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This is to certify that the

dissertation entitled

GENE EXPRESSION ASSOCIATED WITH

VIRULENCE IN THE AMOEBA

NAEGLERIA FOWLERI
presented by

 

Wang-nan Hu

has been accepted towards fulfillment
of the requirements for '

Ph . D degree in ZOOLOGY

R.Neal Band

Major professor

 

Date 8-31-1992

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

 

LIBRARY
Michigan State
University

 

 

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.—___.

GENE EXPRESSION ASSOCIATED WITH VIRULENCE
IN THE AMOEBA EAEGLERIA EOWLERI

BY

Wang-nan Hu

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

DEPARTMENT OF ZOOLOGY

1992

ABSTRACT

GENE EXPRESSION ASSOCIATED WITH VIRULENCE
IN THE AMOEBA NAEGLERIA EOWLERI

BY

Wang-nan Hu

Naeqleria fowleri is an opportunistic pathogen causing
primary amoebic meningoencephalitis (PAM) in humans. Protein
synthesis patterns of low-virulence amoebae from axenic
culture, after mouse brain passage to increase virulence,
and after growth on bacteria were analyzed by two-
dimensional gel electrophoresis. Comparisons of accumulated
proteins, in yiyg-synthesized proteins, and 13 yit;_-
synthesized proteins translated from poly(A)+ mRNA were
made. Differences between amoebae from the different
treatments were noted. The increase in virulence was
correlated with numerous specific changes in protein
synthesis. To identify transcripts differentially expressed
in high virulent versus weakly virulent amoebae, a cDNA
library was made using mRNA from amoebae recovered from
mouse brains. By differential screening the cDNA library
with probes made from total RNA from axenic amoebae and from
high virulence amoebae, three cDNA clones were isolated. The
transcripts homologous to clone Nf314 and Nf116 were
preferentially expressed in high virulent cells whereas the
transcript homologous to clone Nf435 was preferentially

expressed in weakly virulent cells. Further sequence

analysis showed that Nf314 cDNA had an open reading frame
for a 53-kDa protein 94% similar and 19% identical over 194
amino acid residues to serine carboxypeptidase from yeast
cells, barley, and wheat. Southern blot analysis of Nf314
suggested that there was a single gene in the genome with
unknown copy numbers. The increased Nf314 transcript levels
were present in cells fed on mouse brain, liver, or NIH3T3
fibroblasts but not in cells fed on bacteria or in axenic
culture. Thus, the inducer of the increased gene expression
correlated with the use of mammalian cells as a food source
without regard to the level of virulence. Clone Nf435 had a
short open reading frame encoding 107 amino acid residues.
This sequence was 34% identical to troponin C in 47 amino
acids. Clone Nf116 had an open reading frame of 375 amino
acid residues encoding actin. These clones Nf116, Nf314, and
Nf435 were associated with virulence but not responsible for
virulence. Amoebae fed on dissociated mouse brain cells
expressed the above genes although they were weakly
virulent.

In addition, a new cDNA cloning method was described.
In this method, adding adapters, linkers or enzyme digestion

are no longer necessary after cDNA synthesis.

DEDICATION

To
My grand parents,
Dad and Mom,

Jimei and Jin

iv

ACKNOWLEDGEMENTS

I am most grateful to thank Dr. R. Neal Band, my major
professor, for his guidance, encouragement and support for
the successful completion of my research. I would like to
thank Dr. Will Kopachik for his guidance, allowing me to
work in his Lab, teaching me molecular biology techniques
and data analysis. I also would like to thank my other
committee members: Dr. R. Brubaker, Dr. T. Friedman and Dr.
D. Jump for their excellent guidance and assistance. Special
thanks to Mrs. Band for her kindly help to me and my family.

I would like to thank my grand parents, Xiang-gai Hu
and Jian Wang, who I regret passed away before they could
see me to earn my doctorate: my parents, Bin Ru and Xian-
ying Song, my uncles Bun-choi Wu, Bill Pu-choi Woo and their
wives, who made my dream come true to study in the United
State of America and for their love, encouragement and
support during my study. I would like to thank my wife,
Jimei and my daughter, Jin for their love, understanding and
support. I would like to thank my cousin W. Wayne Wu and
his wife for their great help during my first two years in
the Michigan State University. Without all of people I

have mentioned, the work would be impossible to complete.

TABLE OF CONTENTS

Page

LIST OF TABLES.... ...................... .. ............... ix

LIST OF FIGURES ........ ...................................x

INTRODUCTION....................................... ..... ..1
I. CHAPTER ONE: Virulence-related protein synthesis

in u; fowleri....................... ..... 12

A. Introduction.................... ....... . ....... ..12

B. Result and Discussion............................16

II. CHAPTER TWO: Cloning and characterization of transcripts
showing virulence-related gene expression

in u; fowleri...........................31

A. IntrOductionOOOOOO0.00.0.0...O OOOOOOOOOOOOOOOOO .031
B. Resu1t0000000000900....OOOOOOOOOOOCOOOOOOOC.0...I33

1. Cloning of transcript sequences preferentially
expressed in highly virulent amoebae............33

2. expression of transcripts in weakly and highly
Virulent amoebae.0.0.0.0....00.0.00000000000000034

3. Southern blot and sequence analysis of Nf314....38

4. Induction of Nf314 gene expression by mammalian
cells in culture................................45

5. Sequence analysis of clone Nf435......... ..... ..51
C. DiSCUSSionOOO0.0I.OOOOOOOOOOOOOOOOOOOOOOOOOOO0.0055
III. CHAPTER 3. A RAPID METHOD To MAKE A cDNA LIBRARY....60

A. IntrOductionOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.060

B. Materials and Methods................ .......... ..61
C. Result and Discussion. ......................... ..62
SUMRY AND DISCUSSION .......... O O O O O O O I O O O O O O O O ......... 73

APPENDIX 1. ELECTROPHORETIC KARYOTYPE OF N; fiowle;i......89

vi

APPENDIX 2. SEQUENCE ANALYSIS OF AN ACTIN CDNA INSERT....93

APPENDIX 3. MATERIAIS AND METHODS...0.00.00.00.00...0.0.098

1.

2.

9.

10.

11.

wLTURE OF L £0Wler1 O O O I O O O O O O O O I I O O O I O O O O O O O O O O O O I 98
DNA ISOLATION. O O I O O O O I O O O O O O O O O O O I O O O O I O I O O O O O O O O O O O 100
A. Plasmid DNA iSOIation. O O O C O O O O O O O O O O O O I O O O O O O O O I O 100

a. Large scale plasmid DNA preparation
Without CSCIOOOOOOOOOOOOOOOOOOOO0.0.0.0....00.100

b. Large scale plasmid DNA preparation with CsCl.101

c. Small scale plasmid DNA preparation...........103
B. N; fowleri genomic DNA isolation.................105
SOUTHERN BLOT.......................................108
DOT BLOT......... ..... ..............................112
RNA ISOLATION.......................................113
NORTHERN BLOT.......................................121
RADIOACTIVE PROBE PREPARATION.......................128
SEQUENCING..........................................130
A. Single-stranded sequencing using M13 phage.......130
B. Double-stranded sequencing using plasmid.........132
C. Gel preparation..................................134
CONSTRUCTION OF A CDNA LIBRARY .....................135
TRANSFORMATION OF BACTERIA E. lei..................136
TWO DIMENSIONAL GEL ELECTROPHORESIS.................139
1) 2-D gel sample preparation.......................139
2) Two dimensional gel electrophoresis..............140
3) Fixation for autoradiography.....................l44

4) Silver staining of gels..........................144

vii

12. PROTEIN IN VITRO TRANSLATION
USING RABBIT RETICULOCYTE...........................146

13. SHALL AMOUNT DNA PRECIPITATION WITH ETHANOL.........147

LISTOFREFERENCES.O.....OOOOOOOOOOOOOOOOOOOOO0....0.0.0.148

viii

Table

LIST OF TABLES
Page

Chapter One.

1.

3.

Differences in the expression of protein at
accumulated level (silver stain) and in
yivg-synthesized level (L-“S-methionine

incorporation) between LEE and LEEmp amoebae........19

Differences in the expression of protein in yitro
translation from poly(A)+ mRNA among LEE, LEEmp
and LEEObOOOOOOOOO..OOOOOOOOOOOOOOOOOOOOOO0.0.0.0...23

Partial characterization of some proteins translated
in Vitro from pOIY(A)+ mRNAOOOOOOOOOOOOOOO0.0.00.0..29

Chapter Two.

1.

Comparison of predicted Nf435 amino acid sequence with
troponin C from various species.....................53

Appendix 2.

1.

2.

Codon usage by N; fowleri and Aganthamoeba
castellani;0000000OOOOOOOOOOOOOOOOOOOOOOOOO.I.0.... 96

Third-codon-position nucleotide frequency for actin
gene COding regionOOOOOOOOOOO0.00.00.00.00.I0.00.0009?

ix

LIST OF FIGURES
Figure Page
Introduction
1. Three stage life cycle of Naegleria................2
2. n; figwleri LEE strain in axenic culture.. ..... .....5
3. Strategy of differential screening.. ............... 11
Chapter One

1. Silver-stained electropherograms of
accumulated proteins................. ......... .....18

2. Autofluorograms of 2-D gel of rotein
radiolabelled in vivo with L-3S-methionine.. ...... 21

 

3. Autofluorograms from 2-D gel electrophoresis
of proteins translated in vitro....................25

4. Comparison of silver-stained electropherograms of
accumulated protein extracted from low virulent
and high virulent amoebae after six month in axenic
enltureoo...0......OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0.28

Chapter Two

1. Autoradiographs of Northern blots of RNA from
axenically grown amoebae and amoebae from
mouse brain.0......OOOOOOOOOOOOOOOOOOOOO... ........ 37

2. Autoradiograph of Southern blot probed with
Nf314 CDNAOO0.000...00...OOOOOOOOIOOOOOOOOO. 0000000 40

3. DNA sequence of the insert to clone Nf314..........43
4. Comparison of the predicted Nf314 amino acid sequence
with the carboxypeptidase sequences of yeast, barley
and Wheat...0......OOOCOOOOOOOOOOOOO0.0.00.00.00.0047

5. Autoradiographs of Northern blots of RNA from amoebae
in cell culture..0.00.0.0...OOOOOOOOOOOOOOOOOOOO0.050

Chapter Three
1. Diagram of vector-primer preparation ............... 67

2. Diagram of cDNA recombinant construction ........... 68

3. Mini-prep analysis of N; figwlgri cDNA
inserts.0......0..OOOOOOOOOOOOOOOOOOOOOO. ....... .0070

4. Double-stranded DNA sequencing of a randomly selected
CDNA CloneOOOOOOOOO0.00.000.00.00...0.00.0.0...0.0.72

Appendix 1.

1. Electrophoretic karyotype of N; fowler'.. .......... 92

xi

INTRODUCTION

The small, free living amoebae, that are
pathogenic to man, belong to two common genera, ae er'a
and Acanthamoeba (Page, 1988). They occur world-wide and
have been isolated from a variety of habitats. Both are
regarded by clinicians and microbiologists as exotic
organisms in infections such as primary amoebic
meningoencephalitis (PAM) (N; figwleri), granulomatous
amoebic encephalitis (GAE) and non-fatal but painful,
infections of the human cornea, keratitis (Acanthamoeba
spp.) (Ma gt a;., 1990, Lambert, 1991, Visvesvara, 1990).
Although both infect the central nervous system, the routes
of invasion and penetration by amoebae are different. Pre-
acquisition of most human infections of PAM are aquatic-
related due to free-living amoebae or cysts entering the
nasal cavity. Invasion and penetration in the case of GAE
appears to be hematogenous, probably from a primary focus in
the lower respiratory tract or the skin (Ma, gt a;., 1990).

The genus Naegleria (Sarcodina, Vahlkampfiidae)
consists of free-living amoebaflagellates widely distributed
in soil and freshwater where they feed on bacteria and fungi
(Page, 1988). The life cycle includes three stages:
trophozoite, flagellate and cyst (Fig. 1). Naegleria can
transform from trophozoite to flagellate (Fulton, 1970,

Schuster, 1979) while in contrast Acagthamoeba has two

2

AMOEBA

 

ENCYSTM TRAN FORMATION

   

CYST F LAGELLATE

Figure 1. Three stage life cycle of Naegleria

3

stages and cannot transform to a flagellate stage. There

are six species of Naegleria: N. austtaliensis, N. towleri,
N. gngeri, N. Lovaniensis, N. iggini and N. thorntoni
(Marciano-Cabral, 1988). Naegleria are studied because the
flagellate transformation serves as a model system in gene
regulation and developmental biology while some species are
of medical importance as facultative pathogens.

Species N. towleri (Fig. 2) is an opportunistic
pathogen, causing PAM, a rapidly fatal disease of the
central nervous system (CNS). The disease was detected in
man in Australia (Fowler gt gt., 1965). The amoeba was named
in honor of Dr. Malcom Fowler who first recognized the
disease. The term "primary amoebic meningoencephalitis" was
used to distinguish infection of the CNS in humans from the
rare invasion of the brain by the intestinal amoeba
Entgmggtg histo;ytigg (Duma, 1991: Marciano-Cabral, 1988) or
by Acgnthamoeba. The conditions under which N1 fowleri
becomes an opportunistic pathogen of men are not known. The
disease usually occurs in healthy children and young adults
with a history of swimming in freshwater lakes or ponds
(John, 1982). The infection possibly results from the
introduction of water containing amoebae into the nasal
cavity and subsequently amoebae penetrate the nasal mucosa
to the CNS via the olfactory nerves. The disease is rapidly
fatal, usually producing death within 72 hr after the onset

of symptoms (John, 1982) and to date, an effective treatment

Figure 2. Amoebae of Naegleria fowleri LEE strain in axenic

culture viewed with Ziess microscope. A. X 2,000 B. X 5,000

 

6

is still lacking (Ma gt g;., 1990). Although it is a rare
disease, PAM has been reported in many countries. Human PAM
infections have resulted from contact with amoebae from a
variety of environmental sources such as swimming pools
(Cerva and Novak, 1968), freshwater lakes (Butt gt g;.,
1968: Callicott gt g;., 1968), thermal streams (Cursons gt
31., 1976), mud puddles (Apley gt g1., 1970) and a household
water supply (Anderson and Jamieson, 1972). Isolation of
Nt fowleri from thermal effluents, hot springs, and waters
with naturally or artificially elevated temperatures up to
42°C are common. Since the first report of PAM, there have
been more than 140 cases reported (Visvesvara, 1990).

Identification of Nt fowleri isolated from fresh water
has utilized a variety of methods. For example, after
presumptive NaegLeria spp. are identified by characteristic
morphology and flagellate formation, the next step is to
grow the sample in enrichment culture medium (Aufy, gt g;.,
1986) at 439C. Testing the pathogenicity with mice is a
another critical step. If the isolate can kill mice, it
probably is Nt fowleri. Other methods can be added as an aid
for further identification, such as isoenzyme analysis
(Adams, gt gl., 1989: De Jonckheere, 1982: Kilvington, gt
31., 1985), restriction fragment length polymorphism of
genomic DNA (McLaughlin, 1988) or mitochondria DNA
(Milligan, 1988) and electron microscopy (Stevens, gt al.,

1980).

7
Unlike the obligate parasite fit histolytica, Nt
fggleti is a free-living amoeba. Therefore, what makes Nt
figglgti pathogenic, what triggers the switch from a free-
living amoeba to an invader in this species but not in other

species such as N.

gruberi, and what molecules are key to
the process are of major interest in this field. A clear-
cut answer to these problems are not only of great biologic
significance, but also have public health and economic
consequences. A variety of approaches, such as cytological
and biochemical methods have been applied to the problem of
understanding the capacity of N. fowLeti to become
pathogenic either it ytyg or tn yittg. The cytopathic
effects of Nt fowleri might be due to release of cytolytic
substances by amoebae (Chang, 1978) or the result of a
combination of phagocytosis and the cytolytic action
(Visvesvara and Callaway, 1974). Phospholipase A enzyme was
suggested as one of the cytolytic factors. Higher levels of
this enzyme were produced by pathogenic strains compared
with low levels of the enzyme in non-pathogenic strains.
Reduction of cytolytic activity was observed in the presence
of the inhibitor (Rosenthal’s reagent or dimethyl-DL-2,3,-a-
distearoyloxypropyl-2’-hydroxyethylammonium acetate)
(Fulford, gt g;., 1987). Recent studies (Marciano-Cabral,
1988) indicated a different mechanism of cytopathogenicity
between axenically cultivated, low virulent Nt fowleri LEE

strain and mouse brain passaged amoebae. Axenic cultured

8
amoebae ingested nerve cells mediated by an apparatus called
an amoebaestome. In contrast, the highly virulent amoebae
lysed cells by cell-cell contact without cytolytic factors
secreted into the culture medium. The ability of the
amoebae to attach to and penetrate the nasal mucosa or to
exhibit an increased rate of locomotion or both might be
determinative parameters (Marciano-Cabral, 1988). However
the precise nature of the mechanism(s) employed by Nt
figglgti to penetrate the nasal mucosa and to invade the CNS
is still little known. In contrast to previous works which
concentrated in cytology and biochemistry, my research
focused on epigenetic changes in virulence through molecular
and genetic techniques to investigate the basic mechanisms
accounting for PAM.

LEE strain of Nt fiowlgti was used in this study because
of its experimental advantage for molecular studies. The
virulence1 of L fowleri LEE strain can be varied (Wong gt
g1., 1977). When amoebae are axenically cultured for a long
period, virulence is attenuated but can be restored by
serial mouse brain passages (John gt g1., 1985, Marciano-

Cabral, 1988). The ability to manipulate virulence makes it

 

1 It is necessary to distinguish between the terms
pathogenicity and virulence. I will use the term
"pathogenicity" to mean the capacity to cause disease by a
given strain of amoeba: therefore there are two types of
amoebae, pathogenic and non-pathogenic. On the other hand,
“virulence" reflects the degree to which pathogenicity is
expressed. The grade of virulence may depend on many
factors.

9
a good model to study the molecular mechanisms of virulence
in parasitic protozoa.

Cell interactions between Nt figwleti amoebae and host
cells are complex and involve an unusually effective
cytolysis and phagocytosis which may involve with the
expression of many genes. Because Nt fowleri is always
pathogenic but virulence can be easily varied in the LEE
strain, my research emphasizes a study of epigenetic changes
in virulence and not differences in pathogenesis between
species or strains. To begin a molecular and genetic
analysis I first assessed the protein synthesis patterns and
identified proteins whose abundance was altered during the
transition of amoebae from low to high virulence, by two
dimensional gel electrophoresis. This tested the hypothesis
that the increased virulence was a result of altered gene
expression in response to contact with the nasal-mucosa
epithelium and invade the CNS. The next step was to clone
these cDNAs encoding these proteins. The study of eukaryotic
gene structure and expression relies on the availability of
probes. The successful strategy toward isolating a
particular eukaryotic gene is first to isolate a DNA copy
(cDNA clone) of the mRNA encoded by that gene. The cDNA
clone also offers a direct rout towards cloning the
corresponding chromosome gene. In order to do that, a cDNA
library was constructed from mRNAs. The reverse

transcriptase (RNA-dependent DNA polymerase) was then used

10
to reverse transcribe mRNAs isolated from mouse brain
passaged amoebae into cDNA. This pool of cDNAs was inserted
into an appropriate cloning vector, then transformed into an
appropriate bacterium strain. A differential screening
method was used to screen the cDNA library to identify
possible cDNA clones (Fig. 3). After a few rounds of
isolation, we picked up a few cDNA clones. The cloned cDNAs
was used as probes to hybridize with Northern blots to
further confirm the specific mRNAs differentially expressed
in highly versus weakly virulent amoebae. Sequencing the
cloned cDNAs and comparing the deduced amino acid sequence
to the Swiss Protein Database was used to search for clues
as to the possible role of the protein.

In the following chapters, I describe: changes of
protein synthesis pattern and the isolation and
characterization of three cloned cDNAs whose corresponding
transcript levels were altered when the virulence was
changed. In addition, a new cDNA library construction method

was presented.

'NFECT / \ AXENIC GROWTH

 

 

 

 

 

{ 3 x
AMOEBAE
FROM —-—‘
BRAIN
ll
PREPARE RNA \ PREPARE RNA
HYBRIDIZE cDNA UBRARY HYBRIiDIZE
32mm wrm
PcDNA 32 PcDNA
PROBE PROBE
® @ ¢ REPUCA PLATES ¢ ® ®
® ® OF COLONIES ® ®

AUTORADIOGRAPHY AUTORADDGRAPHY

CHARACTERIZE CLONES

Figure 3. Strategy of differential screening.
11

 

CHAPTER ONE: Virulence-related protein synthesis
in mac-mien; mm "
A. Introduction

The free living amoeboflagellate Nggglgtig fiowler; is a
virulent pathogen of man, causing primary amebic
meningoencephalitis (PAM) (Fowler, gt gl., 1965). Studies on
the pathogenesis of N. fongri have been done in an attempt
to discover the determinants of pathogenesis (John, 1982,
Marciano-Cabral, 1988). The mouse is a useful model to
investigate PAM, because basic features of the disease are
the same for mouse and man (John, 1982).

