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LIBRARY
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University

 

 

 

 

This is to certify that the

thesis entitled

CLONING AND CHARACTERIZATION OF GENES INVOLVED IN THE
REGULATION OF THE ALTERNATIVE OXIDASE AND MITHOCHONDRIAL
DNA REPLICATION IN NEUROSPORA CRASSA

presented by

Tak Ko

has been accepted towards fulfillment
of the requirements for

M. S. degree“, Microbio‘oac‘

+6). Mag.

Major professor

Dateguflfl (10% 2000

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CLONING AND CHARACTERIZATION OF GENES INVOLVED IN THE
REGULATION OF THE ALTERNATIVE OXIDASE AND MITHOCHONDRIAL

DNA REPLICATION IN NE UROSPORA CRASSA

By

Tak K0

A THESIS

Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology

2000

ABSTRACT
CLONING AND CHARACTERIZATION OF GENES INVOLVED IN THE
REGULATION OF THE ALTERNATIVE OXIDASE AND MITOCHONDRIAL DNA

REPLICATION IN NE UROSPORA CRASSA

Tak Ko

The mitochondria of Neurospora crassa contain two respiratory pathways, the
normal electron-transport chain and an inducible cyanide-resistant alternative oxidase
pathway. In Neurospora, the assembly of the alternative oxidase pathway requires the
expression of two genes, aod-I, which encodes the alternative oxidase, and a0d-2, which
is involved in the transcriptional regulation of aod-I. The objective of this work was the
cloning and characterization of the aod-Z gene. Crosses between strains that carried
markers in linkage group IIR (thr-3 and arg-5) suggest that aod—2 is located 14 map units
from thr-3 and 3 map units from arg-5, and to the right of a translocation, TALSl76. An
attempt was made to isolate the (rod-2+ DNA in two different ways: chromosome walking,
and by complementation of an aod—Z mutant using aod-I promoter-driven expression of
the bacterial hygromycin resistance gene as a method for selecting transformants. An
expression vector that contains the putative promoter upstream of a bacterial
hygromycin-resistance gene as a reporter has been constructed. However, a working
selection system that can be used to select aod-2+ by complementation of an aod-Z mutant

is not available yet. Similarly, a contig of cosmids was constructed for the region of

linkage group IIR covering the vicinity of the a0d—2 locus. Transformation of the a0d-2

mutant with cosmids from this contig, however, so far has failed to detect the gene.

Most of the proteins involved in mitochondrial gene replication and expression are
encoded by nuclear genes. Included in this group of proteins is DNA polymerase-y, which is
part of the complex involved in the replication of mitochondrial DNA. I have cloned,
sequenced and partially characterized the gene (mip-I) that codes for the presumptive
mitochondrial DNA polymerase of Neurosopra crassa. Sequence analysis showed that
mz'p-I includes a 3918-nucleotide open reading frame encoding a 1305 amino-acid, 146 kDa
protein that has a mitochondrial targeting element at the amino terminus. The gene is
transcribed to a 4.5-kb mRNA. RF LP mapping located the gene in linkage group III between
pro-1 and ad-2. A comparison of the nine available DNA polymerase-y sequences revealed
several highly conserved sequence blocks, and that the polymerase domain is more highly
conserved than the exonuclease domain. The N. crassa and S. cerevisiae polymerase-y
polypeptides have long C-terminal extensions that are not found in any of the available

homologous proteins from other species.

Dedicated in the fond of memory of my father

iv

ACKNOWLEDGEMENTS

I would like to thank my family for providing me with all the means necessary to
complete this thesis. Special thanks to all members of my committee, Dr Larry Snyder
and Dr. Donna J. Koslowsky, for the caring and thoughtfiil guidance. I would like to
thank my mentor Dr. Helmut Bertrand for helping me by continuously encouraging my
scientific curiosity contributed enormously to my growth as a scientist. Without his
mentoring and support, none of this would have been possible. I would like to thank
Kathy Nummy, Georg Hausner, Dipnath Baidyaroy for their friendship as well as long
valuable conversation about science and all other my friends in the Bert's Lab for helping
me with experimental work and friendship.

I am thankful to all the faculty and staff of the Department of Microbiology,

Michigan State University, for their assistance during the years of my study.

TABLE OF CONTENTS

 

 

LIST OF TABLES viii
LIST OF FIGURES ix
CHAPTER 1.

Approach to clone the aod-Z alternative oxidase regulatory gene of Neurospora crassa

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 . 1 Introduction 1
1.1.1 Respiratory Pathways in Plants and Fungi 1
1.1.2 Structure of the Alternative Oxidase 3
1.1.3 Possible Functions of the Alternative Oxidase 4
1.1.4 Regulation of the Activity and Synthesis of the Alternative Oxidase --------- 6

1.2 Materials and Method 5 11
1.2.1 Neurospora and E. coli strains and growth conditions 11
1.2.2 Preparation of competent E. coli cells for transformation 13
1.2.3 Recombinant plasmids and vectors 13
1.2.4 Rapid mini-scale plasmid/cosmid DNA isolation 15
1.2.5 Storage and plating of N. crassa genomic DNA libraries 15
1.2.6 Chromosomal walking of N. crassa genomic cosmid library 16
1.2.7 Measurement of 0; consumption by N. crassa mycelium 16
1.2.8 Induction of the alternative oxidase by inhibitors of oxidative

phosphorylation 1 7
1.2.9 Purification of DNA fragments from agarose gels 17
1.2.10 PCR approach to clone arg-5 from N. crassa 18
1.2.11 DNA Sequencing and Gene Analysis 19
1.2.12 Restriction Fragment Length Polymorphism (RFLP) mapping -------------- 20
1.2.13 Pulse-field gel electrophoresis analysis of YAC clones 20
1.2.14 Genetic crosses of N. crassa 21
1.2.15 N. crassa spheroplasts preparation and transformation 22

1.3 Resultsand Discussion 24
1.3.1 Construction of an aod—I expression vector and approach to cloning aod—2- 24
1.3.2 Genetic crosses 30
1.3.3 Chromosome walking 37
1.3.4 Attempted cloning of the arg-5 gene using degenerate primers --------------- 53

1.4 Literature Cited 59

 

vi

CHAPTER 2.

Cloning and nucleotide sequence of the catalytic subunit of DNA polymerase-y of
Neurospora crassa

2. 1 Introduction

 

 

 

 

 

 

 

63

2.2 Material and Methods 65
2.2.1 Storage of N. crassa genomic DNA library 65
2.2.2 Design of primers and amplification conditions 65
2.2.3 Cloning and analysis of PCR products 66
2.2.4 Screening N. crassa genomic library 66
2.2.5 RNA electrophoresis and Northern blotting 69
2.2.6 Restriction fragment length polymorphism (RFLP) mapping --------------- 69
2.2.7 DNA Sequencing and Gene analysis of the DNA polymerase gamma ----- 70

2.3 Result and Discussion 71

2.3.1 Cloning of the catalytic subunit of N. crassa DNA polymerase-y ----------- 71
2.3.2 Nucleotide sequence of mip—I

 

 

 

 

 

76

2.3.3 Transcription of mip-I 90
2.3.4 Map location of mip-I 90
2.4 Summary and Conclusions 94
2.5 Literature Cited 95

 

vii

LIST OF TABLES

CHAPTER 1

Approach to clone of the and-2 alternative oxidase regulatory gene of Neurospora
crassa

 

 

Table 1-1 N. crassa strains used in this study 12
Table 1-2 Recombinant plasmids constructed and the cosmid clones isolated

during chromosome walks in this study 14
CHAPTER 2

Cloning and nucleotide sequence of the catalytic subunit of DNA polymerase-y of
Neurospora crassa

Table 2-1 Sequences of degenerate PCR primers and N. crassa specific primers used
in this study 68
Table 2-2 Percent similarity among 9 different DNA polymerase-y 82

 

 

viii

LIST OF FIGURES

CHAPTER 1

Approach to clone of the and-2 alternative oxidase regulatory gene of Neurospora

crass a

Figure 1-1 Electron transport pathway in N. crassa

 

Figure 1-2 Construction of expression vector, pTAK, which contains his-3
and 6 kb upstream region of aod—I

+, hygB’,

 

Figure 1-3 Restriction digestion of the pTAK construct to confirm the orientation

 

of each DNA fragment
Figure 1-4 Genetic crosses

 

Figure 1-5 Genetic cross between 128 (a a0d—2 arg-5) and 2102 (A TALSI 76) -------
Figure 1-6 Partial genetic map of Linkage Group II (5 megabases) in N. crassa -----
Figure 1-7 Southern hybridization screening pMOcosX cosmid library G and X
plate pool DNAs with T3 end RNA probe of G8: 1 1H cosmid CHAPTER 2 -
Figure 1-8 Southern hybridization screening pMOcosX cosmid library G and X

plate pool DNAs with T7 end RNA probe of G8:1 1H cosmid

 

Figure 1-9 Southern hybridization screening pMOcosX cosmid library
X25b half plate row and column pool DNAs with T7 side RNA

 

probes of G8:1 1H cosmid
Figure 1-10 Partial sequence of T3 end of cosmid X227C and locations
of primers used to identify the YAC clone

 

 

Figure 1-11 Pulse-field gel electrophoresis of YAC 2:6D

Figure 1-12 Southern hybridization of YAC 2:6D with identified cosmids

Figure 1-13 Southern hybridization screening pMOcosX cosmid library
G3 plate row and column plate pool DNAs with single-strand DNA
NL probe of YAC 2:6D

 

Figure 1-14 Chromosomal walking in LG 11 near org-5

 

Figure 1-15 Generation of degenerate primers for the isolation of the arg-5
DNA from N. crassa

 

Figure 1-16 PCR amplification of acetylomithine aminotransferase using
degenerate primers

 

Figure 1-17 Restriction Fragment Length Polymorphism (RFLP) mapping
of the G5:2D cosmid

 

ix

10
27
28
32

34
36

44

46
47

48
49

51

52

54

56

CHAPTER2

Cloning and nucleotide sequence of the catalytic subunit of DNA polymerase-y of
Neurospora crassa

Figure 2-1 Amino acid homology comparison for the DNA Polymerase—y gene
Homo sapiens (HS), Xenopus laevis (XL), and Saccharomyces

 

 

 

 

 

cerevisiae (SC) 72
Figure 2-2 Screening Neurospora crassa genomic library (Orbach/Sachs) by PCR

with specific primers 5 and 6 designed from the sequence of pGaml ----------- 73
Figure 2-3 Detection of mip-I 74
Figure 2-4 Nucleotide and amino acids sequences of the N. crassa mip-I gene ------- 78
Figure 2-5 Linear map of the N. crassa mip-I gene 83
Figure 2-6 Multiple alignment of DNA polymerse-y sequences 84
Figure 2-7 Detection of a transcript from mip-I by Northern blot hybridization ------ 92
Figure 2-8 Restriction Fragment Length Polymorphysm (RFLP) mapping of the

DNA polymerase-y 93

(Images in this thesis are presented in color)

CHAPTER 1.

Approach to clone the and-2 alternative oxidase regulatory gene of Neurospora
crassa

1.1 INTRODUCTION

1.1.1 Respiratory Pathways in Plants and Fungi

Mitochondria are the sites of oxidative metabolism in eukaryotes. They carry out
the energy capturing reactions during cellular respiration. While the mitochondria of
animal cells contain a single, cytochrome-dependent respiratory system which is
inhibited by cyanide and antimycin-A, higher plants, fungi, algae, and some protistes, are
known to have two respiratory pathways. One of those two systems is the normal
cytochrome pathway, which is cyanide and antimycin-A sensitive, and the second
pathway is an oxidase that is insensitive to cyanide and antimycin-A, but is inhibited by
hydroxamic acids (Henry and Nyns 1975). This pathway is commonly known as the
“alternative pathway”.

The alternative oxidase has been demonstrated to exist in some yeast, including
Hansenula anomala, (Henry and Nyns, 1975; Sakajo et al., 1991; 1993) and Moniliella
tomentosa (Hanssens et al., 1974), protists, particularly in Trypanosoma (Evans and
Brown, 1973; Hill, 1976; 1978), green algae, such as Chlamydomonas (Sargent and
Tylor, 1972), filamentous fiingi, including Neurospora, Cryphonectria and Aspergillus

among others, and in higher plants, including Sauromatum guttatum, Arabidopsis,

soybean, potato, rice, petunia and tobacco (Rhoades and McIntosh, 1991; McIntosh,
1994; Vanlerberghe et al., 1995). However, there are no reports of the altemative-
oxidase activity in animal mitochondria.

The cytochrome chain of N. crassa is similar to that of higher eukaryotes (Figure
1-1) in that it contains enzymes and multiprotein complexes that perform key functions in
oxidative phosphorylation. These multiprotein complexes are responsible for electron
transfer between the various TCA cycle substrates and molecular oxygen. The four
major complexes are the NADqubiquinone reductase (complex I), succinatezubiquinone
reductase (complex 11), ubiquinolzcytochrome c reductase (complex III), and cytochrome
c oxidase (complex IV). Complex 1, III and IV are sites of proton pumping across the
inner mitochondrial membrane. The proton gradient is subsequently dissipated by the
FoFl-ATPase (complex V) and this process is coupled with ATP production (Hatefi,
1985). When electrons flow through the cytochrome pathway, energy is conserved in the
form of an electrochemical gradient across the inner mitochondrial membrane which is
impermeable to protons (Elthon and Stewart, 1983). The alternative pathway diverges
from the cytochrome pathway at the ubiquinone pool and is not linked to oxidative
phosphorylation (Palmer, 1976).

Electron flow through the alternative pathway involves the shunting of electrons
from the main pathway at the level of the ubiquinone pool (Moore and Siedow, 1991).
The altemative-oxidase pathway consists solely of a ubiquinone oxidase that transfers
electrons from reduced ubiquinone to oxygen in a reaction that produces water. The
alternative-oxidase pathway bypasses two energy-conservation sites of the standard

electron transport chain; thus electron flow through the alternative pathway is considered

energetically wasteful. The alternative oxidase activity is tightly associated with the
inner mitochondrial membrane and appears to represent an integral membrane protein or
protein complex, but a protonmotive force does not develop during electron flow through

this pathway (Moore et al., 1978).

1.1.2 Structure of the Alternative Oxidase

The recent years, there has been a considerable advance in the understanding of
the structural features of the altemative-oxidase. The elucidation of the structure of this
respiratory pathway began with the generation of an antibody that inhibited
cyanide-insensitive respiration in isolated mitochondria from Sauromatum guttatum
(Elthon and McIntosh, 1987; Elthon et al., 1989). Immunoblotting experiment showed
three proteins having molecular mass of 35, 36.5, and 37 kDa, in S. guttatum (Elthon et
al., 1989). Experiments using an inducer, chloramphenicol, which inhibits mitochondrial
proteins synthesis, revealed that the antibody recognized two polypeptides (37 and 36.5
kDa) in the mitochondria when the alternative oxidase is induced in the N crassa
(Lambowitz et al., 1989). The specificity of the antibody was confirmed through the
observation that the polypeptides were missing in some of the alternative oxidase
deficient mutants of Neurospora. The S. guttatum antibodies also recognized one single
polypeptide of 36 kDa in Hansenula anomala treated with Antimycin-A (Sakajo et al.,
1993). At present, neither the nature nor the physiological significance of the multiple
bands that were observed in plants and Neurospora is understood clearly.

The antibody against the alternative oxidase protein was also used to isolate a

cDNA clone that encoded the alternative oxidase protein in Sauromatum guttatum

(Rhoades and McIntosh, 1991). Subsequently, cDNA and/or genomic DNA sequences
encoding the alternative oxidase were obtained from Arabidopsis (Kummer and 8011,
1992), soybean (Whenlan et al., 1993), tobacco (Vanlerberghe and McIntosh, 1994), the
yeast Hansenula anomala (Sakajo et al., 1991), and N. crassa (Li et al., 1996). The
alternative oxidase polypeptides are approximately 350 amino acids long, and has an
N-terminal extension that targets the protein to mitochondria. The alternative oxidase of
N. crassa is a chain of 362 amino acids and the mitochondrial targeting sequence is
cleaved by the mitochondrial import machinery in front of the leucine residue at protein

65, which the predicted to be the start of the mature protein (Li et al., 1996).

1.1.3 Possible Functions of the Alternative Oxidase

Although the cyanide-resistant altemative-oxidase pathway was first described
over 65 years ago (Genevois 1929), and much progress has been made towards a clear
understanding of the mechanisms involved in the synthesis and activity of the complex,
its physiological role remains obscure. There are at least five hypotheses that have been
formulated to explain the physiological role of alternative oxidase pathway.

The first hypothesis is therrnogenesis during flowering in S. guttatum, the voodoo
lily (Meeuse, 1975; Raskin et a1. 1987). This is the only confirmed role for the
altemative-oxidase pathway. In this plant, the aroid spadix tissue contains mitochondria
that have a high amount of a cyanide-resistant oxidase that results in a very high rate of

respiration during anthesis. The energy that is released as heat during this respiration

results in volatilization of compounds that attract pollinating insects. The thermogenic
activity is induced by salicylic acid (Raskin et al. 1987).

The second hypothesis is that the altemative-oxidase pathway may provide an
energy overflow mechanism which allows operation of the TCA cycle independent of
ATP synthesis for the production of intermediates required in other metabolic processes
(Lamber, 1982).

The third hypothesis postulates that alternative oxidase might generate heat to
allow maintenance of respiratory fimction at low temperatures which would impair the
operation of the cytochrome pathway (Laties, 1982). This suggestion was based on the
observation that plant mitochondria increased the activity of alternative oxidase in
response to low, non-freezing temperatures, and that electron flow through the
altemative-oxidase pathway is less sensitive to reduced temperature than the main
cytochrome pathway (Yoshida and Tagawa, 1979). This notion has been supported
recently by the finding that the capacity of the alternative-oxidase pathway was increased
in tobacco cells that were transferred from higher to lower temperatures (Vanlerberghe
and McIntosh, 1992).

The fourth possibility is that the alternative oxidase defends organisms against
respiratory inhibitors that are produced by competing organisms in the environment
(Lambowitz and Zannoni, 1978). Many organisms have been shown to be able to
produce cyanide and other compounds as secondary metabolites, which can act as
respiratory inhibitors. These organisms might themselves be expected to have a

respiratory pathway which is insensitive to these compounds (Lloyd and Edwards, 1977).

The fifth possible role of alternative oxidase is that it operates as an antioxidante
defense mechanism (Purvis and Schewfelt, 1993; Popov et al. 1997). It was shown that
inhibition of alternative oxidase with salicyl hydroxamte and propyl gallate stimulates
H202 production by mitochondria oxidizing succinate. Thus, the alternative oxidase
might be involved in limiting the level of reactive oxygen species produced in stressed

and senescing plant tissues.

