Genetic and biochemical studies on the urease enzyme system of Schizosaccharomyces pombe : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University

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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.

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SCHIZOSACCHAROMYCES POMBE

A thesis presented in partial fulfillment of the requirements for the deg ree of Doctor of Philosophy in

Genetics at M assey U niversity

MARK WI LLIAM LUBBERS 1 993

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1 2 residues of the N -term inal sequence, extending the N-termi n al sequence to 1 8 residues. The 1 8 N-terminal am ino acids had 55.6% identity and 83.3% similarity (exact plus conservative replacements) with the jack bean u rease N-termi n al

sequence. The seven amino acids of T21 had 42.9% identity and 1 00% similarity with

the u rease from Klebsiella aerogenes. Peptide T40 (25 amino acids) had only very

poor identity with other sequenced u reases.

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ACKNOWLEDGEMENTS

I than k my supervisors, Dr Roy J . Thornto n , D r. Neville K . Honey, and D r Susan B . Rodrig uez for their guidance and continual encourag e m ent.

The D epartment of Microbiology and Genetics for providing the space and equipment for this work.

The U n iversity Grants Council and M assey University for awarding scholarships to s upport this work.

I also thank:

Dr Takashi Toda (Kyoto U niversity) for the kind gift of the S. pombe gene bank.

D r Neil Haggarty (Separation Science U nit, Biochem istry Department) for hel p a n d advice o n affinity resin manufacture.

D r J ohn W . Tweedie (Biochemistry Department) for i nitial advice o n protein purification and many helpfu l discussions.

D r Christopher Moore (Biochemistry Department) for advice on making tryptic peptides.

Mr Dick J. Poll (Biochemistry D epartment) for help and advice on F P LC pu rification of proteins and HPLC pu rification of peptides.

Dr G raham Midwinter and Mr J ulian Reid (Biochemistry Departm ent) for help with protein sequencing .

The many people in the D epartments of Microbiology & Genetics and Biochemistry who

have given me helpful advice .

To Rob van Duivenboden for making the lab a more pleasant place to be and for proof

reading my references , many thanks.

Thanks also to Pete r Lockhart for proof-reading my i n t roduction .

A v e ry special thank-you to my wife, Vanessa, and our childre n , Fran ia, Chantal, Isaac, Portia, and Ruby, who have been very understanding , patient and supportive .

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ABSTRACT

Two i ndicator media were developed to d etect urease activity in Schizosaccharomyces pombe colonies. These m edia were more sensitive than previously published m edia, permitted the rapid identification of u rease mutants, were s uitable for identifying urease positive transformants and were not affected by amino acid and nucleotide suppleme nts .

Fou r g enes, designated u re 1 , ure2, ure3, and ure4, are required for urease activity i n S . pombe. Each of the genes was mapped to a n approximate genetic location by induced h aploidization and meiotic recombi n atio n : ure 1 on the left arm of chromosome I I I , 32 cM from tur 1 and 50 cM from ade 6; ure2 on the right a rm of c h romosome I, 69 cM from ura2 and 1 00 cM from ade4; ure3 on the right arm of chromosome I , 31 c M from ade4 a n d 9 1 c M from ura2; ure4 on t h e left arm of chromosome I , 1 00 cM from Iys 1 .

The lithium chloride method for S. pombe transformation was modified to improve the transformation frequency up to 1 00-fold by using carrier DNA and res uspending the cells in 0 .9% NaCI after tra nsform atio n . Urease mutants for each of the four ure genes were transformed with a S. pombe gene bank. Three different plasmid clones, each of wh ich specifically complemented one of the ure 1, ure3, or ure4 mutants, were isolated by complementation of the ure- phenotype. A gene bank clone complementing the ure2 mutant was not fou nd .

S. pombe u rease was purified and characterized. The enzyme was intracellular and only o n e urease enzyme was detected by non-denaturing PAG E. The u rease was purified 3 ,939-fold, with a 34% yield, by acetone precipitation , ammo n i u m sulfate

precipitation and DEAE-Seph arose ion exchange column chromatography. The n ative e nzyme had Mr = 21 2,000 (Sepharose C L6B-200 gel fi ltration ) . One subunit was detected, with Mr=1 02,OOO ( S OS-PAG E) , indicating the u ndissociated e nzyme contains two identical subun its. The specific activity was 709 f.lmo l u rea per m i n/mg protei n . The e nzyme was stable between pH 5.0 and pH 9 .0. The optimum pH range for enzyme activity was pH 7.5 - pH 8 . 5 . The Km for urea was 1 .03 m M . The sequences of the am ino-terminus and three tryptic peptides of the e n zyme were dete rmined : N

term i n u s - Met Gin Pro Arg Glu Leu H is Lys Leu Thr Leu His Gin Leu Gly Ser, peptide T21 - Phe lie Glu Thr Asn Glu Lys, peptide T 40 - Leu Tyr Ala Pro G l u Asn Ser Pro Gly Phe Val Glu Val Leu Glu Gly Glu l ie Glu Leu Leu Pro Asn Leu Pro, peptide T 43 - Glu Leu His Lys Leu Thr Leu His Gin Leu Gly Ser Leu Ala. The sequence of T 43 overlaps the last