Several proteins, such as phospholipase (Fulford, gt g;.,
1986), membrane-associated pore-forming protein (N-PFP)
(Young, gt gl., 1989) and Nggglgtig amoeba cytopathogenic
material (NACM) (Dunnbacke, gt gl., 1989), may play some
role in pathogenesis of Nggglgtig. However, none of these
proteins are unique to high virulence amoebae of N. fowlgti.
Therefore, virulence of N. towleri may be a result of
numerous protein changes not yet identified. Virulence
varies in some strains of N. fgglgti (Wong, gt gl., 1977).
The LEE strain of N. towleri isolated from human
cerebrospinal fluid, loses virulence when cultured
axenically for extended periods of time. However, virulence

can be restored by serial mouse brain passage (John, gt gt.,

 

1Chapter one was published by W. Hu, R. N. Band and W.
Kopachik in Infect. & Immun. 59:4278-4282 in 1991.

12

13
1985, Marciano-Cabral, 1988). Virulence persisted for
approximately one month in axenic culture (Marciano-Cabral,
gt 31., 1990).

We used two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE) to observe global changes in
protein synthesis by the LEE strain of N. fowleti.
Comparisons of low and high virulent amoebae were made with
silver stained, it 2129 synthesized and it ytttg translated
protein. Protein patterns were done after growth on
Esghetichia gg1i K-12 to test the effect of diet on
virulence. To test the stability of the pathogenic
phenotype, protein patterns were analyzed between the LEE
strain after mouse brain passages to increase virulence, and
after axenic cultivation for 6 months.

Low virulence amoebae, LEE strain of N. gowleri were
obtained from Dr. David J. John, grown in H4 liquid medium
(peptone medium supplemented with hemin) (Band, gt g1.,
1974) at 37”C and recloned before use. For a clone
isolation, we used migration on H4-agar plates at 37°C. High
virulence (LEEmp) amoebae were obtained by serial
inoculation of the LEE strain into mouse brains for four
consecutive passages. Ten, three to four week old male mice
(Hug musculus strain: BALB/C) were used for each passage.
Amoebae were harvested and washed by centrifugation three
times with LS saline (50 mM NaCl, 4.6 mM MgSO‘ and 0.36 mM

CaC12)(Band, gt g1. 1969). A series of 2 ul of LS saline

14
containing N. fowleti (10" amoebae were used for the first
passage and 10‘ for subsequent passages) were applied into
the nares of each mouse, using a narrow bore 0-200 ul
microcapillary pipet tip (Western Coast Scientific, Inc.).
Within 4-6 hr after death pieces of brains were placed in
250 ml tissue culture flasks (Corning Science Products)
containing 10 m1 H4 medium with Penicillin-G (1000
units/ml), Streptomycin (100 units/ml) and incubated at
37°C. Two hr later, mouse brain tissues were removed and
fresh H4 medium without antibiotics was added to the
residual amoebae. The amoebae were then cultured for 24 to
36 hr before protein analysis. Amoebae were also fed with E.
gg11 (K-12) (1 mg/ml LS saline) and incubated at 37%:
(LEE.b). Before protein analysis, the amoebae were cultured
in LS saline overnight without bacteria.

To radiolabel cellular proteins, 300 uCi L-“S-methionine
(>1000 Ci/mmol) (Amersham) was added to each culture (10 ml)
for 6-8 hr at.37”C before the cells were harvested. About
JJV amoebae for each sample were harvested and washed three
times with LS saline, and prepared by the methods described
by Kopachik (Kopachik, etc., 1985) for 2-D PAGE analysis
(See Appendix 3 for detailed protocol). Total cellular RNA
from 103 amoebae was prepared according to the methods of
Chirgwin (Chirgwin, gt g1., 1979), and Kopachik (Kopachik,
gt n1., 1985) (See Appendix 3 for detailed protocol). Poly A

(+) mRNA was selected from 100 pg total RNA with HYBOND-mAP

15
paper (Amersham) following the manufacture’s instruction.
Then mRNAs were translated i3 gittg in a rabbit reticulocyte
cell-free system (Amersham) with 40 uCi L~”S-methionine for
1 hr at 37°C.

Two-dimensional polyacrylamide gel electrophoresis (2-D
PAGE) was performed according to the methods of Garrels
(Garrels, 1979) and modified by Kopachik (Kopachik, gt g1.,
1985) (See Appendix 3 for detailed protocol). The first
dimension gel contained the ampholyte (Pharmacia LKB) of pH
range 5 to 7 for in ytttg translation gels and pH 3.5 to 10
for other gels. The second dimension gel was a uniform 11%
polyacrylamide with sodium dodecyl sulfate (SDS). Proteins
(200 pg) were stained with silver by the methods described
by Morrissey (Morrissey, 1981) (See Appendix 3 for detailed
protocol). Gels with samples labelled by 3"is-methionine were
fixed and soaked in EN3HANCE (New England Nuclear) for 60
min. Dried gels were exposed at. -70°C to Kodak XAR-S film.
To eliminate inconsistencies, each experiment was repeated
three times under the same conditions and three replicas
were done with each experiment. Only the proteins that
qualitatively changed and were consistently viewed on gels
were labelled.

To compare the virulence of LEEmp after 6 months growth
in axenic culture versus the virulence of LEE amoebae grown
either in axenic or bacterized (LEE.b) cultures, 7 mice per

group were inoculated with 10‘ amoebae. A completely random

16

block design (Gill, 1978) was used to minimize the variation
between different litters of mice.

B. Result and discussion

The steady state level of proteins present in LEE low
virulence amoebae, in comparison to LEEmp, high virulence
amoebae, was analyzed by silver staining of accumulated
proteins. We sought both quantitative and qualitative
differences (Fig. 1). Ten proteins were detectable only in
the LEEmp whereas twelve proteins were detectable only in
LEE cells (Table 1). Quantitative differences were also
observed, such as the protein just above spot #6 which had a
lower level in LEEmp versus LEE amoebae (Fig. 1). The
protein spot numbers are only comparable between similar
treatment. Thus numbers in Fig. 1, 2 and 3 cannot be
compared.

We analyzed the incorporation of L~”S

-methionine during
growth in axenic broth. Here again numerous proteins showed
qualitative and quantitative differences (Fig. 2). Some of
these proteins were preferentially to LEEmp or to the LEE
cells (Table 1). In addition, many proteins were synthesized
at a lower rate in the LEEmp versus the LEE amoebae. This
suggests that the synthesis as well as accumulation, of many
proteins is affected by differences in pathogenesis. Changes
in protein synthesis or accumulation could be the result of

corresponding changes in mRNA transcripts encoding these

proteins. To determine if transcript abundance changes can

17

Figure 1. Silver-stained electropherograms of accumulated
proteins extracted from low-virulence LEE amoebae (A) and
from high-virulence LEEmp amoebae (B). The proteins which

differed between them were identified by numbers.

18

 

 

 

 

 

19

Table 1. Differences in the expression of protein at
accumulated level (silver stain) and in vivg synthesized (L-

:”S-methionine incorporation) level between LEE and LEEmp

 

 

amoebae.
§g11 typg Qualitative chagges gt Etoteins' 31g. Ng.
Silxer stain
LEE 12 (1,2,3,7,B,9,10,11,13,14,2o,21) 1A
LEEmp 1o (4,5,6,12,15,16,l7,18,19,22) 13
lg vivo labelling

LEE 11 (1,5,6,7,9,11,12,13,14,15,16) 2A
LEEmp 5 (2,3,4,8,10) 2B

 

'The proteins are detectable only in either LEE or LEEmp.

20

Fig. 2. Autofluorograms of two-dimensional gel of

proteins radiolabelled i

vivo with L-[BS]-methionine from

 

low virulent LEE amoebae (2A) and from high virulent LEEmp

amoebae (2B). The proteins which differed were identified by

numbers.

21

 

 

 

22
be detected, poly A (+) mRNA was extracted from LEE and
LEEmp cells and translated in yittg in rabbit reticulocytes
(Fig. 3). We found that eleven translation products were
detectable only when LEEmp mRNA was used: this suggests that
higher levels of some transcripts appeared in virulent
amoebae (Table 2, LEE vs. LEEmp amoebae). The change from
the LEE to the LEEmp pathogenic state also included a
changein diet from the axenic culture to a diet of cells
ingested by phagocytosis. Changes of transcript level
occurring in the pathogenic amoebae may be a result of
altered diet and feeding behavior. Therefore, amoebae fed
with bacteria (LEE.b) might show some of the same changes
apparent in pathogenic amoebae. A comparison of LEEmp to
LEE.b translation products (Table 2) showed that this did
occur: 1g ytttg products 1,8,10,12,14,15,19 and 25 were
translated from LEEmp and LEE.b mRNAs but not from LEE mRNAs
(Fig. 3). There were in spite of these similarities
extensive differences both qualitatively and quantitatively
between the LEEmp and LEE.b translation products (Table 2
and Fig. 3).

Twenty-five of the main characteristic, ifl gtttg gene
products of LEE, LEEmp and LEE.b transcripts are shown with
apparent molecular weights and isoelectric points (Table 3).
There is a correlation between the numerous changes in the
accumulation and synthesis of proteins observed and the

susceptibility of mice to infection. The virulence of LEEmp

23

Table 2. Differences in the expression of protein 13

 

vitro translation from poly A (+) mRNA among LEE,

LEEmp and LEE.b.

 

§g11 tygg Qualitative changes gt prgteins' (fig. Ng.

LEE XS LEEEP

LEE 6 (2,3,6,13,17,21) 3A

LEEmp 11 (l,4,8,10,12,14,15,l6,19,20,25) 33
LEE rs LEEIQ

LEE 10 (3,6,11,13,17,18,21,22,23,24) 3A

LEE.b 11 (1,5,7,8,9,10,12,14,15,19,25) 3C
LEEEP X§ LEELD

LEEmp 7 (4,11,16,18,20,22,23) 33

LEE.b 4 (2,5,7,9) 3c

 

“The proteins detectable only in either LEE, LEEmp or LEE.b.

24

Fig. 3. Autofluorograms from two-dimensional gel

electrophoresis of proteins translated it vitro. Poly A (+)

 

mRNA from low virulent LEE amoebae (3A), high virulent LEEmp
amoebae (3B) and LEE amoebae after growth on bacteria (3C)
was translated in rabbit reticulocytes with L-[”S]-
methionine. The proteins which differed were identified by
numbers. Approximate molecular weight range (vertical axis)

and the pH range were used to construct Table 3.

25

 

 

 

 

26
amoebae increased after the amoebae were passaged through
mouse brain three times. About 10‘ LEEmp cells killed a
mouse in approximately 5 days after three passages, while
it” axenic cultured LEE cells were required to kill a mouse
in about 10 days in the first mouse brain passage. However,
after 6 months in axenic culture, LEEmp lost its virulent
state and its protein pattern resembled the LEE patterns:
the time required to kill infected mice was not
statistically different from LEE or LEEmp after 6 months in
axenic culture and on silver-stained gels none of the LEEmp-
specific proteins were observed (Fig. 4).

The virulence of N. fowleri LEE strain varies in
prolonged axenic culture and by mouse brain passages. It is
this unique feature that constituted the basis of our study
so we could investigate the regulation patterns of protein
synthesis during this transformation. The results, different
patterns of protein synthesis between low (LEE) and high
virulence (LEEmp) amoebae, are a necessary prerequisite for
future attempts to clone high virulence-specific genes by
differential screening of a cDNA library made from LEEmp
amoebae. The partial characterization of the gene products
in this experiment (Table 3) may facilitate the correlation
of cloned genes to characterize translation products.
Numerous changes in protein synthesis occur during the
transformation of LEE strain from low to high virulence.

Some of the changes in protein synthesis might be directly

27

Fig. 4. Silver-stained electropherograms of accumulated
proteins extracted from low virulent LEE amoebae (4A) and
from high virulent LEEmp amoebae after 6 month removal from

mouse brain (48).

 

 

28

 

 

 

 

29
Table 3. Partial characterization of some proteins

translated lg vitto from poly A(+) mRNA.

 

WEstimated Etesent- in Etgtgin Estimateg Etesent-in
pI‘

 

#‘e M.W. pl
1 10.5 6.7 LEEmp,LEE.b 14 20.0 6.3 LEEmp,LEE.b
2 10.0 6.0 LEE,LEE.b 15 21.5 5.4 LEEmp,LEE.b
3 10.6 6.1 LEE 16 30.0 5.2 LEEmp
4 12.0 5.5 LEEmp 17 20.0 6.0 LEE
5 11.5 5.5 LEE.b 18 21.0 5.7 LEE,LEEmp
6 10.6 6.2 LEE 19 23.0 5.5 LEEmp,LEE.
7 11.0 6.8 LEE.b 20 44.0 5.2 LEEmp
8 12.0 6.3 LEEmp,LEE.b 21 17.0 5.0 LEE
9 11.5 6.3 LEE.b 22 32.0 5.6 LEE,LEEmp
10 12.5 6.3 LEEmp,LEE.b 23 32.0 5.6 LEE,LEEmp
11 11.5 6.1 LEE,LEEmp 24 23.0 5.2 LEE,LEEmp
12 13.0 5.6 LEEmp,LEE.b 25 30.0 6.0 LEEmp,LEE.b
13 14.0 6.5 LEE

 

'See Fig. 3A, 3B and 3C and table 2 for spot numbers.
bM.W., Molecular weight (x103Da).
cpI, isoelectric point.

30
responsible for virulence while others could reflect
indirect relationships to virulence. For example,
carbohydrate moieties of surface glycoproteins have been
implicated in pathogenesis of Entamoebg histOLytica (Petri,
gt g1., 1990). Many proteins were lost in the LEEmp amoebae;
the decreases were qualitative (#6 in Fig. 1) and
quantitative (the protein above #6 for example). Similarly
the loss of proteins may be important in converting a cell
into a more virulent state. Several low virulent and
avirulent amoebae from various genera will kill mice when
directly injected into the brain (John, 1982). Penetration
of the nasal mucosa and related barriers is necessary under
natural conditions for invasion of the central nervous
system. Thus, in addition to interaction with brain tissue,
other factors related to invading the central nervous system

may be inducers of increased virulence in the amoebae.

CHAPTER TWO: Cloning and characterization of transcripts

showing virulence-related gene expression

in Naegletig fgwleti‘

A. INTRODUCTION

Naggleria towler; is a free-living amoeba of soil and
freshwater which feeds on bacteria present in these
environments. It also exists as a virulent pathogen causing
primary amoebic meningoencephalitis in humans, mice and
other mammals (Fowler, gt g1., 1965: John, 1982). Infection
of the brain occurs after amoebae reach the nasal cavity,
attach to and invade the nasal mucosa and olfactory nerve.
Invasive amoebae able to enter the nervous system digest
neuronal tissue and other mammalian cells by unusually
effective cytolysis and phagocytosis as observed in culture
or in sections of infected brain tissue (Marciano-Cabral,
1988, 1990). Highly virulent amoebae exhibit faster movement
in yittg than do weakly virulent amoebae (Cline, gt g1.,
1986, Thong, gt g1., 1986). A mechanism to evade the host
immune system defenses has also been suggested as a
necessary determinative change in the pathogenic form
(Marciano-Cabral, 1988). However, the molecular mechanisms
responsible for the capacity to be pathogenic are still

poorly understood.

 

1Chapter Two was published by Wang-nan Hu, Will Kopachik and
Neal Band in Infect. & Immun. 60:2418-2424 in 1992.

31

32

Four proteins, a phospholipase (Fulford, gt g1., 1986),
a neuraminidase (Elsen, gt g1., 1987), cytopathogenic
material (Dunnebacke, gt g1., 1989) and a membrane-
associated pore-forming protein (Young, gt g1., 1989) have
been implicated in pathogenesis but the exact role that
these markers play is unknown since they are also found in
non-pathogenic species. Recently, this transition from the
free-living to the highly virulent state was found to be
associated with many additional quantitative as well as
previously unknown qualitative changes. Increases and
decreases in protein synthesis or stability were found by
two dimensional gel electrophoresis (Hu, gt g1., 1991).

The LEE strain has experimental advantages for
molecular studies of the transition from free-living to
highly virulent forms. Virulence can be varied in this
strain (Wong, gt g1., 1977) because it loses virulence when
cultured axenically. Virulence can be restored by serial
mouse brain passages. The ability to manipulate virulence
by serial mouse brain passage made it possible to examine
gene expression during the transition from low to high
virulence. The changes in protein synthesis could arise by
transcriptional or post-transcriptional regulatory
mechanisms. Changes in the levels of specific transcripts
were observed by examination of the translation products
from poly (A)+ mRNA in a rabbit reticulocyte extract (Hu, gt

g1., 1991). This suggested that clones of the genes for

33
these regulated mRNAs might be found by differential
screening of a cDNA library made to virulent cell mRNA.

Here we describe the isolation and characterization of
two cloned cDNAs whose corresponding transcript levels are
altered between weakly and highly virulent amoebae. Having
such clones allowed an examination of the possible role of
the genes in pathogenesis through DNA sequence analysis. The
nature of the inducer of the gene expression changes could
be sought by examination of mRNA levels in amoebae incubated

in culture with mammalian cells.

8. RESULTS

Cloning of transcript sequences preferentially
expressed in highly virulent amoebae. A cDNA library of
1,000 clones in the vector pUC18 was made from poly (A)+
mRNA from LEEmp amoebae which had been isolated from the
brains of infected mice. Initial screening attempts by
‘methods in which bacterial colonies (105) on replica filters
were lysed gave variable results. Consequently, dot blots of
the plasmid DNA ( z 1 pg prepared by a rapid preparation
method) were made on replica filters in order to increase
the sensitivity of detection and make sure that equal
amounts of the plasmid DNA were immobilized on the replica
filters. One set of filters were hybridized with single
stranded cDNA probe made from 25 pg of oligo dT-primed total

RNA of highly virulent amoebae and the other was hybridized

34
with a comparable probe prepared from weakly virulent
amoebae grown axenically. The autoradiographs of the
filters were examined to find cDNA clones which differed in
quantity of hybridization between the two probes. Only two
clones exhibited reproducible and significant differential
hybridization signals by visual screening. Clone Nf314
exhibited higher hybridization to the cDNA probe made to
highly virulent amoebae RNA whereas clone Nf435 exhibited
lower hybridization to that probe. The other 998 clones
showed either negligible hybridization ( z 10% of the
library) at or near background levels or approximately equal
hybridization ( z 90% ) with the two cDNA probes. We
analyzed a randomly chosen set of 15 clones and found that
all had inserts: these inserts ranged in size from w 100 bp
to over 2 kb (data not shown). We concluded that moderate
to high abundance transcripts representing 8 90% of the
library of 900 clones had only 2 differentially hybridizing
clones. The other 10% of the clones without a signal above
background probably contained inserts homologous to low
abundance transcripts such that they are below the level of
detection by this differential screening method.

Expression of transcripts in weekly and highly virulent
amoebae. We used Northern blot analysis to confirm the
apparent differential hybridization of the clones and to
determine when during the transition of the amoebae from low

to high virulence the steady state transcript levels change.

35
Inserts prepared from the three plasmids were labeled by
random priming. The probes were hybridized to gel blots of
total RNA from axenically growing amoebae and from amoebae
taken from brains of mice after one, two, three or four
serial infections of mice. The results (Fig. 1 A) show that
although equal amounts of intact RNA were isolated in the
five samples as assessed by equal ethidium bromide staining
of 18 S ribosomal RNA, there is a strong differential
expression of clone Nf314. The Nf314 transcript levels
dramatically increase to high levels after only one passage
in the mouse. Quantitative analysis of transcript levels
with a Betascope showed that the induced levels of Nf314
mRNA are seven times the basal level found in axenic cells.
The levels stay high after subsequent passages (compare m1
RNA to m2, m3 and m4 RNAs). This autoradiograph was
overexposed to show the minor transcript levels hybridizing
to the Nf314 probe in axenically growing cells. RNA size
markers run on the same gel as the Nf314 transcript allowed
us to estimate the single transcript size as 1.7 kb: this
length was confirmed by primer extension of the mRNA (data
not shown). As a further control an insert from one of the
998 unregulated clones from the library, designated Nf279,
was used to probe the same blot. As shown the Nf279
transcript is present at about the same level in all cells
so the reduction in the Nf314 transcript level is neither

the result of blotting artifacts nor degradation of the RNA

36

Figure 1. Autoradiograph of Northern blot of RNA from
axenically grown amoebae and amoebae from mouse brain.
Total RNA (10 pg) was electrophoretically size separated on
an agarose gel containing formaldehyde. An ethidium bromide
stained gel shows that the 188 rRNA is intact and appears to
be of similar intensity in all of the samples: Ax (axenic
amoebae): m1,m2,m3 and m4 amoebae after 1,2,3 and 4 passages
of infection in mouse brains. A. The blot of the gel was
probed with the cDNA inserts from the Nf314 and Nf279
clones. The location of hybridizing mRNA is shown. Size
markers on the left side are from endogenous rRNA and 13
yittg synthesized transcript size markers. B. An identical
blot was probed with an 32P-end labeled oligonucleotide

homologous to a actin mRNA sequence.