1.1.4 Regulation of the Activity and Synthesis of the Alternative Oxidase

In plants, there are two mechanisms that are involved in the regulation of
alternative oxidase activity. One occurs at the protein level and depends on two factors,
metabolites and the redox status of an intermolecular disulfide bond in the homodimer
complex (Umbach and Siedow, 1993). In the presence of pyruvate, alternative oxidase
activity is stimulated (Umbach et al., 1994). Other metabolites, such as hydropyruvate,
glyoxylate, or-keto acid, succinate, and malate have the same effect on the activity
(Umbach et al., 1994). In plant mitochondria, the alternative oxidase polypeptide appears
to exist as a mixed population of covalently associated and noncovalently associated
dimers. Dimers in which the two subunits are linked by a disulfide bond are less active
than dimers in which this disulfide bond has been reduced. However, disulfide bridging
in dimers does not occur in the fungal alternative oxidases (Li et al. 1996).

The second type of regulation varies the amount of alternative oxidase protein that
is present in the membrane. This is the primary means of regulation of alternative

oxidase activity in fungi. In these organisms, the alternative oxidase pathway is not

active unless the cytochrome pathway is impaired (Slayman, 1977; Lambowitz and
Zannoni, 1978). The biogenesis of the alternative oxidase pathway in N. crassa requires
at least two genes, aod-I, which encodes the alternative oxidase subunit polypeptide, and
aod—2, which is involved in the transcriptional regulation of aod—I (Bertrand et al. 1983;
Li et a1, 1996). In N. crassa, the existence of an alternative oxidase was first postulated
through the observation of cyanide insensitive respiration in the [poky] cytoplasmic
mutant, which is deficient in a- and b-type cytochromes (Tissiers et al. 1953; Lambowitz
et al. 1972). The alternative oxidase was found to be inducible in wild-type strains of N.
crassa by treatments that impair the cytochrome pathway (Lambowitz and Slayman,
1971), such as chloramphenicol, which inhibits mitochondrial protein synthesis
(Lambowitz et a1. 1972); oligomycin, an inhibitor of mitochondrial ATPase activity
(Szakacs and Bertrand, 1976; Szakacs, 1978); antimycin A, an inhibitor of electron
transport (Lambowitz and Slayman, 1971); and starving wild-type cultures for copper,
which is an essential factor for the formation of cytochrome c oxidase (Schwab 1973).
These observations suggest that the regulation of the alternative pathway must be
achieved in N. crassa at the level of transcription and/or translation rather than by
regulation of the enzymatic activity (Li et a1, 1996). Experiments using an inhibitor of
nuclear RNA synthesis, actinomycin-D, suggested that the induction of the alternative
pathway is dependent on the transcription of nuclear genes (Edwards and Unger 1978).
The induction of altemative-oxidase activity in N crassa requires at least two
genes. From circumstantial evidence and nucleotide sequence data, it was deduced that
aod-I encodes the alternative oxidase and aod-Z is involved in the regulation of the

expression of the aod-I gene (Bertrand et al. 1983). The aod—I gene is located at 23 map

units to the left of trp-4 gene in linkage group IV (Bertrand et al. 1983). This gene has
been cloned recently (Li et al. 1996) and was shown to encode a polypeptide that is
homologous to alternative oxidase proteins from other organisms. The aod-Z gene is
located on linkage group II (Bertrand et al. 1983). It was previously suggested that the
function of aod-Z was to encode either a component that regulates the synthesis of the
alternative oxidase or a protein required for the stable accumulation of the aod-I
polypeptide (Bertrand et al. 1983; Lambowitz et al. 1989). Subsequently, it was shown
that the level of aod-I mRNA remains low in the a0d-2 mutant when it is grown under
inducing conditions (Li et al. 1996). This result confirmed that the aod—Z gene product is
a factor involved in the transcriptional control of the aod-I gene.

The regulation of the alternative oxidase gene is of interest because it provides a
model system for studying the mechanism of communication between mitochondria and
the nucleus. Thus, cloning and characterization of the aod-Z gene, which is known to
affect the transcription of aod—I in response to the functional state of mitochondria, will
provide information about this mechanism of mitochondrial/nuclear communication. N.
crassa is an ideal organism for the study of alternative oxidase because wild type strains
normally respire exclusively through the cyanide-sensitive, cytochrome-mediated
pathway, but the alternative pathway can be induced by chemical or genetic inhibition of
the cytochrome pathway (Lambowitz and Slayman, 1971). Furthermore, the genetic
system of N. crassa has been characterized and several mutants deficient in alternative
oxidase have been isolated (Szakacs and Bertrand, 1976) and in 1978 Edwards and Unger
discovered that the transcription of nuclear gene is required for the induction of

alternative oxidase activity.

The a0d—2 gene is not easily cloned because it is a regulatory gene and no
equivalent gene has been identified or cloned from any other organism, thus eliminating
the possibility of generating a PCR product by amplification of part of the gene with
degenerate primers. Furthermore, mutations in this gene do not produce a deleterious
phenotype, making it virtually impossible to clone it by gene replacement. Thus, an
attempt was made to obtain the (rod—2 gene in two different ways: complementation of an
aod-2 mutant using aod—I promoter-driven expression of the bacterial hygromycin

resistance gene (hph) and chromosome walking.

Figure 1-1. Electron transport pathway in N. crassa.

 

1.2 MATERIALS AND METHODS

1.2.1 Neurospora and E. coli strains and growth conditions

Wild type N. crassa 74-R23-1A and aod-2 mutant strains (Table 1-1) were used
in this study. All N. crassa strains were maintained on Vogel’s medium (Vogel, 1956;
Davis and Serres 1970) solidified with 1.5% w/v agar. Liquid cultures were grown in a
reciprocating-shaker incubator. Vegetative cultures of all N. crassa strains were grown at
room temperature (26°C) in the light to induce conidiation. Mycelia for respiration
studies were grown from a conidial inoculum of 106 to 107 conida/ml in 25-ml shaker
flasks containing 5 ml of Vogel’s liquid medium. The flasks were shaken for 12 to 14 hr,
and then 2-ml aliquots were removed from each flask for respiration studies.

E. coli DHSOL strain was used as a host for all pBluscript-derived recombinant
plasmids (Table 1-2). The bacterial strain was grown in L-broth (Maniatis et al., 1982) at
37°C in a shaker incubator. Ampicillin was added to a final concentration of 50 ug/ml as

required for selection of antibiotic resistance.

11

Table 1-1. N. crassa strains used in this study. Ant : antimycin sensitive

 

 

 

 

 

 

 

Strain Isolation no. Genotype
aod-l-l 7001 NSA-95 aod-I, a
7002 NSAK-33 aod-I, pan-I, a
7003 NSAK-44 aod-I, pan-1, A
7004 NSA-MA 6.40 aod—I, nic-I, al—2, A
aod-1-2 7021 NSK-l aod—I, a
7022 NSK-3 aod—I, inos, al-3, a
7023 NSKZ-39 aod—I, pan-I, A
7024 NSK-MA 18.2 aod—I, nic-I, al-2, A
aod—l-3 7041 NSG-9 aod—I, A clumpy conidiating
7042 NSG-13 aod-I, a clumpy conidiating
7043 NSG-lOS aod—I, pan-1, A
7044 NSGK aod-I, pan-I
aod-2-4 7061 NSBA-19 aod-Z, A slow conida
7062 NSBA-24 aod—Z, a slow conida
7063 NSBAN-9 aod—Z, nic-I, al-Z, A
7064 NSBAN-4 aod—Z, nic-I, aI-2, a
7065 NSBAN-12 aod-Z, nic-I, ((1-2, 0
ant 7201 ant-3 inos
7202 ant-1-4 aod-Z, pan, A
7203 ant-l
7204 ant-7 aod-I, al-3
7205 ant-7 aod-I, 01-3
7206 ant-7 aod-I, (11-3
7207 ant-7-7 frameshift
7208 ant-6-122 aod—I, trp—4
7209 ant-6-122 aod—I, trp-4
7210 ant-1-28 pan-2, trp-4
7211 ant-l-ll
7212 ant-l
7213 ant-1-27 pan-2, trp-4
7214 ant-l-27
7215 ant-7-40 pan-2, al-3
7216 ant-6-59 aod—I, pan-2, trp-4 aa substitution
aod+ 4044 met-1, A
4045 pdx—I, Lye-4, A
4046 pyr-I, org-2, A
4008-50 a
4115 27947 Y8743m Ltd37 org-5, pe, fl, trp-3, A
4116 27947 Y8743m Ltd37 org-5, pe, fl, trp-3, a
7194 27947 C167 org—5, aro-3, A
7196 UM107 F29 P2420 org—12, are-3, arr-20, a
2000 T28-M2 Y7655 true-2, aro-I, A
462 his-3, A
7626 RLM his-3 mtr, a

 

12

1.2.2 Preparation of competent E. coli cells for transformation

Bacterial transformation was done by standard procedure described by Maniatis et
al (1982). Competent cells were preserved at —80°C in 1.5-ml micro tubes containing 0.5

ml of L-broth supplemented with 36% glycerin, 12%PEG (MW8000), 12

mM-MgSO47HzO and sterilized by filtration.

1.2.3 Recombinant plasmids and vectors

The plasmid pBluscript KS+ (Strategene) was used as a cloning vector in this
study. Table 1-2 provides the lists of the recombinant plasmids obtained from others or
constructed in this study. The cosmid clones isolated during chromosome walks are
shown in Table 1-2. Cosmid clones are named by their position in the microtiter plates of

the library.

13

Table 1-2. Recombinant plasmids constructed and the cosmid clones isolated during
chromosome walks in this study. The Orbach/Sachs cosmid library of N. crassa
genomic DNA in pMOcosX vector, which has a dominant selectable markers for fungi
(hygromycin resistance) and E. coli (ampicillin resistance), and pSV50 cosmid library of
N. crassa genomic DNA (Volmer and Yanofsky 1986) in vector pSV50 carrying a
selectable markers for fungi (benomyl resistance) were used for chromosome walking.

 

 

pTAK54, pTAK61

Cosmids or palsmids Description
pAOGE-l 8 kb EcoRI fragment of the aod—I gene cloned in
pBluscript
pDV8H+ Contains 2.5 kb N. crassa his-3 gene
pCSN43 Contains hygromycin resistance gene (hph) and
transcription termination sequence of trpC from
Aspergillus nidulans
pHPH pBluescriptKS with 2-kb segment of the hph gene and
terminator sequence of trpC
pHH pHPH with N. crassa his-3 gene.
pHHm pHH with mutated SpeI site.
pHHma pHHm with 20 bp-long adaptor at ClaI site
pTAK28, pTAK29 Final construct contains sense orientation of 6-kb upstram

region of aod-I , hph and his-3 in pBluscriptKS.

Final construct contains antisense orientation of 6 kb
upstram region of aod—I, and sense orientation of hph and
his-3 in pBluscriptKS.

G3:10C pMOcosX cosmid library
G21 :12E pMOcosX cosmid library
X24:12B pMOcosX cosmid library

G1 21C pMOcosX cosmid library

01 :1 1H pMOcosX cosmid library

25:1D pSV50 cosmid library

X2:7C pMOcosX cosmid library
X2527A pMOcosX cosmid library
X10:12G pMOcosX cosmid library
G8:11H pMOcosX cosmid library

G723D pMOcosX cosmid library
X12:5D pMOcosX cosmid library

G5:2D pMOcosX cosmid library

YAC 2:6D N. crassa YAC library

 

14

1.2.4 Rapid mini-scale plasmid/cosmid DNA isolation

Mini-scale plasmid/cosmid DNA preparations from 1.5 ml culture samples were
done by alkaline lysis (Sambrook et. al., 1989) or by using the Quantum Prep Plasmid
Miniprep Kit (BIO-RAD) or the Wizard Mini-prep kit (Promega) as described by the

manufacturers.

1.2.5 Storage and plating of N. crassa genomic DNA libraries

Two cosmid libraries and/or YAC library were used in this study. The two
cosmid libraries were the Orbach/Sachs cosmid library of N. crassa genomic DNA in the
pMOcosX vector, which has a dominant selectable markers for fungi (hygromycin
resistance) and E. coli (ampicillin resistance) and the pSV50 cosmid library of N. crassa
genomic DNA (Volmer and Yanofsky 1986) in vector pSV50 carries selectable markers
for fungi (benomyl resistance) and E. coli (ampicillin resistance). Cosmid libraries were
maintained at —80°C in separate 96-well microtiter plates containing LB medium
supplemented with arnpicillin.

For the wild-type Neurospora crassa genome, a yeast artificial chromosome
(YAC) library (Centola and Carbon 1994) was obtained from the Fungal Genetics Stock
Center (FGSC). The YAC library was stored in 24 microtiter plates in YPD medium (see
appendix) at —80°C. The library contained 2204 clones with inserts of N. crassa genomic
DNA averaging about 170 kb and representing about 8.7 genome equivalents (Centola

and Carbon 1994).

15

1.2.6 Chromosomal walking through the N. crassa genomic DNA cosmid library

The Orbach/Sachs pMOcosX cosmid library of N. crassa genomic DNA was
replica plated in 50 microtiter dishes. Each cosmid clone was grown in 200 pl selection
medium (LB-amp) at 37°C for about 16 hr. DNAs were prepared from the 50 pools
cosmid, each containing 96 clones in a microtiter plate. The pool DNAs were digested
with EcoRI restriction endonuclease. The restriction fragments were separated by
electrophoresis through 0.8% agarose gels and transferred to nylon membranes for
Southern blot analysis to screen for homology to probes of relevant sequences. Plates
containing positive clones were replicated onto new microtiter plates, and 12 column
pools and 8 row pools of clones from each plate were used to prepare DNA for
identification of the specific address of the desired clone by hybridization. The walk was
extended with end-specific riboprobes transcribed from the T3 or T7 RNA polymerase
promoters contained in the vector. RNA probes were labeled by using the Dig-RNA
Labeling Kit (Boehringer Mannheim). Cosmids were isolated as described in section

1.2.4.

1.2.7 Measurement of 0; consumption by N. crassa mycelium

Respiration by intact mycelia was measured using a Clark electrode with an YSl

Model 53 Biological Oxygen Monitor (Yellow Spring Instruments Co.) as described by

16

Lambowitz and Slayman (1971). KCN (0.1 M) was dissolved in water and SHAM (0.33
M) was dissolved in 95% ethanol. Stock solutions of these inhibitors were prepared
freshly just before their use. 25 ul of each stock was added to 3 ml cultures in liquid

Vogel’s medium in the closed oxygen monitor vessel.

1.2.8 Induction of the alternative oxidase by inhibitors of oxidative

phosphorylation

For the induction of the alternative oxidase, chloramphenicol (final concentration
of 5 mg/ml, Sigma) and different final concentrations (0.05-1.0 ug/ml) of alcoholic
solutions of oligomycin (Sigma) and antimycin A (Sigma) were added as alcoholic
solution to 5 ml of 8-hr shaking liquid cultures and 4-hr further growth was allowed for

the induction of the alternative oxidase. Respiration studies were done after a total of

twelve hours of growth.

1.2.9 Purification of DNA fragments from agarose gels

DNA fragments were purified from agarose gels by the GlassMaxTM DNA

isolation Matrix system (GIBCO BRL) or by a modified gel electrophoresis method. In

the modified gel electrophoresis system, purification was performed through the use of

high melting point agarose. After the DNA was electrophoresis through 0.8-1.5% gels,

l7

the DNA was stained with ethidium bromide. Then some TBE buffer was removed from
the gel box until it reached the top of the agarose-gel. A piece of 3MM paper and a
dialysis membrane were inserted 5 m away and on the cathode side of the desired
fragment. These 3MM paper and dialysis membrane were cut 1 mm longer width of the
DNA band on both sides. The 3MM paper was inserted into the gel close to the DNA
fragment and the dialysis membrane was placed behind the 3MM paper. Subsequently,
electrophoresis was performed for 30-60 minutes. The desired DNA fragments were

eluted from the 3MM paper in 150 pl of TE solution.

1.2.10 PCR approach to cloning of arg-5 from N. crassa

Genomic DNA was prepared from N. crassa according to the method of Lee and
Taylor (1990). Figure 2 shows the amino-acid sequence comparison of the
acetylomithine aminotransferases from Alnus glutinosa (Y08680), Synechocystis sp.
(D90904), E. coli (M32796), Saccharomyces cerevisiae (M32795) that were used to
design degenerate primers corresponding to the conserved EANEAA (5’-primer) and
QGEGGV (3’-primer) amino—acid sequences. These primers were expected to produce a
PCR product of about 280 bp if no intron is present in the portion of the N. crassa org-5
gene. PCR reactions were initially carried out using varying concentrations of MgClz
(0.5-5 pl of a 25 mM stock), and varying annealing temperatures. In general, the best
results were obtained with the following reaction conditions: 100 ng of N crasssa

genomic DNA; 5 pl of 10X reaction buffer (Gibco); 2-3 pl of MgClz (25 mM); 1 pl of

18

dNTP mix (10 mM) and lpl of each primer stock (20 pM); 0.5 pl of Taq polymerase
(Gibco-S U/pl); distilled water to give a total volume of 50 pl. Amplification was
performed in a program consisting of 1 min at 94°C, annealing at 52°C for 1 min, and
extension at 72°C for 1 min. This program was repeated through 35 cycles, followed by
5 min at 72°C to ensure completion of products. Amplified fragments were analyzed by
electrophoresis in a 1.5 % agarose gel (Figure 1-16) and used for the cloning.

Bands of the appropriate sizes (over 1 kb, about 0.6 kb, and below 400 bp) were
recovered from agarose gels as described in section 1.2.9, precipitated with ethanol and
used for cloning. These gel purified PCR products were ligated into the plasmid vector
pBluscript KS+ (Stratagene) using standard protocol (Sambrook et. al., 1989) and
sequenced using T3/T7 primers.

The Orbach/Sachs pMOcosX cosmid library of N. crassa genomic DNA was
screened by Southern hybridization and by PCR with degenerate primers. Cosmids were

isolated using the Quantum Prep Plasmid Miniprep Kit (BIO-RAD Inc).

1.2.1] DNA Sequencing and Gene Analysis

DNA sequencing was performed through the use of a ABI Prism DNA sequencer.
Analysis of DNA and protein sequences was performed by using the program of
DNASTAR and the amino acid sequences of the acetylomithine aminotransferase from
different species were aligned using the CLUSTAL program of DNASTAR Megalign.

Some minor adjustments in the alignment were made by visual inspection.

19

1.2.12 Restriction Fragment Length Polymorphism (RFLP) mapping

The location of isolated cosmids on N. crassa was determined by RF LP mapping
(Nelson et al. 1998). Genomic DNA was prepared from N. crassa strains #4450—4488
(Fungal Genetics Stock Center) and the Mauriceville and Oak Ridge parents according to
the method of Lee and Taylor (1990) and 5 pg of each DNA was digested with EcoRI
enzyme. Electrophoresis carried out to separate the restriction fiagments through 0.8%
agarose gels and their transfer to nylon membranes for Southern blot analysis. The whole

cosmid was used as a probe, which was labeled with Dig (Boehringer Mannheim Inc.).