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TABLE OF CONTENTS

ABSTRACT ... . . . .. . . .. . . .. . . .. . . ... . . . ... i i ACKNOWLEDGEMENTS . . . .. . . ... . . ..^...^..... . . i v

TABLE OF CONTENTS ... . . . .. . . .. . . ..... . . . ... v

LIST O F FIGU RES . . . .. . . .. . . xii LIST OF TABLES . . . .. . . .. .... . . .. . . .. . . .. . . x i v INTRODUCTION ... . ... . . . .. . . . .... . . .. . . .... . . ... . . . ....^{. . .}1

1 T H E ETHYL CARBAMATE P ROBLEM . . . .. . . . ... . . ... . . . . ....... 1

1 .1 I n troduction ^{. . . . .}.. . . ..^..^{. .}.. . . ... . . .. . . .. ..^...1

1 .2 Toxicology of Ethyl Carbamate . . . .. . . ... . . ... 1

1 .3 Ethyl Carbamate i n Wine . . . .. . . ... . . 2

1 .4 Ethyl Carbamate Formation .. . . ..... . . . .. ... . . 3

1 .5 Urea Formation in Wine . . . ... .4

1 .6 The Solution . . . ... . . ... . . ...... . . .. . . . ..^{. .}.^{. ....}6

2 SCHIZOSACCHAROMYCES POfV1BE ^{. . . .}.. . . ... . . .. . . ..^{. .}8 3 UREA AND UREASE ^{. . .}.^{. . . .}.. . . ... . . ... . . .... . . .. . . ..^... . . ..^{. . .}1 0 3.1 I ntroduction ^{. . .}.. . . ..^{. . . . .}.. . . ... . . .. . . .. . . .. . . .. . . .. . . 1 0 3.2 Urease Structural Properties . . . ... . . ... . . 1 4 3.3 Urease Kin etic Characteristics ^{. . . . .}.^{. . .}... . . .. . . ... . . . .. . . 1 8 3.4 Urease Genes . . . ... . . ... . . ... . . . .. . . 1 9 3 .5 Urea breakdown in Saccharomyces cerevisiae . . . .. . . .. . . 2 0 3.6 Schizosaccharomyces pombe urease . . . ... . . . ... . . .. 2 3 4 A I M OF THE PRESENT STU Dy . . . . .. . . ... . . . ... . . . .. . . ... . . .. . . ... . . . 2 4 MATERIALS AND METHODS . . .. . . ... . . ... . . ... . . . ... 2 5

M IC ROBIOLOG ICAL M ET HODS ... . . . ... . . ... . . ... ... . . . ... 2 5 1 . 1 Microbial strains ^{. . . . .}.. . . ..^{. . . . .}.^{. . . . .}.. . . . ... . . . 2 5 1 .2 Media . . . .. . . ... . . .... . .... . . .. . . .. . . ..... . . 2 5 1 .2.1 Auxotroph supplements ^.. .. . . '" . . . .. . . 2 5 1 .3 Strain maintenance . . . .. . . .. . . ... . .. . . .. . . . 2 5

1 .3. 1 Schizosaccharomyces pombe strain storage . . . ... . . ...^....2 5

1 .3.2 Escherichia coli strain storage . . . .. . . ... . . 2 5 1 .4 Growing S. pombe celis . . . ... . . . .. . . .. . . .. . . 3 2 1 .5 Growing E. coli cells . . . .. . . ..^{. .}.. . . 3 2 1 .6 Aseptic technique . . . ... . . ..^{. . .}.. . . ... . . .. . . 3 2 1 . 7 Filter sterilizatio n . .. . . ... . . .. . . .... . . 3 2 1 .8 Testing the phenotype of S. pombe strains . .... ....... . . . ..^...3 2

