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thesis entitled

Molecular Study of Actin in
Naegleria fowleri

presented by

Jonghee Ahn

has been accepted towards fulfillment
,of the requirements for

Master's degree in Science

Major professor

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MOLECULAR STUDY OF ACTIN IN HAEGLEBIA EQHLEEI

BY

Jonghee Ahn

A THESIS

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

MASTER OF SCIENCE

Department of Zoology

1992

(5/8- 5/2 ’7’

ABSTRACT

MOLECULAR STUDY OF ACTIN IN NAEGLEBIA EQHLEBI

BY

Jonghee Ahn

Naegleria flawleri is a free-living amoebo-flagellate,
and the causative agent of primary amoebic
meningoencephalitis (PAM). This organism like many other
species has multiple actin genes. Actin genes are present in
most organisms and conserved throughout evolution. Analysis
of Northern blots showed the existence of actin genes and a
possible role in virulence of the amoebae. The multiplicity
of actin genes was demonstrated by Southern blots. Two cDNA
actin genes were isolated, sequenced and compared with many
other actin genes from various organisms. The results
suggested that n. figulgri is more closely related to
{Aganthamgeba than to Trxnanoanma, which is contradictory to
speculations on data from small subunit ribosomal RNA
sequencing which place N,£gnleri closer to Irynangsgma than
.Aganthamgeha in the evolutionary tree. H. figuleri may be a_

more advanced organism than thought previously.

Dedicated to my family

ii

ACKNOWLEDGEMENTS

I wish to express my thanks to Dr. R. Neal Band, my
major professor, for his guidance and encouragement during
the course of this study.

I also wish to thank Dr. Will J. Kopachik, my advisor,
and Dr. Donald B. Jump, my committee member for their helpful
suggestions in preparing my thesis.

Special thanks are extended to Dr. H. T. Band and Wang-
nan Hu for their kind help.

Finally, I would like to thank all the people who have

assisted me in completion of my research.

Page
LIST OF FIGURES .......................................... v
LIST OF TABLES ......................................... vi
INTRODUCTION .......................................... 1
MATERIALS AND METHODS .................................... 3
Northern blot ............................ 3
Screening of cDNA library ................ 4
Southern blot ............................ 4
Preparation of DNA for sequencing ........ 5

Denaturation for double
strand sequencing ........................ 6
DNA sequencing ........................... 6
RESULTS .......................................... 7
Northern blot analysis ................... 7
Southern blot analysis ................... 7

Dot blot and nucleotide
sequence analysis ........................ 8
DISCUSSION ......................................... 11
LITERATURE CITED ....................................... 19

TABLE OF CONTENTS

iv

Figure

Figure

Figure

Figure

LIST OF FIGURES

Autoradiograph of Northern blot of
RNA from axenically grown amoebae
and amoebae from mouse brain ............ 22

Autoradiograph of southern blot
probed with the act2 cDNA ............... 23

Nucleotide and deduced amino acid
sequence of Ni figulgri .................. 24

Amino acid sequence alignment and
comparison of the actin sequences in
N... fouleri (actl), Dicticstelinm
disggidgum, human b-actin,

LIST OF TABLES

Page
Table 1 Condon usage in u; fignleri actin
gene .................................... 35
Table 2 Amino acid comparison of Naeglgria
fowlgri with other species .............. 36

vi

INTRODUCTION

The free-living, pathogenic amoeba Naegleria,fignleri, is

widely distributed in soil and freshwater throughout the
world. These organisms have the unusual ability to undergo
transformation from an amoeboid trophozoite to a temporary,
non-feeding, non-dividing biflagellate. The life cycle of
Naeglgria also includes a dormant cyst. Although Naegleria
flagellates do not encyst, amoebae are able to encyst
(Marciano-Cabral, 1988).

Primary amebic meningoencephalitis (PAM) is a rapidly
fatal human disease of the central nervous system caused by
this organism. Unlike a "true" parasite, this is an
opportunistic pathogen whose virulence is affected by several
factors. Although determinants of pathogenicity are largely
unknown, it has been suggested that phagocytosis,
phospholipolytic enzymes and catalase are responsible for its
virulence (John, 1982).