37
Axrmm2m3m4

—Nf 279

1”" "4:314
1.4m.— .
133-

- A

m1 m2 m3m4
18$- ‘
—N fActin1
B

38
in the Ax lane. Since this method of mRNA analysis allows
detection of differentially expressed mRNA of unknown
function we wanted to confirm the apparent difference by
using an actin gene as a probe. An oligonucleotide was made
to a conserved region of a N. fowleti actin cDNA clone and
end radiolabelled for use as a hybridization probe. As
mentioned high virulence amoebae exhibit increased motility
(Cline, gt g1., 1986: Thong, gt g1., 1986). Interestingly,
actin transcript levels were slightly higher in high
virulence versus low virulence amoebae (Fig. 1 B). Thus
actin mRNA levels which might be expected to increase, did
increase, albeit to a much lesser extent than do Nf314
transcript levels. As will be reported (Fig. 5) the Nf435
clone exhibits dramatically reduced hybridization to RNA
isolated from LEEmp.

Southern blot and sequence analysis of Nf314. We chose
to examine clone Nf314 in more detail because of the
interesting regulation of the transcript. Southern blot
analysis was performed to determine the number of copies of
this gene and to determine if other closely related genes
were present in the genome. Total nuclear DNA was digested
individually with four different restriction enzymes and the
digestion products size separated by agarose gel
electrophoresis. The blot of the gel was probed with
radiolabelled Nf314 insert at moderate stringency. From the

autoradiograph (Fig. 2) we conclude that Nf314 is probably a

39

Figure 2. Autoradiograph of Southern blot probed with
the Nf314 cDNA. Genomic DNA (10 pg) digested with BamH I
(B), EcoR I (E), Hind III (H) and Pst I (P) restriction
enzymes was electrophoretically size separated, blotted onto
Gene Screen and hybridized to radiolabelled Nf314 insert.
Size markers on the left were from lambda DNA digested with

Hind III.

4O

BEHP

23.1—
9.4— p; 'I
t
6.7— ~
0
4..- I

2.3—
2.0-

n.

41
single copy gene because there is only one band of EcoR I
digested DNA of 5 kb hybridizing with the probe. We have
not excluded the possibility that two tandem copies are
present on this 5 kb fragment. The results also show that
at least one BamH I and Hind III site are present in the
gene. Even at a reduced stringency no other DNA hybridized
to the probe. We conclude that the 1.7 kb transcript
detected by Northern blot analysis is transcribed from a
single gene. The low steady state level of transcripts in
axenic cells is from the same gene transcribed to give a
high level of transcripts in the highly virulent cells.

The insert for clone Nf314 was determined to be
approximately 1.6 kb by agarose gel electrophoresis. Since
this insert was too long to sequence and obtain unambiguous
base determination we made subclones of the insert by taking
advantage of the restriction enzyme sites which the cDNA
shares with the gene. Sequence data was obtained both from
single and double stranded templates of an overlapping set
of subclones from both strands: the cDNA insert sequence of
1584 bp is shown (Fig. 3). Only one long open reading frame
was present extending 1449 bases from three ATG putative
start codons (nucleotides 34-42) to a TAA stop codon
(nucleotides 1480-1482): two additional possible stop
codons are at nucleotides 1492-1494 and 1510-1512. A
possible poly A addition signal is underlined 26 bases after

the stop codon. A polypeptide of 53 kDa with a isoelectric

42

Figure 3. DNA sequence of the insert to clone Nf314.
The sequences of both strands of the insert were determined
by use of universal primers and specific internal
oligonucleotide primers. An open reading frame starting from
three ATG codons to a single TAA codon is shown. A potential

poly(A) addition sequence is underlined.

GTC

ACT

TCC

TTT

AAT

CCT

CCA

TTA

CCA

GCA

GAT

TTA

GAT

GGA

CCT

L
TTA

I
AAC

R
CGT

A
GCC

A
GCC

TTT GTC

GTG GGT

P H
CCA ATG

C G
TGT CAA

S S
AGT AGT

G i
GGA ATC

P K
CCA AAG

U S
TGG AGT

C V
TGC GTT

G V
GGA GTG

Y T
TAC ACT

1 L
ATT CTT

I D
ATT GAC

L G
TTA GGA

TCT

g. g. g.

AGT

ATT

GTG

CAA

ATT

A
GCT

l
ATT

H
ATG

G
GGT

N
AAT

I
AAC

T
ACT

K
AAG

P
CCA

R
AGA

G
CAG

V
GTC

K
AAG

T
ACT

CTC

CAT

TAT

AGA

TTG

ACA

GGT

GCT

GGA

TAC

TTA

GAT

GCC

TTG

AGA

AAT

AGA

GTG

TAC

TTG

GTT

AGA

GTT

GCT

AGA

AAC

GTT

TAT

TTT

AAG

TAT

TCG

GTT

TTG

GTG

TTG

CCA

GGT

AAT

GGA

TCT

GTT

ACT

GCA

TCT

CCC

TTT

GCG

ATT

GCC

ACT

GAT

GGA

TCC TTC

CTT

AAT GCC

CAG GAC

GCC AGT

TAT TCT

TCA TAT

ATG AAT

TAT TTG

GGT

TTG

I G
AAT

I N
ATT

L K
CTC

K 1
AAA ATT

E R
GAA AGA

A K
GCA AAC

0 P
GAT CCA

I L
ATT TTA

T 0
ACT GAC

E T
GAA ACT

Y 1
TAT ATT

ATC

CCT

ACT

CCA

TGG

TCC

AAT

GCA

AAT

ACT

TCT

AAT

CCC

ACA

CGT

I
ATG

GGT

CGA

CTT

CAT

AAT

AAT

CCA

GGT

GGA

GAT

ATC

TTG

TAT

ACA

CCA

TTT

TTC

GGA

TTC

GTT

TCT

I
ATG

TTG

GGT

GTC

GGT

CGT

TCG

TTG

AAG

GCT

AAG

CCA

TAT

CTC

GCA

CGT

TGG

TAT

GTT

TGG

TTT

43

N
ATG

AGT

CGA

ATG

TTA

GTT

ACC

4

TAT

GCT

ATT

I
AGA

GGA

TAC

TGG

TTC

TCT

GAT

GAT

TAT

GCG

TAT

TGT

TAT

ATG

CGT

TTG

ATT

C
TGC

AAC

TTG

ACC

CTT

TTC

GGT

a g. gm 5. g.

GAT

ACA

ATG

TTC

GAC

TCT

TTA

CTT

TGG

ACT

V
GTG

ATC

TTT

AAT

GTC

ATT

TAT

TTG

GGA

TAT

GGT

AAT

CTC

CAA

ACT

GTT

TAT

AAT

GGA

TTT

TGT

GGA

CCT

TAT

AAT

CGA

TAT

GTG

AGT

TAT

TTG

CCA

GTG

CAT

GAT

ATT

CAT

GAT

GGG

ATT

CTC

TTT

GTT

AAT

ATT

AAC

ACA

TGG

TTG

GGT

GCT

GCA

ATT

ATT

GCT

TCA

TTG

60

120

180

240

300

360

420

480

540

600

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

Figure 3. cont'd

T F i T V R G A G N N V P L V K P O S A
ACC TTT ATA ACT GTT CGT GGA GCA GGT CAC ATG GTG CCT CTT GTT AAG CCT GAT AGT GCC 1440

F Y N F K I F l D G T C C End
TTT TAC ATG TTT AAG AAT TTC ATT GAT GGA ACA TGT TGT TAA GAGAATGCATAAATGTTCATTCC 1505

AAAAIAAAATCACACAGGAATTCACACTGTGATTTTATTTTGGAATTTATCACAGTGTGAATTCATTTGTCAAAAAAAA 1584

AAAAAAAAAAAAAAAA

44

45

point of pH 7.8 was predicted from the cDNA sequence.
Comparison of the predicted amino acid sequence to the
GENEMBL database sequences resulted in identification of
possible related peptide sequences (Fig. 4). The Nf314
sequence is shown in comparison to serine carboxypeptidase
sequences from yeast (carboxypeptidase Y: Svendsen, gt g1.,
1982), barley (carboxypeptidase I: Sorensen, 1986 ) and
wheat (carboxypeptidase II: Breddam, gt g1., 1987): within
194 amino acids (starting at residue 19) the Nf314 sequence
is 94 % similar and 19 % identical to the other sequences.
Outside of this area of high sequence homology, amino acid
residues 1 through 18 and 214 to the carboxyl terminal end
there is negligible homology of sequences (not shown). The
active site of the yeast carboxypeptidase Y has been
determined to be at serine 146 (Svendsen, gt g1., 1982).
There are five invariant amino acids in a peptide domain
including the serine at the yeast residue 146, A-G-E-S-Y,
that are shared by all four sequences. A carboxypeptidase
role for the Nf314 gene product is conceivable since there
are probably different proteins ingested by amoebae invading
the nasal mucosa, olfactory nerve or brain of the host
animal in comparison to amoebae growing axenically.

Induction of Nf314 gene expression by mammalian cells
in culture. The Nf314 gene expression gene product might
play a role in phagocytosis and feeding on the host cells or

for invasion of the host or evasion of the host immune

46

Figure 4. Comparison of the predicted Nf314 amino acid
sequence (amino acid residues 19 to 213 ) optimally aligned
by use of the FASTP program (7) with the carboxypeptidase
sequences of yeast, Cpbyy (25), barley, Cpbhs (24) and
wheat, C29639 (4). The amino acid residues that are
identical in all four sequences are shaded. The amino acid
residues of the Nf314 sequence that are similar (identical
or with conservative amino acid replacements) to any of the

other sequences are in boxed areas.

 

ut314 L P c L s c u i c v K s 1 IgG t L L A u A T R c R Y Li? v u

 

r rfEIs K R 54

 

P—

Cpbyy P K I L c 1 o P u v T 0;y ran 2 . L 0[:]E DCE]D K n r FEF u 1 39

 

 

 

 

Cpbhs L P a r 0 c A L P s K Hyfifc t v T v o E c u c R u Lgf v v v
c29639 L P a Zip Afjlv o r

45

 

45

O
I

 

 

 

 

 

5! sic i 1 1 v o E c :10 R s LiFiY L

........

 

 

 

89

 

 

Eff-1}”; '1

Cpbyy o P A K Dig v 1 L39 LjR 0 0 P B 2;; i L T .§¢ L r stills P
Cpbhs 0 P 0 K o é’v v Lip L R ch39 e c sfis F 0 .39 r v v E P36 P r

L

.l

-~--. :3.7:* m “T’- T 12>. . ' fl

uf314 n P s 0 ogg L v KER 1 R Ego Reef; 8 s L c .gg E A Sgggu§o L:4
1‘ s 74

 

80

 

 

 

81

 

 

 

 

 

.29... 0 .m. n L v m ”was: v . m . s elég‘lulei . .

 

 

Nf314 . . . . . A T 1 T REP pg; 3 9;. R v s I 1 L r 1 E a P v a v c r s v s 120

 

 

c
Cpbyy L . . . . . EflP’i 5 .ER Pig 5 9}» S[:]A t v i r L o 0 v E s E R 0 P c K 104
CpbhsG
CZ9639G.....
L.

  

pR‘Pyrgiffiis K v s t u 1 Y L o s P A a v c L s v s 106

 

 

 

1R E;r R Bin K v A u v L r L o s P A a v c r s v 1 112

 

 

 

Nf314 u s 1 o 0;: 0 u L N o v 0 A A s§§ n u u Afr R 0 r L 1 R PEP 0 r 1 . . c 154

 

 

u T v A A c K 0 vi; u rfoe L F r o x PEP E v v n K c 138

 

 

 

 

 

 

 

 

. c o L K 1 A T Bis u 1 F71 L K H r 0 L YEP E r L . . s 149

c o’uln 1 Alulo.s le FiL A K v F E R PPR H Y K . . v 146
: . . l. ,

190

 

 

 

 

 

 

 

 

 

 

 

 

If314 R E 1 v LiK‘§.Ejsgt c 0 v:TVV£ET T A v u 1 v

 

 

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029639 R o F v 1 11¢ é;s i A c HZPEvJP E L s . 0 L v

ht

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ur314 v c i L vie K‘s v 15051 E A 0 s u s 1 P P n u 213

 

 

 

Cpbyy T s v L zit R 9 L T §¥P L Ttlllilt’t E P u A 192
Cpbhs IKCWIIH v}cR B v[:]o~1 1 r 0 c u A L v P r A 208
029639 K c r n viB RIB L i §{o v u o v v c t r ElF u 201

 

 

 

 

 

 

 

 

 

47

48

defenses. To determine what might be the inducer of
increased Nf314 gene expression in amoebae we incubated
amoebae with mammalian cells in cell culture. In this
experiment amoebae might be exposed to an inducer of
increased gene expression even though the amoebae have not
been in contact with the nasal mucosa. Northern blot
analysis of RNA showed that amoebae incubated with mouse
brain cells, NIH 3T3 fibroblasts, or liver cells, but not
bacteria, E. gg11 or EacilLus gubtilis (not shown), had a
dramatic increase in gene expression (Fig. 5A). The
increase was of the same magnitude to that induced in fourth
passage amoebae isolated from mice brain. Thus all of the
induction can be accounted by some interaction of amoebae
with several types of mammalian cells in yittg. This
induction does not correlate with a simple change in
nutrients or ingestion of particulate foodstuffs as the
inducer of the gene expression since amoebae fed on gram
negative bacteria, E. gg11, or on gram positive bacteria, E.
ggpt111g, showed no elevation in Nf314 transcript levels.

The Nf435 transcript levels, in contrast, showed a
completely different response to co-culturing amoebae with
mammalian or bacterial cells. For Nf435 gene expression
axenic cell culture or culture with E. gg1i provided an
inductive stimulus to maintain high steady state transcript
levels. In contrast the interaction of amoebae with any of

the three types of mammalian cells caused a large decrease,

49

Figure 5. A. Autoradiograph of a Northern blot of RNA
from amoebae in cell culture. RNA was analyzed as described
in the legend to Fig. 1: The source of the RNA was
axenically growing cells (Axenic), amoebae fed with bacteria
(E. gg11), mouse liver (liver), 3T3 fibroblasts (NIH 3T3),
dissociated mouse brain (brain) and amoebae from brains
after the fourth infection of mice (M4). The blot was
probed with the cDNA insert to clone Nf314. B. An identical

blot probed with the cDNA insert to clone Nf435.

1“.

——Nf314‘

188- .

“4

L4Hy_

. 1
w - . —Nt435l
l
l
i
l

188—
1AKB—

51
not an increase, in transcript levels (Fig. 5B). Here too
all of the change in level could be entirely explained by
the interaction with mammalian cells in culture.
Transcripts from Nf435 are also not affected by a simple
change from axenic culture conditions because a substantial
change in diet, growth on bacteria, does not affect levels.
As a further control the Nf279 constitutively expressed
transcript levels did not vary with any of the conditions
which affect Nf314 or Nf435 transcript levels (data not
shown).

Sequence analysis of clone Nf435. Gene Nf435 was
differentially expressed during transition from low to high
virulence. Northern blots showed that the estimated size of
this transcript was about 1.5 kbp. However, sequence
analysis showed that this cDNA insert was incomplete and
only contained 324 base pairs and one open reading frame of
107 amino acids was present. The following is the

nucleotide sequence and deduced amino acid sequence.

K S G O D E I R l K R R K i K E 16
GA AAT TCA CAG CAA GAT GAA ATT CGA ATC AAG CGG AGA AAA ATT AAG GAG 50

K H N S S l D D I 1 G K T D L D G 33
AAA ATG ATG TCT TCT ATT GAC GAT ATT ATT CAA AAA ACA GAT CTC GAT GGT 101

D G I l N T G E F L E F H K I F D 50
GAT GGT AAT ATT AAT TAT CAA GAA TTT TTA GAA TTT ATG AAA AAC TTT GAT 152

R S E H L T S i E P S G V I G 1 L 67
CGA AGT GAA ATG TTG ACG AGT ATT GAA CCT TCT CAA GTC AAT CAA ATC TTG 203

S G P F R L L E E D L S l D I S T 84
TCA CAA CCT TTT AAT CTT TTA GAG GAG GAT TTA TCT ATT GAT AAT TCA ACA 254

I S E L T N E S N R I T F K F S H 101
ATT TCA GAA CTT ACA AAT GAA TCA AAT CGC AAT TAC TTC AAG TTC TCT CAC 305

I L N L G i 107
AAT CTG CAT CTC CAG ATT T 324

52

Comparison of the predicted amino acid sequence to the
Swiss protein database resulted in the following possible
related peptide sequences:

1. Of 40 best matched scores 17 are troponin C, e.g. from
human, pig, chicken, rabbit, mouse ranging from 22% to 36%
identical in 38 to 82 amino acid residues (Table 1). The
following is the comparison of the predicted Nf435 amino
acid sequence aligned with troponin C sequences from slow
skeletal and cardiac muscles of rabbit, human, chicken and
mouse. Asterisks(*) indicate positions of calcium-ligating
structures. Calcium binding domain III of troponin C is
highly conserved in rabbit, chicken, mouse and human and
located from 140aa to 153aa. Calcium is bound in the domain
by 6 amino acid, 141(0), 143(N), 145(D), 147(R), 149(0),
152(E) and 153(F). Alignment showed that Nf435 has high
similarity in this calcium binding region (77%). It has an
identical match at the positions 30(0) vs. 141(D), 34(0) vs.
145(0), 41(E) vs. 152(E) and 42(F) vs. 153(F), and similar
(with conservative amino acid replacements) at 32(D)(Asp)
vs. 143(N)(Asn), 36(N)(Asn) vs. 147(R)(Arg) and 38(N)(Asn)
vs. 149(D)(Asp). We noted that there was a segment of 7
amino acids E-F-L-E-F-M-K outside of the calcium-binding
region, which was identical with all of the troponin C

segments.

53

10 20 30 40 50
Nf435 NSQQDEIRIKRRKIKEKMM-SSIDDIIQKTDLDGDGNINYQEFLEFMKNFD
I'

TpCC_R YIDLDELKIMLQATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE

120 130 140* * * * * ** 160
TpCC_H EELKIMLQATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE
TpCC_C EELKIMLQATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE
Tpcc_M DELKMMLQATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE

Table 1. Comparison of predicted Nf435 amino acid sequence
with troponin C from various species.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Species Acch Troponin c in Identity“) Overlapus) In Nf435
Rabbit P02591 SLOU SKELETAL AND CARDIAC HUSCLES 36.2 ‘7 4'50
Human P02590 SLOU SKELETAL ARD CARDIAC HUSCLES 36.0 ‘7 5-50
House P19123 SLOU SKELETAL AND CARDIAC HUSCLES 35.0 67 5'50
Turkey P10246 SKELETAL MUSCLE 34.2 38 9'66
Chick P072588 SLOU SKELETAL MUSCLE 34.0 47 5-50
Barnacle P2179? ‘I’roponin C, isoform 27.6 58 20-77

P19 P02587 SKELETAL MUSCLE 22.0 82 9-88

L J l l I l J

 

2. Calmodulin from Q; discoideum (P02599): 40% identical in
42 amino acid residues ranges from 4 to 46 aa of Nf435
verses 106 to 147 aa of calmodulin. Following is the
comparison of the predicted Nf435 amino acid sequence
aligned with calmodulin sequence of Q; discoideum (Marshak,
gt al., 1984). The calmodulin molecule contains four
structural domains that are homologous to each other and to
the four domains of troponin C (Watterson gt al., 1980).
Calcium binding domain IV (Asterisks (*) represent the
positions of calcium-ligating bonds) is located from 129aa

to 140aa of calmodulin and binding bond at positions of

54
129(0), 131(0), 133(0), 135(0), 137(N) and 140(0), which are
identical to Nf435 at the position of 30(0), 32(0), 34(0),
38(N) and 41(E), similar at 36(N)(Asn) vs. 135(Q)(Gln). It
is interesting that the possible calcium ligating site in
the product of Nf435 matched with calmodulin of

Dictyostelium is the same as the site matched with troponin

C.
10 20 3O 40
435f.P DEIRIKRRKIKEKMMS-SIDDIIQKTDLDGDGNINYQEFLEFM
::| 000 '|0 0 00l::'000.'|||'00'|0:l000:
000 "0 0 00'0 0|000"|"'00|'0| 000
Calm_0 AELRHVMTSLGEKLTNEEVDEMIREADLDGDGQVNYDEFVKMM
110 120 * * * * * * 145

3. Also, there was a match with another calcium-binding

protein (SMZO) from fighigtgggma manggni (P15845): 44.4%

identity in 27aa overlap ranges from 22 to 48aa of Nf435.