1 .8.1 Haploid/Diploid . . . ... . . ... . . . ....^...3 2

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1 .8 . 2 M ating Type . . . ... . . ... . . ..^..^..^{. .}. ..... . . 3 2 1 .8 .3 Auxotrophy and fluorou racil resistance . . . ... . . ..^... . . 3 2 2 UREASE ACTIVITY TESTS FOR S. POMBEC U LTURES . . . ... . . ... . ... 3 3 2.1 R U H test. . . . .. . . ... . 3 3 2.2 I ndicator plates .. . . .. . . .. . . .. . . 3 3 2.3 M I N H plates . . . .. . . .. . . .. .. . . . ... 3 3 3 S. POMBE GENETIC MAPPiNG . . . .. . . .. . . .. . . .. 3 3

3.1 Genetic crosses . . . .. . . .... . . .. . . .. . . .. .. . . 3 3 3.2 Tetrad Dissection . . . .... . . ... . . ...^{. .}.. . . ... . . 3 4 3.3 Spore suspensions . . ... . . .. . . .... . .. . . .. . . ... . . ... . . . ... . . 3 4 3.4 Analysis of tetrad data . . . .. . . .. .. . . 34 3.4 . 1 Genetic linkage ^{. . . . .}. .. . . ... . . ... . . .. 3 4 3.4.2 Linkage to a centromere . . . .. . . 34

3.4 .3 Chro mosome assignment by induced

h aploidization ^{. . .}.^..^{. . .}.^{. .}.^{. . . .}.. . . ... . . ... . . 3 5 4 PURI FICATION AND CHARACTERIZATION OF S. POMBE UREASE . . . ..... . . ... 3 6 4.1 Buffers for urease pu rification . . . ... . . ..^..^{. . . .}.. . . .. . .. . . .. . . 3 6 4 . 1 . 1 PEB .. . . ..^.. . . .. . . .... . . ..^.. . .^{. . . .}.. . . 3 6 4 . 1 .2 PEBS ^.. .^{. . . .}. . . .^..^..^{. . . . .}... . . ..^{. .}.^... . . ... . . ... . . ..^{. .}3 6 4 . 1 .3 O.2 M PEBS . . . .. .. . . .. . . .. . . .. 3 7 4 . 1 .4 O.35 M PEBS . . . .. . . 3 7 4.2 Urease assay ^.. .. . . ..^.. .. . . ..^{. .}.^{. . .}.. . . ..^... . . 3 7 4.3 Protein assay . . . ..^{. . . .}.^{. . . . .}. .. . . ..^{. .}.^{. . . .}.^..^{. . . .}.^{. .}.^..^{. . . . .}.. . . 3 7

4.4 Preparation of S. pombe crude cell extracts for u rease

pu r i f i c a t i o n . . . ... . . ... . . ..^{. . .}.^{. . . . .}.. . . .. . . 3 7 4.5 Preparation of jack bean u rease crude extracts . . . 3 8 4.6 Preparation of affinity adsorbents . . . ..^{. . . .}. .. . . 3 8 4 . 6 . 1 Oxirane Hydroxyurea Agarose (OHA) ^{. .}.. . . ... . . .. . .. . . .. . . 3 8 4 . 6 .2 Aminocapry l ic acid hydro xyurea ag arose (AHA) . . . .. 3 8

4 .6 .3 Ethylenediam ine-ethylenediamine hydroxyurea

agarose ( EHA) . . . ..^{. . . . .}.. . . ..^... . . .. .^... . . .. . . . 3 9 4.6.4 Substitution of the affi nity resins with

hydroxy u r e a ^.^{. . . . .}.^.... . . 40 4.7 Purification of S. pombe urease . . . ..^... . . ..^..^... . . 4 0 4 . 7 . 1 Aceton e precipitation .^{. .}.^... . . ..^... . . 40 4 . 7.2 Ammo n i u m su lfate precipitation ^{. .}.. . . ... . . .. . . 4 1 4.7.3 Diethylaminoethyl ( DEAE) Sepharose ion

exchange . . . ..^... . . ... . . .... . . 4 1

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4.7.4 F P LC purification of urease . . . .. .... . . 4 1 4.8 Ultrafiltration of protein solutions . . . 4 2 4.9 Preparation of dialysis tubing . . . ... . . . 4 2 4.1 0 Polyacrylam ide gel electrophoresis (pAGE) . . . 4 2 4.1 1 Staining polyacrylam ide gels for protein . . . .. . . .. . . 4 3 4 . 1 1 . 1 Coomassie Blue R-250 stain . . . 4 3 4 . 1 1 .2 Silver sta i n .. . . .. . . 4 3 4.1 2 Urease activity stain for n ative-PAGE gels . .. .. . . 4 3