N,£gnleri, like all other eukaryotic cells, keep their
shape and motility with actin filaments. Actin also
participates in cytokinesis. Many organisms have multiple
isoforms of actin that often exhibit developmentally
regulated and cell-type-specific expression (Lees-Miller e;

a1. 1992). At least N» grnheri, translatable actin mRNA

disppears rapidly during the differentiation of amoebae to
flagellates ( Sussman at al, 1984).

Here, I reported the existence of a multigene family of
actin genes

in Nagglgria,£gnlezi. Amino acid and nucleotide

sequence comparisons show an extremely biased codon usage.

MATERIALS AND METHODS

Northern blot

DNA-RNA hybridization was carried out for 2 days at 50°C
in 50% formamide, 5x Denhardt's solution (10% Ficoll/10%
polyvinylpyrrolidone/10% bovine serum albumin), 1% ultrapure
sodium dodecyl sulfate (SDS), 10% dextran sulfate, looug/ml
denatured salmon sperm DNA, 1M NaCl, SOmM Tris-HCl (pH 7.5)
and 3.2x105 cpm/ml of an end-labelled DNA probe. The probe
was end-labelled to reduce the background.

The probe was prepared with the synthetic oligomer (5'-
TAGAAGCATTTTCTGTGCAC-B'). This conserved sequence was
determined by comparison of available actin gene sequences in
various species. This oligonucleotide was synthesized by the
Michigan State University Macromolecular Synthesis Facility.
The 20mer oligonucleotide was radiolabelled with _f32P ATP by
the polynucleotide kinase (PNK) reaction (Sambrook, at 31.,
1989).

The blot was washed at room temperature, 15 min twice

with 2x sodium chloride/sodium citrate (SSC).

Screening of cDNA library

Poly (A) + mRNA purified from 200ug total RNA was used
as a template for oligo dT-primed cDNA synthesis by using a

cDNA synthesis kit (Amersham). Blunt ended double stranded

cDNA was ligated into pUC18 which had been cut with Sma I and
dephosphorylated with calf intestinal phosphatase. After
Escherichia ggli DHS'a cells were transformed, 1000 mini-
plasmid preparations of cDNA clones were made and dot blotted
onto GeneScreen membranes (Dupont). The library was screened
with the same probe used in the Northern blot, but the
washing step was done at reduced stringency (6x SSC/1% SDS at

40°C for 20 min, 3x SSC/1% SDS at 40'C for 20 min).

Southern blot

Southern blot transfer was done on Naggleria DNA,
digested with restriction enzymes (BamH I, Hind III, EcoR I,
Pst I) for 5 hr and separated by electrophoresis in 0.7%
agarose (IBI) gel. Denaturation of the gel is accomplished
with 0.4N NaOH/0.6N NaCl, and then transferred to GeneScreen.

BamH I and EcoR I was from Stratagene, Hind III and Pst
I was purchased from BRL. The buffer used for restriction
enzyme digestion was Stratagene universal buffer at
concentrations recommended by Stratagene.

The oligolabelled probe for the Southern blot was first
prepared by cutting the insert from actin cDNA plasmid with
EcoR I, followed by agarose gel electrophoresis and
electroelution of DNA into a dialysis membrane. This insert
went through a further purification step by running with low

melting agarose (FMC) and a second electroelution. This

double purified probe was labelled with a-32P dCTP by random
primer method (Feinberg, et al., 1983).

Hybridization was done for 2 days at 65‘C in 1M sodium
chloride, 1% ultrapure SDS, 10% dextran sulfate, 5x
Denhardt's solution, 50mM Tris-HCl (pH7.5), 100ug/ml
denatured salmon sperm DNA with oligolabelled probe.

The Southern blot was washed with 2x SSC for 10 min at
room temperature twice, 1xSSC for 20 min at 60‘C twice, 0.5x
SSC for 20 min at 60’C twice. The blot was exposed to Kodak

X-ray film overnight.