All of these genes encode a protein that binds calcium.
Calcium binding proteins, such as calmodulin are important
regulatory proteins. With bound calcium, they can then bind
to and regulate the activity of many different cell
proteins. This includes calcium-calmodulin-dependent protein
kinase, which, in turn, phosphorylates target proteins and
alters their activity levels. The function of troponin C in
non-muscle cells is unknown. It is interesting that
repression of transcript encoded the calcium binding protein

was related with high virulence of amoebae.

55

The above alignment analysis suggested that there was a
possible calcium binding function in the product of gene
Nf435. Northern blot analysis showed that the transcript of
gene Nf435 was repressed during the transition from low to
high virulence. However, since the expression also was
inhibited when amoebae were fed with mammalian cells, like
gene Nf314, the altered expression of this gene was related

to high virulence but not related excluding to virulence.

C. DISCUSSION

Complex changes occur in n. fiowleti amoebae when these
cells undergo a transition from low to high virulence. One
approach to try and determine the molecular basis of this
transition has been to compare high and low virulent amoebae
by cytological or biochemical methods and to search for
differences. In most studies negligible differences have
been noted but highly virulent LEE strain differ from weakly
virulent amoebae in relative absence of phagocytotic food
cups (John, gt gl., 1985), in more rapid movement in gittg
(Cline, gt g;., 1986, Thong, gt g;., 1986) and in contact-
mediated lysis of cultured mammalian cells. Specific
proteins, a phospholipase (Fulford, gt g_., 1986), a
neuraminidase (Elsen, gt gl., 1987), cytopathogenic material
(Dunnebacke, gt gl., 1989) and a membrane-associated pore-
forming protein (Young, gt g1., 1989) may play a role in
establishing degrees of virulence. Whether these

quantitative changes are necessary and sufficient to convert

56
amoebae has been difficult to determine.

A second approach using molecular and genetic
techniques can be complementary to the cytological and
biochemical work. To begin a molecular and genetic analysis
we assessed the protein changes which occur during the
transition from low to highly virulent LEE strain by two
dimensional gel electrophoresis (Hu, gt g1., 1991). Many
more quantitative and most importantly new qualitative
changes in protein accumulation and synthesis were found
than had been previously thought to exist. The mechanism
for increasing or decreasing protein levels in the cells was
in part ascribed to changes in levels of specific
transcripts. lg gtttg translation of poly (A)+ mRNA in
rabbit reticulocyte extracts revealed many changes in steady
state levels of individual transcripts (Hu, gt g1., 1991).
We have now extended our results based on the previous
knowledge of variable transcript levels by isolating cDNA
clones of some of the differentially expressed mRNAs. Clones
representing increases (Nf314) and decreases (Nf435) in
levels have been found but as expected from the previous
work most clones are constitutively expressed transcripts
(Nf279 for example).

Here we have concentrated on the analysis of the Nf314
clone. The deduced sequence of the amino acid residues
shows it to be related to a carboxypeptidase from yeast,

barley and wheat. This role for the gene product would be

 

 

57
consistent with the previously noted importance of digestive
enzymes, elastase (Ferrante, gt g1., 1988) or hydrolytic
enzymes or cytolytic factors (Fulford, gt g1., 1986; Lowrey,
gt 91., 1985) for the increased virulence. The overall
similarity of identical and conservative changes of amino
acid residues is high when the FASTP program is used for
alignment of the sequences (Devereux, gt g1., 1984). A 194
amino acid section of the predicted amino acid sequence is
94 % similar to the known serine carboxypeptidase sequences.
Little information is currently available to assess whether
this percentage is within the range expected for comparisons
of the sequences of g. fowleri. In Qictyogtelium amoebae
similarity to related proteins of the yeast figggngtgmyggg
ggtgxigigg is not common even for highly conserved ribosomal
proteins (Kopachik, unpublished). Data on the active site
sequences of carboxypeptidases suggests that the serine 146
of yeast carboxypeptidase Y is at or near the active site.
It is perhaps therefore significant that the deduced Nf314
gene product shares the high level of identical or conserved
amino acids found in all three carboxypeptidases in the
putative active site. If a transformation vector can be
developed further knowledge about the function of the Nf314
gene product might be obtained by over-expression of the
gene product in u. fowleti. Increased levels of
carboxypeptidase in the expressing cells would be consistent

with the role of Nf314 gene product predicted from sequence

58
analysis.

The current work showed that Nf314 transcript levels
are regulated by mammalian cells in culture. The inducer
for maximum increased mRNA levels was determined to be
mammalian and not bacterial cells. This finding is not
consistent with an inductive signal associated with
invasiveness of nasal mucosa or response of amoebae to the
host soluble or cellular immune defense mechanisms.
Macrophages can target and kill amoebae in culture and
antibodies are present against H. fgwleti in many
individuals (Fisher-Stenger, gt gl., 1990; Marciano-Cabral,
1988). It is conceivable that these immune responses could
induce changes in amoeba gene expression but we have not
found this to be the case for any of the two regulated genes
cloned. Given this background the biochemical nature of
the inducer of increased Nf314 gene expression remains to be
determined but we have shown that the inducer is not unique
to nerve cells. The induction also occurred when liver or
fibroblast cells were used. It will be interesting to
determine if transcripts from other genes are coordinately
induced along with Nf314 gene expression. We expect this
might occur because the synthesis of numerous proteins is
coordinately increased or decreased (Hu, gt gl., 1991). The
Nf435 transcript levels are oppositely regulated to Nf314
transcript levels: when Nf314 transcript levels are high,

Nf435 transcript levels are low and when Nf314 transcript

59
levels are low Nf435 transcript levels are high. It is
conceivable that the same induction signal from mammalian
cells is transduced in cells by a common second messenger
pathway, but the signal is interpreted differently to result
in either increases or decreases of mRNA levels. Having
found regulated genes we are interested to know if the
mechanism of response to increase Nf314 mRNA levels includes
increased transcription rates and increased mRNA stability,
or both. Transcriptional regulation of gene expression can
be determined by nuclear run-off experiments and post-
transcription regulation could be assessed by examination of
mRNA half lives.

A variety of approaches, cytological, biochemical and
molecular-genetic have now been applied to the problem of
understanding the capacity of n. fogleti to become
pathogenic. All of these approaches are rapidly increasing
our knowledge of this complex change and interaction of the
amoebae with host cells. With more information on the
biology of Naegleria improved strategies for prevention and
treatment of primary amebic meningoencephalitis should

emerge.

CHAPTER THREE: A simple, efficient method to create a

cDNA library‘

A. INTRODUCTION

Making a successful cDNA library is the one of the key
steps in molecular cloning mRNA expressed from the
eukaryotic genome. Currently, two popular techniques are
used. The first "Classic method” (3) uses oligo(dT) while
the second uses a "vector-primer” (Okayama-Berg method)
(1,2), with an oligo(dT) at one end to prime poly (A)+ mRNA
for cDNA synthesis. Although these two methods have been
widely used, the chief drawbacks are the large amounts of
poly (A) + mRNA necessary and the number of steps needed. In
the "Classic method" either adapters or linkers are added to
cDNA ends before inserting into an appropriate vector. The
cDNAs must be separated from the excess unattached adapters
which results in loss of cDNA. In the Okayama-Berg method,
much less poly (A) + mRNA is needed but an oligo(dC) tail
has to be added. Second, if the first strand cDNA is not
completely synthesized a recessed 3' end will be difficult
to tail and some potential inserts will be lost. Third, a
Hind III enzyme has to be used to remove one oligo(dC) tail,

but incomplete enzyme digestion may be a problem. Fourth,

 

1Chapter 3 has been accepted for publication in
BioTechinique, December issue of 1992. (Hu, W., W. Kopachik,
and R. Neal Band. 1992. A simple, efficient method to create
a cDNA library. BioTechinique.)

60

61

the oligo (dC) tail of the non-cDNA end must be removed to
prevent interference with ligation. The extra steps and
additional manipulations taken increase the chances of
losing samples and making errors. Therefore, even though
large amounts of mRNAs may be available the yield of clones
is sometimes low. This may be part of the reason why many
beginners often fail to make a good cDNA library. Here we
describe a quick simplified "All In One Tube" procedure to

construct a cDNA library.

B. MATERIALS AND METHODS

yggtgtzztimgt Brepatation (Eigt 1). We used pBluescript
II KS plasmid (Stratagene) to create a vector-primer. In the
poly-cloning site, following complete digestion of 10 pg of
the vector to form a blunt end with the restriction enzyme
EcoR V (GAT'ATC) , we added oligo(dT) tails to both ends of
the vector with terminal deoxynucleotidyl transferase
(Pharmacia) according to the Okayama and Berg method (1,2).
The sample was extracted with phenol-chloroform and
precipitated with ethanol and then digested with Sma I
(CCC'GGG) . The vector-primer was removed from the small part
of the other oligo(dT) end by electrophoresis on a GTG
SeaPlaque low melting agarose gel(1%) (FMC) and
electroelution. After ethanol precipitation, the vector-

primer was dissolved in 40 pl 10 mM Tris (pH 8) buffer.

62

gDNA gigging (Eigt z). Vector-primer 0.5 pg was mixed
with the poly(A)+ mRNA selected with an oligo(dT) cellulose
column (Pharmacia) from 200 pg total cellular RNA of soil
amoebae Nggglgtig fiowleti and heated 2 min. at 65W3. It was
slowly cooled to room temperature to allow annealing. The
first and second strand cDNA was synthesized using the "cDNA
synthesis system plus" kit (Amersham) following the
manufacturer's procedures. The second strand cDNA synthesis
was labelled with 1 pl (10 pCi a-‘ZP-dc'rp) (3000 Ci/mmol)
(DuPont/NEN) and used as a tracer. After double-stranded
cDNA synthesis, the sample was extracted with phenol-
chloroform and ethanol precipitated overnight. The next
step was self-ligation of the two intra-molecular blunt end
arms. The DNA was resuspended in 28 pl ligation buffer with
2 pl (20 units) ligase (Boehringer Mannheim) and incubated
overnight at 15°C (Fig. 2). 01150 cells (BRL) were
transformed with the re-circularized recombinants following

the manufacturer's directions.

C. RESULT AND DISCUSSION

The cDNA library made by this method contains about
23:105 clones/pg vector and has 85% white colonies. Ten white
colonies were selected and plasmids prepared (3). After
digestion with restriction enzymes BamH I and Hind III the
DNA was separated on an agarose gel (1.2%) (Fig.3). The cDNA

insert sizes ranged from 200 bp to 2,000 bp, and are similar

63
in size to the inserts synthesized when using oligo(dT) as a
primer (data not shown). This cDNA insert size range is
representative of the mRNA size range of fit fowleri amoebae.
Double-stranded DNA sequencing of one plasmid (4) confirmed
its cDNA insert was correctly connected to the vector EcoR V
site (Fig. 4).

The cDNA orientation can easily be constructed in the
opposite direction if in the "vector-primer" preparation the
restriction enzyme Sma I is first used,

The procedure described here has the following
improvements over the two other methods:

1) It has the advantages of vector-primed double-stranded
cDNA synthesis which include the cDNA inserts with a known
orientation and low background of plasmids without inserts.

2) It is simpler because addition of linkers, adapters
and C-tails is not necessary. It is therefore quicker and
especially useful when only small amounts or valuable mRNA
samples are available. The vector-primer synthesized cDNA
needs only to be extracted with phenol-chloroform, ethanol
precipitated and self-ligated.

3) This method overcomes the disadvantages of bi-
molecular blunt end ligation while adding linkers or
adapters in the "Classic method". Here a more efficient
intramolecular ligation occurs.

4) In addition, the cDNA clones are already in

pBluescript II KS with T3 and T7 promoter sites flanking the

64
insert. Once cloned, RNA transcripts can be generated

directly by in vitto transcription systems.

A limitation of this method is that it misses the genes
whose transcripts do not have poly(A) tails, such as histone

gene.

Recommended Protocol:
Egtt 1. yector-Ptime; Erepatation Lfiigt 1):

1. Completely digest 10 pg pBluescript KS with EcoR v
in a 100 pl volume.

2. Extract the DNA with phenol-chloroform once and
chloroform once. Add 1/10 vol 3 M NaAc and 2 vol cold
ethanol to precipitate the DNA on ice for at least 0.5 hr.
Spin down at 13,000 rpm for 15 min at.4PC, rinse with 70%
ethanol, dry and dissolve in 16 pl H53.

3. Add poly(dT) tails to both ends with terminal
transferase (TdT) (1,2). Add 2 pl 10x TdT buffer, 1 pl of 5
mM dTTP and 40 units of TdT at 37°C for 15 min. The DNA is
purified as in step 2.

4. Digest the poly(dT) tailed DNA with Sma I in'a 100
pl volume.

5. Remove the vector-primer from the small dT tailed
partial cloning site by running the DNA in a 1% GTG
SeaPlaque low melting gel (FMC) at.4PC following the

manufacturer's protocol.

65

6. The vector-primer is electro-eluted in a dialysis
bag at.4PC and repurified as in step 2 and dissolved in 40
pl TE buffer.

Egtt 11. QDNA gigging Lfiigt 21: Adapted from the Amersham's
cDNA synthesis Kit procedure.

7. For first strand cDNA synthesis: Mix 2 pl (0.5 pg)
vector-primer with 9 pl poly (A)+ mRNA (~1 pg) at 65%: for 2
min and let tube cool down for 15 min at room temperature:
add 4 pl 5x reaction buffer, 1 pl Sodium pyrophosphate, 1 pl
RNAse inhibitor, 2 pl dNTPs (lOmM) and 1.5 pl (30 units)
reverse transcriptase and incubate for 1 hr. at 42°C. Keep
on ice.

8. For second strand cDNA synthesis: Add 37.5 pl second
strand synthesis buffer, 1 pl E; 9911 RNAse H (0.8 unit), 7
pl E; 9211 DNA polymerase I (24.5 units) and 34.5 pl Hg)
(total 100 pl) and incubate at 12%: for 1 hr (optional: add
1 pl 32P-dCTP for tracer) and then at 22°C for 1 hr: transfer
to 70°C for 10 min. After spinning down for 15 sec. in a
microcentrifuge, add 1 pl T4 DNA polymerase (4 units) at
37°C for 10-15 min.

9. Purify the DNA as in step 2. Add an equal vol. of 4
M ammonium acetate and 2 vol. of ethanol to precipitate
overnight at -20°C. Spin down at 13,000 rpm at 4°C for 15
min. Aspirate the supernatant, rinse the cDNA with 70%
ethanol, dry and dissolve in 25 pl H53.

10. Blunt end ligation. Add 3 pl 10x ligation buffer

66
and 2 pl T4 DNA ligase (20 units, Boehringer Mannheim) and
incubate at 15°C overnight.
Egtt 111. Transformation:
11. Take 5 pl of the ligation mix and add to 100-200 pl
DHSa competent cells (BRL) following the manufacturer’s

procedure.

67

a. Partial Multiple Cloning Sites of Plasmid
pBluescript II KS (Stratagene)

 

 

 

 

 

 

is... H
H HH
110506 I:
coouma
w-IUUUIEGI
5’ mmmmwm
3.1111111111
b. Eco RV digestion & Oligo(dT) tail
H E P S B
5’ TTT
3.1:]:T TTTT111111
c. Sma I digestion & purification
H B
5’ TTT 3’
3.1:]:T 111 5,
E P

TTTT:I::I: (Discard)

Figure 1. Diagram of vector-primer preparation.

3’
SI

3’
5’

Abbreviations: H: Hind III: E: EcoR I; P: Pst I: S: Sma I:

B: BamH I.

68

 

 

a. b. Annealing & first strand
AAAAAA ---------- mRNA cDNA synthesis
H H
5’ TTTTT 5’ TTTTT—-—-———
3._:_I:l 3.3m .......
vector
c. Second strand cDNA synthesis d. Ligation
H H B
5' 1—|TTTTT———— 5'—(—.'r'r'r'r'r , 1
3!
3' J—‘AAAAA— 3'—1—‘AAAAA 1 1
5!

Figure 2. Diagram of cDNA recombinant construction.

69

Figure 3. Mini-prep analysis of Naeglerig fowleri cDNAs.
cDNA inserts were released with double-digestion of
restriction enzymes BamH I and Hind III and electrophoresed

on 1.2% agarose gel. Lanes 1 to 10 were cDNA clones: M, size

markers.

7O

12345678910

—Vector
2.0kb—

1.3kb—

0.5kb—
0.3kb—

 

71

Figure 4. Double-stranded DNA sequencing of a randomly
selected cDNA clone to show that cDNA insert was connected
at the Eco RV site. Sequencing was performed with Sequenase
kit Version 2.0 (United State Biochemical) according

manufacture’s procedure and labelled with 35S-dATP (DuPont).

e
5
m

 

1t).

 

SUMMARY AND DISCUSSION

My research emphasized a study of epigenetic changes in
virulence of fit fowleri and not differences in pathogenesis
between species or strains among Naegleti . This contrasts
with the work on pathogenesis in 5t histolytica. Much of the
work on fit tistolytica now focuses on distinguishing
pathogenic from non-pathogenic isolates (Ravdin, 1989). As
mentioned, fit fowleri is always pathogenic but virulence can
be altered. Therefore, I focused on differential expression
of genes responsible for virulence changes during the
transition of LEE amoebae from axenic growth to growth in
the mouse brain. The goal of this work was an initial step
to uncover the molecular signals of high virulence by fit
tgglgti. By inoculating long term, axenically cultured LEE
strain amoebae into the mouse brain to trigger high
virulence, I was able to investigate the mechanism of the
process. Changes of gene expression of amoebae were observed
by two-dimensional gel electrophoresis analysis during the
transition of amoebae from low to high virulence. To
identify the transcripts differentially expressed in highly
virulent versus weakly virulent amoebae, a cDNA library was
made to highly virulent amoebae. Three cDNA clones were
isolated by differential screening of the cDNA library. The
transcripts homologous to Nf314 and Nf116 were
preferentially expressed in highly virulent cells whereas

the transcript homologous to Nf435 was preferentially

73

74
expressed in low virulent amoebae. The results were
important because mRNA levels of these genes correlated with
changes in virulence. However, because the Nf116 (actin),
Nf314 and Nf435 gene expression correlated with feeding on
mammalian cells as a food source, these genes might be
required but are not sufficient for increased virulence.

We eliminated the possibility that the initial low
virulence of at fowleri was due to a mixture of low or non-
virulent species or variants. We used a cloned amoeba of Hg
figylgti for our experiment so that this potential problem
was not possible.

Is it possible that the induced gene expression
responsible for high virulence is terminated after amoebae
entered the brain? I believe this is unlikely since the
virulence of amoebae after brain passage has been increased
which indicates the gene(s) are still being expressed.
However, it is possible that transient gene expression is
needed to penetrate host barriers (e.g. the nasal mucosa)
and these would not be detected in virulent amoebae isolated
from the brain.

Could the changes in protein synthesis be due to
stress proteins? Stress protein synthesis inhibits other
protein synthesis and cell growth (Atkinson, gt gl., 1985).
LEEmp amoebae in mouse brain and in axenic culture grow very
well. The changes associated with virulence noted in two-

dimensional gels, persisted for weeks or for months and

 

75

conserved patterns of stress proteins was not observed in
two-dimensional gels. Therefore, there is no reason to
believe the induced proteins are simply stress proteins.

Protein synthesis changes could have arisen by
transcriptional and by post-transcriptional regulatory
mechanisms. My research mainly focused on the changes in
transcript levels. Therefore, what genes would I miss by
technical limitations or by the way I screened for the cDNA
library? In the current cDNA screen method, I only detect
relatively high abundance transcripts at about 5%. I will
miss some epigenetic changes in gene expression. Here are
representative examples. We would not detect a change: if
the mRNA level does not change, but the translatability
does: if the protein abundance does not change, but the
protein is post-translationally activated or inactivated and
undetectable on two-dimensional gels; if a change in
glycosylation of a glycoprotein or glycolipid is important
for increasing virulence: if membrane surface rearrangements
increase virulence: if physiological(e.g. chemotaxis,
chemokinesis, phagocytosis) and metabolic changes are
important for virulence and do not result in protein or mRNA
change. Fortunately other investigators using immunological,
biochemical and other cell biological methods will address
some of what we miss.