1.2.13 Pulse-field gel-electrophoretic analysis of YAC clones

In order to perform pulse-field gel electrophoresis, DNAs were extracted from
yeast clones and embedded in agarose plugs as described by Nelson and Brownstein
(1994). YAC clones were grown to saturation for about 2 days at 30°C in 50 ml YPD
minimal medium lacking uracil and trytophan. Yeast cultures were harvested and
resuspended in 0.8 ml of SCEM containing Yeast Lytic enzyme (2 mg/ml), and mixed
with 1 ml of 2% low-melting-temperature Seaplaque agarose dissolved in SCE. The
hardened plugs were incubated at 37°C for 12 hr to remove the yeast cell walls. Then,

these plugs were incubated in 0.5 M EDTA, 10 mM Tris-HCl (pH 8.0), 1% sodium

20

N-lauroylsarcosine, protease K (0.5 mg/ml) for 16 hrs at 50°C for lysis of the
spheroplasts. The plugs were then dialyzed overnight in 10 mM Tris-HCl (pH 8.0), 50
mM EDTA at 4°C and stored in the same solution at 4°C.

YACs were separated from the large yeast chromosomes by pulse-field gel
electrophoresis (Hula Gel system, Hoefer Scientific Instruments Inc.) in 1.2% SeaKem
GTG agarose gels in 0.5X TBE containing ethidium bromide (1 ug/ml) (Sambrook et. al.,
1989) was used as the electrophoresis buffer. Pulse field electrophoresis was performed

for 48 hr at 130V and with 110° gel-rotation angles.

1.2.14 Genetic crosses of N crassa

Genetic crosses were performed as described by Davis and de Serres (1970). The
female parent was inoculated onto agar slants containing Watergaard’s crossing medium
and incubated at room temperature for 1 to 2 weeks until protoperithecia were formed. A
suspension of fresh conida from the prospective male parent was spread over the
protoperithecia and incubated at room temperature for about 2 weeks to mature the asci,
which eject their ascospores by light induction. Ascospores were collected using sterile
wooden sticks, and transferred to sterile water. The ascospores were activated at 60°C
for 60 min and plated on supplemented Vogel's medium and incubated for 12 hr at room
temperature until germination was observed. Individual germinated ascospores were

collected and transferred to supplemented Vogel's slants. Phenotypic determinations

21

were carried out after conidia were formed by the single-ascospore isolated on the agar

slants.

1.2.15 N. crassa spheroplast preparation and transformation

The preparation and transformation of N. crassa spheroplasts was performed as
described previously (Akins and Lambowitz 1985). A suspension of fresh conidia from
the appropriate N crassa strain was prepared in sterile water. The suspension was
inoculated into 250 ml of Vogel's medium containing appropriate supplements. The final
concentration of conidia was about 1.0-1.5 X 107 per ml. The culture was incubated with
gentle shaking (about 150 rpm) until 90% of conidia were germinated. The conidia were
harvested by centrifugation at 5000 rpm for 10 min at 4°C in a Sorvall GS-3 rotor and
washed twice with sterile distilled water and once with l M sorbitol. The conidial pellet
was resuspended in 1 ml of 1 M sorbitol at 1 X 107 conidia per ml. The suspension of
germinated conidia was incubated with lysing enzyme (3 mg/ml, Sigma Chem. Co.) or
Novozym 234 (2 mg/ml, Novo Inc.) at 30°C for 30 min with gentle agitation, to generate
protoplasts.

The spheroplast suspension was transferred to a 50-ml screw cap centrifuge tube
and centrifuged at the low speed (1000 rpm) in a Sorvall 34 rotor for 10 min. The
spheroplasts were washed twice with sterile l M sorbitol and once with MCS. Then,

spheroplasts were resuspended in MCS as the concentration of 5 X 108 per ml. Then, 13

pl of DMSO, 65 pl of sterile heparin (5 mg/ml) and 275 pl of sterile PMC were added to

22

each milliliter of spheroplast solution. This solution was gently mixed and dispensed into
sterile Eppendorf tubes to be stored at —80°C

The frozen spheroplasts were thawed on ice and then 1 to 5 pg of DNA in 50 pl
deO was added for transformation. The mixture was gently shaken and incubated on
ice for 30 min. Then, 9 volume of sterile PMC solution was added to this mixture and
incubated at room temperature for 20 min. This mixture was added to pre—warmed 50°C
top agar and layered onto proper Vogels agar plates. When the top agar plate was
hardened, the plate was incubated in a 4°C cold room for 48 hours. Then, the top agar,
which contains hygromicin was layered onto this agar plate. After the top agar has

solidified, the plate was incubated at 30°C for 2-5 days to allow colonies to form.

23

RESULTS AND DISCUSSION

1.3.1 Construction of an aod-I expression vector and approach to cloning aod—Z

The first strategy for cloning aod—Z was based on the observation that this gene is
required for the induction of aod-I. Thus, it was assumed that aod—I-promoter-driven
expression of a bacterial hygromycin resistance gene (hph) could be used as a method for
selecting aod—Z+ transforrnants. For this purpose, a new expression vector, the plasmid
(pTAK) was constructed. In this vector, the 6 kb of DNA located upstream of the aod-I
gene in wild-type Neurospora was positioned in the right orientation in front of hph in a
pBluescriptKS+ vector, which also contains the N crassa his-3+ gene (Figure 1-2). As a
negative control, a construct in which the promoter is located in the wrong orientation in
front of the hph gene also was generated. During the initial phase of creating both
constructs, the pCSN43 plasmid (Staben et al. 1989) was partially digested with ClaI and
BamHI to remove the hph gene and transcription-termination sequence of the Aspergillus
nidulans trpC gene. This 2-kb segment of DNA was ligated into pBluescriptKS which
had been digested with ClaI and BamHI. For the screening of the construct (pHPH),
which has the hph gene and terminator sequence of trpC, double digestions with ClaI and
BamHI or ClaI and MluI were used. After moving the hph and terminator sequence into
pBluscriptKS, the his-3 gene of N crassa was moved into the plasmid. To obtain this
construct, the pDV8H plasmid, which contains the his-3 gene of N. crassa, was digested

with NotI to remove the 2.5 kb his-3 DNA. This his-3 DNA was purified from an

24

agarose gel and then ligated into the pHPH construct, which was cleaved with Natl. For
the screening of the new construct (pHH), NotI digestion and hybridization with a probe
of his-3 DNA were used. Then, the pHH plasmid was digested with SpeI, blunted by S1
nuclease and religated. SpeI digestion was used for screening the mutant pHH (pHHm)
to give uncut DNA. In order to move the putative promoter sequence of the aod—I gene,
the pHHm was opened with ClaI and a 20-bp synthetic adaptor, which contains a SpeI
site, was inserted. SpeI digestion was used for screening the construct(pHHma). Then,
the pAOPB-29 plasmid, which contains the promoter of aod-I (Paod-I) was digested
with SpeI to remove the 6-kb promoter. This 6-kb DNA was purified from an agarose gel
and then ligated in the pHHma plasmid, which was opened with SpeI to make the
expression vector (pTAK). The digestion with Spel or Not I (Figure 1-3) and
hybridization were used to screen the DNA construct (data not shown).

The his-3+ gene in pTAK serves as a target for the integration of the construct
into linkage group I at the his-3 locus. For this purpose, a his-3 aod—2 double mutant has
been constructed by crossing the appropriate strains. Integration of pTAK into the his-3
locus of this mutant will generate his-3+ autotrophs, which can be selected on minimal
medium. We will use these engineered nod-2 [Paod-I hph] his-3+ strain of N crassa to
select the dad-2+ DNA from 3 cosmid libraries of wild-type N crassa DNA. Protoplasts
of aod-2 [Paod-I hph] his-3+ will be transformed with pools of 96 cosmids and
transforrnants will be selected by hygromycin resistance on medium containing
oligomycin, which is an inducer of the alternative oxidase. Presumably, spheroplasts that
were transformed by a cosmid containing the aod—Z+ DNA will express the hph gene

because the aod-I promoter has been activated by the Aod-2 protein, which should be

25

induced or activated by oligomycin. Once a group of 96 cosmids that yields (rod-2+
transformants has been identified, this group will be subdivided into smaller groups.
Then, DNAs from the smaller groups will be used to transform the aod-Z [Paod-I hph]
his-3+ recipient. The process will be repeated until the cosmid that contains the DNA
with the (rod-2+ allele has been identified. Then the smallest segment of DNA that
transforms the aod—Z+ recipient will be subcloned from the cosmid.

The cloning of aod—2 by expression of the bacterial hph gene from Paod-I by
inducers of the alternative oxidase in cells that had been transformed by (rod-2+ DNA
proved to be impractical. During the construction of the pTAK, it was discovered that
prolonged exposure of the aod—2 mutant to inducers of the alternative oxidase resulted the
expression of aod-I. Hence, expression of hph occurred in all transformants, regardless
of whether or not they received aod—2+ DNA. Consequently, this project was abandoned

in favor of alternate methods for cloning the aod—2 gene.

26

Figure 1-2. Construction of expression vector, pTAK, which contains his-3+, hygB',
and 6 kb upstream region of and-I.

  
 
 

  

pCSN43
(5.35 kb)

  
 
   
 
 

pAOGE-l
(1 1 kb)

   

hph' (2 kb)

  

Paod-I (6 kb) pDV8H+

(8.5 kb)

    
   
  
 

Adapter

his-3+ (2.5 kb)

   

 

27

Figure 1-3 Restriction digestion of the pTAK construct to confirm the orientation of
each DNA fragment. Lane 1; 1 kb ladder DNA, lanes 2, 3, 4, 5, 6; A029, pTAK28,
pTAK29, pTAK54, pTAK61 digested with SpeI to check the presence of promoter of
aod-I gene, lanes 7, 8, 9, 10, 11; pHH, pTAK28, pTAK29, pTAK54, pTAK61 digested
with Not] to confirm the presence of the his-3 gene and check the orientation of promoter
region of the aod-I gene, lanes 12, 13, 14, 15, 16; pHH, pTAK28, pTAK29, pTAK54,
pTAK6l digested with BamHI.

28

lé- SpeI -) | (- NotI 9 I 6- BamHI -)l

Lanel 2 34 5 6 7 8910111213141516

6kb
5 kb

4kb

3kb

2 kb

1.6 kb

lkb

 

29

1.3.2 Genetic crosses

In order to generate mutant strains that could be used for the identification of
cloned DNAs of the aod—Z and org-5 genes by complementation, several crosses were
performed. Initially, strains 7064 (a aod-2 nic-I al-3) and 462 (A his-3) were crossed to
generate aod—Z his-3 double mutants. The individual progeny of this cross were
identified as TakD followed by a number, and among 45 ascospore isolate TakD 31, 39
and 40 showed the aod—2 his-3 double mutant phenotype in respiration and requirement
tests. These three progeny were kept for screening genomic libraries for cosmids that
complement the aod-2 mutation.

To map aod—2, the mutant was used in a number of crosses with strains that have
well-defined markers in linkage group IIR (Table 1-1). Random ascospore progeny were
collected from each cross and tested for induction or lack of induction of
cyanide-resistant respiration by chloramphenicol, antimycin-A or oligomycin, as
described in Material and Methods. Initially, 7061 (aod—2 A) or 7063 (aod—Z nic-I a1-3
A) were crossed with 4116 (org-5 pe fl trp-3 0). J28 (cod-2 org-5 A) was crossed with
105 (bal a). The results of these crosses showed that bal, pe and fl produced high levels
of lethality in ascospores, and thus could not be used for the precise mapping of aod—2.
Consequently a strain, TAKH#15, whose phenotype is org-5 thr-3, was generated from a
cross between org-5 and thr-3 strains (Figure 1-4). This strain was crossed with the aod—2
mutant, and progeny from this cross were tested for the induction of alternative oxidase
activity. The results indicated that aod—Z is located in linkage group IIR, about 3 map

units to the left of org-5 and 14 map units to the right of thr-3 (Figure 1-4). To obtain a

30

more precise map position for aod—2, crosses were made between I 28 (aod—2 org-5 a) and
2102 (TALSI 76 A), which has a translocation (TALSI 76) between the centromere and
org-5 (Figure 1-5) to check for location of aod-2 to the left or right of the translocation
site (TALSI 76). Due to chromosomal pairing at meiosis, either a 2:1 or a 1:2 (Aod+
Arg+ : Aod- Arg-) phenotypic ratio should appear among the progeny from this cross,
depending on the location of the aod-Z gene to the right or left of the translocation point
respectively. The cross yielded 54 progeny of the Aod+ Arg+ phenotype, and 27 progeny
with the Aod- Arg- phenotype. Thus, the aod—Z gene is located to the right of the
translocation site, narrowing its position to a location between TALSI 76 and org-5 in

linkage group 11 (Figures 1-5 and 1-6).

31

Figure 1-4. Genetic crosses (A) Genetic cross between 7063 (A dad-2‘ nic-I' al-2') and
4116 (a org-5' pe' fl trp-3') strains and genetic map of the region in LGII near the
centromere. (B) Genetic cross between J 28 (bal+ aod-Z' org-5' a) and 105 (baf
aod—2+arg-5+A) strains. (C) Genetic cross between 7061 (aod—2 A) and H15 (org-5 thr—3
0). Partial genetic map of Linkage Group II (5 megabases) in N. crassa. Genetic
mapping of aod-Z revealed that it is located 3 map units from org-5 and 14 map units
from thr-3. Black dot indicates centromere.

32

(A) Genetic cross between 7063 (A aod-Z nic-I' aI-Z') and 4116 (a arg-5' pe’fl trp-3')

 

 

7063 thr-3Jr aod-2' org-5' pe+ fl+
° scor I soon
-..-.I....-.IIIIII.“.I .‘I...-‘... ..*.-.--.-...l......-...-..-..-.
4116 thr-3" (rod-2+ org-5+ pe' fl

(B) Genetic cross between .1 28 (balT amt-2' arg-5’ a) and 105 (bar aod—ZTarg-STA)

 

 

J 28 bal+ (rod-2' org-5'
ESCOI XS‘COII
IIIIII-IIIIIIIIIII.IIIII“.I III-IIIIIIIIIII‘.‘ III-I'IIIIIIIIIIIIIII
105 baf dad-2+ org-5+

(C) Summary of genetic Cross J 7061 (aod—2 A) x H15 (org—5 thr—3 a).

 

 

7061 thr-3+ aod-2' org-5+
'° scorx scon

III-IIIIIIIIIIIIII.“.I IIIrIII‘DI II‘IIIIIIIIIIIIIIIIIIIIIIIIIIIII
H15 thr—3' aod—2+ org-5'

 

Genotype of progeny No. of progeny Frequency

 

 

 

 

 

thr—3 acct-7" org-5 73

thr-3+ aod-2 org-5+ 66 83.7% Parental type

thr-3 aod-2 org-5°r 10

thr-3+ aod-2+ org-5 12 13.3% Single cross over at I

thr-3 aod-2+ org-5F 3

thr-3+ aod—Z org-5 1 2.4% Single cross over at 11

thr-3 (rod-2 org-5 0

thr-3+ aoal-Z+ org-5+ l 0.6% Double cross over I and II
Total 166

 

33°

Figure 1-5. Genetic cross between J28 (a and-2 arg-5) and 2102 (A TALSI 76).
Yellow arrow indicates the possible location of the aod—2 gene. The centromere is
indicated by a black dot. The translocation site is indicated by a small black line. (A)
Chromosomal difference between the J28 and 2102 strains. (B) Chromosomal pairing at
meiosis. (C) Possible segregation during meiosis. i) this progeny has a normal LG 11 and
truncated LG V which has a duplication of the right side of LG 11. ii) LG II and V come
from strain J28 and will show same phenotype as the parent. iii) LG 11 and V with
translocations and this progeny show the same phenotype as strain 2102. iv) normal LG
V and most of LG 11 is missing. This condition is lethal and not show in the progeny.

 

 

 

Phenotype Number of % Expected ratio Expected ratio
progeny (I) (II)
aod+ arg+ 54 55 1/3 2/3
aod+ arg- 1 1 0 0
aod- arg+ 2 2 0 0
aod- arg- 27 27.5 2/3 1/3
Total 98

 

I: if the aod-2 gene is on the left side of the translocation site, 11: if the aod-2 gene
is on the right side of the translocation site.

34

(A) Chromosomal difference between J28 and 2102 strains.

Strain J 28
(aod-2°) org-5'

11 fi‘T’

 

 

 

Strain 2102
119 W +
”a? (dad-2 )
V’ M
TALI 76 org-5+

(B) Chromosomal pairing at meiosis.

II’

 

 

(0 Possible segregation during meiosis

 

 

 

 

i) ii)
aod-2' org-5' aod-2' org-5'
11 —O . 11 —'.
W org-5+
V l-nfi V M
iii) TALSl76 iv) TALSI76 (Dead)
———.-—_ —+—
M org-5+ M
w

35

Figure 1-6. Partial genetic map of Linkage Group II (5 megabases) in N. crassa.