Preparation of DNA for sequencing

Plasmid DNA from the cDNA clone was prepared for double
strand sequencing. Bacteria grown overnight (100ml) were
harvested, resuspended in lysis buffer (25mM Tris-HCl, 10mM
EDTA, 5mg/ml lysozyme) and incubated in ice for 10 min. The
cells were lysed in 0.2N NaOH/1% SDS. After neutralization
with 3M potassium acetate (pH4.8), the plasmid DNA was
recovered by standard procedures. Ethanol precipitation,
washing, RNA digestion, phenol-chloroform extraction, second
precipitation and washing were carried out as previously
described (Sambrook, at al., 1989). The plasmid DNA was

dissolved in nanopure water before further experiment.

Denaturation for double strand sequencing

DNA (10ug, measured by spectrophotometer) was denatured
with 0.4M NaOH at 37'C for 30 min. An 0.1 vol. 3M sodium
acetate (pH 4.5-5.5) was added to neutralize and 4 vol. 100%

ethanol was added to precipitate the DNA at -20'C overnight.

DNA sequencing

Naegleria fonleri actin cDNA clones were sequenced by
the dideoxynucleotide chain termination method according to
the method provided by US Biochemical with USB Sequenase
version 2.0. For labelling, 33P dATP is used as well as 35$
dATP .

Two sequence primers (5'-CCAATTGAACACGGTAT-3', 5'-
CACAACCTTAATCTTCA-B') were synthesized to get the full length

actin cDNA.

RESULTS

Northern blot analysis

The blot of long of total celluar RNA was made by Wang-
nan Hu. The RNA was size separated on agarose gels with
formaldehyde as the denaturant. After electrophoresis the
RNA was electroblotted onto GeneScreen. The hybridization of
end-labelled probe to Northern blot occurred in the 1.2kb
region of the blot which was determined by running RNA size
makers on the same gel. The level of hybridization increased
after subsequent mouse brain passages (compare m1 RNA to m2,
m3 and m4 in Figure 1) while the axenically grown RNA showed
the lowest hybridization (lane Ax). An equal amount of
intact RNA was present in the five samples beCause the rRNA
had equal ethidium bromide staining, I conclude that there is
a higher expression of actin gene in virulent amoebae than

non-viruelnt amoebae.

Southern blot analysis

Southern blot analysis was performed to determine the
number of copies of actin genes (Figure 2). Total nuclear
DNA was digested individually with four different restriction
enzymes (BamH I, EcoR I, Hind III and Pst I) and the
digestion products size separated by agarose gel

electrophoresis. First, the hybridization was done with the

end-labelled probe but the signals were too weak to be
clearly visualized. Therefore a random primer oligo-labelled
probe of cDNA N,fgnleri actin gene was made to give stronger
signal. The 4-7 bands shown on Southern blot suggested that

actin genes are a multigene family.

Dot blot and nucleotide sequence analysis

A cDNA library of 104 clones was made from LEE strain
with SmaI digestion and pUC 18 ligation by Wang-nan Hu. This
library was screened with the end-labelled probe.

Three positive clones were found. The inserts were
sequenced but one of them had the SmaI site within the coding
region of actin gene and it couldn't represent the full
length cDNA actin. The actual insert sizes were 1120 base
pair and 1177 base pair, designated actl, act2 respectively.

In the actl sequence, one long open reading frame was
present extending 1116 bases from the ATG putative start
codon to TAA stop codon. A possible poly A additional signal
was located 25 bases after the stop codon. The other
sequence, act2, 1128 bases were identified within the coding
region, missing 12 bases from start codon. It contained 24
base pairs of the 5' upstream region, 68 base pairs of the 3'
region after the stop codon including poly A tailm as well as
coding sequence. Also actl had a possible poly A additional

signal at 36 bases after the stop codon.