4.1 3 Determination of protein molecular weight by gel

filtration . . . 4 4 4.1 4 Purification of peptides by High Perform ance Liquid

Chromatography ( H PLC) . . . ... . . .... . . . ... . ... . ... . . 4 5 4.1 5 Protein sequ encing . . . .. . . 4 5 5 POLYMERASE CHAIN R EACTION (PCR) AMPLIFICATION OF DNA . . . .. . . 4 5 5 . 1 Oligonucleotide primer manufacture . . .. . . .. . . 4 5 5.2 PCR amplification of DNA . . . .. . . .. . . 4 5 5.3 Construction of T -vectors . . . 4 6 6 GENERAL DNA MANIPULATION TECHNIQUES . . . 4 6

6 . 1 Preparation of plasmid DNA . . . .. . . .. 4 6 6 . 1 . 1 Smal l-scale plasmid isolation . . . 4 6 6 . 1 .2 Large -scale plasmid isolation . . . 4 6 6.2 Purification of DNA . . . .. . . _. 4 6

6.2. 1 Cesi u m ch loride density gradient equilibrium

ce n t rifugation . . . 4 6 6.2.2 Phenol/chloroform extraction of D NA . . .. . . .. . . .. 4 7 6.3 Ethanol precipitation of DNA . . . .. . . 4 7 6.4 Agarose gel electrophoresis .. . . 4 7 6.5 Restriction enzyme digestion of DNA .. . . . .. . . .. . . 4 7 6.6 Gel purification of DNA frag ments . . . 4 8 6.7 DNA quantitation . . . .. . . .... . . .. . . 4 8 6.7.1 Absorbance at 260 nm . . . .. . . 4 8 6.7.2 Ethidium bro m ide dot quantitation . . . .. . . 4 8 6.7.3 Fluorescence i n agarose gels . .. . . 4 9 6.8 Ligation of DNA fragments . . . .. . . 4 9

6.9 Preparation of sing le-stranded DNA fro m phagem ids

(pU C 1 1 8/1 1 9) for sequencing . . . .. .. . . 4 9 6 . 1 0 DNA sequencing . . . .. . . ... . . .. . . .. .. . . 5 0 6 . 1 1 Southern transfer of DNA .. . . .. . . .. . . 5 0

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6.1 2 Hybridization of DNA probes to Southern blots . . . .. 5 0 6.1 3 Hybridization of o ligonucleotide probes to Southern

blots . . . ..^{. . . .}.^... . . ..^{. . . . .}. .^{. . .}. . . .^... . . ..^{. . . . .}. .^{. .}.^{. . . .}. . . .^.5 1 6.1 4 Nick-translation labeling of DNA probes .^..^{. . .}.. . . ..^.. . .^.. . .^...^.. . . .^{. . .}5 1 6.1 5 End- labeling of oligonucleotide probes .. . . ... 5 2 6 .1 6 Preparation of S. pombe chromosomal DNA ^{. .}. . . .^...^{. .}. . . .^... 5 2 6.1 7 Plasmid vectors .. . . .. . . .. .. . . 5 2 7 T RANSFORMATION WITH PLAS M I D DNA .^{. . . . .}. . . .^.. .^{. .}. .^{. . .}. . .^....^....^....^... 5 2

7.1 Schizosaccharomyces pombe transformation .. . . .. . . .. . . 5 2 7.2 Stability test for S. pombe transformants . . . ..^...^.. . . .^..^.. . . 5 4 7.3 Recovering plasm ids from Schizosaccharomyces pombe ^{. . .}.^.. . . 5 5 7.4 Electro-transformation of E. coli . . . . .. . . ..^.. . . ..^{. .}.^{. . .}.. . . ... . . ..^..^..^..^.5 5

7 .4 . 1 Preparation o f competent E. coli cells for

electro-tran sformation . . . .. .^{. .}.^{. .}.^..^{. . .}. .^.. .^{. . .}. . . .^.. . .^.. 5 5 7 .4 .2 Electro-transformation procedure ^.. . .^{. .}. . .^{. . .}.^..^{. .}.^.. . . 5 6 8 A DDITIONAL BUFFERS, STOCK SOLUTIONS AND NOTES . . . ...^..^{. .}.^{. . . . .}.^{. . .}.^{. . . .}.^{. . . .}5 6