 

 

thr-3 arg-5 nuc-Z org-12
aro-3 pe aro-I un-20 fl trp-3
ure-3
TALS176
CEN
4"
aod-2

36

1.3.3 Chromosome walking

Chromosome walking is another strategy for identifying the aod—Z gene. This
requires the determination of a reasonably precise map position for aod—Z and
identification of a very well defined marker DNA which is close to aod-Z in linkage
group II. In this study we used the org-5 gene as a marker and two known cosmids,
68:11H from the pMOCosX library and 25:1D from the pSV50 library, as the starting
points for a chromosomal walk. The 68:11H cosmid was digested with EcoRI, and T3 or
T7 primers used to generate the end-specific RNA probes. These probes were hybridized
to the 50 plate-pool DNAs of the pMOcosX cosmid library as described in Material and
Methods. The results of the hybridizations revealed that the G7, G8, X10, X12, and X25
plate pools were detected by the T3 probe of G8:11H, and the G8 plate pool gave the
strongest signal (Figure 1-7). The T7 probe indicated that G3, G7, G8, X10, X12, and
X25 plate pools contained cosmids that overlap G8:11H (Figure 1-8). The row and
column plate-pool DNAs from the X25 plate were prepared, cut with EcoRI and
electrophoresed on an agarose gel as described in Materials and Methods. The
hybridization showed that the X25:7A cosmid was the next on the contig (Figure 1-9).
The result of the hybridization with the T7 probe of X25:7A cosmid showed that the 62,
G7, X10, X2, X3, X10, X16, X17, and X25 plate-pool DNAs were hybridized (data not
shown). The T3 probe of X25:7A cosmid identified plates G3, G7, G8, G11, X10, X12,
X22, X25. Plate X10 was selected and the row and column pool DNAs were prepared
and separated by electrophoresis on an agarose gel. Cosmid X10:12G was identified by

both the T3 and T7 probes of the X25:7A cosmid. The T3 end-probe of the X10:12G

37

cosmid gave strong hybridization with X25:7A and the G8:11H cosmids. However, the
T7 end-probe of X10:12G hybridized with X25:7A, but not with G8:11H. Thus, the
cosmid X10:12G yielded a small extension of the contig from G8:11H. Nonetheless, the
hybridization experiment with T3 and T7 probes of X10:12G did not yield a new plate-
pool of DNAs that could be used to extend the walk. Thus, the X2 plate was selected for
further chromosomal walking from the cosmid X25:7A. The row and column pools of
DNAs of the X2 plate revealed that cosmid X2:7C hybridized with the T7 probe of
X25:7A but not with the T3 probe. Both the T3 and T7 end-probes of X10:12G did not
hybridize with any row and column pools of DNAs from the X2 plate. The T3 end probe
of X25:7A strongly hybridized with cosmid G8:11H but the T7 end-probe did not
hybridize with this cosmid. Thus, the T7 end probe of X25:7A was used to extend the
contig by screening the plate-pool DNAs. The hybidization experiment showed that G2,
G7, G8, X2, X3, X10, X12, X16, X17, X22, and X25 plate-pool DNAs hybridized with
the T7 probe of X25:7A. The hybridization with the cosmids of the contig revealed that
cosmid 25: 1D (pSV50 cosmid library) was recognized by the T3 probe of X2:7C but not
by the T7 end-probe of X2:7C. Thus, 3 cosmids (X10:12G, X25:7A, and X2:7C) fill the
gap between the cosmids G8:11H and 25:1D. The G and X plate pools DNAs were
screened with the T3 and T7 probes from the X2:7C cosmid and the T3 probe of X2:7C
pulled out G1, G2, G3, G7, G8, X1, X2, X3, X4, X6, X8, X10, X17, X22, and X24 plate
pools. Another cosmid was pulled out by probes from the X25:7A cosmid. Both the T3
and T7 probes of X25:7A hybridized with the G7:3D cosmid, but only T3 and not the T7
probe of X10:126 hybridized with G7:3D. Thus, the G7:3D cosmid extended the contig

further from the X10:12G cosmid. Hybridization experiments with probes from this

38

G7:3D cosmid revealed that the T7 probe did not hybridize with X2:7C but hybridized
with the G8:11H, X25:7A, and X10:12G cosmids. However, the T3 probe of G7:3D
cosmid hybridized with all cosmids (X2:7C, X25:7A, X10:12G, and G8:11H) in this
contig except 25:1D. Screening G and X plate pool DNAs with the end probes of G7:3D
showed that G3, G7, G8, X10, X12, X22, X25 plates were detected by the T3 probe,
whereas the T7 probe detected G7, GS, X10, X12, X25 plate-pool DNAs. Extension of
the contig with the T7 probe of G7:3D identified the X1225D cosmid. From the X1225D
cosmid, G3, G7, G8, X6, X8, X10, X12, X24, and X25 plate—pools were identified by
using the T3 probe, whereas the T7 end probe identified the G3, X10, X12, and X25
plate-pools. RNA probes could not be generated from the 25:1D cosmid with T3 or T7
primers. Thus, this cosmid was digested with EcoRI and 7.5 kb, 6 kb, 5 kb, and 3.8 kb
DNA bands were purified fi'om an agarose gel. Probes were made from the purified
restriction fragments as described in Materials and Methods. A hybridization experiment
with the 7.5-kb probe pulled out G1, X10, X12, and X24 plate—pools, and the screening of
G1 row and column pool DNAs showed that G1:1C and G1:11H were recognized. The
T3 probe of the G1:1C cosmid identified a new plate-pool DNA, G3, but the T7 probe
did not recognized any plate-pool DNAs. The T7 probe of Glzl 1H hybridized
specifically with the X24:12B cosmid. However, RFLP mapping revealed that the
X24:12B cosmid is located near the centromere of the linkage group I, and not in the
linkage group II (data not shown).

A partial sequence was obtained fi'om the T3 end of cosmid X2:7C and 2 sets of
primers, setl (HB337 and HB338) and set 2 (HB385 and HB386), were designed to

identify the YAC clone by PCR (Figure 1-10). The PCR reaction with set 1 primers did

39

not amplify any of the genomic DNA of N. crassa, but a 190-bp long fragment was
amplified with primer set 2 from the X2:7C cosmid and N. crassa chromosomal DNA
templates. Thus, the HB386 and HB387 primers were used for screening YAC
plate-pool DNAs, and YAC 2:6D was identified to overlap cosmid X2:7C. Pulse-field
gel electrophoresis was performed to purify the YAC DNA as described in Materials and
Methods (Figure 1-11). A hybridization experiment was performed to identify the
portion of the YAC (Figure 1-12) that overlapped with the established contig. The
purified YAC DNA was used to make end-specific probes by using the NL and NR
primers as described in Material and Methods. Screening of the cosmid library was
continued with the NL and NR probes by Southern hybridization. The G3:10C cosmid
was identified by the NL probe, but the NR probe did not identify a new cosmid (Figure
1-13) in the library.

The chromosome walk has generated a contig which consists of 10 cosmids and
one YAC (Figure 1-14). We know that the genomic DNAs in 2 cosmids (25:1D in the
Vollmer and Yanofsky library, and G8:11H in the Orbach/Sachs library) are located
close to the org-5 gene (overlapping or similar RFLP patterns). These were used as
starting points for a chromosome walk to aod—2 by hybridization with other cosmids. The
10 neighboring cosmids were used to transform spheroplasts of the org-5 aod—2 mutant
strain 128. Transforrnants were selected by growth on media containing either benomyl
or hygromycin, depending on the selectable marker provided by the cosmid. To
determine whether or not the transforrnants that had received aod-2+ DNA, respiration
tests were performed to see if the alternative oxidase was induced by oligomycin.

However, the complementation tests showed that none of the cosmids on the contig

40

(G3:10C, GlzllH, G1:1C, 25:1D, X2:7C, X25:7A, X10:12G, G8:11H, G7:3D, X12:5D)

did complemented either the aod-2 or org-5 defects.

41

Figure 1-7. Southern hybridization screening pMOcosX cosmid library G and X
plate pool DNAs with T3 end RNA probe of G8:11H cosmid. All plate pool DNAs
were digested with EcoRI enzyme. Lanes 1, pSV50; 2, G811H; 3, 1 kb ladder DNA; 4
through 20, G plates #1 through 17. Lanes 21, pSV50; 22, G811H; 23, 1 kb ladder DNA;
lanes 24 through 40 represent X plate pool DNAs #1 through 17. Lanes 41 through 47
represent to G plate pool DNAs #18 through 25; and lane 48, 1 kb ladder DNA; 49,
pSV50; 50, GSllH; 51, lkb ladder DNA; lanes 53 through 60 represent X plate pool
DNAs #18 through 25.

42

12345 67891011121314151617181920

\

    

2122 23 24 25 26 27 28 293 31 32 33 34 35 36 37 38 39 40

 

43

Figure 1-8. Southern hybridization screening pMOcosX cosmid library G and X
plate pool DNAs with T7 end RNA probe of G8:11H cosmid. All plate pool DNAs
were digested with EcoRI enzyme. Lanes 1, pSV50; 2, G811H; 3, 1 kb ladder DNA; 4
through 20, G plates #1 through 17. Lanes 21, pSV50; 22, G811H; 23, 1 kb ladder DNA;
lanes 24 through 40 represent X plate pool DNAs #1 through 17. Lanes 41 through 47
represent to G plate pool DNAs #18 through 25; and lane 48, 1 kb ladder DNA; 49,
pSV50; 50, G811H; 51, lkb ladder DNA; lanes 53 through 60 represent X plate pool
DNAs #18 through 25.

44

12345 67891011121314151617181920

 

2122 23 24 25 26 27 28 29 3O 31 32 33 34 35 36 37 38 39 40

 

4142 43 44 45 46 47 48 49 50 5152 53 54 55 56 57 58 59 60

n
u cc -- = w O-ib ‘ ”I M ”W “awe a“
. . . .. .0

INS

45

Figure 1-9. Southern hybridization screening pMOcosX cosmid library X25b half
plate row and column pool DNAs with T7 side RNA probes of G8:11H cosmid. Lane
1,pSV50; 2, G8:11H; 3, 1 kb ladder DNA; lanes 4-12, column pool DNAs 7-12; lanes
11-18, row-pool DNAs A—H.

12345 6 7 89101112131415161718

 

46

Figure 1-10. Partial sequence of the T3 end of cosmid X2:7C and locations of
primers used to identify a YAC clone. The underlined sequence is ORF for unknown.

 

 

ccccagccctcttgcttggctttcttctcccgttccttctgcatcttgtgc 51
HB 385
atcacttcagccgtgagctcctgcttcagtfiftcgctccaagacatccttc 102
HB337
ttgcgagtacgcctatcgccgatggcaatgagcaaaccggcagtactgttg 153

 

 

gcagaaatggagcagcaaagaacaggggggggtcactgcacttttcctggt 204

 

 

 

Ifl3386
gtggtaatcggtagaccaggtataagaacgagaghgatctggagacactgt 255
HB338
gcagtccacagcaacatgcatggcagcggcggtggtcactgcacttttctg 306
tgtgtatogagaccg 357

 

47

Figure 1-11. Pulse-field gel electrophoresis of YAC 2:6D. Lanes 1 and 3 are wild type
yeast and lanes 2 and 4 through 20 are YAC 2:6D. The arrow indicates YAC.

123456789mnunmwmnmwm
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48

Figure 1-12. Southern hybridization of YAC 2:6D with identified cosmids. Lane 1,
hybridization result with T3 end probe from X24:123; 2, hybridization result with T7
G1:11H probe; 3, hybridization result with T3 G1:11H probe; 4, hybridization result with
T3 G1:1C probe; 5, hybridization result with T7 G1:1C probe; 6, hybridization result
with 25:1D probe; 7, hybridization result with T3 X2:7C probe; 8, T7 X2:7C probe; lane
9, hybridization result with T7 side probe of G7:3D; 10, hybridization result with T3
G7:3D probe; 11, hybridization result with T3 G8:11H probe; 12, hybridization result
with T3 G8:11H probe; 13, hybridization result with T7 X10:12G probe; 14,
hybridization result with T3 X10:12G probe; 15, hybridization result with T7 X25:7A
probe; 16, T3 X25:7A probe.

49

 

50

Figure 1-13. Southern hybridization screening pMOcosX cosmid library G3 plate
row and column plate pool DNAs with single-strand DNA NL probe of YAC 2:6D.

ABCDEFGH123456789101112

 

51

Figure 1-14. Chromosomal walking in LG II near org-5. Arrow indicates T7 site of
cosmid. The contig could be localized in one of 3 regions: A, which is to the left side of
aod—Z; B which is located between aod—2 and arg-5; and C, which is located to the right

side of org-5.

TALSI76
f-

I-

J Linkage group II
1 ?(B) : I?(C) : 7‘

 

til

Contig

G3:10C

elm

E

a.

NL -> 4— NR
NL primer 5'—GAAGAAAGAGTATTACTACATAAC

NR primer 5'-CATTCACTTCCCAGACTTGC

1.3.4 Attempted cloning of the arg-5 gene using degenerate primers

An alignment of acetylomithine aminotransferase from Alnus glutinosa (Y08680),
Synechocystis sp. (D90904), E. coli (M32796), Saccharomyces cerevisiae (M32795) is
shown in Figure 1-12 and revealed two highly conserved regions that were selected to
generate degenerate primers for amplification of a portion of the N crassa org-5 gene by
PCR from genomic DNA. The distance between the two degenerate primers was
anticipated to be less than 300 bp (Figure 1-15). An initial PCR reaction using these
primers resulted in a product of the expected size (~300 bp, data not shown). Various
conditions were used to amplify the internal region of the org-5 gene of N crassa.
However, the reaction did not yield an obvious PCR product from genomic DNA (Figure
1-16). Thus, the three sets of DNA fragments were purified from an agarose gel to
generate Dig-labelled probes, which where used to screen cosmid libraries of N. crassa
genomic DNA. The probe generated from band A did not hybridize distinctly with any
cosmid in the library, whereas the probe generated from band C hybridized with a large
number of different cosmids. In contrast the probe generated from band B hybridized
strongly with a single cosmid, G5:2D. However, transformation revealed that this cosmid
did not complement the org-5 mutation. Moreover, RFLP mapping showed that this
cosmid probably is located between vma-I and aI-3 in the LG V (Figure 1-17), rather
than on LGII. Hence, org-5 could not be used as an anchor for the chromosome walk to

aod-Z.

53

Figure 1-15. Generation of degenerate primers for the isolation of the rug-5 DNA
from N. crassa. A) Multiple alignment of acetylomithine aminotransferase sequences.
Amino acid-sequences were aligned by the clustal method of the DNASTAR program.
The highly conserved residues are shown in the green shaded boxes. Identical residues
are marked with asterisks above the sequences. The location of degenerate primers is
indicated by red arrows above the sequence. Ag, Alnus glutinosa (Y08680); Sy,
Synechocystis sp. (D90904); Ec, E. coli (M32796); Sc, Saccharomyces cerevisiae
(M32795). B) Sequence of 5’and 3’ degenerate primers. Abbreviations represent the
following combinations of nucleotides: N=A, C, G, or T; Y=C or T; I=deoxyinosine;
R=A or G. The standard one letter code is used for the amino acids.

 

 

A)
I I
Ag MTSLQYFSLNRPVFPATHLHRPGIRHLQVSACANVEVQAPSSVKKQGVSKEVMEAAGRVLVGTYARVP-V 69
Sy MT ————————————————— YSPVVESVEAQAFAVTDLSPAAEFKTADFDTYVMN ———————— TYGRFP—I 44
Ec :13 QPITRENFDEW~HIP ——————— VYAPAP—F 23
Sc M —————————————————— FKRYLSSTSSRRFTSI ——————— LEEKAFQ ——————————— VTTYSRPEDL 34
II I I I I III IIII I I I
Ag VLSRGKGCKLYDP-EGREYLDLSAGIAVNVLGHADSDWLRAVTEQAATLTHVSNVFYSIPQVELAKRLVA 138
Sy AIARGQGSTLWDT-EGKSYLDFVAGIATCTLGHAHPALVRAVSDQIQKLHHVSNLYYIPEQGELAKWIVE 113
EC IPVRGEGSRLWDQ-QGKEYIDFAGGIAVNALGHAHPELREALNEQASKFWHTGNGYTNEPVLRLAKKLID 92
Sc CITRGKNAKLYDDVNGKEYIDFTAGIAVTALGHANPKVAEILHHQANKLVHSSNLYFTKECLDLSEKIVE 104
"r (H8412)
I III III IIIIII I I IIIII
Ag ssr ------- ADRVFFSNSGTEANEAAIKFARKFQRFTRPDEKQPATE—F‘VSFSNSFHGRTMGSLALTSK 200
Sy HSC ——————— ADRVFE‘CNSGAEANEAAIKLVRKYAHTVLDFLEQPV—-~ILTAKASEHGRTLATITATGQ 173
EC ATE‘ ——————— ADRVFFCNSGAEANEAALKLARKFAHDRYG—SHKSG--—IVAFKNAFHGRTLFTVSAGGQ 151
Sc KTKQFGGQHDASRVFLCNSGTEANEAALKE‘AKK ----- HGIMKNPSKQGIVAFENSFHGRTMGALSVTWN 169
“I (HB 413)
I I II IIIIII I
Ag ENYRSPFEPVMPGVTFLEYGNIEAATQLIQ ----- RRKIAAVF’VEPIQGBGGVYSATKEE‘LYALRKACDD 265
5y PKYQQYE‘DPLVPGFDYVPYNDTDQT :‘nrlunnr. TFLEPLQGEGGVRPGDLAYFKRVREICDQ 243
Ec PAYSQDFAPLPADIRHAAYNDINSASALI DD —————— STCAVIVEPIQGEGGWPASNAFLQGLRELCNR 215
Sc SKYRTPFGDLVPHVSFLNLND—-EMTKLQSYIETKKDEIAGLIVEPIQGEGGVFPVEVEKLTGLKKICQD 237
IIIIIII I II IIII IIII II I
Ag SGTLLVFDEVQCGLGRTGYLWAHEIY~—DVFPDIM‘I‘LAKPLAGGLPIGAVLVTERVASAITYGDHGTTF‘A 333
Sy NDILLVFDEVQVGVGRTGKLWGYEHL--GVEPDIFTSAKGLAGGVPIGAMMCKKFCD—VFEPGNHASTFG 310
Ec HNALLIFDEVQTGVGRTGELYAYMHY--GVTPDLLTTAKALGGGFPVGALLATEECARVMTVGTHGTTYG 283
Sc NDVIVIHDEIQCGLGRSGKLWAHAYLPSEAHPDIFTSAKALGNGFPIAATIVNEKVNNALRVGDHGTTYG 307
I II II I I I I II II I
Ag GGPLVCKAALTVLDKILRPGFLASVSKKGHYFKEMLINKLGGNSHV—REVRGVGLIVGIELD——-—VSAS 398
Sy GNPLACAAGLAVLKTIEGDRLLDNVQARGEQLRSGLAEIKNQYPTLFTEVRGWGLINGLEISAESSLTSV 380
EC GNPLASAVAGKVLELINTPEMLNGVKQRHDWFVERLNTINHRYG-LFSEVRGLGLLIGCVLNADYAGQAK 352
SC GNPLACSVSNYVLDTIADEAFLKQVSKKSDILQKRLREIQAKYPNQIKTIRGKGLMLG----AEFVEPPT 373
I II I I I I

Ag PLVNACLNSGLLVLTAGKGNVVRIVPPLIITEQELEKAAEILLQCLPALDRHG 451
Sy EIVKAAMBQGLLLAPAG-PKVLRFVPPLVVTEAEIAQAVEILRQAIATLV 429
EC QISQEAAKAGVMVLIAG—GNVVRFAPALNVSEEEVTTGLDRFAAACEHFVSRGSS 389
SC EVIKKARELGLLIITAGKSTV-RFVPALTIEDELIEEGMDAFEKAIEAVY—---A 423

B) Degenerate primers

S’Egion primer 5’-GARGCIAAYGRGCIGC : l7 mer (H8412)

 

 

3’Region Primer 5’ -ACICCICCYTCICC!TG: l7 mer (HB413)

 

Figure 1-16. PCR amplification of acetylomithine aminotransferase using
degenerate primers. Left lane contains 100 bp ladder DNA and the right lane showed
the PCR products obtained by PCR with degenerate primers. Three (Band A, B and C)
sets of DNA fragments were purified from agarose gel and used to make probes.

2 kb
1.5 kb

600 bp

 

 

Figure 1-17. Restriction Fragment Length Polymorphism (RFLP) mapping of the
G5:2D cosmid. Genomic DNAs were isolated from the N crassa strain Mauriceville,
and Oak Ridge #4450—4488 (Fungal Genetics Stock Center) strainsand 5 pg of each
DNA was digested with EcoRI, electrophoresed on an agarose gel and transferred to a
nylon membrane. Cosmid G5:2D, was labeled with Dig (Boehringer Mannheim Inc.) and
used as a probe. The restriction pattern in each lane is designated as 0 (Oak Ridge) or M
(Mauriceville). The location of the cosmid G5:2D is probably between vma-I and al—3 in
LG V.