9

Comparison of the nucleotide sequence to the GENEMBL
data base sequence showed a homology with 241 actin gene
sequences. 'The highest homology shown here was 78.1% for
actl with a Candida albigans actin gene (accession number
X16377). ’Other organisms, Digtygstglinm (73.8%, accession
number X03281), Saggargmygga (77.3%, accession number L00026)
and Entamggha (76.7%, accession number M16396) actin genes
showed relatively high homology with that of N,fnnl£zi. Also
the deduced amino acid sequence was compared with other actin
peptide and showed the highest homology (83.4%) for actl with
Ehysarnm pglygephalnm (accession number P02576).
Dictygstelinm diaggideum (accession number P02577) human
(accession number P02570) and Acanthamgeba (accession number
P02578) showed more than 80% homology with Nflfgwleri actin
amino acid sequence (Table 2).

Because the structure of actin protein is now available
from x-ray crystallography, we can deduce the ATP and
divalent metal ion binding site as well as actin-myosin and
actin-actin interaction site. The amino acid residues which
involves ATP, metal ion binding and actin-myosin interaction
are largely conserved while actin-actin contacts are not as
highly conserved as other interaction sites.

There were 10 nucleotide differences out of 1116 base
pairs between actl and act2 genes in coding region. Six
amino acid changes were predicted from the nucleotide

difference, 4 of them had different side chain polarity.

10

The A+T content inside the coding region was 58.3% while
in the 5' and 3' region, it was 67.4% (actl both 5'and 3'
ends),and 78.7% (act2, 3'end only). For translation, actin
gene showed the preference of purine base at third position

(Table 1).

DISCUSSION

Actin is present in the form of filaments, small
filament bundles and meshworks in a wide variety of organisms
(Taylor at al. 1979). Thick and thin microfilaments,
morphologically similar to actin and myosin, have been seen
in the cytoplasm of Naeglgria,figwlgri (Lastovica at al.,
1976). The Northern blot showed that there is at least one
actin transcript of moderately high abundance in this
organism (Fig. 1). The probes were hybridized to gel blots
of total RNA from axenically growing amoebae (Lane Ax) and
from amoebae taken from brains of mice after one, two, three
or four serial infections of mice (m1, m2, m3, m4). This
results showed that there is a higher expression of actin
gene. Because of the increase in hybridization, the
increased mRNA of actin may be necessary for the virulence in
N. fonleri. Wong et_al. (1977) noted that after prolonged
periods of maintenence in axenic medium, strains of N.
foulgri lost their original pathogenicity in mice Virulence
was restored after serial mouse brain passage. The highly
virulent amoebae exhibit faster movement in xixg than do
weakly virulent amoebae (Cline at al. 1986). The increase of
actin mRNA is correlated with increased motility possibly for
host cell invasion and phagocytic activity.

The actin gene generally belongs to a multigene family,
but there are exceptions such as in Sagghargmycea,gerexisidae
and Tetrahymena spp. (Amar at al. 1988). The size of the

11

12

multigene family may be small, as in Aganthamggha spp. and
Physarum spp. which have three or four actin genes, or large
as in Dictybstelinm spp., mice or human which have 20 to 30
actin genes (Amar e; al.1988). Cloned actin cDNAs hybridize
to at least four fragments in genomic DNA which were digested
with restriction endonucleases (Fig. 2) indicating that actin
may be present in many copies in Naggleria genome. In
addition, faintly hybridizing bands were detected which might
be due to cross-hybridization to similar sequence of actin or
parts of actin genes which have restriction sites within
their genomic sequences.

The construction and identification of cDNA clones
containing sequences of actin provides an understanding of
actin protein structure as well as gene evolution. Actins
are widely assumed to be evolutionary conserved proteins.

The sequences determined here are between 65-80% for both
nucleic acid and amino acid homology to other organisms.

This is lower than comparisons of Sghizgsanghargmyces,pombe
actin genes to other actin sequences in various organisms
(Lees—Miller at al. 1992). I have sequenced two actin genes
in N, fgnlgri, one of which is 12 base pairs short of the
start codon (Fig. 3). There are 13 nucleotide differences
which cause 3 silent and 5 amino acid changes in actin
protein. This is a big difference (79.6% amino acid homology)
compared with Entamgeha,histglytiga (Edman at al. 1987) which
was previously thought to be closely related to N..£Qfllfinir

13

that showed just 4 nucleic acid changes, all of which were
silent changes when translated into amino acid sequence.