We knew that the changes in gene expression during

transition from low to high virulence were complex (Hu, gt

 

76
31., 1991). To simplify the complexity and to clarify the
future work, it is necessary to make a classification of
genes in order to focus on those regulated genes and gene
products which are related to virulence. Genes whose
expression is altered in response to feeding on mammalian
cells and tissues without significantly increasing virulence
are referred to as LEE:CELL genes. We will refer to those
genes, whose expression is changed due to introducing
amoebae intranasally in mice and coinciding with increased
virulence, as LEE:MP (mouse brain passage) genes. LEE:MP
gene products could be used to overcome host barriers and
participate in the pathogenic action of amoebae on brain
tissues together with LEE:CELL gene products.

To clone LEE:MP genes, we will use the cDNAs made from
mRNA of amoebae fed with mouse brain cells (LEE:CELL) as
probes, instead of using cDNAs from mRNA of axenic amoebae
(LEE:AX). The other probe will be cDNAs from mRNA of mouse
brain passaged amoebae (LEE:MP). This will permit us to
isolate the virulence-specific genes. As an alternative, we
could also construct a subtracted cDNA library from LEE:MP
mRNA. We would isolate LEE:CELL mRNA and bind them onto a
poly(T) column. First strand LEE:MP cDNAs would be passed
through the poly(T) column a few times to hybridize with
homologous mRNAs. The un-hybridized cDNAs will be used to
construct a cDNA library. With small amounts of mRNA, we can

use the method described in Chapter three to make and then

77
differentially screen the library.

Increased virulence could be a result of either
increased or decreased gene expression in response to
contact with the nasal-mucosa epithelial matrix and cells.
To clone transcripts present at a lower level when amoebae
enter the mouse brain, we could make a cDNA library from
axenic cultured amoeba LEE:AX mRNAs. Those low level
transcripts could be isolated by differential screening the
cDNA library with cDNAs made from LEEzMP and LEE:AX mRNAs.

The increased protein synthesis changes may be due to
transcripts regulated to a high level. To clone those genes,
we could run two-dimensional gels using protein samples
prepared from LEE:MP and LEE:CELL amoebae to observe changes
in protein synthesis. The negative control would be to run a
two-dimensional gel with a protein sample made from mouse
brain cells to eliminate the possibility of contamination.
We can then, isolate the specific spots and micro-sequence
the protein (Matsudaira, 1987). Oligonucleotides based on
that sequence could be used to isolate the cDNA by screening
of a cDNA library.

Many micro-organisms such as viruses, bacteria, fungi
and protozoa can cause CNS diseases. The routes of infection
are diverse depending on the microbes. For example,
bacteria may gain access to the subarachnoid space via
hematological invasion or local spread. Virus-induced

diseases are mostly due to infection of skin, usually by the

78
bite of insects, or via the exposed mucous membranes of the
respiratory, gastrointestinal or urogenital tracts (Adams,
gt 11., 1988, Chadwick, gt gl., 1989). Once penetration
occurs, viruses may reach the CNS by direct neural spread or
by hematogenous or lymphatic spread to cause disease. Fungi
may give rise to a variety of neurological syndromes causing
a subacute meningitis. Some diseases of the CNS are also
caused by amoebae such as Nggglgtig fiowleti or Acanthamoeba
spp. The port of entry may be different between them.
Infection from Nt fowleri is via swimming in warm polluted
lakes or pond, whereas Aggnthgmggbg spp. may gain entry by
lungs, urogenital tract, or skin (Ma, gt gl., 1990, Lambert,
1991). However, the precise nature of the mechanisms
employed by many micro-organisms to gain an entry is still
not well known. Factors involved in parasite entry may be
mediated by one major protein or multiple gene products. The
molecular and genetic study to understand the precise nature
how at jgglgti gains the entry into the host is the area of
the present research. However, to date, none of those genes
have been identified.

The host-parasite interaction is a very complex
process. Signaling between microbe and host involves
reciprocal interaction. For example, virulence modulation
occurs for poliovirus serotype 3 in host cells (Wick, gt
31., 1991). It is neurotropic but is attenuated after

passage through cell cultures and monkeys. The genetic basis

79
of viral modulation is several nucleotide differences in the
region upstream of the poly-protein encoded by the viral
RNA. Position 472 in the 5’ untranslated region of the viral
RNA is particularly related to neuro-virulence. Virulence
correlates with Cytosine phosphate-472 whereas attenuation
correlates with Uridine phosphate-472. This was supported by
site mutagenesis (Wick, gt g1., 1991). We do not know
whether there is a similar process involved in the amoebae,
but it does serve as one example of virulence modification
at the molecular level.

One essential factor for infection by a particular
microbe is that the host cell must have specific receptors
on its plasma membrane. For several bacterial genera,
specific proteins encoded by the microbe appear to play a
critical role in their ability to invade eukaryotic cells
(Falkow, 1991). The bacterial pathogen Xersinig
pgguggtubetculosis is a well-studied example of an invasion
process mediated by a membrane protein encoded by a so-
called "invasion gene" (Miller, 1992). This invasin protein
interacts with integrins (host surface receptor) to mediate
the bacterial entry of the host.

Entgmggtg histolyticg is an obligate human gut
parasite. The invasive amoebae penetrating the intestinal
mucosa and phagocytosis of host tissues is a multifactorial
process. The first step is surface contact between

trophozoites and the target cells. This interaction is

80
believed to be mediated by specific molecules present in the
surface of the parasite. The adherence is mediated with a
galactose-specific cell surface lectin (Tachibana, gt g1.
1991). It is not known if the invasion and penetration of
the nasal mucosa requires a similar receptor-ligand binding
mechanism in Ht fgylgti.

In the future, with LEE:MP genes which are likely to
play a role in virulence, the question may be: what is the
biological function of the gene(s)? In our case, a cloned
gene may be of interest because of its differential
expression at a specific developmental stage (necessary and
specific for high virulence). To test possible roles of
cloned LEE:MP genes, a method of "Reverse genetics" may be
used. The logic is simple in principle: isolate a gene, then
test the gene's role by molecular genetic techniques. One
of the advantages of this method is that without knowing the
role of a particular DNA sequence beforehand or reference to
a specific protein, we could determine its function or
developmental significance by "reverse genetics" according
to an altered developmental program. For example, the
experimenter can place a gene under the control of
regulatory elements that will alter the temporal or spatial
regulation of gene expression. The resulting phenotype may
suggest how the gene product functions in normal
development. This technique has been widely and successfully

used in analyzing the functions for many genes in many

81
organisms such as human genetic diseases (Orkin, 1986),
mouse (Landel, gt gl., 1990), thggpgilg (Ballinger and
Benzer, 1989) and yeast (Campbell, 1988). Here is an
example of ”reverse genetics" in human genetic disease.
Transgenic animals that display phenotypes similar to human
disorders have been useful for defining gene function. The
generation of animal models for human disease has used the
insertion of genes encoding mouse homologue of human
dominant mutation into the mouse germline in attempts to
create a mouse analogue of a human disorder. Osteogenesis
imperfecta (OI) type 2 is a autosomal dominant disorder
caused by the substitution of a single glycine residue in
the triple helix of the al—procollagen gene COLIAI. This
substitution in a repeating Gly-X-Y structure alters the
molecule so that collagen assembly is affected and results
in abnormalities of bone formation. A similar phenotype was
induced in a transgenic mouse strain by performing in vitro
mutagenesis on the mouse homolog of the human al-procollagen
gene to induce the same amino acid substitution. The mutated
gene was then used to produce transgenic mice by micro-
injection into isolated mouse embryos. All of the resulting
transgenic animals died shortly after birth due to severe
developmental defects in the formation of the skeleton.
Analysis of these mice for the mutant procollagen
demonstrated that expression of the mutation gene at levels

as low as 10% of normal was sufficient to disrupt bone

82
formation and lead to death. This suggests that the
defective protein inhibits collagen assembly rather than
forming a less stable molecule that is susceptible to
degradation or altered secretion (Landel, gt gl., 1990).

In our case, we would develop a transformation
expression vector carrying the cloned LEE:MP gene and re-
introduce this into weakly virulent amoebae. The gene can be
over-expressed at the transcriptional level under the
control of a strong promoter (e.g. actin gene).
Transformants will then be tested for virulence. Site-
directed or deletion mutagenesis could also be used to
define the bases or sequences necessary for regulation of
transcription. 1

One limitation to study the function of introduced gene
is the presence of the wild counterpart of the gene in the
cell. This requires that both alleles be inactivated prior
to testing. To overcome this limitation, "gene targeting or
homologous recombination" could be used, by which the
endogenous functional gene can be precisely replaced with an
engineered derivative. This is a very powerful method for
altering and testing gene functions. This has been
successful in Qictyosteligm (Witke, gt gt., 1987: De
Lozanne, gt al., 1987; Manstein, gt g1., 1989), yeast and
mice (Bollag, gt al., 1939, Capecchi, 1989). In diploid
organisms with a sexual cycle, a single heterozygous

replacement is first obtained, and then rendered homozygous

83
by sexual crossing. However, many diploid unicellular
organisms, or cultured mammalian cells lack a sexual cycle
and they offer superb systems for studying biological
phenomena not readily accessible in whole organisms. Gene
sequences in mammalian cells have been targeted by
homologous recombination. This is a strategy to obtain a
double knockout of the 2 copies of a targeted gene, creating
null mutants (Riele gt gl., 1990, Mortensen, gt gl., 1991).
It has also been used in the asexual protozoan parasite of
Lgighmgnig mgjgt (Cruz, gt gl., 1991). In this method,
homologous recombination was used to sequentially inactivate
both alleles of an endogenous gene. Two markers (neomycin
phosphotransferase [NEO] and hygromycin phosphotransferase
[HYG]) were used in their studies. Briefly, a targeting
vector that contains the targeting gene interrupted by the
marker gene (NEO), was constructed and introduced into cells
by electroporation. Stable transformants (+/neo) were then
selected with G418. The remaining normal allele was
inactivated by a similar process. The same vector containing
the target gene but interrupted by a different marker gene
(HYG) was used to knock out the second allele. The '
transfection and selection were the same as for the +/neo
except hygromycin B was substituted for G418. Both Southern
blot analysis and/or PCR were used for analysis of targeted
event. In the PCR approach, two primers were synthesized:

one was designed to anneal and prime at the site of the

84
target gene and the other at the site of G418 resistant
gene. With non-homologous recombination, no fragment was
amplified. In targeted insertion, the primers were annealed
to each other and produced an amplified fragment at a
predicted size.

Nggglgtig fowleri is at least a diploid organism. This
is strongly suggested by the occurrence of heterozygous
electrophoretic patterns of many isozymes (Pernin, gt g1.,
1985, Cariou, gt gl., 1987 and Adams, gt gl., 1989). Sexual
recombination in this organism is unknown. Therefore, the
above strategy could be used to inactivate both alleles of
the endogenous gene (LEE:MP) before testing its function.
Availability of antibiotic markers is another important
issue. Resistance of N; fowleri LEE strain to G418 has
been tested previously in our laboratory. The LEE strain is
not sensitive to this drug. Resistance to Hygromycin-B and
some other antibiotics such as Bleomycin could be tested in
the future.

Other methods could also be used to obtain homozygous
mutant lines from a heterozygous parent in the absence of a
sexual cycle. Parasexual crossing has been utilized in
Digtyggtgligm, but has not been demonstrated in Naegleria.
Another approach may be the use of radiation or other agents
to induce mutations. The disadvantage of this method is that
radiation or mutagenic agents may create many unpredictable

mutations which are not easy to analyze.

85

An alternative approach for shutting off the functional
existing gene is called the "antisense" method. The idea is
to target the gene's mRNA rather than the gene itself. We
could transfect amoebae with an expression vector that
carries a portion of the target gene (LEE:MP) downstream of
a strong promoter. But the orientation of the target gene in
the vector would be backward, so that the RNA transcribed
from the vector is complementary in sequence to the mRNA
transcribed from the corresponding cellular gene. The
antisense RNA is present in large excess, its bases pair to
the functional mRNA to form a double-stranded RNA that
cannot be translated into protein. Then the transformed
amoebae would be tested for virulence.

Much of the above depends on the availability of a
transfection vector. Therefore, constructing a transfection
vector used for above tasks will be another important
project in the future. This vector will have an EL ggli ori
and an antibiotic (e.g. ampicillin) resistance gene for
propagation in bacteria. A strong expression promoter (e.g.
actin gene) would be included to overexpress the transgene
in a sense or antisense configuration. Multiple cloning
sites with three translational reading frames would be used
to insert into a gene of interest in a proper position.
Finally, translation stop codons in all three reading frames
and a transcription termination signal (AUAAA) segment would

be also included in the vector.

86

There are several things we could do in the analysis of
gene Nf314. First, to further understand more about the
structure, function and possible regulatory mechanism of the
gene Nf314, a genomic clone of Nf314 is needed. A genomic
library could be constructed by using bacteriophage lambda
Zap II (Stratagene) vector. The cDNA insert of Nf314 would
be used as a probe to screen a genomic DNA library of Nt
figglgti to obtain the genomic clone of gene Nf314.

To further verify that a serine carboxypeptidase is the
product of gene Nf314, an oligo-peptide from the deduced
amino acid of gene Nf314 could be synthesized and used to
generate an antisera in rabbits (Reilly, gt gl., 1991). The
immunoglobin fraction is precipitated from the sera,
dialyzed and redissolved in a proper buffer. The antibodies
would be then used for immuno binding to: a) a known
carboxypeptidase: b) the amoeba extract fractionated on
SDS/PAGE gel and transferred to a nylon-membrane (Western
blot analysis). An additional control would use a known
carboxypeptidase antibody generated from another species
(e.g. from mosquito, Cho, gt g1., 1991) to hybridize with
the blot of amoeba extract. We expect that the hybridization
band in b) experiment would be the same as that of control
experiment if the product of Nf314 is a serine
carboxypeptidase.

A binding analysis to an inhibitor could also be

performed to determine the molecular size and amounts of

87
serine carboxypeptidase between LEE:AX and LEE:MP amoebae.
Amoeba extract would be incubated with a labelled serine
protease inhibitory [3H1-diisopropyl fluorophosphate (DFP)
which binds to the active site of serine protease (Cho gt
g1., 1991) as a marker. The mixture would then be separated
by SDS/PAGE. The gel would then be dried and exposed to a X-
ray film. We expect to see thicker and darker band in LEE:MP
comparing with LEEzAX amoeba extract line.

Serine carboxypeptidase purification for partial
sequencing compared with the deduced amino acid sequence of
Nf314 may be another way to verify the gene Nf314 product.
The purification of the enzyme could be performed according
to the methods described by Cho, gt g1.(1991). Partial
amino acid sequencing could be done by using the protein
sequencer from the Department of Biochemistry, Michigan
State University.

We know that the increased Nf314 expression was induced
by co-culturing amoebae with mammalian cells. However, we do
not know where the inducer is located. We could incubate
amoebae with mammalian cell surface membrane or cytoplasm
individually (Clarke, gt g1., 1988). Then RNA would be
extracted from amoebae and Northern blot analysis used to
see the response of Nf314 transcript to the different
feeding studies. After determining the possible location of
the inducer, its ultimate identification would be possible.

There are still many other questions to be answered

88
such as how many LEE:MP genes are really involved in the
amoebae’s virulence? how are those genes expression
regulated? what is the inducer of the virulence? How do the
signal transmitted from environment to genes? what are the
immune functions of the host to the microbe? why amoebae fed
by an mouse brain cells (LEE:CELL amoebae) did not increase
in virulence? We are just beginning to explore the nature
of the molecular mechanism of virulence. With more
information available, better methods for prevention and

treatment of PAM may be revealed.

Appendix 1.
Electrophoretic Karyotype of Naegletig fowleri

The analysis of chromosomal organization has been
difficult in Ngggtgtig. The exact number and size of
chromosomes are not known. This controversy is due to the
difficulty in counting the chromosomes cytologically since
they are small and closely packed to accurately enumerate
even with electron microscopy (Fulton, 1970).

Pulsed-field gel electrophoresis (PFGE) allows
separation of chromosome-sized DNAs. Previous PFGE in at
Igglgti (De Jonckheere, 1989) did not give a good separation
pattern. The bands were smeared and blurry. It was reported
that between 15 to 23 bands resolved in size ranged from a
few hundred kb to about 1.5 Mbp.

I attempted to establish an electrophoretic karyotype
for fit fgwlgti. It would be possible to localize the cloned
genes onto specific chromosomes, to study linkages with
other genes of this organism.

Materials and Methods

1. Equipment for PFGE: CHEF-DR II Megabase DNA pulsed
field electrophoresis system from BioRad was used.

2. Preparation of Chromosome Sample for PFGE: 10°1cells
were harvested and embedded in 1% low melting agarose gel
(Sea Plaque) in the mold on ice for 1 hr. The agarose
blocks were incubated in lysis buffer (1% SDS, 0.5 M EDTA pH

9.5, and 2 mg/ml proteinase K) at 54°C for 3 days. Blocks

89

90
were stored in the same buffer at.4PC until electrophoresis.

3. Pulsed Field Gel Electrophoresis: The gel was run
in 1% SeaKem Gold agarose (FMC Inc.) in 0.5 X TBE buffer at
180 V with pulse times of 80 to 100 sec. for 24 hr along
with the standard DNA molecular weight marker (Lambda ladder
and Yeast chromosomes).

Results and Discussion
The gel resolved about 14 bands and 15 Yeast
chromosomes (Fig.1). Bands having stronger intensity may
contain more than one chromosome with similar molecular
weights. The size ranged from about 50 Kbp to more than 1.6
Mbp.

In comparing my result to De Jonckheere's work, a
clearer banding pattern was observed. This can be seen by
the sharp resolution of yeast chromosomal and lamba markers.

This was a preliminary experiment. Many factors such as
different pulsed times, voltages, concentration of agarose
and temperature affect the migration and resolution of
banding pattern (Lai, gt g1, 1989). Further improvement
could be done by adjusting the above factors to achieve the
best resolution. For example, using a lower range of pulsed
time (50 to 70 sec. instead of 80 to 100 sec.) to increase
the mobility of low molecular weight chromosomes and repress
larger ones to get a better resolution of smaller sized

chromosomes.

91

Figure 1. Electrokaryotype of E; fowleri LEE strain. Yeast

(Sggghgtgmyggg getgvisiae) chromosomes (line 1) and lambda

DNA (Line 2) were used as size markers (Kbp). Line 3 is at

fowleti karyotype pattern.

 

2,200—

1:020‘
850—
700-—

460——
245——
486——

 

92

Appendix 2. Sequence analysis of an actin cDNA insert‘.

The rapid penetration of amoebae into the brain implies
that actin, one of the components governing cell mobility,
may play an important function for pathogenesis of fit
1221911. in addition to participating in maintenance of cell
structure and cytokinesis. A study of four species of
Nggglgtig under agarose showed that pathogenic spp. are more
motile (Thong and Ferrante, 1986). EL fowleri and fit
gggttgligggig (pathogenic to mice) exhibited more rapid
locomotion than nonpathogenic Ht lgvaniensis and fit gruberi
(Marciano-Cabral, 1988). The species differences in motility
were also observed when these four species were placed in
proximity to nerve cells (Marciano-Cabral, 1988). Highly
virulent amoebae of E1 fiowleti LEE strain (LEE:MP), but not
axenic cultured, weakly virulent amoebae (LEE:AX) were
chemotaxic it yittg to neuroblastoma cells extract.
(Brinkley and Marciano-Cabral, 1992). Northern blot analysis
showed that the level of actin transcripts in LEEmp amoebae
increased at least 3 fold over the LEE amoebae (Hu, gt gl.,
1991). An increase of actin transcripts and motility might
be associated with enhanced amoeba virulence. We isolated a
few positive clones from the cDNA library by hybridization

C G G G
with a 20mer oligo-nucleotide (5'-TAGAAGCATTTTCTGTGCAC-3’),

which was complementary to the sense strand near the

 

1This work was in part of Jonghee Ahn’s master thesis.