57

LGV

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A AlB BIC
l 416 7|l

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leu-S M 010 OIM
mfa M 010 OIM
11v-2 - 01- -|-
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cog-6 M MIO OIM
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inl M MIO OIM
cit-2 M M10 01M
cya-Z M MIO 01M
hspe-l M M10 010
cmd M MIO OIM
hsp83 M MIO OIM
tom70 - Ml- OIM
Fer-20 0 M10 OIM
AP34b.3, R42.3 0 MIO OIM
8:9A 0 M10 OIM
exp-4 0 M10 010
clone 129 0 M10 MIM
AP16.2 O MIO MIM
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IIJ
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O O O O O O I O O O O O O O O O O I O I O O 0 Z O K 3 Z A m

0

O O O O O O O O O 0 0'0 0 O OIO O O O O O O O OIO

1.4 Literature Cited

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cyanide insensitive respiration in Neurospora crassa, pp. 495-507 in Mitochondria,
edited by R. J. Schweyen, K. Wolf, and F. Kaudewitz. Walter de Gruyter Co., Berlin.

Edwards, D. L., and B. W. Unger, 1978 Induction of hydroxamate-sensitive respiration in
Neurospora mitochondria. Transcription of nuclear DNA is required. FEBS Letters
85:40-42.

Elthon, T. E and R. C. Stewart, 1983 A chemiosmotic model for plant Mitochodria.
BioScience 331687-692

Elthon, T. E., and L. McIntosh, 1987 Identification of the alternative terminal oxidase of
higher plant mitochondria. Proc. Natl. Acad. Sci. 84:8399-8403.

Elthon, T. E., R. L. Nickels, and L. McIntosh, 1989 Monoclonal antibodies to the
alternative oxidase of higher plant mitochondrial. Plant Physiol. 89:1311-1317.

Evans, D. A. and R. C. Brown, 1973 m-Chlorobenzhydroxamic acid- an inhibitor of
cyanide-insensitive respiration in T rypanasoma brucei. J. Protozool. 20:157-160

Freitag, M., and M. S. Sachs, 1995 A simple blot assay to measure hygromycin B
phosphotransferase activity in whole cell extracts of Neurospora crassa. Fungal Genet.
Newslet. 42:26-28.

Genevois, M. L., 1929 Sur lafermmtation et sur la respiration chez les vegetaux
chlorophylliens. Rev. Gen. Bot. 41:252-271

Gessert, S. F., J. H. Kim, F. E. Nargang, and R. Weiss, 1994 A polyprotein precursor of
two mitochondrial enzymes in Neurospora crassa: Gene Structure and precursor
processing. J. Biol. Chem. 269:8189-8203.

Hanssens, L., E. d'Hondt, and H. Verachtert, 1974 Cyanide-insensitive respiration in
Moniliolla tomentosa and effect of drugs on respiration and polyol biosynthesis. Arch.
Microbiol. 98:339-349.

Hartl, F-U., N. Pfanner, D. W. Nicholson, and W. Neupert, 1989 Mitochondrial protein
import. Biochim. Biophys. Acta 988: 1-45.

Hartefi, Y., 1985 The mitochondrial electron transport chain and oxidative
phosphorylation. Ann. Rev. Biochem. 54:1015-1069

59

Henry, M. F., and E. J. Nyns, 1975 Cyanide-insensitive respiration: an alternative
mitochondrial pathway. Sub-Cell. Biochem. 4:1-65.

Hill, G. C. 1976 Electron transport systems in kinetoplastida. Biochim. Bi0phys. Acta.
456:149-193

Hill, G. C. 1978 Characterization of the electron transport systems present during
differentiation of African T rypanasomes. In: Functions of Alternative Oxidases (Degan,
H., Loyd, D. and Hill, G.C eds) FEBS meeting Vol 49, Pergamon, Oxford. pp67-77

Kumar, A. M., and D. 8011 1992 Arabidopsis alternative oxidase sustains Escherichia coli
respiration. Proc. Natl. Acad. Sci. USA 89:10842-46.

Lambowitz, A. M., and C. W. Slayman, 1971 Cyanide-resistant respiration in
Neurospora crassa. J. Bacteriol. 108: 1087-1093.

Lambowitz, A. M., J. R. Sabourin, H. Bertrand, R. L. Nickels, and L. McIntosh, 1989
Immunological identification of the altematve oxidase of Neurospora crassa
mitochondria. Mol. Cell. Biol. 9:1362-1364.

Lambowitz, A. M., E. W. Smith, and C. W. Slayman, 1972 Electron transport in
Neurospora mitochondria: studies on wild type and poky. J. Biol. Chem. 247 :4850-4858.

Lambowitz, A. M., and D. Zannoni, 1978 Cyanide—insensitive respiration in Neurospora.
Genetic and biophysical approaches. Pp. 283-291 in Plant Mitochondria, edited by G.
Ducet and C. Lance. Elsevier/North-Holland Biomedical Press, Amsterdam.

Laties, G. G, 1982 The cyanide-resistant, alternative pathway in higher plant respiration.
Annu. Rev. Plant Physiol. 39:519-555.

Li, 0., R. G. Ritzel, L. L. T. McLean, L. McIntosh, T. Ko, H. Bertrand, and F. E.
Nargang, 1996 Cloning and analysis of the alternative oxidase gene of Neurospora
crassa. Genetics 142:129-140

Lloyd, D. and S. W. Edwards, 1977 Electron transport pathways alternative to the main
phosphorylatory chain. In: Functions alternative terminal oxidases. FEBS 11th Meeting,
Copenhagen, Vol. 49.

McIntosh, L., 1994 Molecular biology of the alternative oxidase. Plant Physiol. 105:781-
786.

Meeuse, B. J. D., 1975 Thermogenic respiration in aroids. Annu. Rev. Plant Physiol.
26:117-126

60

Moore, A. L., W. D. Bonner, Jr., P. R. Rich, 1978. The determination of the proton-
motive force during cyanide-insensitive respiration in plant mitochondria. Arch.
Biochem. Biophys 186:298-306

Moore, A. L., and J. N. Siedow, 1991. The regulation and nature of the cyanide-resistant
alternative oxidase of plant mitochondria. Biochim. Biophys. Acta 1059: 121-140.

Nelson, L. D. and H. B. Brownstein, 1994. YAC libraries: a user's guide. W. H. Freeman
and Company, New York

Palmer, J. M 1976 The organization and regulation of electron transport in plant
mitochondria. Ann. Rev Plant Physiol 27: 133-157

Popov, V. N., R. A.Simonian, V. P. Skulachev, , and A. A. Starkov, 1997 Inhibition of
the alternative oxidase stimulates H202 production in plant mitochondria. F EBS Letters,
415287-90.

Purvis, A. C. and R. L. Schewfelt, 1993 Does the alternative pathway ameliorate chilling
injury in sensitive plant tissues? Physiol. Plant. 84:80-86.

Raskin, I., A. Ehmann, W. R. Melander, and B. J. D. Meeuse, 1987 Salicylic acid: a
natural inducer of heat production in Arum lilies. Science 237:1601-1602.

Rhoades D. M., and L. McIntosh, 1991 Isolation and characterization of a cDNA clone

encoding an altenrative oxidase protein of Sauromatum guttatum (Schott). Proc. Natl.
Acad. Sci. USA 88:2122-2126.

Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989 Molecular cloning: a laboratory
manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Sakajo, S., N. Minegawa, T. Komiyama, and A. Yoshimoto, 1991 Molecular cloning of
cDNA for antimycinA-inducible mRNA and its role in cyanide-resistant respiration in
Hansenula anomala. Biochim. Biophys. Acta 1090: 102-108

Sakajo, S., N. Minagawa, and A. Yoshimoto, 1993 Characterization of the alternative
oxidase protein in the yeast Hansenula anomala. FEBS Lett. 318:310-312.

Sargent, D. F. and CBS. Tylor, 1972 Terminal oxidases of Chlorella pyronoidosa. Plant
Physiol. 775-778

Schwab, A. J ., 1973 Mitochondrial protein synthesis and cyanide-resistant respiration in
copper-depleted, cytochrome oxidase deficient Neurospora crassa. FEBS. Lett. 35:63-
66.

61

Slayman, C. W., 1977 The function of an alternative terminal oxidase in Neurospora, pp.
159-168 in Functions of Alternative Terminal Oxidase, edited by H. DEGN, D. LLOYD
and G. C. HILL. Pergamon Press, Oxford.

Szakacs, N., 1978 The isolation and characterization of alternate oxidase deficient
mutants of Neurospora crassa. Ph. D. Thesis, University of Regina.

Szakacs, N. and H. Bertrand, 1976 Selection of mutants of Neurospora crassa defective in
the mitochondrial alternate oxidase system. Can J. Genet. Cytol 18:574

Tissieres, A., H. K. Mitchell, and F. A. Haskins, 1953 Studies on the respiratory system
of poky strain of Neorospora. J. Biol. Chem 205:423

Umbach, A. L., and J. N. Siedow, 1993 Covalent and noncovalent dimers of the cyanide-
resistant alternative oxidase protein in higher plant mitochondria and their relationship to
enzyme activity. Plant Physiol. 103:845-854.

Umbach, A. L., J. T. Wiskich, and J. N. Siedow, 1994 Regulation of alternative oxidase
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Vanlerberghe, G. C., and L. McIntosh, 1994 Mitochondrial electron transport regulation

of nuclear gene expression: studies with the alternative oxidase gene of tobacco. Plant
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Vanlerberghe, G. C., D. A. Day, J. T. Wiskitch, A. E. Vanlerberghe, and L. McIntosh,
1995 Alternative oxidase activity in tobacco leaf mitochondria: dependence on

tricarboxilic acid-mediated redox regulation and puruvate activation. Plant Physiol.
109:353-361.

Vollmer, S. J ., and C. Yanofsky, 1986 Efficient cloning of genes of Neurospora crassa
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Wang, Z., M. Deak, and S. J. Free, 1994 A cis-acting region required for the regulated
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Whelan, J and L. McIntosh, D. A. Day, 1993 Sequencing of the soybean alternative
oxidase cDNA clone. Plant Physiol 103: 1481

Yoshida, S. and F. Tagawa, 1979 Alteration of the respiratory function in chill-sensitive
callus due to low temperature stress. I. Involvement of the alternate pathway. Plant Cell
Physiol. 20: 1243-1250.

62

CHAPTER 2.

Cloning and nucleotide sequence of the catalytic subunit of DNA polymerase-y of

Neurospora crassa.

2.1 INTRODUCTION

Though its discovery mitochondrial DNA (mtDNA) four decades ago (Nass et al.,
1963; Reich and Luck, 1966), it was recognized as an essential genomic component in
eukaryotic cells and consolidated many of the existing basic concepts about cellular and
organelle biogenesis, non-Mendelian inheritance, and the molecular basis of
mitochondrial diseases (Griffiths, 1992; Shoffner et al. 1994; Bourgeron et al. 1995).
The functional state of mitochondria depends on nuclear-gene encoded factors for
mitochondrial gene expression, and mtDNA replication, repair and recombination.

The Neurospora crassa mitochondrial genome is approximately 62 kb in size. It
encodes 13 mRNAs required for oxidative phosphorylation and two ORFs of unknown
function. In addition, it also encodes two ribosomal RNA molecules and 25 tRNAs that
are required for translation of mtDNA-encoded mRNAs in the mitochondrial matrix
(Mishra, 1991). However, it does not encode any protein involved in the replication of
mtDNA.

The replication of mitochondrial DNA appears to be the result of the collaboration
of several enzymes, but the basic mechanisms of this event are not fully understood at

this time. While replication of the chromosomal DNA is strictly coupled to cell cycle

63

controls, mitochondrial DNA replication is not coupled in the same way. It is regulated
to accommodate variations in the rate of mitochondrial biogenesis (Clayton, 1992). The
mitochondrial genome is replicated by proteins encoded by nuclear genes, including
DNA polymerase-y (mip-I), which is the only polymerase involved in mtDNA
replication (Clayton, 1982). DNA polymerase-y has been cloned from 8 different
organisms, including Saccharomyces cerevisea, Schizosacchromyces pombe, Pychia
pastoris, Homo sapiens, Mus musculus, Xenopus laevis, Drosophila melanogaster, and
Gallus gallus (F oury, 1989; Ropp and Copeland, 1995, 1996; Ye et al.,l996; Lewis et al.,
1996). It is characterized by its resistance to aphidicolin and sensitivity to
dideoxynucleotide triphosphates (Bolden et al.1977; Knopf et al., 1976; Bertazzoni et al.,
1977; Wemette and Kaguni, 1986). It has been shown in vitro that purified y polymerase
has 3’ -) 5’ exonuclease activity (Kunkel and Soni, 1988; Kunkel and Mosbaugh, 1989;
Insdorf and Bogenhagen, 1989; Olson and Kaguni, 1992).

Here I report the molecular cloning and partial characterization of mip-I from N.

crassa, the gene coding the DNA polymerase-y of this fungus.

64

2.2 MATERIALS AND METHODS

2.2.1 Storage of N. crassa genomic DNA library.

The Orbach/Sachs cosmid library of N. crassa genomic DNA in pMOcosX
vector, which has a dominant selectable markers for fimgi (hygromycin resistance) and E.
coli (ampicillin resistance). Cosmid libraries were maintained at —80°C in separate

96-well microtiter plates containing LB medium supplemented with ampicillin.

2.2.2 Design of primers and amplification conditions.

The degenerate primers for amplification of a segment of the DNA polymerase-y
gene by PCR from N. crassa genomic DNA where designed on the basis of information
derived from highly conserved regions in the corresponding proteins of Homo sapiens,
Xenopus laevis, and Saccharomyces cerevisiae. These degenerate primers contain a
BamHI restriction site for cloning of the PCR product (Table 2-1). Amplification
reactions were initially tested using various concentrations of MgClz (2-10 pl of a 25 mM
stock), and varying annealing temperatures. Denaturation was for 1 min at 94°C:
annealing at 45°C for l min; and extension at 72°C for 2 min. This program was repeated
through 35 cycles, followed by 5 min at 72°C to ensure completion of the products.
Amplified DNA fragments were analyzed by electrophoresis in a 2% agarose (Seakem

ME) gel using TAE buffer at 95 volts for 1.5 hr. 5’- and 3’-specific primers (Table 2-1)

65

where subsequently designed on the bases of the nucleotide sequence of the cloned DNA
obtained by PCR with degenerate primers from the genomic DNA of N. crassa. These
two specific primers were used for locating the gene in the cosmid library of N. crassa

genomic DNA.

2.2.3 Cloning and analysis of PCR products.

Gel-purified, BamHI-digested PCR products were ligated into the plasmid vector
pBluescript SK+ (Stratagene) using standard protocols (Sambrook et. al., 1989).
Sequencing of DNA was performed by using an ABI Prism DNA sequencer. Analysis of

DNA and protein sequences was performed by using the DNASTAR software.

2.2.4 Screening of the N. crassa cosmid library.

PCR based screening was performed to identify a single cosmid which contains
the DNA polymerase gamma. The Orbach/Sachs pMOcosX cosmid library of N. crassa
genomic DNA was replica plated in 50 microtiter dishes. Cosmid clones were grown in
200 pl of selection medium (LB-amp, see appendix) at 37°C for about 16 hr and prepared
as 50 DNA pools, each containing 96 clones from one of the 50 plates. The pool DNAs

were first screened by PCR using primers specific for N. crass mip-l DNA (Table 2-1).

66

Individual positive cosmids were located by PCR and Southern hybridization with row-

and column-pool DNAs from the appropriate plates.

67

Table 2-1 : Sequences of degenerate PCR primers and N. crassa specific primers
used in this study. Abbreviations represent the following combinations of nucleotides:
N=A,C,G, or T; Y=C or T; I=deoxyinosine; K=T or G; H=A or T; M=A or C; D=A,T, or
G. The standard one letter code is used for the amino acids. Degenerate primers contain a
BamHI site (underlined) for cloning.

 

 

Degenerate PCR Primers
Amino acid Sequence Primer sequence
1 GT DLH 5’- primer GCGGGATCCGGNACNGAYCTNCAY
(2) WTRAM FC 3’- primer GCGGGATCCRAACATNGCICKNGTCCA
(3) RIYGA 5’- primer GCGGGATCCCGNATHTAYGGNGCN
(4) HDEIR 3’- primer GCGGGATCCMCGDATYTCRTCRTG

 

N. crassa specific primers

 

(5) 5’- specific primer AAGTTCGCATCTCAGCTTCTC

(6) 3’- specific primer GAAGCGGCGAGTGAGATAGTC

 

68

2.2.5 RNA electrophoresis and Northern blotting.

N. crassa mycelium was generated from conidia by 14-hr incubation on a rotary
shaker at 25°C, harvested by filtering through Schleicher and Schuell #480 filter paper,
and then washed with water and frozen in liquid nitrogen before pulverization. The
RNeasy total RNA purification protocol from QIAGEN was used to prepare total RNA
from N. crassa mycelium. RNA electrophoresis and Northern blotting were performed as
described by Sambrook et al. (1989). A 3.3-kb ClaI fragment of X25210C which is an
internal part of the mip-I gene was purified from an agarose gel as described previously
(Materials and Methods in Section 1). This gel-purified DNA fragment was used to
generate a 32P-labeled probe by using the Random DNA labeling kit from Boehringer

Mannheim, Inc.

2.2.6 Restriction Fragment Length Polymorphism (RFLP) mapping.

The location of cosmid DNA on the RFLP map of N. crassa was determined as
described by Nelson et al. (1998). Genomic DNA was prepared from N. crassa strains
#4450-#4488 (Fungal Genetics Stock Center) and the Mauriceville and Oak Ridge strains
according to the method of Lee and Taylor (1990), and 511g of each DNAs were digested
with EcoRI. Electrophoresis was canied out to separate the restriction fragments through

0.8% agarose gels before their transfer to a modified nylon membrane for Southern blot

69

analysis. Whole cosmids were used to generate Dig-labeled probes with the kit provided

by Boehringer Mannheim, Inc.

2.2.7 DNA sequencing and analysis of the DNA polymerase gamma gene.

DNA sequencing was performed by using an ABI Prism DNA sequencer.
Analysis of DNA and protein sequences was performed by using the programs of
DNASTAR. The amino acid sequences of the DNA polymerase gamma from different
species were aligned using the DNASTAR Megalign program. Some minor adjustments

in the alignments were made by visual inspection.