Even though 3 of the changed amino acids have the same
side chain polarity, one of them is polar/non-polar change
and the other is non-polar/polar change. Thus, the overall
charge of these two proteins remains the same from 4 to 375
amino acid but the isoelectric point can be different because
the N-terminus is a very variant region in actin protein. It
is now established that various forms of actin, differing
from one another by certain changes in primary strcture of
protein, exist in different cells and tissues and within a
given cell (Taylor :1 a1. 1979) or in an amoebae (Nellen gt
al. 1982). The two different actin proteins deduced from the
actin gene cloned here can coexist in a anguleri, excuting
different roles structurally and functionally.

The comparison of both actl and act2 3' untranslated
regions did not show high homology (23%), but they have 11
A's in the polyA tail as well as the eukaryotic
polyadenylation signal (Fig3, underlined). From GENEMBL data
base sequences, a comparison of both 5' and 3' untranslated
region was done with various species. Unlike the coding
region, there were no detectable similarities between N.
fnnleri and other organisms that have high homology in coding
sequences. The 3' untranslated regions including poly A tail
in N, fgnleri actin genes are very short (50 and 57 base pair
long, shown in Fig.3) which is also true for a- and B-tubulin

genes of Naegleria (Clark. 1990). This short untranslated

14

portion of actin mRNA was also observed in Aganthamggha,
Digtygstelinm, and yeast (Nellen at al. 1982). It,
therefore, Seems that the group of smaller actin mRNA is
confined to the simpler eukaryotesfi

Deduced amino acid sequence comparison is contradictory
to that of nucleic acid sequences (Fig 4). Among 8
organisms, Dictygatelinm and Sagchargmyges exhibit the
highest homology (77.3%) followed by Entamggha (76.7%) in
nucleic acid comparison. However, in amino acid alignment,
Aganthamgeba, Digtygstelinm, and human show higher homlogy
than Entamgeha (Tabel 2). No matter how high the homology
is, there are two parts of amino acid sequences in N. fowleri
which are very distinct in terms of side chain polarity. The
region of 192-194 and 216-220 amino acids are quite different
from other actin sequences while all other eight genes shared
those sequences or have few replacements. As N-terminal
amino acids showed high degree of replacement in other
comparison (Lees-Miller a; a1. 1992), it is not remarkable
that there is a variety of amino acids in this comparison.
In one species of Naegleria, N. gznhazi. an antibody for
actin did not recognize determinants in actin of
Aganthamgeba, Dictygsteinm and Rhysarum (Fulton et.al. 1986)
This antibody defines a region possibly species specific.
Lack of N-methylhistidine in N, gruheri (Fulton at. al.
1986.) may also participate in the uniqueness of actin. The
reason for specific antibody specificity may be clarified by

sequence analysis.

15

Recently, the atomic structure of actin was determined
by X-ray crystallography in rabbit skeletal muscle (Kabsch a;
a1. 1992). Actin generally consists of two domains which can
be further subdivided into two subdomains. It is suggested
that a five-stranded a sheet consisting of B-meander and a
right handed BaBunit appears in each domain. Because
Naegleria actin is homologous to their actins, possibly it
has the same atomic structure.

Naegleria actin, although different enough to have
unique antigen determinants, is conserved in many properties
like calcium-, nucleotide-, myosin-, actin-binding sites
(Lees-Miller at al. 1992). As shown in Fig 4, calcium-
binding sites are completely conserved while nucleotide-
binding sites are relatively well conserved in N. fnnlafi.
The most variable replacements are found in actin-actin
binding sites, however, those replacements have the same side
chain polarity except 40, 41, 43, 110, 196 amino acids. The
myosin binding sites are highly conserved throughout the
peptide exept the variant N-terminus region where no homology
was found among 9 species. DNAseI contact sites, composed of
50, 53, 61, 68, 69 amino acid residues (Edman gt a1. 1987)
was conserved in N, fowleri actin. Therefore the N,fguleri
actin polypeptide possesses the common structure of actin
which allows the functional properties of it.