8 . 1 Tris stock sol utio n s . . . .. . .. . . ..^{. . . .}.^{. .}.. . . .. . . .^.. 5 6 8.2 Ethylenediaminetetraacetic acid ( E DT A, pH 8 .0) . . .. . . .. . . 5 6 8.3 TE (Tris EDT A) .. . . .. . . 5 7 RESULTS . . . 5 8 UREASE ACTIVITY SELECTION M ETHODS FOR S. POMBESTRAINS ^{. .}. . .^.. . .^.. . . 5 8 1 . 1 Nitrogen Source Plates . . . ..^..^{. . .}.^... . . .. . .^{. .}. . . .^.. .. . . 5 8 1 .2 I ndicator Media ^{. .}.^{. .}.. . . ... . . ..^..^... . . .. .. . . .^{. .}. . .^{. .}.^{. . . .}.^{. . . .}. . .^{. . .}.^.6 0 1 .2 . 1 Standard methods ^{. . .}.^{. .}.^{. . .}.^{. . . . .}.^.. . .^.. . .^.. ..^{. . .}. . . .^.. . . .^.. . . .^{. .}. . . .^..^.6 0 1 .2.2 I ndicator plates .. . . ..^{. . .}.^{. . .}..^{. . .}.^{. . .}.^{. .}.^..^{. .}.^.. . . . .^.. . .^{. . .}. . . . .^.. . . 6 1 2 CHARACTERIZATION OF T H E UR EASE GENES . . . ..^{. .}...^{. . .}.^{. . . . .}...^.. .^...^{. . . .}....^..^{. .}.^{. .}.^{. . .}6 8 2 . 1 Characte rization of ure mutants . . . .. .. . . ..^{. . .}.. . . ..^{. . . . .}.^..^..^{. . .}6 8 2 . 1 . 1 Urease m utant complementation g roups . .^{. .}. . .^{. . . . .}.^{. .}... . . .. .^.6 8 2 . 1 .2 The urease activity of cell-free extracts .^.. .^{. .}. . .^.. . . . .^{. .}. . . 6 8

2.1 .3 Effect of n ickel and manganese on u rease

act i v i t y .. . . ... . . ..^{. . . .}.^{. .}. . . .^.. . . .^..^{. .}.^.. .. . . .^.. . . 6 8 2.2 Chromosome assig nment and linkag e relationships of ure 1 ,

ure2, u re3, and ure4 ^.. .^{. . .}..^{. . . . .}. .^{. .}.^{. .}. . . .. . . .. .^.. . . .^{. .}. . . 7 0 2.2.1 Linkage to Iys 1, the mat locus , or a centromere . . .. . . .^.. 7 0

2.2.2 Assign me nt to linkage groups by induced

haplo idization ^{. . .}.. . . .... . . ... . . .. .^.. . . . .^...^{. . .}.^{. .}.^{. .}. . . .^.. . . . .^.. . 7 0 2.2.3 Mapping by m eiotic recombination .. . . .^.. . . .^... .^.. . . .^...7 3

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3 S. POMBE UREASE PURI FICATION AN D CHARACTERIZATION ... ... 8 1 3.1 Factors affecting the urease activity assay . . . ... . . .. . . 8 1

3 . 1 . 1 Urea concentration o f t h e substrate buffer . . . 8 1 3 . 1 .2 Urease a ssay incubation time .. . . .. . . .. 8 1

3 . 1 .3 Effect of 2-mercaptoethanol, E DTA, and sodium

azide on u rease activity . . . .. . . 8 3 3.2 Preparation of the crude extract . . . .. . . ... . . .... . . .. . . 8 3

3 . 2 . 1 Location of Urease Activity . . . .. . . 8 3 3 .2.2 S. pombe u rease isozymes . . . .. . . .... . . .. 8 5

3 2.3 Effect of u rea, nickel sulfate, and m anganese

su lfate o n the yield of u rease activity . . . ... 8 5 3.2.4 Growth phase of the culture . . . .. . . .... . . ... 8 5 3.2.5 M ethod of cell disruption . . . 8 8

3.2.6 P h e ny l m ethylsulfonyl fluoride ( P M S F ) protease

i n h i b i t o r . . . . .. . . .. . . .. . . 8 8 3.3 Affinity col u m n pu rification of S. pombe u rease . . . 8 8

3.3.1 Oxirane Hydroxyurea Agarose (OHA) . . . .. . . 8 9 3.3.2 Aminocaprylic acid hydroxyurea aga rose (AHA) . . . .. . . . 9 1 3 .3.3 Ethylenediamine-ethylenedia m i n e Hydroxyurea

Aga rose ( E HA) . . . 9 1

3.4 Precipitation and ion-exchange purification of S. pombe

urease . . . .. . . 9 8 3.4.1 Acetone P recipitation . . . .. . . 9 8 3.4.2 A m mo n i u m Sulfate Precipitation .. . . 9 8