70

2.3 RESULTS AND DISCUSSION

2.3.1 Cloning of the Catalytic Subunit of N. crassa DNA Polymerase-y

Alignment of mitochondrial DNA polymerases from several species revealed
highly conserved regions of amino acids (Figure 2-1). Four of these regions were used
for the design of degenerate primers (Table 2-1) for PCR amplification of a portion of the
N. crassa mip-l gene from genomic DNA. An initial PCR reaction using degenerate
primers 1 and 2 on genomic DNA from N. crassa resulted in a product of the expected
size (~6OO bp, data not shown). A second round of PCR was performed using degenerate
primers 3 and 4, and it yielded a more obvious product of a lower size (~440 bp, data not
shown). This PCR product was isolated from the gel, and ligated into the BamHI site of
pBluescript SK+ (Stratagene) and cloned in E. coli. One clone, named pGaml, contained
an insert of the expected size, and, upon sequencing, was found to encode an amino-acid
sequence which showed 63.6% identity to the S. cerevisiae DNA polymerase-y. This
fragment was used as a probe to hybridize genomic DNA, and the result confirmed the
existence of a corresponding sequence in the N. crassa genome (data not shown). When
the Orbach/Sachs N. crassa genomic library was screened by PCR with specific primers
5 and 6 designed from the sequence of pGaml, plate pools G3, G13, X7, X14, X22, and
X25 gave positive results (Figure 2-2). When PCR and hybridizations were performed on
row and column pool DNAs from the X25 plate, a single cosmid (X25:10C) was

identified as containing the mip-I gene (Figure 2-3).

71

Figure 2-1 Amino acid homology comparison for the DNA polymerase-y genes of
Homo sapiens (HS), Xenopus laevis (XL), and Saccharomyces cerevisiae (SC).
Sequences used for primer design are indicated in bold letters and by asterisks.
Functionally conservative residues are shown as black letters in a yellow background.
The 3’-primer sequence WTRAMF C is highly conserved in all three yeast species
examined by Ye et. a1 . (1996).

HS GTDLHSKTATTVGISREHAKIFNYGRIYGHGQPFAERLLMQFNHRLTQQEAAEKAQQMYAATKGLRWYRL 997
X1 GTDIHSKTASTVGISREHAKVFNYGRIYGBGQPFAERLLMQFNHRLTQEQAAEKAKQMYAVTKGIRRYIL 967

Sc GTDLHTKTAQILGCSRNEAKIENYGRIYGAGAKFASQLLKRFNPSLTDEETKKIANKLYENTKG-K---- 795
* i i it iv 1' i * k *
Degenerate S’primer (l) Degenerate 5’ primer (3)

HS SDEGEWLVRELN—LPVDRTEGGWISLQDLRKVQRETARKSQWKK—WEVVAERAWKGGTESEMFNKLESIA 1065
X1 SKEGEWLVEELG-ISVERGEENSVNLQDLRKIQKDATKRSRRK--WNLVSRRIWTGGTESQMFNKLETIA 1034
SC TKRSKLFK ------- KFWYGGSESILFNKLESIA 821

 

Hs TSDIPRTPVLGCCISRALEPSAVQEE---FMTSRVNWVVQSSAVDYLHLMLVAMKWLFEEFAIDGRFCIS 1132
X1 MSPSPKTPVLGCRISRALEPTAVKGE—--FITSRVNWVVQSSAVDYLHLMLVAMKWLFEAYDIDGRFCIS 1101
SC EQETPKTPVLGCGITYSLMKKNLRANS--FLPSRINWAIQSSGVDYLHLLCCSMEYIIKKYNLEARLCIS 889

HS IHDEVRYEYREEDRYRAALALQITNLLTRCMFAYKLGLNDLPQSVAFFSAVDIDRCLRKEVTMDCKTPSN 1202
X1 IHDEVRYLVHSKDRYRAALALQITNLLTRCMFASRLGIQDVPQSVAFFSAVDIDKCLRKEVTMDCSTPSN 1171
SC IHDEIRFLVSEKDKYRAAMALQISNIWTRAMFCQQMGINELPQNCAFFSQVDIDSVIRKEVNMDCITPSN 959

*kfii’i’ ****‘k*

Degenerate 3’primer (4) Degenerate 3’primer (2)

72

Figure 2-2. Screening Neurospora crassa genomic library (Orbach/Sachs) by PCR
with specific primers 5 and 6 designed from the sequence of pGaml. Plate pools G3,
G13, X7, X14, X22, and X25 gave positive results.

L G1 G2 G3 G4 GS G6 G7 G8 G9 G10GllGlZGlSG14G15GléGl7

2kb

   

 

L G19 GZOG21G22 G23 024 G25 ”(13 X11 X2 X3 X4

.- n! . u- ‘ org A ‘1.“ . ‘"

2kb

600bp

100bp

73

Figure 2-3. Detection of mip-I. (A) by Southern blot hybridization in column and (B)
row pools DNAs fi'om cosmid DNA plate X25. The 350-bp mip-I specific probe detected
the column 10 and row C pool DNAs. (C) PCR screening of plate X25 row- and
column-pool DNAs was done with specific primers 5 and 6. It also detected row C and
column 10 in the X25 plate.

(A) X25 plate column-pool DNAs

1 2 3 4 5 6 7 8 9 10 11 12

 

74

(C) PCR detection of mip-I fiom row— and column-pool DNAs of plate X25.

LABCDEFGH1234567891011

7 Iris-D

600 bp,

300 b1»

 

75

2.3.2 Nucleotide Sequence of mip-I

The nucleotide sequence of the mip-l gene was determined by directly
sequencing cosmid X25:10C outwards with a series of synthetic primers from the
segment corresponding to the DNA cloned in pGaml. The sequence of mip-l is
deposited in GenBank under accession number AF111068. The gene has a
39l8-nuc1eotide open reading frame (ORF) encoding a protein of 1305 amino acids (146
kDa), and contains no introns. A mitochondrial targeting sequence and a cleavage site
for a mitochondrial presequence were found in the N-terminal region of putative protein
(Figure 2-4).

The DNA polymerase-y from Neurospora has a high degree of homology with the
other eight polymerases of its type that have been characterized so far (Table 2-2 and
Figure 2-5). These proteins all contain the three exonuclease motifs (Figure 2-4 and 2-5)
identifying an editing domain and the three motifs characteristic of the family-A DNA
polymerases. Ropp and Copeland (1996) reported five sequence insertion sites in higher
eukaryotes. However, the alignment shown in this paper (Figure 2-6) demonstrates that,
relative the fungal proteins, higher eukaryotic gamma DNA polymerases have 8 insertion
sites (inserts A-H) totaling approximately 200 amino-acid residues. Insertion sites A and
B are located between the Exo I and Exo II domains, inserts C, D, E, and F are located
between the last exonuclease domain and the first polymerase domain, one insertion site,
G, is situated between polymerase motifs B and C, and the last insert, H, is located after
polymerase motif C near the N-terminus of the insect and vertebrate polymerases. In

addition, the N. crassa and S. cerevisiae polypeptides have C-tenninal extensions of

76

approximately 200-270 amino-acids relative to all the other gamma DNA polymerases.
Nonetheless, the similarity between the amino-acids sequences in the extensions of the
yeast and Neurospora protein is relatively low when compared to the similarity in the
regions spanning the polymerase and exonuclease domains. The significance, if any, of
this extension is unclear. However, two potential cAMP- and cGMP-depentent protein
kinase phosphorylation sites, KKTT(1248-1251) and KKET(1259-1262), are found in the
extension of the N. crassa DNA polymerase-y. The S. cerevisiae polymerase-y
polypeptide also has four potential cAMP- and cGMP-dependent protein kinase
phosphorylation sites in the C-terminal extension, which are KKLT(1137-1140),
KKNT(1142-1145), KKPS(1204-1207), and RKSS(1232-1235). All the y-DNA
polymerases share potential phosphorylation and glycosylation sites in the middle of
proteins (Figure 2-6), but it is not clear whether or not the proteins are phosphorylated or

glycosylated in vivo.

77

Figure 2-4. Nucleotide and amino acids sequences of the N. crassa mip-I gene.
Putative regulatory elements including CAAT box (red), TATA box (blue), N. crassa
transcription consensus starting point (TCATCANC; Bruchez et al. 1993) are bold and
indicated with asterisks below the sequence. The cleavage site for the prepeptide is
highlighted in yellow in the amino acid sequence and the cleavage point is indicated by
an arrow. The locations of the degenerate primers are highlighted in pink in the amino
acid sequence and two mip-I specific primers are highlighted in green in the nucleotide
sequence.

grraraargrrargargrrrrr LanrnAPLAruafhfnnartanaffar

J 2 2 2 2 22 22 2 J:

J gLCdLuatgacaCt

 

 

 

 

frfarrrnanafrarfaarrnnrrarraarrf Luanrarnfrrafnrrrui L nrfnrnraf

2 2 J 22 2 2 J 2 2 2 J J JJ 2 2 2
rrrarrfrrnfff L dltl LL L LuananaLL Lnauut

2 2 222 2 2 J 2 J2 J 2 J J J J 2 2
LL 2 L L I L I _
Pfgd' J JJ 2 J J J "‘339*J J J J
L L Lnrnanrnrnarnarnaarrnafraaananr Lanrarrrnaarararnnnraraaar

J 2 2 2 2 2 22 2 2 2 2 2 2 J 2 2 2 J22 2

L L urrrrr L L ounrnnaarrrrrrraaarrrrrrrrrrrrrrrrrr
2 2 2 2 J JJJJ 222 22 22 22 2

 

.1 .12 J J 2 2 2

rrrrrrrrrrrrrrrrrrrrrrrrgrrggarorgogaargrnrarga;Jr

 

E:?: , LPgardL rxdfrragfrt tataaccagtcaccgtggcgtatcnccact

‘0 *‘k ******* fii‘ifi'fii‘i

rrrranarr Larrarnarrrnaaarrrnarrr L Udll L L LP
2 J 2 J J J J 2 22 2

 

M L T P V

J riffrrrrgararrrr
R V L R R A N L F S R Y P

(‘gr 1
J 2 2 2

R C R T V P N A T V A T A A

 

L nnnar‘r ( nm 1.1? J JLgr‘f J J 2 JJJ J J J J

R Q )L G H L R W Ii+8 T I A O V L E R K G L G V P S T A
CgCCatadLgdgdthgth J J J J L J J L J J L L
G V Q Q L S E H L Y K Q L F P R G N T P P
J J a: L J rnr Laagarrrrrrrf

A P E L I E L A K H D L L G K T T D K T P P

afrnrafrr J J J J J
I A P Q L V D A A E P
ffPFffarrrniJ I (I

F L T H A K O F A D A H L P P K P T S W V R R S G W T

 

aaar J J JJ J J J J rrrJ JJJ grgrrrrgafgrggaggrrargraf
K Y N R D G T T E N D V L P Q G N M M C F D V E V M Y

a n a n rnrnrrnnrarrrrnnarnrrrnararnrrrnnrrnrrarrarnnrrnrfrnna
JJ 2 2 2 22 22 2 J2 2 J2 2 2 2 22 2 22
K D N P Y A V M A C W

A G T P D A W A W L S

 

 

 

99 ““‘"' JJ J 2 2 J ‘“ J JJJ 22
E T E N K A Q L V P G D P T V D R I I V G H N I G Y
aarrnrgrraanararr 93994“? J L L LffnrfJ L erghargrrgrr
D R A K I L E E Y D L K Q T HR N F F L D T M S L H V A

 

 

 

 

 

933d9'grragiglagad“J J J J JJJ J 22 J JJ JJ J 222 J “fl"
E S A S V E L Q E V L Q G G S L T A E E A D L W V D K
agorrrarraarfrgrr J irrarrrraargrraanar J JJ a: J rgrg
S S I N S L R D V A Q F H L N V K I D K D I R D V F A
rgaaargfgafrrrJ JLg RP arrrrrrgarrrarrgu J Lnrr adnf J

(Y n t
D R E T N V I L N Q L D D L L T Y C A A D V Q V T H O

L r L LgrrrrrafrrrgfragrrrrgrrgrrrI n
P N F L G V C P H P V A A L R H L A S

arrrarrannrrnrnff
2 JJ J 2
V Y O V V F

 

 

 

grrarfrfgrrmn J rgggai’af‘ffafal J J J JJ LAt ( rgr‘aaargrr JJ J‘i‘
V I L P V N K T W D T Y I T A E A T Y L Q N L H G V
J J Ud'L J J J H" J. L LL 9” JJ J
Q E R L F T L M E R T L D Y K A D P E K Y L S D P W L
2 2 22L L‘””9”f J J ZLJJ 22 2 naaf
S Q L D W S G Q E I K M A K P K K K G D V E R P A L N

79

810
32

891
59

972

1053
113

1134
140

1215
167

1296
194

1377
221

1458
248

1539
275

1620
302

1701
329

1782
356

1863
383

1944
410

2025
437

2106
464

2187

cagaaactaccaggctatccccaatggtacaaggatctgtttgtaaaggttcccaaggagctctcgggcttggacgaaccc
0 K L P G Y P Q W Y K D L F V K V P K E L S G L D E P

gacaaagagcaggaaaatagaaaggctcgacatgaattcatcaatctcaccgtccgttcacggatagccccgttgctcctc
D K E Q E N R K A R H E F I N L T V R S R I A P L L L

aaattatcatgggaaggctaccccttattctggtccgatcaatttggctggacgttccaggtcccacgagaaaaagcagag
K L S W E G Y P L F W S D Q F G W T F Q V P R E K A E

acctttattcagcgccagatgacacctgttcaatttgaggacccagatgtcgatgatcgacttaggatggatgttgatcac
T F I Q R Q M T P V Q F E D P D V D D R L R M D V D H

aagtacttcaaacttcctcacaaagatggcccgaacgctagatgtgtcaacccaatggccaagggttatcttccctacttt
K Y F K L P H K D G P N A R C V N P M A K G Y L P Y F

gaaaagggcatcctttcttccgagtacccctacgcaaaggaagccctggaaatgaatgcttcttgttcatactggatcagt
E K G I L S S E Y P Y A K E A L E M N A S C S Y W I S

gctcgagagagaatcaagaatcagatggttgtctatgaagatcagcttcctccgtctcagagatttgtcaacaaggatgca
A R E R I K N 0 M V v Y E D Q L P P S Q R F V N K D A

gacagcaacacccctattggcggctttgttcttccccaggtcattcctatgggtaccattactcgtcgtgctgtcgagaga
D S N T P I G G F V L P Q V I P M G T I T R R A V E R
acatggttgacggcatccaatgccaagaagaaccgtgttggatcagagctcaaagccatggttcgcgcaccaccaggttat
T W L T A S N A K K N R V G S E L K A M v R A P P G Y

gtctttgtcggtgccgatgttgactcggaagaactttggattgcctctgttgttggagacgctactttcaaactgcatggt
V F V G A D V D S E E L W I A S V V G D A T F K L H G

ggtaatgccatcggcttcatgacccttgagggtaccaaatcccagggcactgatctccacagtcggacggcttccattctg
G N A I G F M T L E G T K S Q kiL_’m‘ “1236 S R T A S I L

-+Degenerate S'primer (1)

 
 
 

ggcatcacccgtaatgacgcaaaagtgtttaactatggccgtatctacqchccggtct
GITRNDAKVrNYGs-r,.ez~r:--,,GJ,_..JNGLKFASQLL
—+ Degenerate 5’primer(3) -§ S'specific primer

 
 
 

cggcagttcaaccctagcttgaccgaggcggaaacgactgccatcgcgacaaagctctacgatgccaccaaaggtgccaaa
R Q F N P S L T E A E T T A I A T K L Y D A T K G A K

actaaccgcaagagcctttacaagcggcccttctggcgaggcggtaccgagtccttcgtgtttaacatgcttgaagagttt
T N R K S L Y K R P F W R G G T E S F V F N M L E E F

gccgagcaagagcgtcctcgtaccccggttctcggtgcgggcatcacagaggccctcatgagccgctgggtcagcaaaggc
A E Q E R P R T P V L G A G I T E A L M S R W V S K G

2268
518

2349
545

2430
572

2511
599

2592
626

2673
653

2754
680

2835
707
2916
734

2997
761

3078
788

3159
815

(5)

3240
842

3321
869

3402
896

gggttcctgacttcgcgtatcaactgggccatccaatctagtggtgtcgattaccttcacttgctcatcatcgctatgll' 3483

G F L T S R I N W A I Q S S G V D Y L H L L I I A M D

aacctcgcctgccggttggccatcactgtgcacgacgaaatccgctatcttgccgaggaacct
Y L T R R F N L A C R L A I . . :

+— 3’ specific primer (6) e— Degenerate 3’primer (4)

 

 

gacaaataccgggtagccatggcactgcagattgccaacctgtggactcgtgtcatgttcgcgcagcaagtcggcattcaa
D K Y R V A M A L Q I A N L -"w- ; -

 

 

e-Degenerate 3'primer (2)

gatctaccgcagtcctgcgccttcttcagcgctgtggacatcgaccacgtgcttcggaaggaggtcgacatggattgtatc
D L P Q S C A F F S A v D I D H v L R K E V D M D C I

acgcctagtaacccgatacccattgcgcacggcgagagcatcgatatcttccagatcctggagaagggagacgacgcaaag
T P S N P I P I A H G E S I D I F O I L E K G D D A K

ctggatgacagcattgtccctcagtctcaatatgcacctcgcctggagaacatcccgtatacgcctcgtgtgcctgtgatg
L D D S I V P Q S Q Y A P R L E N I P Y T P R V P V M

cagaggcttcgcgagagggccgaggccggcgatcaccaagccttccttcggttcatccgggcacagattaccaattccgat
Q R L R E R A E A G D H Q A F L R F I R A Q I T N S D

80

923

3564
950

3645
977

3726
1004

3807
1031

3888
1058

3969
1085

gaagagctgaagaggatcatcgcagagacaaggtatagtgacccatatggtgccttttccctggcttcgaatggaagagta
E E L K R I I A E T R Y S D P Y G A F S L A S N G R V

tcaggcaatccacatcagcggcatgccgctgtacatgcttcgacaaagacggctgcggctccttcaaaaccttccatcgca
S G N P H Q R H A A V H A S T K T A A A P S K P S I A

agtcgtttcgattccgtctcgcaggcatcgaggatcaagtccgtcgcagctggcagtgatgagcccaccatcagagcgacc
S R F D S V S Q A S R I K S V A A G S D E P T I R A T

aaagcgcagggcaaagccatggcaaaagccagtggtacaaaacttgctgcctccacgaaggataccgtcctcaacgtaacg
K A Q G K A M A K A S G T K L A A S T K D T V L N V T

atcaagaagaaggtggcggcccccgagatggcggctgttccgtcaacctcttctgaatctaagtccaaggcttcagccaca
I K K K V A A P E M A A V P S T S S E S K S K A S A T

acaagcacgacaaccaccgagaacgccaccgcatccccttcatcctcatcgaacgtcgatgcaaagaaaaccacatccaaa
T S T T T T E N A T A S P S S S S N V D A K K T T S K

accaagccgacccacaagaaggagactgaaggggaaccatttccctcccttgacgaccccgtcatcgccgcccgcctcgaa
T K P T H K K E T E G E P F P S L D D P V I A A R L E

gccgtctccaagacttcaccagggaccagggcttccgtcgccgcgaagctagacgcccttgcctccttttgccatgcgagc
A V S K T S P G T R A S V A A K L D A L A S F C H A S

tgctgcggctgctgaggctgctgtcaccaccaccactactccagagcacgccaaccaaccctcccccgttgctcccaaggc
C C G C ***

aaaggaacccacaaccaccaactggcagaaaaggtctactgaaacctaccgccgtccccaaaaaccccactcctactctca
cgcccactcaccaaaaagtctaatccacattcacaccaaccacaccaaaacccgtcggtcgtcctcgtacactcccccatc

ctcggctacaccgctcccaagaaaaag

81

4050
1112

4131
1139

4212
1166

4293
1193

4374
1220

4455
1247

4536
1274

4617
1301

4698
1305

4779

4860

4887

Table 2-2. Percent similarity among 9 different y—DNA polymerases. Nc, Neurospora
crassa ; Pp, Pichia pastoris; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces
pombe; Hs, Homo sapiens; Mm, Mus musculus; X1, Xenopus laevis; Dm, Drosophila
melanogaster; Gg, Gallus gallus.