The relative frequency of synonymous codons for any
amino acids suggests that codon usage differs among species

(Starner at al. 1989). The preference of particular codons

16

implies that a functional role of codon usage may differ in
many organisms as a consequence. As shown in Table 1, the
amino acid codon usage in N. fouleri actin is very biased.
Every amino acid is encoded by a preferred codon with
exceptions of threonine, asparagine, histidine and serine.
N. fauleri shows a strong preference for substitution of an
adenine or thymidine residue in the third position of a
codon. The adenine or thymine occurrence at the third
position presents at a frequency of 64.5% in contrast to 30%
in human actin. Comparison of the codon usage of N, fowleri
actin with E. 1113mm, S. minions and A. nastellani
actin (Edman et a1. 1987) revealed that N, fowleri actin
codon usage is more smilar to S, cerexisidae than to E.
hiatglytiga and very different from A. gastellani. Also,
this bias will prove useful in producing probable DNA
sequences for oligonucleotide based gene isolation and for
confirming the proper reading frame when genes are being
sequenced.

As amoebas have long been considered the most difficult
protozoa to place satisfactorily in a taxonomical scheme,
actin is extremely valuable for probing phylogenic
relationship. Many regions of actin remain conserved over
large periods of time, and the rate of molecular evolution in
general is relatively constant with time (Baverstock er a1.
1989). Most of the molecular work to determine evolutionary
relationships are by comparison of small-subunit ribosomal

RNA sequences. These sequences are considered highly

17

conserved in evolution and because of slow divergence (Clark
gt a1. 1989). According to rRNA sequencing, Nagglgtig is more
closely related to Tgttahymgna or Irxpannanma than
(Aggnthgmgghg (Baverstock gt gl. 1989). This is contradictory
to the conclusion based on actin nucleotide and amino acid
sequence which place Nagglgtia more closely to D,digggidgum
and Aflgastgllanii. Table 2 shows the similarity between
Nagglgria4fgnlgri and other species. Amino acid sequence
comparison instead of nucleotide was used because Loomis gt
g1. (1990) had suggested that amino acid sequence might be
more reliable than untranslated nucleic acid sequences for
evolutionary comparisons. N,fgwlgti has a higher H-value
(defined by Sogin gt,gl, 1986) with Arcaatellanii and
D.digggidgnm than with Erhigtglxtiga. This discrepancy might
be due to the species used for the rRNA sequencing. N,
grnbgti is considered to be the most divergent species in the
genus Nagglgrig (Clark gt al. 1988). Restriction endonuclease
analysis of mitochondria DNA also showed the most divergent
nature of N, grubgti_within the genus as well as the most
distant relatedness to N, fgulgti (Milligan gt g1. 1988).
Furthermore, ribosomal DNA digestion demonstrated that N.
exuberi‘is very variant within the species (Clark gt a1.
1989). Therefore the possibility remains that N, finalezi is
a more advanced eukaryote than N, gtnbgti and is closely
related to Aganthamggbg. Similar contradiction in phylogenic
analysis have been reported in Digtyggtglinm.diggg1dgnm
(Loomis gt al., 1990). Cladistic analysis of amino acid

18

sequence in actin from a variety of eukaryotes shows that
D.diacoideum and E.higtglytiga are closely related and are
closer to metazoa than are yeast. But in distance matrix,
Digtygstglinm is grouped with the metazoa, and Entamggba
forms a separate line. Therefore although rRNA sequencing
could be a useful method to determine the phylogenic
relationships in evolution, the comparison of highly
conserved protein sequences can come to a different
conclusion. One remarkable characteristic of Aggnthgmggba is
to form a cyst in the absence of exogenous nutrients. Also,
Dictxnfitelinm aggregates and form spores when staring
(MacLeod gt al., 1980). .Aaanthamneha, Di£L¥QfiL£linm and
Nagglgtia transform their shape when starving and the actin
is an organelle which is resposible for the shape of an
organism. Therefore among these three organisms, the
unexpected high homology of actin might be due to the ability

of transforming their shape in starvation.