3.4 .3 Acetone precipitation followed by a m monium

su lfate precipitation . . . .. 9 8 3.4.4 lon-exchange purification of S. pombe urease . . . .. 1 0 1

3 .4 . 4 . 1 D EAE-Sepharose pu rification . . .. . . 1 0 1 3 . 4 . 4 . 2 F P LC-Mono-Q purification .. . . 1 0 1 3.5 Urease Enzyme Characterization . . . .. . . ... . . . 1 0 6

3.5.1 N ative M olecular Weight.. .. . . 1 0 6 3.5.2 Subunit Size . . . ... 1 1 2 3 .5.3 p H Stability . . . 1 1 2 3.5.4 p H opti m u m . . . 1 1 6 3.5.5 Km for u rea . .. . . .. . . .. . . .. . . .. . . .. . . 1 1 6 3 .5.6 Partia l sequence of S. pombe u rease . . . 1 1 6 4 PCR AMPLIFICATION USING PRIMERS TO THE UREASE PROTEIN

SEaJ�E . . . .... . . 1 2 2

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4.1 PCR primer design . . . .. . . ... . . ... . . ... . . .... . . 1 2 2 4.2 Optimization of PCR . . . .. . . .. . .. . . ..^... . . ... . . ..^.1 2 5 4.3 Cloning of the PCR product. . . . ..^..^{. .}. ... . . ... 1 2 8 5 ISOLATION OF URE GENES BY COMPLEMENTATION . . . .. . . ..^{. . . .}. . . .^.1 3 5

5. 1 Construction of ure - strains for transformation . . . ..^{. .}.. . . ..^{. .}. . . 1 3 5 5.2 I mprovement of the lithium chloride tra nsformation

method .^..... . . ... . . ... . . .. . . ... . . ..^... . . ...^.1 3 6 5.3 T ransformation of ure- strains with a S. pombe gene

bank ^... . . ...^{. . . .}.. . . ... . . ... . . .. . . .. . .. 1 4 1 5.3. 1 S. pombe g ene bank amplification , insert size and

frequency, and DNA preparation ^{. . . . .}.. . . 1 4 2 5.3.2 Transformation of ure- strains . . . ... . . ... . . 1 4 3 DiSCUSSiON . . . .. . . .. . . 1 5 1

G ROWTH AND DIFFERENTIATION OF S. POMBE URE+ AND URF

STRAINS ON HYPOXANTHINE, UREA, AND INDICATOR MEDIA . . . .. . . 1 5 1 2 C HARACTERIZATION OF URE MUTANTS . . . ... . . ..^.1 5 3 I ntroduction . . . 1 5 3 2.1 Urea transpo rt. . . 1 5 4 2.2 Urease I nducibi l ity ^{. . .}.. . . ... . . 1 5 5 2.3 N ickel tran sport/cofactor . . . ... . .. . . .. . . 1 5 6 2.4 Urease subu nits . . . ... . . .. . . ... . . . .. . . 1 5 7 3 GENETIC MAP OF THE S. POMBE URE GENES ^{. . .}.. . . .. . . ..^{. . . .}.^{. . ..}1 5 8 4 PURIFICATION AND CHARACTERIZATION OF S. POMBE UREASE . .. . . ... . ... 1 6 2 4.1 U rease assay method and inhibitors . . . .. ... . . 1 6 2 4.2 Cellular localization of urease .^{. . . .}.. . . ... . .. . . . ..^{. . ..}.. . . .. . .^..^{. .}1 6 3 4.3 Urease isozymes . . . ... . . ... . . .. . . .. . . 1 6 3 4.4 Effect of n ickel and manganese on u rease activity ^{. .. . . .}.^{. . . . .}.. . . 1 6 3 4.5 Urease stability . . . ... . . 1 6 4 4.6 Affinity chro matog raphy . . . ... . . ..^{. .}.. . . . .. . . ..^..^...^.1 6 4

4.7 Purification of u rease by precipitation and ion exchange

chromatog raphy . . . .. ... . . ... . . ... . . .. . .. . .. ... . . 1 6 6 4.8 Urease structural properties . . . ... . . ..^{. . . .}.. . . 1 6 6 4.9 pH stability and pH optimum . . . .. . . .. . . ..^{. . . . .}.. . . ..^.1 6 8 4 . 1 0 Urease sequence analysis . . . .. . . ...... . . .. . . ..^{. . . .}1 6 9 5. AMPLIFICATION OF S. POMBE DNA USING PRIMERS TO THE UREASE