93

94
carboxyl terminus end of actin genes according to a
consensus sequence from various species. The longest insert
1220 bp was sequenced and had an open reading frame of 375
amino acids (Jonghee Ahn, 1992, M.S. thesis, unpublished).
Sequence analysis comparison revealed a difference in
genetic codon usage between fit fowleti and A; castellanii
(Table 1.). For example, Nt fowleri (actin and
carboxypeptidase) uses only TAA as the stop codon, but TGA
and TAA were both used in A; castellanii. Table 1
illustrated that CGT (30%) and AGA (59%) were the preferred
codons for arginine in Nt fowleri, while CGC, CGG and AGG
were not used. In contrast, CGC (54%) was used by At
gggtgllgnii. In the case of leucine, TTG (63%) was the
dominant codon in Nt fgwleti but CTC (56%) and CTG (36%)
were used by A; castellanii. Other comparisons included
glutamine in which fit fowleri used CAA (92%) and GAA (89%)
for glutamic acid, instead of CAG (90%) and GAC (95%) in A;
gggtgllgnii. We compared the third-position nucleotide
frequency codon usage for actin cDNA with A; castellanii and
several other species (Table 2). Interestingly, there is a
significant difference in G+C usage between these two
species. Actin cDNA in fit fowleti used (40%) G+C: which was
comparable to other eukaryotic microorganisms. However, A.
gggtgllgnii exhibited a much higher G+C content (85%),
similar to ptosgphilg melanogastet (G+C, 85%). This result

suggests that those two species may have diverged early in

95
evolution. This codon usage information will be useful in
designing oligonucleotides for probes and for the polymerase
chain reaction.
The GenBank accession number for the cDNA clone of

action gene transcript was #M90311.

Table l. Codon usage by Naegleria fowleri and Acanthamoebg castellanii.*

Amino Codon
ac id
ARC CGT
(R) CGC
CGA
CGG
AGA
AGG
ALA GCT
(A) GCC
GCA
GCO
VAL G‘IT
(V) GTC
GTA
GT G
ASN AAT
(N) AAC
GLN CAA
(Q) CAO
LYS AAA
(K) AAG
MET ATG
(M)

‘ S codon frequency given for & fowleri (NF) actin I (GenBank accession number M9031!) and serine

Nf

30

5|

21

47

27

72

28

92

19

mo

62

29

5|

43

97

IO

Amino
acid

LEU

(L)

GLY

(0)

ASP

(D)

GLU

Codon

GGC

GGA

GOG

ACT

ACC

ACA

ACG

GAT

GAC

GAA

GAG

TGG

NF

63

55

42

50

29

80

20

89

47

53

56

36

25

65

69

24

93

95

94

Amino
ac id

SER

(5)

PRO

ILE

CYS

(C)

HIS

339

END

Codon

TCT
TC C
TC A
[TCG
AGT

AGC

ccr
ccc
CCA
cco
ATT
ATC
ATA
TGT
TGC
‘CAT
CAC
TAT
TAC
TGA
TAG

TAA

carboxypeptidase (M88397); A castellanii (AC) actin (V00002. IOl0l6) and myoein IB heavy chain

002974, M13564. MI3565). Codon frequency calculated with the Genetics Computer Group Sequence

Analysis aoflware package (Devcreux gt 51.. I984).

96

NT

27

30

63

78

20

79

2|

76

24

I00

Ac

38

59

27

92

IO

9|

92

50

50

Table 2. Third-Codon-Position Nucleotide Frequency for Actin Gene Coding

Region“

N aegleria fowleri

(carboxypeptidase)

Dictyostyelium discoideum

film mlycephalum
Saccharomycec cerevisiae
[etrahymena thermophila
Entamoeba bistolytica
Elasmodium falcigmg
Acanthgmoeba casteILa_n_ii

Droso ila melanogaster

96A

20

27

32

27

21

16

38

45

 

%T

40

33

17

36

15

40

10

11

96C

20

16

35

l4

13

29

31

%C

20

I3

29

21

29

36

14

56

52

%G + C Reference

40

29

35

S6

43

49

22

15

85

83

this paper

Hu, e_t 11., 1992
Warrick, 1987

Hamelin e_t 91., 1988
Edman, g §_l_., 1987
Martindale, I989
Edman, gt a_l. 1987
Wesseling, I989

Nellen & Gallwitz, I982

Starmer & Sullivan, 1989

*Carboxypeptidase from E; fowleri was included since it is the only other gene

sequence published for this organism, except ribosomal nucleotide sequences

(Baverstock g 3L, 1989).

97

Appendix 3
Note: The following methods (or protocols) used during my
research, were developed by Dr. W. Kopachik, or they were

commonly used procedures available in his laboratory.

MATERIALS AND METHODS

1. CULTURE OF N; gowleti

Low-virulent amoebae of the LEE strain of fit fowlgti
were obtained from Dr. David John, grown in 10 ml H4 liquid
medium supplemented with 1 ml hemin in 250 ml culture flask
(Band and Balamuth, 1969) at 379C.

High virulent amoebae (LEEmp) were obtained by serial
inoculation of the LEE strain amoebae into mouse brains for
four consecutive passages. Ten 3 to 4-week-old male mice
(Mtg musgglus BALB/c) were used for each passage. Amoebae
were harvested and washed by centrifugation three times with
LS saline (50 mM NaCl, 4.6 mM MgSO‘ and 0.36 mM CaClz). A
series of 2 pl of LS saline containing N. fowleri (10°
amoebae were used for the first passage and 10‘ for
subsequent passages) were applied into the nares of each
mouse, using a narrow bore 0-200 pl microcapillary pipet tip
(Western Coast Scientific, Inc.). Within 4-6 hr after death
pieces of brains were placed in 250 ml tissue culture flasks
(Corning Science Products) containing 10 ml H4 medium with

Penicillin-G (1000 units/ml), Streptomycin (100 units/ml)

98

99

and incubated at 37°C. Two hr later, mouse brain tissues
were removed and fresh H4 medium without antibiotics was
added to the residual amoebae. The amoebae were then
cultured for 24 to 36 hr before protein analysis.

Bacterized (LEE.b) amoebae were grown in E. ggli
(strain K-12) (1 mg/ml LS saline) and incubated at 37W3.
Before protein analysis, the amoebae were cultured in LS
saline overnight without bacteria.

Amoebae were co—cultured with 3T3 fibroblast tissue
culture cells, with dissociated mouse brain cells and with
dissociated mouse liver cells in H4 medium without hemin at

37°C.

2. DNA ISOLATION
A. Plasmid DNA isolation (for high copy number plasmid,
e.g. pUC18, pBluescript).
a. Large scale plasmid DNA preparation without CsCl.
The plasmid DNA isolated with this procedure is good
for sequencing, restriction enzyme digestion, etc.

1. Grow 50 m1 gt coli DHSa bacterial culture with 50

 

pg/ml ampicillin overnight.

2. Harvest bacteria with 50 ml tube.

3. Resuspend cells in 3 ml lysis buffer (50 mM Tris,
10mM EDTA, pH 8 and 20% Sucrose) with 20 mg lysozyme and
keep on ice for 10 min.

4. Carefully dropwise add 6 ml freshly made 0.2 N
NaOH/1% SDS buffer while swirling and pipeting them up and
down with 10 ml pipette until mixed, then incubate on ice
for about 5-10 min. (after cell lysis, the solution will be
clear.)

5. Add 3.8 ml 3 M KAc (pH 4.8 or 5.2). Carefully mix by
inversion and incubate on ice for about 15 min, or until all
the white staff floating on the surface.

6. Centrifuge at 12,000 rpm for 10 min. at room
temperature.

7. Filter the supernatant with No.1 Whatman filter into
30 ml Corex tube.

8. Add 2 vol. 100% cold ethanol, mix and keep the tube

on ice for 30 min.

100

9. Spin 15 min at.4PC at 12,000 rpm using HB-4 rotor.

10. Discard the supernatant, rinse with 10 ml 70%
ethanol and dry.

11. Dissolve the pellet in 3 ml TE buffer.

12. Add 150 pl RNase A (10 mg/ml) and incubate at 37%:
for 1-2 hr.

13. Transfer the solution into a 15 ml orange cap tube;
add 2 ml phenol/chloroform, vortex and pour into a 15 m1
Corex tube.

14. Spin 10 min as in step 9.

15. Repeat step 13 and 14.

16. Extract DNA with equal volume of chloroform and
spin 10 min. at.4PC at 12,000 rpm using HB-4 rotor.

17. Transfer DNA into a new 15 ml Corex tube and add
1/10 vol. 3 M NaAc (pH5.2), 2 vol. 100% ethanol. Keep on ice
for 30 min.

18. repeat step 9 and 10.

19. Dissolve DNA in 1 ml TE and Rinse with 1 ml TE
buffer. Combine them together and check 0.0 at 260/280.

b. Large scale plasmid DNA preparation with CsCl.

(Vertical rotor preparation of plasmid).

1. Grow 100 ml Et ggti DHSa bacterial culture with 100
pg/ml ampicillin overnight.

2. Harvest bacteria using a 50 ml tube with HB-4 rotor
at 7,000 rpm for 5 min. and resuspend in 3.8 ml sucrose

buffer (50 mM Tris, 10mM EDTA, pH8 and 20% Sucrose) with 20

101

102
mg lysozyme. Keep on ice for 5 min, then add 100 pl RNAse
(10 mg/ml) for anther 5 min.

3. Add slowly with a pipette while swirling 5 ml of 2%
triton X-100/50 mM Tris/10 mM EDTA. Draw up and down the
solution several times in the pipette to make sure all of
the cells are exposed to the detergent. Leave on ice for 10
min (or until solution is clear).

4. Transfer the solution into a clear T865 tube. Spin
in ultracentrifuge T865 rotor at 25 K for 20 min. Pellet
should be slightly fluffy but doesn't have to be.

5. Transfer supernatant into a 15 m1 orange cap tube
and dilute to 10 ml with water containing 0.4 ml of 5 mg/ml
ethidium bromide.

6. Dissolve 9.6 g CsCI in the solution by warming to
55°C for 15 min or longer. Spin in table top centrifuge at
maximum speed for 10 min. Carefully take out the red protein
floating on the top and load the solution into 15 ml
polyallomer TV856 tubes. Balance to within 0.1 g and spin
for at least 8 h at 50 K at 20°C.

7. Localize the lower band with the long wave-length UV

 

lamp (366nm) in a dark room. Remove the lower band in about
1 ml volume. Extract with 5 M saturated isopropyl alcohol 4
times to remove ethidium bromide (the solution will be clear
without any pink color). Dialyze against TE buffer in 2
liters with 2 changes in overnight.

8. Extract dialysate with phenol/chloroform, chloroform

103
alone. Precipitate with 1/10 vol. 3 M NaAc and 2 vol.
ethanol.
9. Dissolve in 200 pl TE buffer. Take 5 pl for 0.0.
260/280 nm reading to measure the concentration, and also

check on gel.

c. Small scale plasmid DNA prep. (alkaline-SDS method)
The plasmid DNA isolated with this procedure is good

for sequencing, restriction enzyme digestion, subcloning,
etc.

1. Pick a single bacterial colony with the plasmid
interest from a LB-agar plate containing ampicillin (50
pg/ml), inoculate in 5 ml of SOB medium with 100 pl
ampicillin (10 mg/ml) and grow overnight at 37W:.

2. Spin down 1.5 ml culture in a Eppendorf tube.

3. Aspirate all the medium as much as possible.

4. Add 100 pl GTE buffer and vortex to mix.

5. Add 200 pl 0.2 N NaOH/1% SDS buffer and shake to mix
well. Keep on ice for 2-3 min or solution clear.

6. Add 150 pl 3 M KAc buffer, mix and keep the tube on
ice for 5 min.

7. Spin down at 12,000 rpm for 10 min. at room
temperature.

8. Transfer supernatant to a new tube with a pipette.

9. Add 800 pl 100% cold ethanol, mix well and keep on

ice for 30 min.

10.
11.
ethanol.

12.

104
Spin down at 13,000 rpm at 4°C for 15 min.

Aspirate the supernatant and add 0.5 ml of 70%

Spin down the DNA for 3 min in microcentrifuge and

aspirate the ethanol and dry.

13.

Dissolve DNA in 200 pl TE buffer and add 10-15 pl

RNAse (10 mg/ml) at 37mc for 1-2 hr.

14.
15.
16.
speed.
17.
step 16.
18.

19.

Add 250 pl TE buffer.
Add 300 pl phenol/chloroform and vortex 1 min.

Spin down in microcentrifuge for 5 min in high
Transfer the supernatant into a new tube and repeat

Extract DNA with 300 pl chloroform.

Transfer the supernatant into a new tube and add

1/10 vol. of 3 M NaAc (pH5.2), 2 vol. 100% cold ethanol.

Keep on ice for 30 min.

20.

21.

22.

Repeat steps 10, 11 and 12.
dissolve DNA in 50 pl TE buffer.

Take 3 pl to check on gel and 0.0. at 260/280 for

concentration.

Solutions for plasmid preparation:

10

SOB medium LB medium

20 g 10 g bacto typone
5 g 5 g yeast extract

2.5 ml - 1 M KCI

2.5 ml 10 g 4 M NaCI

110 to 1000 ml

105

adjust pH to 7.6, autoclavg, thgn ggg filter stezile 10
ml of 1 M MgCI2 and 10 ml of 1 M MgSO‘ for SOB.

2. SOC medium: Add 10 ml of filter sterilized 2 M
glucose per liter or 18.6 ml of 20% glucose to SOB solution.
3. LB-agar plate: Add 15 g bacto-agar/ 1 liter.
4. Alkaline-SDS lysis buffer (ptgpgtg ttggh):
8.5 ml H20, 1.0 ml 808 (10%) and
0.5 ml NaOH (4 M) ----- Total 10 ml
5. 3 M KAc buffer: Dissolve 14.72 g KAc in 35 nu.150 in

50 ml orange cap tube, add 5.75 ml Acetic acid, 1120 to 501111.

B. H1 fowleri Genomic DNA isolation (CsCI method) (Nellen,
gt al., 1987)
1. Grow 4 liters of L fgwlgti cells (5-6 x 10°/ml)
2. Harvest cells using GSA rotor at 5,000 rpm for 5
min.
3. Dissolve the cells in a 1:40 vol. ratio of ice cold
HMN buffer 0.1% NP-40, and on ice for 5 to 10 min.
(the solution should be clear)
4a. Check cell lysis under the microscope, if no
lysis, add more NP-40.
4b. After cell lysis, spin at 8,000 rpm for 5 min to
pellet nuclei.
5. Repeat the step 3.
6. Repeat the step 4b, and dissolve the pellet in 5 ml
HMN buffer.

7. Drip into 0.1 M EDTA (pH8.2)/4% Sarcosyl

106

8. Put the tube in 65°C water bath for 5 min, solution

should be clear.

9. Measure the amount of solution, add ethidium bromide

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

to 0.4 mg/ml.

Add 0.95 g CsCI per ml (including ethidium bromide
solution).

Put the tube into 55°C until all the solid CsCI
dissolved.

Using table centrifuge to spin for 5 to 10 min at
high speed to get rid of the upper protein film.
Transfer the solution into an ultracentrifuge tube.
Ultracentrifuge at 35 K at 15°C for 36 hr using the

fixed angle rotor.

Take off the tube from the ultracentrifuge, look it
under the long wave-length UV light. Sometime it
will be seen 2 bands. The upper band may be the
mtDNA etc. The lower one will be genomic DNA.

Carefully cut off the top of the tube with a sharp
razor blade and take off the top red protein layer
and other solution using a wide-bore pipet tip.

Then transfer the band from the top into a 50 ml
orange cap tube and add 1.5 vol water to mix well.

Add 1 vol. phenol/chloroform and gently mix.

Spin down and transfer the upper aqueous phase into
a dialysis tubing against TE buffer (4 L) for

overnight and change once.

20.

21.

22.

23.

24.

107

Transfer into 30 ml Corex tube and add 1/10 vol. 3M
NaAc, 2 vol. ethanol to precipitate at -20°c for
overnight.

Spin down using HB-4 rotor at 13,000 rpm for 15
min.

Discard the supernatant and rinse with 70%

ethanol.

Dry with speed vacuum and add 1 ml TE.

Rock for a day at room temp. to dissolve the DNA.

3. SOUTHERN BLOT

1. Completely digest 10 pg of genomic DNA in a total
reaction volume of 50 pl with 5 pl restriction enzyme at
37°C for 3 hr or overnight. Use only a cut off pipette tip
with a wider bore so the DNA is not sheared excessively. Mix
the reaction well by pipetting and flicking the tube because
the high M.W. DNA is very viscous.

2. Prepare 0.7% agarose gel with 1X TBE buffer. Need
400 ml of gel in the 20 X 30 cm gel box and 800 ml running
buffer. Use the large comb slots (0.9 cm X 1.5 cm) or else
the DNA may smear during the run. Cover the gel with a thin
(2 mm) layer of buffer.

3. Add 5 to 10 pl loading buffer to the sample and mix
well. Then load the sample in the slots and use lambda DNA
(lpg) cut with Eco RI or Hind III or both as a size marker
at the ends of the gel. Run EC 500 at 80 V. for 10 min. to
make all of the DNA stack in slot. Then lower voltage to 40
V. and run overnight for about 15 hr until the dye has run
about 10 cm. Run all gels the same way so that blots done on
different days could be easily compared later.

4. Shut off power and cut away the excess gel. Transfer
the DNA in gel using a X-ray film as a support because the
gel breaks easily. Cut off a small bit of one edge of gel as
an orientation mark. Place in a large plastic box and add
about 1 liter of buffer then about 2 to 3 drops of ethidium

bromide until the solution is just pink. Shake for about 15

108

109
min. or until the DNA is stained. View on the
transilluminator and take a picture with a ruler set along
the side of the gel. At the same, turn on the Haake Cooler
set at -10°C.

5. Replace the buffer with 1 liter of 0.2 N NaOH (8
g)/0.6 M (35 g) NaCI and shake for 30 min. to denature the
DNA. Replace with 1 liter of 25 mM phosphate buffer pH 6.5.
Change buffer twice within about 30 min.

6. Pour 5 Liters of phosphate buffer into the
electroblot apparatus. Cut 4 sheets of 3 MM filter paper and
1 sheet of Gene Screen membrane to the size of the gel to be
blotted. Soak the gene Screen for about 10 min. Assemble the
blot sandwich: on the plastic grid in a box with 3 liter of
phosphate buffer place a layer of foam, 2 layers of pre-
wetted filter paper, the gel, the Gene Screen, 2 layers of
pre-wetted filter paper, foam and then the other side of the
plastic grid. 00 all of this under the buffer to prevent air
bubbles from lodging in the sandwich. Sandwich will not
transfer in bubble regions. Clamp the sandwich with your
hands and do not release your grip until the sandwich within
the electroblotter slots and under the buffer.

7. Electroblot for z0.4 Amp for 1 hr. Check after 10
min. to make sure the Amp. is stable. Increase Amp. to zl
Amp. and continue for 6 hr. or longer. At the end remove the
filter paper and dry it. View the blot with the short wave

UV light and mark lightly with pencil any lambda size bands

110
or rDNA bands and the slot.

8. Fix the DNA on the blot by baking 80%: for 2 hr.

9. Seal the blot in a seal-a-meal bag with 1X 10 ml
hybridization buffer (depend on the size of membrane).
Prehybridize the blot at 65°C for at least 1 hr using the
agitator set for slow movement of the buffer over the blot.

10. Cut the bag at a corner and pour off the
prehybridization buffer. Then add a very hot inset probe at
5 10 ng/ml for at least 24 hr. Shake at 65%:.

11. For the post-hybridization, wash the blot as

follows:
a. 2 X 2X SSC R.T. for 5 min each
b. 2 X 0.5x SSC/1% SDS at 65°C for 30 min.

Check background count (cpm) over region which
should not have any counts: if less than 50 cpm, stop here.
If the counts still high:
c. 2 X 0.2x SSC/1% SDS at 65°C for 30 min.
Check counts
d. 2 X 0.1x SSC/1% SDS at 65°C for 30 min.
(There should be almost no detectable cpm even over the
DNA which should hybridize.) Wash away excess $08 with a
couple of rinses in 0.1x SSC.
12. Wrap the blot in plastic wrap and mount over 3 MM
filter paper using tape. Mark all size markers, rDNA and
anything other bands you saw on the blot with radioactive

(”8) ink. Put date, orientation marks as X on the filter.

111
Do not get outside wet or else the film in contact will be
ruined. Set up blot against XAR-S Kodak film and lay this
over an intensifying screen in a cassette. Expose at -70%:
for overnight or longer.

13. Keep blot for reuse as many as three times. For
reuse, follow procedure for stripping old probe (0.1x SSC/
1%SDS, boil for 30 min.). Store the dry blot at room temp.
in plastic bag.

Sglgtions tgt Southern Btgt:

1. Phosphate buffer for 1 M stock.

Mix 93.84 g NaHzPO‘ and 45.44 g NaZHPO‘ in 700 ml water,
heat and stir then cool to room temp.: adjust pH to 6.5 and
volume to 1 liter.