 

Nc Pp Sc Sp Hs Mm X1 Dm Gg

 

*** 41.8 37.3 45.6 29.2 29.5 29.6 26.8 36.0 No
1...... 48.4 41.9 30.8 29.9 30.6 26.5 31.2 Pp

*** 41.9 23.9 24.2 22.5 21.7 33.4 Sc

.24 31.7 31.6 32.1 27.4 32.0 Sp

*** 82.4 64.8 39.3 74.7 Hs

*** 63.5 37.4 74.0 Mm

*** 37.0 70.8 x1

*** 39.3 Dm

*Iklk Gg

 

82

Figure 2-5. Linear map of the N. crassa mip-I gene. The ORF is represented by a wide
grey bar. Putative regulatory elements including CAAT box, TATA box, N. crassa
transcription consensus starting point (TCATCANC; Bruchez et al. 1993), and the
possible cleavage site for the mitochondrial presequence are indicated. Conserved
exonuclease and polymerase motifs are shown as labelled black boxes.

ATT Box
[TATA Box

Transcription consensus sequence (TCATCAN C)
(lavage site for mitochondrial presequence

 

 

 

 

15:3

 

1 km 111 LMotifc

Exo II Motif B
Exo I Motif A

250 bp

83

Figure 2-6. Multiple alignment of DNA polymerse-y sequences. Amino
acid-sequences were aligned with the DNASTAR program. Functionally conservative
residues are shown as black letters in a yellow background and the residues that are
identical in all the polymerases are marked with asterisks below the sequences. Amino
acids that are highly conserved, but different in the fungal polymerases from those that
appear at the corresponding position in the animal polymerases, are shown in white
letters in a black background. The locations of exonuclease and polymerase domain
sequences are indicated above the aligned sequences. The mammalian and insect DNA
polymerases have 8 insertion sites (inserts A-H) relative to the fimgal polymerases. The
amino acid sequences that were used to generate of four degenerate primers are indicated
by arrows below the aligned sequences The possible glycosylation site is indicated by
black triangles (A). A possible Y-phosphorylation site is indicated by black dot (O). No,
Neurospora crassa (AF 111068); Pp, Pichia pastoris (1149510); Sc, Saccharomyces
cerevisiae (J05117); Sp, Schizosaccharomyces pombe (Z47976); Hs, Homo sapiens
(U60325); Mm, Mus musculus (U53584); Xl, Xenopus Iaevis (U49509); Dm, Drosophila
melanogaster (U60298); Gg, Gallus gallus (U60297).

84

NC IL-TPVRCRTVPNATVATAARVLRRANLFSRYPRQL-GHLRW DST IAQVLERKGL -------- GVPSTA.
I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pp I---IIR ————— Rrvarr LST sass I —————
Sc: I‘I‘KLMVRFECMLRMVRRRPLRVQ ————— FCA ————————— RWFST ------ KKNA ———————— AEAP-—I
Sp IF- YKA— CPSTLTCSKWIHSIIKTKKFLYCRH ————————————————— YSSKSF ———————— IDNAPLI
Drn IQFHLIRKYAs—IIIJ. . irimoovnIrMVKPPL‘ KVNKPKKPENVENGPTEYA
Hs lsmnWRKVAflATvGPGPVPAPGRIVISSVPASDPSDGQRRRQQQQQQQQQQQQQPQQPQVLSslGGaL:
Mm ISILI'WKKVAEAK'ASGPVPATEGIVISSVLAPVPSDGRP w.
x1 INILIIQK---ITSINPS ——————— IRIRGC ———————— RYRRCSYAPQLH ———————— AKPLEersuR:
69
NC mmwmm“—-znuniDP FI-nwl E: 1|)K'I"HI ur1___ _
Pp _ _ _ n—-———E—nl.u Mlivtmll
Sc m J _ _ ,N m—smmxnl LITDPISFPIPW
Sp- PQQNTQISQLHII- mm TIMSFNFRIPP
Dm EleemmTPQAPRsrSIQQUASAIWY--KDELRRIGVDIESSAPVSMKMAI.RHAII
H5 Mn mm -_ -l-
I... .2... , , , , III-8662A». - -__-- '5: . ,,
X1 7 ___V II'BCKOVQPIAIEDIQ“! ‘‘‘‘‘ _ I “mnvuxmmxuunl.
Gg

~h * i * i- i ii i

 

 

 

Hs amt-AWPYLEAANILLQA .JJJI , KIQAIIERILVFDVEVCI.
x1 8mm ’ " "JNC-WMQTMQSWAT—CKEL'DE‘IDIME‘ «a

 

 

 

** i: ** i * iii!-

NC
PP
Sc
SP
Dm
Hs
Mm muralsmmmsoflPAfiMGGsrsmss"11(ro —————— Q1303.
X1 Im TSMNCNYM——IKNNW —————— TERI.

 

 

G9
to not I:

NC
Pp
Sc
SP --IARIAYIRSQSTETSEDDDS
Dm SKIE
Hs
Mm WGHNVSFDRAHIREQYIEQMLDTMSMHMAISGLSSFQWQEQQ DPAVHKARAEVPE
x1 J Govt-Wal- OFVKQHIKKTRS

 

 

g
on" at "to.

85

198
151
176
176
190
203
186
177

240
197
221
220
254
265
248
239

306
267
285
288
307
335
317
309

Nc

PP
Sc

SP
HS

x1
69

Nc

PP
Sc

SP
HS

x1
G9

NC

Pp
SC

SD
HS

x1
69

Nc

Pp
SC

3?
HS

x1
Gg

NC

PP
SC

SP
HS

x1
69

VELQEVLQGGSLTAE-—EADLWVDKSSINSLRDVA N IDKDIRDV NVILNQLDDLLTY
EELQELLDSTSIEDPMLEDNPWINKSSLNSLERVA CK KKDIRNS PDELRGNFDELMQY
s -------- ISIED--—YDDPWLNVSALNSLKDVA CK DLDKTDRDF s STIIENFQKLVNY
SSFDDDYQN-YLKQE--P---WLAHSSVNSLKDVA CN- LDKSKRDD s PILQKLNELITY
----PAAEDLG ---------- WLEQSSLNSLVELHRLYCGGDTLSKEPRNIFVEGTLEQVRQSFQSLTNY
ARRGPAISSWD ---------- WLDISSVNSLAEVHRLYVGGPPLEKEPRELFVKGTMKDIRENFQDLMQY
ssowssosswo ---------- WMDISSANNLADVHNLYVGGPPLEKEPRELFVKGSMRDIRENFQDLMQY
NFSGSPISSWD ---------- WVNISSINNLADVHALYVGGPPLEKEARELFVKGSMSDIRTEFQELMR!

* *rk'k ** * **
CAADVQVTHQVYQVVFPNFLGVCPHPVSFAALR SVILPVN—KTWDTYIET TYLQMLHGVQERLF

CAKDVFATGKVFQKVYPKFKKLIPHPVTLAALK SCILPTT-TKWEDYIETS YQESRRMIEKNLH
CATDVTATSQVFDKIFPVFLKKCPHPVSFAGLK SKCILPTKLNDWNDYLNS SLYQQSKVQIESKIV
CAHDTYSTHQVFKKVFPQFLEVCPHPATFSAML SVFLPVN-HSWTRYING QYQQMIQLVDQKLS
CASDVEATHRILRVLYPLYAERFPHPASLAGMLEMGSAYLPVN-SNWERYIREAQLTYEDLSIEAKYHLG
CAQDVWATHEVFQQQLPLFLERCPHPVTLAGMLEMGVSYLPVNHQNWERYLAEAQGTYEELQREMKKSLM
CARDVWATFEVFQQQLPLFLERCPHPVTLAGMLEMGVSYLPVN-QNWERYLTEAQNTYEELQRDDEEVVV
CALDVQATHEVFQEQFPLFMERCPHPVTLSGMLEMGVSYLPVN-QNWERYLDEAQTSYEELQKEMKKSLM

 

TLMERTLDYKADPEK—YLSDPWL DWSGQEIKMAKPKK+ -----------------------------
VICEETVKLKDDPTKPWENDPWL DWTIDPIRLTK--—‘ ------------------------------
QIIKDIVLLKDKPDF—YLKDPWL DWTTKPLRLTK—--< ------------------------------

 

 

 

QYAEKAKDLINTKDT—VLKDPWL DWT——PCNLYRKLK ------------------------------

 

 

 

 

 

 

 

 

RRAEEACSLLLDD-~QYRQNLWLWDEDWSVQELKLKQPPKRK ------------------- PLPTVELKD
DLANDACQLLSGE—-RYKEDPWLWDLEWDLQEFKQKKAKKVKKEPATASKLPIEGAGAPGDPM---DQED
VWLMMPGQLLSGE--RYKEDPWLWDLEWDLQEFKQKKAKKVKK-PASASKLPIEGAGPFGDPM---DQED
KLANDACQLLTKD--AYKEDPWLWDLEWDIQESKQKKTKISKKQ ————— KKANEAAESVGNKLVEDHNED
* *' *
C
------ KGDVER--—---------------PALN-QKLPGYPQWYKDLFVKVP-KELSGLDEPDKEQENR
------ KGEI---—-—------—-----—--HKN-QKLPGYPNWYKQLIVK--—NEL——-—-----—--—
------ KGv—------------------—-PAKc—QKLPGFPEWYRQLFPs---KDTVEP—-———-—---
------------------------------ -KAT-QEVPVVPKWYKKAYCKTE-KRAV-------—----
SGNTPEERRLQ-AKFQHLYDQQ ------ ALLPARRPLLPGYPLWYRKLCRKPPAKRADEILE--DDEEPW
LGPCSEEEEFQQDVMARACLQKLKGTT-ELLPKRPQHLPGHPGWYRKLCPRL ------------- beam!
PGPPSEEEELQRSVTAHNRLQQLRSTT-DLLPKRPQHLPGHPGWYRKLCPRL ------------- enema
PGPPTEKEE-SRPSMGKLYLEDLKLKTLPLLPKRNQHLPGHPGWYRKLCPKL ------------- EDPDW
------------------------------------------------------------------ EEG!
‘k * * *
I)
KARHE 112$ IAPLLLKLSWEGYPL DQ FGWTFQVPR- --1.=: - ----------------------
------- TSPLLLKLAWNGNPL IQTQGWCPKVPKNKTE--—---------—-—--------
------- KI I IIPILFKLSWENSPVI KESGWCFNVPHEQVE---—--—-—--------------
-------- PILLRLKWKKHPL DTYGWVFSVERTSKD--------—-------------
SPGASEISTGM--QIAPKLLSLCWEGYPLHYEREQGWGFLVPFRSDSEGVDRL--PMEQLLAHCPVPEFA

TPGPSLLSLQM--RVTPKLMALTWDGFPLHYSERHGWGYLVPGRRDNLAKLPTGTTLESAGVVCPYRAIE
APGPSLLSLQM--RVTPKLMALTWDGFPLHYSDSHGWGYLVPGRRDNLTEPPVSPTVESAAVTCPYRAIE
LPGPGLISLQM--RLTPKLMRLTWDGYPLHYSEKHGWGYLVPGRKNN----KLNNEEEEEIIPCPYRAIE
VPGPSLISLQM--RVTPKLMRLAWDGFPLHYSEKHGWGYLVPGRQDNLP---—AASAEPEGPVCPHRAIE

it at * rk * ** * .....

86

373
336
343
351
362
395
377
369

442
405
413
420
431
464
446
438

482
443
450
458
480
529
510
501

525
469
479
483
541
585
566
557

570
509
519
522
607
653
634
621

68

NC

PP
SC

SD
HS

x1
Gg

NC

Pp
SC

SP
HS

x1
69

NC

Pp
SC

SD
HS

x1
Gg

 

 

 

 

 

 

------——-—--—---—E! ----------------- (AET--FIQRQM TPVQFE---DPD
------------------------------------ EYEKLNFVMVSL KKLSEDPGFESI
------------------------------------ YKAKNYVLAD- PsvsosE-—EEI
— ----------------------------------- .IEM--LLDQGL VPCSRE---E--
RL ----------- SASKAESDMA ----------------- FDMLPGQVEQHLGKREHYKKLSQK ------

SLYRKHCLEQGKQQLMPQEAGLAEEFLLTDNSAIWQTVEELDYLEVEAEAKM-ENLRAAVPGQPLALTAR
SLYRKHCLEQGKQQLEPQEVDLAEEFLLTDSSAMWQTVEELGCLDVEAEAKM-EN---SGLSQPLVLPAA
DIYAEYSKNKTKDGCLSQHSTIPEEFMLTDDNSMWQKVEELSRTEMDLSSEVPATAKAKKRNNSSEHPVK
RLYRQHCLQRGQEQ-PPEEAGVEDELMVLEGSSMWQKVEELSQLELDME-RP-GRAEQSQMQDEDGLPEL
OIO

 

 

F
VDDRLRMDVDHKP -------------- FKLPHKDGPNARCVNPMAKGYLPYFEKGILSSEYP---YAKE
RAEDLK---NFT~ -------------- KVPHPDGPSARVTNCMTKSCLGFFEKGFLNSQYP---LAKD
RMNNLGLQCTGV --------------- FKVPHPNGPTFNCTNLLTKSYNHFFEKGVLKSESE---LAHQ
-DTKLDYN-NYF FKVPHKDGPEARCCSPLSKSYHAYFEEGILQSDYE--—VAKK

 

GGPK-DTQPSYHHGNGPYNDVDIPGCWFFKLPHKDGNSCNVGSPFAKDFLPKMEDGTLQAG-PGGASGPR
CAPK-SSQPTYHHGNGPYNDVNIPGCWFFKLPHKDGNNYNVGSPFAKDFLPKMEDGTLQAG-PGGASGPR
LEMEFDSLPDNHHGNSPCGDVNVSGCWFYKLPHKDGNANNVGSPFAKDFLPKMEDGTLQAS-TGDSSATR
VE—E-SSQPSFHHGNGPYNDVNIPGCWFFKLPHKDGNENNVGSPFAKDFLPRMEDGTLRAA-VGRTHGTR
BQQRLETQ--YQGSGVWCNKVLDDCCFFLKLPHKNGPSFRVGNPLSKDFLNKEAENVLSSGDPSCQAAAR

*** 'k 'k *

  
  
  
 

ALEMNASCSYWI ERIKNQMVVYEDQLPPS-QRFVNKDADSNTPIGGF--VLPQV ITRRAVER

ALQMAVASSYW SRERIMNQFVV -------- FE ------------- DDMGYILPQI TITRRAVEN
ALQINSSGS ERIQSQFVVPNCKFPNEFQSLSAKSSLNNEKTNDLAIIIPKI TITRRAVEN
ALEMSASCSYW DRIRSQMVVWDKD ---------- AELGVPSSVDGFGIILPCI TVTRRAVEN
VIDIARMMSYWRNNRDRIMGQMVVWLDS--QQLPNEFTGEK--CQP-IAYGAICPQVVACGTLTRRAMEP

ALEINKMISFWRNAHKRISSQMVVWLPR--SALPRAVIRHPDYDEE-GLYGAILPQVVTAGTITRRAVEP
ALEINKMISFWRNAHKRISSQMVVWLPR--SALPRVVTRHPAFDEE-GHYGAILPQVVTAGTITRRAVEP
ALEINKMISFWRNAHKRISSQMVVWMKK--NELHRTITRDPEFDEE-NKYGAILAQVVSAGTITRRAVEP
ALEINKMVSFWRNAHKRVSSQVVVWLKK--GELPRAVTRHPAYSEE-EDYGAILPQVVTAGTITRRAVEE

* * t t ** ** **** *

  
   
  
    
 

   
  
   
 

TWLTASN VGSELKAMVRAPP GADVDSEELWIASVVGDATFK-LHGGNAIGFMTLEGTKSQ
TWLTASNAK GSELKSLIEAPKGY GADVDSEELWIASLIGDSVFK~IHGGTAIGWMTLEGTKNE
TWLTASN IGSELKTQVKAPPGY GADVDSEELWIASLVGDSIFN-VHGGTAIGWMCLEGTKNE
TWLTASNSK GSELKAMIRAPDGY GADVDSEELWIVALMGDSQFR-LHGATALGMMTLEGKKSE
TWMTASNSRPDRLGSELRSMVQAPPGYRLVGADVDSQELWIASVLGDAYACGEHGATPLGWMTLSGSKSN

TWLTASNARPDRVGSELKAMVQAPPGYTLVGADVDSQELWIAAVLGDAHFAGMHGCTAFGWMTLQGRKSR
TWLTASNARPDRVGSELKAMVQAPTGYVLVGADVDSQELWIAAVLGDAHLAGMHGCTAFGWMTLQGRKSR
TWLTASNARADRVGSELKAMVQVPPGYHLIGADVDSQELWIAAILGEAHFAGIHGCTAFGWMTLQGKKSS
TWLTASNARADRVGSELKAMVQVPPGYSLVGADVDSQELWIAAVLGEAHEAGMHGCTAFGWMTLQGKKSD

** **** * *‘k‘k‘k * ** ****** *rk'kir ** i 'k 'k * *

  
   
   
 

 

GTDLHSRTA ITRNDAKVFNYGRIYGAGLKFASQLLRQFNPSLTEAETTAIAT DATKGAK
GTDLHSKT ISRNEAKIFNYGRIYGAGIKFTTTLLKKFNPALSDAEAKATAN TATKG-I
GTDLHTKTA CSRNEAKIFNYGRIYGAGAKEASQLLKRFNPSLTDEETKKIAN ENTKG-K
GTDLHSKT VSRDSAKVFNYGRLYGAGLKHTTLLLMQMNPTLKTAEAKELA STKGVK
GSDMHSITAKAVGISRDHAKVINYARIYGAGQLFAETLLRQFNPTFSASEAKAKAMKMFSITKGKRVYRL

GTDLHSKTATTVGISREHAKIFNYGRIYGAGQPFAERLLMQFNHRLTQQEAAEKAQQMYAATKGLRWYRL
GTDLHSKTAATVRIHREHAKIFNYGRIYGAGQSFAERLLMQFNHRLTRQEAAEKAQQMYAVTKGLRRYRL
GTDLHSKTASTVGISREHAKVFNYGRIYGAGQPFAERLLMQFNHRLTQEQAAEKAKQMYAVTKGIRRYIL
GTDLHSKTAATVGISREHAKVFNYGRIYGAGQPEAERLLMQFNHRLTQQQAREKAQQMYAVTKGIRRFHL