AMINO ACID SEQUENCE .. . . ... . . .. . . .. . . ..^{. . . . .}.^{. .}.. . . .. . . 1 7 1 6 TRANSFORMATION OF S. POMBE. ... ... . ... . . ... . ... 1 7 5

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6 . 1 I mprovement of t h e lithium chloride proced u re for

transformation of S. pombe ^{. . . .}.^{. . . . .}.^..^... . . ..^... . .. . .. . . .. . ... . . 1 7 5 6.2 Strain dependence of transformation . . . ... . . ... . . ..^{. . . .}.^.1 7 6 7 ISOLATION OF GENES COMPLEMENTING THE URE MUTATIONS . . . ..^..^{. . . .}.^.1 7 7

7. 1 Complementation of ure- strains with a S. pombe gene

bank ^{. .}.^{. . . .}.^{. .}.^... . . .. . . ..^... .. . . ... . . 1 7 7 7.2 Hybridizatio n of the PCR primers to the u re--

complementing clones . . . .. .^{. .}.. . . ...^..^{. .}.^..^{. . . .}.. . . 1 7 9 SUMMARY AND CONCLUSiONS . . . ... . . ..^..^... . . 1 8 0 FUTUR E WORK . . . ... . . ... . . ... . . ..^... . . ..^{. .}1 8 1 REFERENCES . . . 1 8 3

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LIST OF FIGURES

1 Pathway of purine catabo lism in Schizosaccharomyces pombe ^{. . .}.... . . ..^....^..^... . . . 2 2 2 Growth of S. pombe wild type and ure- strains on M I N H medium .^{. .}.^.. . . . .^....^{... . .}. . . .^{. . . .}. 5 9 3 E ffect of u rea and phenol red on S. pombe g rowth . . . .. . . 6 3 4A D ifferentiation of S. pombe u rease+ and urease- strains on I HG indicator

m edium by spread-plating .. . . .. . . 6 5 4 8 Differentiation of S. pombe u rease+ a nd urease- strains on I LG indicator

medium by patch ing and repl ica plating . . . .. . . ... 6 6 5 Effect of n ickel and manganese on the growth of S. pombe ure- strains on

MINH medium . . . .....^..... . . .. .. . .. . . 7 1 6 Urea saturation cu rve of S. pombe urease . . . ....^...8 2 7 The activity of S. pombe urease over a two minute time cou rse ^.. . . .. . . .... 8 4 8 Native- PAGE of S. pombe crude u rease extract stained for urease activity . . . 8 7 9 Structure of adsorbents made for affin ity chromatog raphy pu rification of

urease . . . .. . . 9 0 1 0 Pu rification of jack bean urease by EHA affinity chro matography with

0 .2 M PB elution . . . .. . . ... . . ... . . 9 2 1 1 Pu rification of jack bean u rease by EHA affin ity chromatog raphy using

u rea g radient elution . . . ..^{. . . .}.^{. . . .}9 4 1 2 S DS-PAGE of jack bean urease pu rified by E HA affin ity chromatography

using urea gradient elution ^.. . . .. . . 9 5 1 3 Pu rification of jack bean urease by D EAE-Sepharose ion-exchange

chromatography ^. . . . .. . . .. . . 9 6 1 4 Purification of partially purified jack bean urease by E HA

chromatography ^.. . . ... . . ..^..^{. . . .}.^.. 9 7 1 5 Purification of S. pombe u rease by D EAE-Sepharose ion-exchange

c h romatography . . . .. . . .. . . .. . . .. . . .. ... . ... . ....^{... . .}.^..^....^.. 1 0 3 1 6 N ative- PAGE of S. pombe urease purified by acetone precipitation ,

ammonium su lfate precipitation a n d DEAE-Sepharose ion-exchange

chrom atography . . . ... . . .. . . .. . . 1 0 5

1 7 Purification of S. pombe urease by using FPLC Mono-Q ion-exchange as

the final pu rification step . .. . . .. . . .. ..^.1 0 7 1 8 Native- PAGE of S. pombe urease purified by using Mono-Q FPLC as the

last purification step . . . ... . . .. . . .... ..^...... . . ..^{. . .}. . . .^.1 0 9 1 9 Elution volume (Ve) of S. pombe u rease determined by g e l filtration

th rough a calibrated Sepharose C L6B 200 col u m n ^{. .}. . . .^....^.... . .^... .^..^{. . . ..}1 1 0 20 Cal ibration curv e of Sepharose C L6B 200 column . . . ..... . . .. .^{. . .}.^{. . .}1 1 1

