thinking, and Dr. Kathryn
Wright for her f5iendly encouragement.
Thanks also to Andre Bergeron for al1 of his technical assistance, to Dr. Dond
Kickey for his help in interpreting the BLAST data, and to Nancy Delcellier for her
mentoring.
To the former and present members of the Garber/Cameron lab (Shannon,
Isabelle, Dino, Wally, Debbie, Josee, Renuka and Kelley (who is an honorary member):
You have al1 touched my hart in individual ways. Thank you so much for your
encouragement, advice, Wendship and good times, and for putting up with me during the
bad times.
Finally, special thanks to my husband and my family for their strength and
support throughout the last four years.
TABLE OF CONTENTS
.
AB STRACT. .........................................................................................
-11
ACKNO WLEDGEMENTS ......................................................................... **-
III
TABLE OF CONTENTS ............................................................................ iv
..
LIST OF FIGURES .................................................................................w i
.
LIST OF ABBREVIATIONS .................................................................. ..m*i l
CRAPTER l-.INTRODUCTION ................................................................. 1
Why is T. vaginalis Important? ........................................................... -1
Taxonomy and Structure. .................................................................. -2
Epidemiology. ................................................................................ 5
T. va@-nalisI nfection. ....................................................................... 6
Treaîment ..................................................................................... -9
Host Defense Mechanisms ............................................................... -13
Pathogenesis ................................................................................. 15
Experinental Rationale/Objectives ..................................................... -26
CWTER2 : MATERIALS AND METHOD S ..............................................- 29
Introduction ................................................................................. -29
Parasites ..................................................................................... 29
Bacteriophage Methodology ............................................................. -30
T. vaginais EMBL3 Genomic DNA Library Screening. ............................ -32
Isolation of T. vaginais Nucleic Acids ................................................. -33
RNA Analysis ..........................................*................... .. ............. -36
DNA Analy sis ............................................................................... -37
Restriction and Modification of DNA ................................................. -38
Preparation of Radiolabelled Probes .................................................... -39
Southern Blomng and Hybridisation Analysis. ....................................... -42
Northern Blotting and Hybridisation Analysis ........................................ -433
Plasmid Methodology ..... .. ....... .. .................*..................................- 44
Oligonucleotides. ......................................................................... -47
Inverse PCR-. ............................................................................... 47
S equencing. ................................................................................ -50
Sequence Analysis ........................................................................ -50
Rapid Amplification of Genomic DNA Ends WGE). . . ........................... -51
Rapid Amplification of cDNA Ends (&%CE). ........................................ -57
Long-PCR Amplification of CDF-ORF. .............................................. -60
RNA Primer Extension Andysis ......................................................... 61
In vitro Transcription. .................................................................... -63
In vitro Translation ....................................................................... -63
Protein Analy sis ............................................................................. 64
CHAPTER 3 : CHARACTERISATION OF A CANDIDATE GENE FOR CDF. ........ 67
Introduction. ............................................................................... -67
Resuits. .....................................................................................- 68
Screening of the T . vagindis genomic DNA library ................... .. ... -68
Northem blotting ................................................................. -68
Southem blotting ................................................................. -69
Inverse PCR ........................................................................ 72
Genome walking and PCR amplification of the fiill-length ORF .......... -73
RACE PCR ........................................................................ 79
Primer extension analysis. ...................................................... -82
Sequence anaiysis.. .............................................................. -82
In G o T ranscrïption/TransIation. ............................................. -89
CHAPTER 4: DISCUS SION AND CONCLUSIONS ......................................... 93
Discussion-. ................................................................................ -93
Conclusions ................................................................................ 107
LIST OF REFERENCES. ........................................................................ 110
APPENDIX A: PRlMERS AND ADAPTORS.. ............................................. -129
APPENDM: B : NUCLEEC AClD SEQUENCE.. ............................................ -132
vii
LIST OF F'IGURES
I . Generation of Probes. ......................................................................... -40
2. Inverse PCR Amplification of EcoRI and Ta@ Restriction Fragments. .............. -49
3 . The Suppression PCR Effect .. ............................................................ .- 54
,.
4. Genome Waflcing .............................................................................. -55
5 . Flow Chart of the Marathon RACE Procedures ........................................... -58
6. Northem Blot Analysis of T. vugikuzZis 220 Total and PolyA'RNA. .................... -70
7. Southem Blot Analysis and Restriction Mapping of T. vagimIis 202
High Molecular Weight Genornic DNA. ..... .. ............................................ -71
8. Primary and Secondary (Nested) RAGE PCR Products .................................. -74
9. Genome Walking Map ..................................... .. ................................... 76
10. PCR Amplification of the CDF-ORF ....................................................... -78
I 1 .R ACE PCR Products. .......................................................................... 80
12. Characterisation of the RACE PCR clones and 5' and 3' Untranslated Regions ...... -81
13. Arnino Acid Sequence of the Translated ORF ............................................. 87
14 .T n vitro Transcription of CDF-ORF ......................................................... -90
15 .I n vitro Translated Produds and Immunoprecipitation. ................................ -92