2. (pre-)Hybridization buffer:

NaCI 2.92 g
1 M tris pH 7.5 2.5 ml
50% dextran sulfate 10 ml
50 X Denhardts 5 ml

10% SDS (ultra-pure) 5 ml
10 mg/ml Salmon DNA 0.5 ml
water to total 50 ml

4. DOT BLOT

1. Dissolve about 1 pg DNA in 10 pl HgL.

2. Add 2.5 p1 of 2 N NaOH (0.4 N final).

3. Put the Eppendorf-tube in 80°C for 10-15 min

4. Add 10 pl of Neutralization buffer.

5. Spot 4 pl at a time on GeneScreen membrane, and
allow to try before adding another 4 pl.

6. Bake the blot at 80°C for 2 hr. or use UV cross
linking method.

7. Ready for pre-hybridization.
Neutralization buffer:

2.5 ml 1M Tris pH 7.5, 1.25 ml 2N HCl and 6.25 ml 20X SSC

112

5. RNA ISOLATION (Chirgwin, gt al. 1979) ,

A. Isolation of total cellular RNA from N1 fowleri
DA! 1

1. For each 10° cells (about 4 ml cell pellet from 200
ml shaking culture) dissolve in 5 to 10 ml of 4M guanidinium
thiocyanate/0.1 M 2-mercaptoethanol/pH 5. Freeze sample in
dry ice or place in -20 or -70°C freezer to make sure that
all cells lyse. Samples can be stored indefinitely when
frozen.

2. Thaw sample, then spin at 3000 rpm or more in a
table top centrifuge to pellet insoluble material if any.
Centrifuge for 15 to 20 minutes is usually enough.

3. Take an RNAse free AH-629 polyallomer tube and add
2.5 ml of 5.7M CsCl/0.1 M EDTA/pH 5 to it to form a CsCl
cushion. Gently layer the sample over the cushion. Fill the
tube up to within a couple of millimeters of the top. The
sample can be up to 13 ml. Balance the tubes to within 0.1 g
and load all of the buckets even if only 2 have samples.
Centrifuge for 24 hrs at 25K at 15'C.
For the Beckman SW 41 use 2.2 ml of CsCl and spin at 33K and
for the SW 28 add 5.5 ml of CsCl and spin at 27 K. Shorter
spin times (18 hr) could be used without significant loss of
RNA yield.
BAX Z

4. Remove all of the sample by aspiration down to the

CsCl layer and then add 3 ml of dep'd water to the tube to

113

114
wash the walls. Aspirate the wash water and most of the
CsCl so that about 1 ml is left. Invert the tube and keep
inverted while cutting the bottom of the tube with a razor
blade. Make a cup of the bottom so that you can get to the
pellet easily.

5. Carefully add about 0.5 ml of dep'd water to wash
the area around the pellet and drain this out. Do not
disturb the small button-like translucent pellet of RNA
which will be in the exact bottom of the tube. The small
aggregates around the pellet are not RNA and should be
avoided. They can be wiped out with a Kim-Wipe if
necessary.

6. Take up 0.5 ml of dep’d water in a blue tip and jab
the pellet to break it up then add the water and take up the
pieces to transfer to a microfuge tube. Add another 0.5 ml
to the cup to rinse and make sure that no RNA is left in the
cup or in the blue tip. Now vortex the RNA continuously for
about 2 min to dissolve the RNA then spin in the microfuge
for 5 min to pellet the insoluble material, if any. Take up
the supernatant and transfer to a baked Corex 13 ml tube and
add 2 ml of dep'd water, 75 pl of 4M NaCl, and 7.5 ml of
cold 100% ethanol. Cap with parafilm, invert to mix and
store at -20°C for a few hours or overnight or indefinitely.

7. Spin the tube at 13,000 rpm in the HB-4 rotor for 20
min at 4'C to pellet the RNA. Drain or aspirate the

supernatant and then gently wash the film of‘a pellet with

115
cold 80% ethanol using about 0.5 ml to remove the residual
NaCl. Remove this wash ethanol by aspirating. Cap the tube
with parafilm and poke holes in it and then place in a
vacuum for 5 minutes or until the ethanol and water have
evaporated. The pellet assumes the appearance of a dry
cracked lake bed. Take up the pellet in 200 pl of dep'd
water and vortex to dissolve the RNA. Transfer to a
microfuge tube and then add another 200 pl of water to rinse
the tube and transfer this to the microfuge tube also. RNA
dissolves easily within 2 min of vortexing and if there is
pellet material that does not dissolve in this time it is

not RNA.

This RNA is good enough for use in Northern blots.

8. To further purify the RNA (for in yittg translation
and construction of a cDNA library) add 0.4 ml of buffered
phenol/chloroform and vortex to make a cloudy emulsion.

Spin for 2 min. in the microfuge to separate the phases.
Remove the upper water phase and leave any interface behind.
Re-extract the water phase in another tube with
chloroform/isoamyl alcohol 0.4 ml and spin as before. Remove
the lower chloroform phase and re-extract with chloroform
again. The small amount of residual RNA left in the
phenol/chloroform phase could be recovered by adding 0.2 ml

of dep'd water to it and vortexing and spinning as before.

116
If done add this to the ca. 0.3 ml first recovered from the
chloroform. Add 4M NaCl to 0.1 M final concentration ( 12.5
pl to 0.5 ml) and 2 volumes of cold 100% ethanol (1.0 ml to
the 0.5 ml). Vortex to mix and allow to precipitate at -20°C
for at least 2 hours or overnight or indefinitely.

9. Spin in the microfuge for 10 min and wash the pellet
with 50 pl of 80% ethanol. Dry in the Speedy Vacuum.

10. Add 1 ml of cold 2 M LiCl to the dry pellet and
break up the pellet. Cap and rotate or rock for about 1
hour at 4'C. Spin again in the microfuge to recover the
pellet and then wash the pellet with 50 pl of 2M LiCl.
Remove the wash and add 0.5 ml of dep’d water to dissolve
the pellet and then precipitate with NaCl and ethanol as
before (step 8). Finally dissolve the RNA in about 100 to
200 pl of dep’d water and determine the 00 at 260 and 280
nm. Add 5 pl of the RNA to 1 ml of water and read the 00.
Get at least 0.1 00 units at 260nm, if necessary add more
sample. Determine the 00 and multiply by the dilution factor
of 200 and multiply by 40 pg/ml to get the RNA concentration
in pg/ml. The usual 260/280 ratio is about 2 to 2.3 but
values as low as 1.6 are still good preparations.

NQIEE

1. This procedure gives total RNA which can be
translated in the reticulocyte assay because inhibitory
material has been removed with the extra phenol/chloroform

and LiCl washes. Do not use more than about 2 x 10°«cells

117
in the 10 ml at step 1 because a large amount of gelatinous
material will spin if the gradient is overloaded.

2. At step 10 the RNA will not dissolve easily
occasionally if the RNA concentration is too high and
especially after phenol/chloroform extraction. These RNA
aggregates will need extra help in dissolving such as
breaking up the aggregates with a blue tip and vortexing for
a long time (over 10 min).

Sglgtigng fig; RNA prgparation:

1. All glassware should be baked to be RNAse free (at
350'F for at least 3 hours).

2. All plasticware is OK if not handled with bare hands
which have RNAse. Wear gloves throughout the procedure.
Have pipet tips and microfuge tubes and glassware that you
will use only for RNA preparation.

3. Water. Use nanopure water that has been treated
with diethypyrocarbonate at a final concentration of 0.2% as
follows. Make a 10% solution of dep in 100% ethanol and
dilute this 500 fold in the water. Leave overnight or at
least 3 hours and then autoclave for 20 min to inactivate
the residual dep. Store the water in small baked bOttles.
Use gloves when handling the dep because it is a strong
denaturing agent.

4. 4 M NaCl. Treat with dep as above.

5. Polyallomer tubes. Treat with dep as above. Place

the tubes in a large beaker with water and add the dep to

118
0.2% and mix up. Autoclave as before and store the tubes in
a closed box.

6. Guanidinium thiocyanate/0.1 M 2-mercaptoethanol.

Add 47.2 g of Fluka G/SCN or Kodak or BRL to about 70 ml of
nanopure water and then in a hood add 7 ml of 2-
mercaptoethanol. Cover and stir and gently warm to dissolve
and make up to a final volume of 100 ml. Filter through a
0.45 pm Nalgene filter and store at -20°C in 50 ml plastic
tubes. Check pH , it should be pH5 and will be if Fluka
G/SCN is used. Adjust if necessary with acid.

7. 5.7 M CsCI/0.1 M EDTA. Add 48 g Of baked CsCl
technical grade to about 30 ml of nanopure water and then 5
ml of 0.5 M EDTA and stir and warm to dissolve at a final
volume of 50 ml. Adjust the pH to 5 with acid. Filter
through a Nalgene filter and store at -20°C or add dep to
0.2% and treat as for dep'd water.

8. Phenol/Chloroform. Shake phenol with 0.1 M Tris pH8
(usually 25 ml of phenol with 25 ml of Tris) to equilibrate.
Spin in 50 ml plastic tube at 3000 rpm for 5 min to separate
the phases. Repeat until the water phase is pH about 7-8.
This may be several times. Add a pinch of 8-hydroxyquinoline
to give the phenol some brown color for visibility and then
add 2-mercaptoethanol to 0.1%. This makes the equilibrated
phenol which can be stored at -20°C tightly capped. Add
equal parts of this phenol to chloroform to make the 50:50

mixture.

119
8. Isolation of Poly(A)+ mRNA
(using Amersham's Hybond-mAP paper)

Materials required:

0.5 M NaCl Clean, sterile (flame) forceps
70% ethanol Clean, sterile (flame) scissors
Dep’d water Disposable gloves

Eppendorf tubes Filter paper, 3MM

Parafilm Water bath, set at 70%:

Wear gloves at all times to prevent contamination of Hybond-
mAP by ribonuclease.

1. Prepare 100 pg RNA in an Eppendorf tube. And cut 1
cm2 Hybond-mAP paper .

2. Wet it in 0.5 M NaCl solution.

3. Place Hybond-mAP on 3MM filter paper, then transfer
onto parafilm. Slowly spot RNA solution on Hybond-mAP, stay
for 2-3 min.

4. Using a pipet tip to transfer RNA solution back into
an Eppendorf tube (if any), then place Hybond-mAP onto 3MM
filter paper, place the RNA solution onto Hybond-mAP paper,
leave for 2 min.

5. Wash Hybond-mAP in 0.5 M NaCl for 5 min (rocking) in
13 ml orange cap tube. Repeat twice, using new solution, to
remove free RNA.

6. Rinse Hybond-mAP in 70% ethanol for 2 min. Then put
Hybond-mAP onto 3MM filter paper to remove excess ethanol.

7. Transfer Hybond-mAP into 200 pl dep’d water (cover

120
the paper) in an Eppendorf tube and incubate at 70°C water
bath for 5 min.
8. Remove the mAP using sterile forceps. The poly(A)+
RNA remains in the water. Using Speed vacuum to move excess
water until 10-20 pl left.

Ready for further processing.

6. NORTHERN BLOT

1. To 5.2 g of agarose in a 500 ml flask add 80 ml of
5X MOPS buffer pH 7 and 250 ml of nanopure water. Put over
a low flame and allow to boil thoroughly to melt all of the
agarose. Meanwhile tape the edges of the 20 x 30 cm gel box
and put it in the hood and fix the 0.7 cm x 2 mm gel comb in
place. When the agarose is melted cool to about 60' and add
69 ml of 37% formaldehyde while swirling to evenly mix the
formaldehyde into the agarose. Pour the mixture in the gel
box and smooth out any bubbles. It hardens in about 30 min.

The concentration of agarose is 1.3 % and the gel is
about 0.7 cm thick. Up to about 60 pl of sample can be
loaded in the slots.

2. While the gel is cooling denature the RNA samples.
Normally about 10 pg of total RNA is run but 50 pg will
successfully separate by this method.

Prepare the following in a microfuge tube:

5X MOPS buffer 4 pl

formaldehyde (37%) 3.5 pl

formamide 10 pl

RNA 2.5 to 4 p1
Mix and place at 55°C for 15 min. Add 2 pl of sterile
loading buffer(50% glycerol/0.05% bromophenol blue). Make
sure that the RNA is assayed correctly for concentration or
else the lanes may have different amounts. It is best to

determine the 00 of all samples then make a dilution to 1 or

121

122
2 mg/ml for all samples. Reassay the 00 of the dilution
then calculate the amount to load. RNA transcripts for size
markers: use 10 pl of SP6 and T7 transcripts.

3. Gently remove the comb so you don’t rip the slots.
Fill up the chambers in the gel box which require about 800
ml per side and add a covering layer of buffer to about 1mm
to just cover the gel and fill up the slots. Do not overfill
because the formaldehyde concentration must remain high.

Add the samples and run the gel at about 40 volts overnight
(ca. 11.5 hr with the EC 500: use 50 V. for the EC 600) so
that the blue dye has run about 10 cm. Run all of the gels
the same way so that in the future you can compare gels run
on separate days.

Optional faster runs: 80 V 5% hrs z 10 cm EC 500

100 V 5% hrs z 10 cm EC 600

Save the running buffer for reuse: can be used at least
3 times.

4. OPTIONAL STEP. Cut out the gel area containing the
lanes and put it in another box filled with 1 liter of 50mM
NaOH/lOmM NaCl. Shake gently for 30 min to denature and
break up the larger RNA species. This is not usually done
except if the RNA is very large (over 7 kb ).

5. Meanwhile turn on the Haake circulator cooling bath set
at -10'C.
FOR GENE SCREEN PLUS: Also prepare 6 liters of 12 mM

Tris/6mM sodium acetate/0.3 mM EDTA/ph 7.5. A 20 X stock

123
solution of the electroblot buffer can be prepared: 29 g
Tris base/40 ml 3 M sodium acetate pH 5/ 12 ml 0.5 M EDTA/z
10 ml concentration. HCl to pH 7.5 and brought up to 1
liter. If using GENE SCREEN, prepare 8 liters of 0.025 M
sodium phosphate buffer(pH 6.5). A 1 M stock solution is
prepared as follows: dissolve 93.84 g sodium phosphate
monobasic and 45.44 g sodium phosphate dibasic in 700 ml
distilled water, heat and then cool to room temperature, and
bring up to 1 liter. Check and adjust the pH with about 9.0
g of NaOH pellets. Usually Gene Screen is used.

6. If used, pour out the NaOH/NaCl and add 1 liter of
the Tris or phosphate buffer solution and shake for 15 min.
and then pour that out and replace with one more liter of
Tris or buffer. If the NaOH/NaCl is not used rinse the gel
with 1 liter of phosphate buffer. Shake again for 15 min
plus add 5 drops of 5mg/ml Ethidium bromide to stain the
RNA. The rRNA on the gel is usually not visible in the
stained gel but it will be stained on the blot.

7. Remove the gel and look at the RNA on the
ultraviolet light box. The RNA will only be visible well if
the Tris buffer had been used. The ribosomal RNA of 26S and
188 should be visible about 5 to 7 cm from the slot. Trim
away any extra gel outside of the lanes and cut off the gel
below the bromphenol blue area at the bottom of the gel.
Good visualization at this stage is not really necessary.

The bands will appear well on the membrane after blotting.

124

8. Cut out 4 pieces of Whatman 3MM paper and one piece
of Gene Screen or Gene Screen Plus the same size as the gel
and soak these in the Tris buffer. Assemble the electroblot
sandwich in a large plastic box. First add about 2.5 liter
of the appropriate buffer and soak one of the sponges. Then
add 2 sheets of the paper making sure that no air bubbles
are trapped between or under them. Add the gel on top of
them while submerged to prevent air bubbles and then the
Gene Screen or Gene Screen Plus. Smooth out the layers and
then add the final two sheets of paper under the buffer as
before. Add the other side of the sandwich and place in the
electroblot apparatus which is filled with 4 liters of the
buffer.

. 9. The Gene Screen or Gene Screen Plus side of the
sandwich is on the positive side of the electroblotter.
Start the electroblot at 0.4 amp for one hour and check the
amperage in about 15 min to make sure that it is stable and
still on. After one hour increase the amperage to ca. 0.9
amp for 3 hr. Remove the blot and air dry or briefly blot
and then bake at 80'C for 2 hours. The air drying step is a
good overnight stopping point. Look at the blot under the
short wave length uv light and mark the slots, the rRNA
bands the date and the samples in pencil on the blot to the
side of the rRNA bands.

10. Prepare the prehybridization buffer in a 50 ml
tube.

125
For Gene Screen Plus:
50% formamide 25 ml Deionized and stored at -20°C

50X Denhardt's sol. 5 ml Do not heat over 37' to thaw

20X SSPE 12.5 ml

10% SDS 0.5 ml Ultrapure

10 mg/ml salmon DNA 1.0 ml Boiled and sheared
nanopure water 7.0 ml

Check pH with paper Should be pH7.

For Gene Screen:
50% formamide 25 ml Deionized and stored at -20°C
50X Denhardt's sol 5 ml Do not heat over 37' to thaw

1 M Tris, pH 6.8 2.5 ml

NaCl 0.92 g Dissolve NaCl first in the aqueous
10% SDS 5 ml ingredients not the formamide or $08.
50% dextran sulfate 10 ml

10 mg/ml salmon DNA 0.5 ml

Check pH with paper. Should be 7 and pale green. If not
and the pH is very green the formamide may need to be
deionized and the buffer should not be used for
hybridization.

50 % dextran sulfate = 100 9 plus 155 ml of nanopure

water: heat and stir to dissolve. May take a while.

11. Seal the blot in the seal a-meal-bag and add 10 ml

of the prehybridization buffer. Place in a 42'C water bath

126
and allow the bubbles to float up to the top. Prehybridize
at least one half hour but can be overnight or longer.

12. Add the probe to 10 ml of the prehybridization
buffer so that the probe concentration is S 10 ng/ml with a
specific activity of at least 1 X 107 per pg. Usually add
one half of the oligolabelling reaction of 100 ng DNA. Heat
the hybridization buffer for about 10 min. at 80'C to
denature the DNA then quick cool on ice briefly. Pour off
the old prehybridization buffer and add the cooled
hybridization buffer to the blot and seal again. Leave in
the 42'C bath for two days before the posthybridization
washes. Turn on the 65°C shaker bath before the
posthybridization so that it heats up in time.

13. Remove the hybridization buffer and save in a
plastic tube at room temperature (can be reused at least 3
times within 2 weeks). Wash the blot in 1 liter of 2 X SSC
twice for 5 min with shaking at room temperature. Follow
with a wash in 1 liter of 0.5x SSC/1% SDS (Sigma 95% SDS) at
65°C for 30 min. with shaking. Check counts of blot with
geiger counter held over the top of blot for background
which should be low and over the lower part below the rRNA
band which should be noticeably higher. Usually necessary
to wash again at 0.5x SSC/1% SDS at 65°C for another 30 min.
Remove excess 808 by several quick washes over 30 min. with
0.1X SSC at room temperature. During the washes do not allow

the blot to dry.

127
14. Wrap the blot in plastic wrap and then mount on 3MM
paper. Do not get the outside wet or it will stick to the
film during exposure. Mark the rRNA band location, the date
and the orientation of the lanes and some other X marks on
the paper with ink spiked with some 35S. Place into light
proof cassette against XAR-S film with the film against an

intensifying screen. Expose at —70’C for at least overnight.

7. RADIOACTIVE PROBE PREPARATION (Feinberg, gt gt, 1983)
A. Random primer method

OLIGO LABELLING

Dilute 60-100 ng of purified DNA fragment to 12 pl
in dHZO .

Boil for 5 min. to denature and then
Quick-cool in ice-water for 10 min.

Add following:
L8 18 pl
BSA 1 pl (16 mg/ml)
dNTPs 3 p1 (dGTP, dATP and dTTP)
(”p)dCTP 5 pl (50 pci)
Klenow frag. 8 unit (DNA polymerase I)

 

total 40 pl
Incubate at room temp. from 5 hr to overnight.

Note: TM : 250 mM Tris.Cl pH 8.0 2.5 ml
25 mM MgCl2 0.25 ml
50 mM B-Mercaptoethanol 36 pl

water to total 10 ml

OL

90 0.0. Units/ml Hexamers
1 mM Tris.Cl pH 8.0
1 mM EDTA pH 8.0

LS : 1 M Hepes pH 6.6 : TM : OL ,
25 : 25 : 7

128

129

B. End labelling

1. Mix the following:
H20 11 pl
oligonucleotide 1 pl (200 mg)
10 X PNK buffer 2 pl
(r-32P}dATP 5 pl

Polynucleotide Kinase 1 pl

2. incubate the mix for 45 min. at 379C, then heat

it to inactivation for 10 min. at 70W3.