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21 S OS-PAGE of S. pombe urease (7.5% acrylamide) . . . 1 1 3 22 S OS-PAGE of S. pombe u rease ( 1 5% acrylamide) . . . .. . .. .... . . 1 1 4 23 pH stability of S. pombe u rease .. . . .. .... . . 1 1 5 24 Activity of S. pombe u rease at various pH .. . . ..^{. . . . .}.^{. .}.. . . ....^..^{. . . .}...^..^{. . .}. .. . . ..^... . . 1 1 7 25 Eadie-Hofstee plot of S. pombe u rease activity .. . . 1 1 8 26 P urification of S. pombe u rease tryptic peptides by High Performance

Liquid Chromatog raphy ( H P LC) . . . .. .. .. . . .. . . 1 2 0' 27 Amino acid sequences of S. pombe urease N-te rm i n u s and tryptic

peptides . .. . . 1 2 1 28 PCR primer sequences and codon usage frequency tables . . . .. . . 1 2 3 29 Agarose gel electrophoresis o f PCR amplified S. pombe DNA using prim ers

ML1 and ML2 .. . . .. . . .. . . .. . . 1 2 7 30 Autoradiograph of S. pombe genomic DNA probed with pML12.4 . . . 1 3 1 31 Autoradiog raph of S. pombe genomic DNA probed with PCR primers ML 1

and M L2 . . . .. . . ... . . .. . . .. . . 1 3 3 32 Ethidium bromide stained agarose gel Southern blotted for pML 12.4

hybrid ization e xperiments . . . .. . . .. . . .. . . 1 3 4 33 Agarose gel electrophoresis of S. pombe gene bank plasm ids which

complement ure- mutants . . . .. . . .. . . 1 4 5 34 Agarose gel electrophoresis of ure--complementing gene bank clo nes

digested with Hind l l l . . . .. . . .. . . . 1 4 6 35 Agarose electrophoresis of ure--complementing g e ne bank clones,

digested with Hindl l l and SaL ^{. .}.^... . . ... . . ... . . ..^{. . .}.. . . 1 4 9 36 Autoradiograph of ure--complementing gene bank plasmid clones probed

with PCR primers ML 1 and M L2 . . . .. . . 1 5 0 37 Genetic map of S. pombe chromosome I and II , showing the positions of the

ure genes . . . .. . . 1 6 0 38 Comparison of S. pombe u rease amino acid sequence with other u reases .. . . 1 7 0

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LIST OF TABLES

1 Strains of Schizosaccharomyces pombe , Escherichia coli and bacteriophage

M 1 3 u sed in th is study . . . 2 6 2 S. pombe media . . . 2 9 3 E. coli media . . . 3 1 4 Antibiotics . . . 3 1 5 E. coli and S. pombe plasm id vectors used in this study . .. . . .... . . .. .. . . 5 3 6 U rease Indicator Plates . . . .. . . 6 4 7 Complementation of ure- strains . . . .. . . .. . . 6 9 8 Analysis of linkage of ure genes to Iys 1 , the ma t locus, or a centromere . . . 7 2 9 ObseNed n u mbers of the g enotypes among the haploid segregants from

i nduced haploidization . . . .. . . 7 4 10 Evaluation of haploidization dataa .. . . .. . . .. . . . .. . . .. . . 7 5 11 Fractional viability of spore tetrads used for the linkage studies .. . . .. . . 7 9 12 Linkage R elationships . . . .. . . 8 0 13 Urease activity in a fractionated S. pombe cell extract. . . 8 6 14 Purification of S. pombe urease by aceton e precipitation . . . 9 9 1 5 P urification of S. pombe urease by a m mo n i u m su lfate precipitation . . . ... 1 0 0 1 6 Ammonium sulfate precipitation of acetone precipitation purified

S. pombe u rease .. . . .. . . 1 0 2 17 D EAE-Sepharose ion-exchange purification of S. pombe urease . . . . ... . . 1 0 4 18 The activity of S. pombe urease fractions obtained from the FPLC-Mono-Q

purification step .. . . .... . . 1 0 8 1 9 PCR thermal -cycl ing conditions . . . .. . . .. . . 1 2 6 20 The effect on the transformation frequency of resuspending tra nsformed

cells i n various isoton ic and hypotonic solutions . . . .... . . ... . . 1 3 9 21 Transformation frequency of S. pombe strains . . . 1 4 0

22 Compleme ntation of ure- mutants by transformation with gene bank

plasmid clones . . . .. . . 1 4 7