* * * *‘k ** *** * **** ** * * ***

[Degenerate 5’primer (I) ~ [Degenerate 5’ primer (3)

 

 

 

 

87

 

588
532
538
539
644
722
700
691
135

640
581
590
588
790
768
760
202
710

707
630
660
648
775
857
835
827
269

776
699
729
717
845
927
905
897
339

843
765
795
784
915
997
975
967
409

 

Nd

PR
80

____________________________________ >TNR---—K---SLYKRPFWRGGTESFVFNMLEEFA
_______________________ 1»SGRYD-KK--—-———-SIWYGGSESIIFNRLEAIA
___________________________________ I TKRSKLFK--—---—KEWYGGSESILFNKLESIA

 

 

 

Hs

x1
Gg

NC

Pp
Sc

SP
HS

x1
69

 

------------------------------------ SKMSKRLQEMGLPKLTFWSQGTESFVFNKLEAMA
R-E—E-FHDELEDRAYSSYEASRLAIQRNRTLA ------------- EVFHRPKWQGGTESAMFNRLEEIA
SDEGEWLVRELN-LPVDRTEGGWISLQDLRKVQRETARKSQWKK-WEVVAERAWKGGTESEMFNKLESIA
SADGEWLVKQLN—LPVDRTEDGWVSLQDLRMIRREASRKSRWKK-WEVASERAWTGGTESEMFNKLESIA
SKEGEWLVEELG-ISVERGEENSVNLQDLRKIQKDATKRSRRK-~WNLVSRRIWTGGTESQMFNKLETIA
SEEGEWLVKELE—LAVDKAEDGTVSAQDVQKIQREAMRKSRRKKKWDVVAHRMWAGGTESEMFNKLESIA

* * ** ** ** *

SSGVDYL IAMDYLTRRFNLAC IT
SSGVDYL ISMDYLIKLFDID IT

   
  
  
 

EQERPRTPVLGAGITEALMSRWVSKGG--FLTSRI
EMAHPKTPVLGAGITAPLQKANLSTNN--FLTSRI
EQETPKTPVLGCGITYSLMKKNLRANS--FLPSRI SSGVDYL CSMEYIIKKYNLE IS
QLPSPRTPVLDAGIQQALSSKNLSKNS--FMTSR SSAVDYL VSMNHLIKKYYL LT
TGSQPRTPFLGGRLSRALEADTGPEQEQRFLPTRINWVVQSGAVDFLHLMLVSMRWLMGS---HVRFCLS
TSDIPRTPVLGCCISRALEPSAVQEE---FMTSRVNWVVQSSAVDYLHLMLVAMKWLFEEFAIDGRFCIS
MSDTPRTPVLGCCISRALEPSVVQGE~--FITSRVNWVVQSSAVDYLHLMLVAMKWLFEEFAIDGRFCIS
MSPSPKTPVLGCRISRALEPTAVKGE---FITSRVNWVVQSSAVDYLHLMLVAMKWLFEAYDIDGRFCIS
LSASPQTPVLGCHISRALEPAVAKGE---FLTSRVNWVVQSSAVDYLHLMLVSMKWLFEEYDINGRFCIS

* ** * rk * ** *** *‘kir ** *** * 'k *

VHDEIRYLAEEP' RVAMALQIAN RVM VGIQDLPQSCAFFSAVDIDHVLRKEVDMDCITPSN
VHDEIRYLVKEE FRAAYALQISN RAMF LGINEVPQSCAFFSAVDLDFVLRKEVDLDCVTPSN
IHDEIRFLVSEK RAAMALQISN RAMF MGINELPQNCAFFSQVDIDSVIRKEVNMDCITPSN
VHDEVRYLSSDK RVAFALQVAN RAFF LGINELPQSVAFFSSVDIDHVLRKDVKMDCVTPSN
FHDELRYLVKEELSPKAALAMHITNLMTRSFCVSRIGLQDLPMSVAFFSSVEVDTVLRKECTMDCKTPSN
IHDEVRYLVREEDRYRAALALQITNLLTRCMFAYKLGLNDLPQSVAFFSAVDIDRCLRKEVTMDCKTPSN
IHDEVRYLVREEDRYRAALALQITNLLTRCMEAYKLGLNDLPQSVAFFSAVDIDQCLRKEVTMDCKTPSN
IHDEVRYLVHSKDRYRAALALQITNLLTRCMFASRLGIQDVPQSVAFFSAVDIDKCLRKEVTMDCSTPSN
IHDEVRYLVQEQDRYRAALALQITNLLTRCMEAYKLGLQDLPQSVAFFSAVDIDRCLRKEVTMNCATPSN

* * * 'k * fl * 'k * **** * *‘lr'k * ****

Degenerate 3’primer (4)] rate 3’ primer (2)]

 

 

 

 

 

PIPb ----- RAHGESIDIFQILEKGDDAKLDDSIVPQSQYAPRLENIPYTPRVPVMQRLRERAEAGDHQA
PDPL ----- IPCGKSLDIYQLLQQ-EDIKGADFPRTMHLNDVHYRKRTPVIEMFDKAVDDKTRKFMVSLQ
KTAP ----- IPHGEALDINQLLDK—PNSKLGKPSLDIDSKVSQYAYNYREPVFEEYNKSYTPEFLKYFLA
KV ----- TPPGEELTIESVLEKLEQSGQSLEPLEQIQCFVDVKATTSAEITEEDKKNIA--YLKAQAF
PHGLRIGYGIQPGQSLSVAEAIEKAGGNDVSQWDWIKKS
PTGMERRYGIPQGEALDIYQIIELTKGSLEKRSQPGP
PTGMERRYGIPQGEALDIYQIIELTKGSLEKRKPAWTLALSGGSVFAPVELYWSGAGS
PNGMEKRYGIPQGEALDIYQILKVTKGVL

PTGMEKKYGIPRGEALDIYQIIEITKGSLEKK

* *

 

 

 

FLRFIRAQITNS—-DEELKRIIAETRYSDPYGA-FSLASNGRVSGNPHQRHAAVHASTKTAAAPSKPSIA
IAQDKTEFTKWKRSRGELIVH
MQVQSDKRDVNRLEDEYLRECTSKEYARDGNTAEYSLLDYIKDVEKGKR -------- TKVRIMGSNFLDG
Yi’

88

870
790
821
817
969
1065
1043
1034
478

938
858
889
885
1036
1132
1110
1101
545

1008
928
959
955

1106

1202

1180

1171
615

1072

981
1022
1017
1145
1239
1238
1200

647

1139
1012
1084
1018

NC SRFDSVSQASRIKS-VAAGSDEPTIRATKAQGKAMA-KASGTKLAASTKDTVLNVTIKKKVAAPEMAAVP 1207
P

P
SC TKNAKADQ--RIRLPV-NMPDYPTLHKI-ANDSAIPEKQLLENRRK--KENRIDDENKKKLTRKKNTT—P 1147
SP
Dm
HS
Mm
X1
99

 

NC S 1 .C. .1 ID 1 1 1 1 ram-um; JJSJII v DAKKI lel KPLHKK—EIEGEPP PSLDDPVIAARLEAV 1276

Sc M---ERKYKRVYGGRKAF-E ------------- AFYECAN-KPLDYTLETEKQFFNIPIDGVIDDVLNDK 1199

NC S----KTSPGTRASVAA-KLDALASFCHASCCGC 1305

 

SC SNYKKKPSQARTASSSPTRKT IDDRV TTNRNLVELERDITISREY 1254

89

2.3.3 Transcription of mip-I

Northern blots of the total RNA from 10- to 12-hr old mycelia of N. crassa were
examined for the presence of mip-I transcripts as described in Materials and Methods. A
4.5-kb mRNA was recognized by hybridization with a radioactively-labelled 3.3-kb C laI
DNA fragment originating from the internal region of mip-I (Figure 2-7). Thus, the
transcript has combined 5’ and 3’ extensions of approximately 600 bp relative to the

mip-I ORF.

2.3.4 Map location of mip-I

The chromosomal location of the mip-I gene was found by restriction fragment
length polymorphism (RF LP) mapping using genomic DNAs from the strains generated
by Metzenberg et al. (1984). The segregation of the RFLPs was detected by
hybridization of Southern blots of the EcoRI-cut DNAs with probe generated from intact
X25:10C cosmid, which includes the complete mip-I gene. On the basis of the RFLP
map compiled by Nelson et al. (1998), the mip-I gene was found to be located on the
right side of linkage group III, somewhere between pro-1 and ad-2 (Figure 2-8).

Because of its location, the uvs-4 mutation in the LGIII was identified as a
possible allele of mip-I. Thus, several PCR products, which cover the whole region of
the mip-I gene, where generated and sequenced from the genomic DNA of uvs-4 mutant.

This comparison of the wild-type mip-I nucleotide sequence with the sequence of the

90

mip-l DNA from uvs-4 revealed no difference. Hence, it is unlikely that uvs-4 is allelic

to mip-I.

91

Figure 2-7. Detection of a transcript from mip-I by Northern blot hybridization. A:
Total RNA from N. crassa (N.c.) mycelium run on an agarose gel. The lane containing
RNA standard is labelled L. B: Northern blot of the NC. lane 80111 the gel in A
hybridized to the radioactively labeled 3.3 kb ClaI internal mip—I fragment of X25210C.

A) Total RNA gel electrophoresis B) Northern hybridization experiment

L NC. NC
A) B)

9.5
7.5

4.4

2.4

1.4

 

92

w-

-\u

 

Figure 2-8. Restriction Fragment Length Polymorphysm (RFLP) mapping of the
DNA polymerase-y.

51 52 53 54 55 56 57 58
A13 BIC C1D 01E

61 62 63 64 65 66 67 68 69 7o 71 72 73 74 7s 76 77 7e 79 so 81 82 83 84 as 56 87
EIF FIG GIH HII IIJ JIK KIL LIM MIN N10 0|P PIQ QIR R

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£98
on:

(.41.)
U1

1 416 711 415 711 711 311 415 716 811 411 411 415 812 312 411 412 411 4

LZCl 0 010 010 01M 010 0 M MIM 01M MIM OIO MIM M10 010 01M M1- M10 OIM 010 OIM M
AP9b.2 O 010 01M MIM 010 0 M MIM 01M MIM 010 MIM M10 010 M10 01M MIM M10 010 01M M
APl2f.2 O 010 010 MIM 010 0 M MIM OIM MIM 010 MIM M10 010 M10 OIM MIM M10 010 01M M
X14:1F 0 010 010 MIM 010 0 - -1M 01M MIM 010 MIM M10 010 M10 01M MIM M10 010 01M M
ace-2 0 010 OIM MIM 010 0 M MIM OIM MIM M10 MIM M10 010 M10 01M MIM M10 010 01M M
con-l 0 MIL 01M MIM M10 0 M MIM Ml' MIM M10 01M 010 M10 M10 010 MIM M10 010 010 M
cyt-8 0 010 01M MIM M10 0 M MIM MIO M1M M10 01M 010 01M M10 01M MIM M10 010 01M M
R15.4 0 010 01M MlM M10 0 M MIM MIM MIM -10 -1M M10 OIM M10 010 01M M10 010 01M M
AP31a.4 O 010 OIM MIM M10 0 M MIM MIM MIM M10 -1M M10 01M M10 010 01M M10 010 01M M
ser-l 0 010 01M -IM '10 0 M MIM MIM MIM M10 MIM M10 01M M10 01M MIM M10 010 01M M
krev-l 0 010 01- -1M -1- 0 M MIM MI- MIM M10 MIM MIO 01M MIO 01M M1- M10 01- 01M M
Cen III, crp-Z 0 010 01M MIM M10 0 M MIM MIM MIM MIO 01M MIO 01M M10 01M MIM M10 010 OIM M
thi-4 O 010 01M MIM 010 0 M MIM MIM MIM MI- OIM MIO 01M M10 01M MIM M10 010 01M M
pSK7—81C 0 010 OIM MIM 010 0 M MIM MIM MIM M10 OIM M10 OIM M10 01M MIM M10 010 01M M
pro-1 O 010 01M MIM 010 0 M MIM MIM MIM M10 MIM MIO OIM MIO 01M -IM M10 010 01M M
LZES 0 010 OIM 01M OIO 0 M MIM OIM MIM Ml- MIM MIO 01M M10 01M MIM M10 010 01M M
RRK 0 010 01M 'IM -1- 0 M MIM Ol- MIM M10 MIM M10 OIM MIO 01M M1- M10 010 01M M
LZE4 - 010 01- -IM 010 0 M MIM 01M MI- -10 MIM MIO OIM M10 01M MIM MIM M10 010 0
X25:10C(mip-I) O OIO -IO MIM OIO O M MIM OIM MIM OIO OIM MIO OIO MIO OIM MIM MIM OIO OIM M
622:1H 0 010 010 MIM 010 0 M MIM OIM MIM OIO 01M M10 010 M10 01M MIM MIM 010 01M M
con-7 0 01‘ 010 MIM 010 O M MIM OIM MIM 010 01M M10 010 M10 01M MIM MIM 010 01M M
ad-2 0 010 010 MIM 010 O M MIM 01M MIM 010 MIM M10 010 M10 01M MIM MIM 010 01M M
trp-l 0 01- 010 MIM 010 0 M MIM OI- MIM 010 MIM M10 010 M10 OIM MIM MIM 010 OIM M
D8002 O 010 010 MIM 010 O M MIM OIM MIM 010 MIM M10 010 L10 01M MIM Ml- 01M 01M M
AP32a.2 M 01M M10 MIM 0|- 0 M MIM OIM -IM 010 MIM M10 010 M10 MIM MIM M10 -10 01M M
32:26 O 010 010 MIM 01' O M MIM OIM MIM 01' MIM M10 010 M10 01M 01M MIM 010 OIM M
Fsr-45 M OIM 010 010 M10 M 0 MIM OIM M10 01M 01M 010 M10 010 M10 010 MIM 010 MIM 0
00032 M 010 010 M10 MIL M 0 MIM 01M 010 01M 01M 01M 010 010 M10 010 MIM 010 MIM O
cat-3 M 01M 010 010 M10 M 0 M10 MIM M10 OIM 01M 01M M10 010 M10 M10 MIM OIO MIM 0
MCI-6 M 01M 01' M10 M10 - 0 M10 MIM M10 -1M -1M -1M MIM M1- M1- M10 MI- 010 M10 0
AP8f.1 M 01M 010 M10 M10 M 0 M10 MIM M10 01M 01M 010 M10 010 M10 010 MIM 010 OIM O
AP12f.4 M OIM 010 M10 M10 0 0 M10 MIM M10 01M OIM 010 M10 010 M10 01M MIM 010 MIM 0
Tel IIIR M OIM 010 M10 M10 M 0 -1- M10 M10 01M OIM 010 M10 010 M10 01M MIM 010 MIM 0

93

2.1 Summary and Conclusions

Duplication of any DNA genome is the result of many enzymes and other
proteins. I have cloned and sequenced the DNA polymerase-y gene (mip-I) from N.
crassa using degenerate PCR primers designed from conserved regions of gamma DNA
polymerase from other species. The N. crassa mip-I gene encodes a protein of 1305
amino-acids and which has a mitochondrial targeting element. The N. crassa mip-l gene
was located on the right side of linkage group 111 between pro-1 and ad-Z. The N. crassa
and S. cerevisiae polymerase-y polypeptides have long C-terminal extensions that are not
found in the homologous proteins from other species. The higher eukaryotic gamma
DNA polymerases have 8 insertion sites totaling approximately 200 amino-acid residues.
The gamma DNA polymerases can be divided in two evolutionary groups on basis of the
presence and absence of inserts A to H. At this time, it is not known whether or not the
y-DNA polymerase that has been cloned is essential for the replication of the mtDNA in
N. crassa. However, the role of the protein in the maintenance of the mitochondrial
genome of this fimgus now can be analyzed through targeted mutagenesis and
observation of the effects of the mutations in homokaryons extracted from sheltering

heterokaryons.

94

2.2 Literature Cited

Bertazzoni, U., A. I.Scovassi, , and G. M. Brun, 1977 Chick-embryo DNA polymerase
gamma. Identity of gamma-polymerases purified from nuclei and mitochondria. Eur J
Biochem. 81 :237-48.

Bolden, A., G. P. Pedrali-Noy and A. Weissbach, 1977 DNA polymerase of mitochondria
is a gamma-polymerase. J Biol Chem. 252:3351-6.

Bourgeron, T., P. Rustin, D. Chretien, M. Birch-Machin, M. Bourgeois, E.
Viegas-Pequignot, A. Munnich, and A. Rotig, 1995 Mutation of a nuclear succinate

dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat. Genet.
11:144-149.

Clayton, D. A. 1982 Replication of animal mitochondrial DNA. Cell 28:693-705

Clayton, D. A. 1992 Structure and function of the mitochondrial genome. J. Inherit.
Metab. Dis. 152439-47.

F oury, F. 1989 Cloning and sequencing of the nuclear gene MIPl encoding the catalytic
subunit of the yeast mitochondrial DNA polymerase. J. Biol. Chem., 264: 20552-20560

Griffiths, A. J. F. 1992 Fungal senescence. Annu. Rev. Genet. 26:351-372

Insdorf, N. F., and D. W. Bogenhagen, 1989 DNA polymerase gamma from Xenopus
laevis. J. Biol. Chem, 264:21498-22503

Knopf, K.W., M. Yamada, and A. Weissbach, 1976 HeLa cell DNA polymerase gamma:
further purification and properties of the enzyme. Biochemistry. Biochemistry,
16:2874-2880

Kunkel, T. A., and A. Soni, 1988 Exonucleolytic proofreading enhances the fidelity of
DNA synthesis by chick embryo DNA polymerase-gamma. J. Biol. Chem. 263:4450-
4459

Kunkel, T. A., and D. W. Mosbaugh, 1989 Exonucleolytic proofreading by a mammalian
DNA polymerase. Biochemistry, 28:988-995

Lewis, D. L., C. L. Farr, Y. Wang, A. T. Lagina III, and L. S. Kaguni, 1996 Catalytic
subunit of mitochondrial DNA polymerase from Drosophila embryos. Cloning, bacterial
overexpression, and biochemical characterization. J. Biol. Chem., 271:23389-23394

Metzenberg, R. L., J. N. Stevens, E. U. Selker, and E. Morzycka-Wroblewska, 1984. A

method for finding the genetic map position of cloned DNA fragments. Neurospora
Newsl. 31:35-39.

95

Mishra, N. C. 1991 Genetics and molecular biology of Neurospora crassa. Advances in
Genetics, 29:1-62

Nass, S. and M. M. K Nass,. 1963 J. Cell Biol. 19:593-629.

Nelson, M.A., M. E. Crawford, D. O. and Natvig, 1998 Restriction polymorphism maps
of Neurospora crassa: 1998 update. Fungal Genet. Newsl. 45:44-54

Olson, M. W., and L. S. Kaguni, 1992 3'-->5' exonuclease in Drosophila mitochondrial
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