3. add 70 pl STE (100 mM NaCI, 20 mM Tris pH 7.5
and 10 mM EDTA pH 8.0) buffer.

4. use Stratagene’s "Nuctrap push columns" to
separate unincorporated dNTPs from labelled

oligonucleotides.

8. SEQUENCING (Sanger, gt gl)

A. Single-stranded sequencing using M13 phage
Preparation of single stranded M13 for sequencing
Gtgg 5 ml gt DHSaF’ ggllg lg §Q§ medium gig, then
transfer 100 pl to 5 ml new media to continue to grow
to exponential phase (00 550 at 0.2 to 0.7) and dilute
100 times (1 ml to 99 ml SOB).

Place 5 ml in 15 ml glass tube and then add a plaque
from a plate by removing an agar with a cut off blue
pipette tip. (using a plaque which are well isolated
from others and have not been growing for more than
overnight)

Shake at 37°C at 200 rpm for about 5 hours, then
transfer to 3 microfuge tubes and spin 5 min. at SH-MT
rotor 13,000 rpm.

Remove 1.3 ml supernatant (avoiding pellet) from each
to 3 new tubes.

Add 200 pl of 20% PEG/2.5 M NaCl to each tube. Invert
to mix several times then vortex gently. Leave at room
temp. for 15 min. Spin again in SH-MT at room temp. at
13,000 rpm for 5 min. Remove supernatant with drawn-out
pasteur pipette then spin 10 sec. in microcentrifuge
(room temp.) to get rest of supernatant. Shoulg ggg g
gggll pellet lg gggg tggg ggg tgg pellet shoulg pg
9109.93: $111-

Add 150 pl of TE to each tube and vortex vigorously to

130

10.

resuspend. (good point to stop)

Extract with phenol once, phenol/chloroform a few times
(maybe up to 5 times) and chloroform once. (vortex 1
min. and spin 7 min in microcentrifuge at room temp.)
After step 7, remove 70 pl upper aqueous from each tube
to one new tube. Add 400 pl of a 25:1 ethanol:3M Na-
acetate (pH 5) solution. Vortex to mix and store at
room temp. for at least 15 min. or overnight.

Spin in SH-MT 15 min at 13,000 rpm to pellet. Remove
most of supernatant. Add 200 pl of room temp. 70%
ethanol to wash the pellet for 3 min, then spin 3 min
in microfuge. Gently remove supernatant. Dry at room
temp. or in Speedy Vacuum. Resuspend in 40 pl of ng)
(not TE).

Run 5 pl of sample against M13 DNA control in 1% mini-
gel. If any contaminants appear below the DNA (bands or
fuzzy RNA) do not use for sequencing. Estimate 2 pg

amount for sequencing and store rest at 4W2.

11. For sequencing, following the USB Sequenase protocol.

131

B. Double-stranded sequencing using plasmid DNA

Double stranded DNA
sequencing is time and labor saving
comparing to ss-DNA sequencing. By
using the following protocol, we can

easily read up to 300 bp.

I. PREPARATION OF DOUBLE-STRANDED

DNA FOR SEQUENCING

see Plasmid mini-prep and large

preparation

132

250-.

150—.

1.0—e

 

 

 

10.

11.

12.

13.

II.DENATURATION OF PLASMID FOR SEQUENCE ANALYSIS
Take 10 pg. plasmid in TE buffer in 0.5 ml micro-tube.
Add up to 0.4 M NaOH, then incubate at 37°C for 15 min.
Add 0.1 vol 3 M NaAc (pH 4.5-5.5) to neutralize.
Add 2 to 4 vol. 100% ethanol (cold), mix well and leave
the tube at ~20°C for 30 to 60 min or O/N.
Centrifuge for 15 min at 4°C at 13,000 rpm.
Washing w/ 300 pl of 70% ethanol for 3 min., then spin
3-5 min. at micro-centrifuge.
Dry the pellet at room temp. for 30 min.
Keep the sample dry at.4PC until ready for sequencing.
Before sequencing, add 7 pl ddHZO to dissolve the DNA.
Add 2 pl 5X sequence kit buffer, 1 pl Primer. (USB kit)
Put the tube in 65°C water bath for 2 min., then cool at
room temp. to <30°C. (about 30 min.)
Continue follow the USB ptlgfi protocol.

We follow the Sequenase Version II protocol supplies by

USB except following:

1) use 1:15 dilution of labelling mix (instead 2:8).
2) the temperature of labelling reaction must be <20%L

3) the termination reaction is done between 45-50%:

(instead of 37°C) for 2-3 min.

4) boil the sample for 2-3 min (instead of at 85%”.

133

C. Gel preparation

1. thoroughly mix the following components:

 

 

8% gel 50.4 g. Urea 6% : 50.4 g. Urea
24 ml 40% acrylamide:bis 6.84 g/
(38 g: 2 g to 100 ml) 0.36 9. bis
12 ml 10 X TBE buffer 12 ml 10X TBE
1.2 ml 10% ammonium persulfate 0.12 9. amp.
44 ml dHZO 68 ml dHZO
120 m1 120 m1

2. filter (Whatman No.1) the solution into a flask on ice.

3. place gel solution on room temp. until the temperature
of the solution reaches 15°C.

4. add 60 pl TEMED and swirl gently to ensure thorough
mixing, and pour the gel immediately.

5. insert the comb at the top of the gel in a horizontal
position and clamp the plates at the top. Leave the
gel flat on the bench horizontally for about 30 min.

6. buffer preparation: 1 X TBE 2000 ml.

7. after the gel is polymerized, tear off the tape of the
bottom and affix plates to the sequencing apparatus.
Pour running buffer (1 X TBE) into upper and lower
reservoirs.

8. remove one comb and flush wells with 1 X buffer: insert
one shark-tooth comb in the same position: then, do the
same for the other comb.

9. pre-run the gel for 1 to 2 hours at 60 Watt (constant
power). (The running power is 50-55 Watt.) The optimal
plate temperature (measured with a thermometer outside
the plate) for a good run is about 40%:.

10. (Optional) check the leakage of the wells by loading 1
pl stop buffer.
(prepare samples while pre-run. see brief protocol
supplies by manufacturer)

11. after gel-running, fix 30 min. using 15% methanol/ 5%
acetic acid with gentle shaking every 5 min. and drying
at 80°C for 40-50 min.

12. checking counts after drying the gel.

134

9. CDNA LIBRARY CONSTRUCTION
A. RNA and poly(A)+ mRNA preparation:
See RNA section for detail.
B. cDNA synthesis:

See Amersham’s "cDNA synthesis system plus" kit
for detail.

C. Vector preparation:

Digest 10 pg pUC18 plasmid with Sma I restriction
enzyme for 3 hr at 379C, then treated with CIP (10 units)
for 30 min. at 379C. Extract vector with phenol/chloroform
once, chloroform once and ethanol precipitated. Ready to
use.

0. Ligation and fit ggll transformation:

Ligation: 0.5 pg of plasmid vector and poly(A)+
mRNA extracted from 200 pg total RNA were used with 20 units
ligase (Boehringer Mannheim) for ligation in a 20 pl at 15%:
for overnight. Add additional 1 pl ligase, plus 9 pl
distilled H20 to continue ligation at room temperature for 3
hr.

E1 ggli transformation: see next section for

detail.

135

10. TRANSFORMATION OF BACTERIA E. 9011

DAY 1.

1.

Grow DH-5a cells directly from —70°C stock on LB plate

overnight at 37°C.

DAY 2.

2.

3.

Pick about 10 colonies to grow in 5 ml SOB medium at
37°C for 1 to 1.5 hr.

Transfer about 0.5 to 1 ml overnight culture to 50 ml
SOB in 500 ml flask. continuously to grow at 37”C to
00550 of 0.5 (about 1.5 to 2 hr, may be longer).

Cool on ice for 10 min, then transfer into a 50 ml pre-
ice cold tube, then spin 5,000 rpm for 5 min to pellet
cells. Resuspend in 10 ml be I, and keep on ice 10 min

Spin at 5,000 rpm for 5 min.

EELS; ELQEQD Qéllé £2: lQESI 0591
SEE next page for details.

5.

7.

it ggking fresh cellg tg pgg plght gway:

 

Resuspend pellet in 2 ml be I. Add 70 pl freshly
thawed DMSO, swirl and ice 5 min. Add 70 pl 2.25 M
DTT/40 mM KAc pH 6.0, Swirl and ice 10 min. Agd gnother
ZflulflfliQéDQiQQiQIfimni

Transfer 200 pl of cells into Falcon 2057 tubes (keep
on ice), and then add 10 to 20 pl of DNA (about 10 ng)
to the tube, leave on ice for 1 hr. (the volume of DNA
solution should not be more than 10% of total volume.)

Heat shock cells at 42°C for 1 min. (critical gtep:

136

10.

11.

12.

137

WmmmmMMMflm
mummmmnmmmm
9229...).

Cool on ice for 1 min.

Add 800 pl SOC medium (prewameg t9 ,37_%_:) into the tube
and shaking for 2 hr at 37°C. (speed should not be more
than 200 cycle/min.)

Spin down at table top centrifuge at max speed (2,100
rpm) for 10 min. Discard supernatant, add 200 pl SOC
and mix well.

Put 50 pl X-gal (20 mg/ml) and 10 pl IPTG (100 mM) plus
40 pl SOC on plate containing 50 pg/ml ampicillin and
spread evenly. Close lid for 10 min. and open it dry in
the hood.

Plate out aliquot to LB plate under the hood. Spread
evenly and leave the plate inversely at 37”C incubator

for overnight. (no more than 20 hr)

ZIQIQR Qfillfi £2; léLQI REEL

5’.

6’.

7’.

Resuspend the pellet in 2 ml be II. Add 70 pl freshly
thawed DMSO, swirl and leave on ice for 5 min. gdg
31191209: 2.9 IL]. mg m £91: 1991.919119]. 5 1.111.111.

Transfer 210 pl aliquot to each Eppendorf tube on ice.
Chill them very quickly in -70°C ethanol, then take
them out and store in -70°C freezer until later use.
When using frozen cells: take out from -70°C, warm it

up in hand. Once thawed, put back into ice bath

138
immediately, then follow the step 6 of making fresh
competent cell protocol.
Notezammmslshemsnamfimmn £9
mmmrmmmiecm

11. TWO DIMENSIONAL GEL ELECTROPHORESIS ( O’Farrell, P.
1975, Garrels, J.I. 1979, Garrels, J.I. 1983, Kopachik, gt
gl. 1985, Morrissey, J.H. 1981.)
1) 2—0 gel sample preparation:
add 60-80 pci 3SS-methionine to 1 x 107 cells

and incubate at 37°C for about 8 hr

1. Harvest cells
2. Add 18 pl 1% 303/20 mM Tris pH 7.2 and
4 pl 55 mM DTT/20 mM Tris pH7.2.
3. Vertex and boil 2 min. in a tightly
capped microcentrifuge tube
4. Spin down 10 sec. in microfuge and cool to
rome temperature.
5. Add 2 pl DNAse/RNAse mixture and leave at
room temperature for 1 min.
6. Freeze 2 min at -70°C, then spin 1 hr in
speed vacuum to dry the sample.
7. Add 20 pl dde, 2 pl Ampholines (pH 3.5-7),
4.4 pl DTT, 4.4 pl NP-40 (44%), 30 mg Urea:
8. Dissolve all with vortexing and warming at
37°C.

(Store the sam le at ~20°C or go to Protein determination)
P

139

H

4.0

140
2) TWO DIMENSIONAL GEL ELECTROPHORESIS

a) Isoelectric focusing gel electrophoresis:

1. Prepare gel. Measure out 0.582 g urea, add 0.35 ml
11.4% NP-40, 0.22 ml 11% acrylamide/0.55% bis and 50 pl
ampholines (pH 3.5 to 10). Vortex and warm to 37‘C to
dissolve completely. Then spin 1 min to get rid of bubbles.

2. Degas for 5 min under vacuum from water aspirator.
(optional: filter the solution by using 0.2 pm filter)

3. Add 10 pl of 10% APS freshly made. Vortex briefly to
distribute but do not make aerated. Quickly load into acid
washed capillary tubes (see notes below) up to a level of 11
cm. Place upright in poster putty. Allow to polymerize 1 hr.

4. Cut putty end so gel is 10.5 cm, total length is 15
cm: and overlay with 8 M urea (0.48 g/ml).

5. Prefocus the gel. Load gel carefully into gel box
containing 1000 ml of HSPO‘ (1.2 ml up to 1 L) in lower
chamber adjust level of gel so that it just touches the
acid. Submerge the upper end in 0.025 M NaOH (1 g to 1 L).

Start electrophoresis at 200 V. for 15 min.,
500 V. for 15 min., 750 V. for 15 min. and note
final current which should be S 52 pAMP per gel.

6. Load sample: thaw sample and gplg gggg fig; 5 gig in
microcentrifuge, then from the supernatant load about 150 pg
of protein up to 10 pl volume. Can load up to 200 pg of
protein for silver stained gels (more protein causes

sticking in tube and stretching) in 20 pl. Keep all samples

141
to be compared later as close as possible in protein amounts
and sample volume. For autoradiography of 3'SS-methionine
labelled samples try to get about 160,000 cpm or more in 10
pl. Remove NaOH until level is below upper gel end then
load sample just above the gel level under the 8 M urea. Add
urea to replace any displaced by the sample. Replace NaOH to
cover upper end.
(Note: load equal amount count for ls8 gel and equal protein
for silver stain gel)

7. Focus samples. Run gels 250 V. constant voltage for
15 hr, then 500 V. 1 hr, 750 V. 1 hr and 1000 V. 2 hr.

8. Remove gels and put in -20°C freezer (actually -16°C
and gels may break if temperature is -20’C) for 2 min. Expel
with lab air into equilibration buffer (5 ml per gel) and
lgt gtggd fig; 5 glgt Catch gel in a tgg strglggr and add to
upper tank of SDS gel box ready to electrophorese samples.
Notes for IEF:

A. Equilibration buffer: 5 ml per gel. For 20 ml 0.169
g DTT, 0.8 g 808, 2.0 ml glycerol, 1.24 ml 1 M Tris pH 6.8
and water to 20 ml.

B. Ampholines are from LKB and are pH 3.5 to 10

mixtures. Other brands will give different spot patterns.

142
b) SDS gel electrophoresis.

1. Pour separating gel of 11% acrylamide using 1.5 mm
spacers. Assemble plates with spacers and clamps and seal
edges thoroughly with molten 1% agarose. Each gel requires
25 ml of gel solution (see notes below for composition).
Degas 5 min then add APS and TEMED. Swirl but do not make
aerated. Add to plates with 25 ml pipette and then overlay
with about a 3 mm layer of water. The gel is 10.5 cm long
with about 2 cm from the top of the slot to the water
overlay. Polymerize for 1 hr.

2. Assemble gel in a gel box and add electrophoresis
buffer (see notes below) to upper and lower tanks. Add 50 pl
of bromphenol blue dye to upper tank for a tracking dye.

3. Manipulate the acidic end (the swollen end) of the
gel to the far left of the SDS gel with spacers or spatulas
and anchor that end. Then brush the rest into the slot with
a wafting motion using the water pressure generated to push
the gel into the slot.

(This step takes practice).

4. Electrophorese at 125 V. per gel until the dye
reaches 10 cm.

Note for 808 gel electrophoresis:

A. Separating gel: 25 ml per gel of 10.5 cm depth and
1.5 mm thickness. For 11% acrylamide:

Make up of stock of 100 ml of 30% acrylamide 0.8% bis

acrylamide and store at 4'C in darkened bottle.

143

Make up at stock of 1.5 M Tris pH 8.8 with 0.4% $08.

For 30 ml of gel solution mix 10.9 ml of acrylamide stock,
11.2 ml of Tris-SDS buffer and 7.8 ml of water. After
degassing add 150 pl of freshly made 10% ammonium persulfate

and 14 pl of TEMED. Pour quickly within 5 min.

B. Electrode buffer. Need 1500 ml for 2 gels boxes. For
1500 ml add 4.5 g Tris, 21.6 g of glycine, and 1.5 g of 808
to 1.5 liter of water. The pH is about 8.3 and does not need
to be adjusted.

144
Fixation for autoradiography.

1. Place gel plates in plastic box and pry away the
plates at any place except the rabbit ears (they break
easily). Let gel fall into 100 ml of methanol/ acetic acid.
Wash 3 times with gently rocking for 40 min each. For gels
from in ylttg translations use 300 ml of fixative to wash
away the greater amount of background counts.

2. Add 60 ml of ENHANCE in hood for 1 hr using rocking
motion. Pour off into container for disposal.

3. Add lots of cold water to precipitate fluors in the
gel and rock for 1 hr. Pick up gel by supporting it from
below with a glass plate or film .

4. Place wet 3MM paper on top and smooth out bubbles.
Dry with vacuum and heat for 1 hr at 60°C. Do not leave to
long (cracks appear) or too short (the undried gel with
shatter if the vacuum is release to soon).

5. Place against XAR 5 film and expose for 1.6 x 10°
cpm times days. (1 day for a gel with that amount of counts
loaded)

Silver Staining of gels:

Use filtered water (0.2 pm) and gel solutions
throughout the 2 D gel procedure because the dust in
unfiltered solutions will stain and give vertical streaks in
the gel. Keep the bromphenol blue tracking dye at minimal

levels for the SDS gel because it will stain . Do not press

145
hard on the gel at any time while changing solutions because
this distortion will cause spots on the gel after
development. Use gloves when handling the gels. Use gentle

rocking throughout the staining procedure.

1. 50% methanol/10% acetic acid (100 ml) for 30 min.
Pour off.

2. 5% methanol/7% acetic acid (100 ml) for 30 min. Pour
off.

3. 10% glutaraldehyde for 1 hr (100 ml) Pour off.

4. Wash extensively with water. Several quick (5 min)
changes using a lot of water, then a few changes over a 2 hr
period and proceed. Or lggyg gygtglggt gt gygtgggkggd. Pour
off.

5. 5 pg/ml DTT for 30 min (100 ml). Pour off.

6. 0.1 % silver nitrate for 30 min (100 ml). Pour off.

7. Quick rinse with about 100 ml water and another 100
ml water for about 1 min. Follow with 2 small rinses with
developer. Developer is freshly made 3% Nagxh with 50 pl of
formaldehyde added per 100 ml.

8. Add 100 ml of developer and develop on light box
until spots appear in about 2 to 5 min. Stop development
with 5 ml of 2.3 M citric acid and rock for about l5 min.
Pour off.

9. Wash with lots of water and then store in sealable

plastic bag.

12. PROTEIN IN YITBO TRANSLATION USING RABBIT RETICULOCYTE

(AMERSHAM’S KIT)

1. 25 pl lysate (Rabbit reticulocyte)
4 pl 35’S-methionine
3 pl poly(A)+ RNA (from total 100 pg RNA)

1) gently mix with pipet, don't vortex.

2) lysate should be thawed on ice.

2. Incubate for 1 hour in 30°C.

3. Add 2 pl DNAse/RNAse at room temperature for 2 min.

4. Freeze (thoroughly) in -70°C for 3 min to
indefinitely.

5. Dry down in Speed Vacuum.

6. Add 50 pl of translation buffer.

7. TCA cpm count on 2x2 pl.

Stock for lg ylttg translation buffer:

9.95 M urea 597 mg urea

4% NP-40 40 pl

2% LKB pH 5-7 20 pl

100 mM. DTT 100 p1 1M DTT
0.3% SDS 30 pl 10% 808

water to 1 m1 work.

146

13. SMALL AMOUNT DNA PRECIPITATION WITH ETHANOL

If the DNA concentration is very low (e.g. below 1,000-
10,000ng/ml in the final volume which including ethanol),
this method should be used if possible.

1. Add 0.1 vol. of 3 M NaAc, 2 vol. of 100% cold ethanol
into 1.5 ml microcentrifuge tube, mix well and stored at

-20°C for overnight to 24 hr. (MgCl2 may be added to 0.01
M final solution of DNA before add salt).

2. Fill the whole microfuge tube with 80% ethanol.

3. Microfuge tubes are then put into ultracentrifuge
tubes which have been filled with ice-gglg ggtgt contgiglng
253 etganol to prevent freezing. Egg ultracentrifuge tgtgt
511911131 be 0w- 1 in the new fer 91910191111...

4. Balance the tubes with no more than 0.1 gram
difference.

5. Ultracentrifuge at 25,000-27,000 rpm (SW27 rotor) for
1 hr. at 4°C.

6. Supernatant should be carefully removed with
micropipet until 10-30 pl is left, then use 80% ethanol to
wash once, dry the sample in speed vacuum and dissolve in

appropriate buffer.

147

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