LITERATURE CITED

1. Amar, M.F.B., A. Pays, P. Tebabi, B. Dero, T. Seebeck, M.
Steinert and E. Pays. 1988. Structure and transcription of

the actin gene of Itypgngsgma htnggi. Mol. Cel. Biol.
8(5):2166-2176

2. Baverstock, P.R., S. Illana, P.E. Christy, B.S. Robinson
and A.M. Johnson. 1989. srRNA evolution and phylogenic
relationships of the genus Nggglgzig (Protista:Rhizopoda).
Mol. Biol. Evol. 6(3):243-257

3. Clark, C.G., G.A.M. Cross and J.F. DeJonkheere. 1989.
Evaluation of evolutionary divergenCe in the genus Nggglgtig
by analysis of ribosomal DNA plasmid restriction patterns.
Mol. Biochem. Parasitol. 34:281-296

4. Clark, C.G. and G.A.M. Cross. 1988. Small-subunit

ribosomal RNA sequence from Nagglgria gtnbgti supports the
polyphyletic origin of amoebas. Mol. Biol. Evol. 5(5):512-

518

5. Clark, C.G. 1990. Genome structure and evolution of
Nggglgtia and its relatives. J. Protozool. 37(4)25-6s

6. Cline, M., R. Carchman and F. Marciano-Cabral. 1986.
Movement of Naecleria.fculeri stimulated by mammalian cells
in vitro. J. Protozool. 33:10-13

7. Edman, U., I. Meza and N. Agabian. 1987. Genomic and
cDNA actin sequences from a virulent strain of Entgmghg
higtglytiga. Proc. Natl. Acad. Sci. USA. 84:3024-3028

8. Feinberg, A.P and B. Vogelstein. 1983. A technique for
radiolabelling DNA restriction endonuclease fragments to high
specific activity. Anal. Biochem. 132:6-13

9. Fulton, C., E.Y. Lai, E. Lamogi and D.J. Sussman. 1986.
Nggglgtig actin elicits species-specific antibodies. J.
Protozool. 33(3):322-327

19

20

10. John, D.T. 1982. Primary amebic meningoencephalitis and

the biology of Nggglgig‘figulgri. Ann. Rev. Microbiol.
36:101-123

11. Kabsch, W., H.G. Mannherts, D. Suck, E.F. Pai and K.C.
Holmes. 1990. Atomic structure of actin:DNaseI complex.
Nature 347(6):37-44

12. Lastorica, A.J. 1976. Microfilaments in Nafiglfiria
fgnlgri amoeba. Z. Parasitenkd. 50:245-250

13. Lees-Miller, J.P., G. Henry and D.M. Helfman. 1992.
Identification of act2, an essential gene in the fission

yeast Sghizggggggtgmyggg ngmhg that encodes a protein related
to actin. Proc. Natl. Acad. Sci. USA. 89:80-83

14. Loomis, W.F. and D.W. Smith. 1990. Molecular phylogeny

of Digtyggtglium.digggidgnm by protein sequence comparison.
Pro. Natl. Acad. Sci. USA. 87:9093-9097

15. MacLeod, C., R.A. Firtel and J. Papkoff. 1980.
Regulation of actin gene expression during spore germination

in Wm disccideum. Dev. Biol. 76:263-274

16. Marciano-Cabral, F. 1988. Biology of Nggglgtia spp.
Microbiol. Rev. 25:114-133 7

17. Milligan, S.M and R.N. Band. 1988. Restriction
endonuclease analysis of mitochondrial DNA as an aid in the

taxonomy of Nggglgtig and yghlkgmgfig. J. Protozool.
35(2):198-204

18. Nellen, W. and D. Gallwits. 1982. Actin genes and

actin messenger RNA in Aggnthgmggha gggtgllgnii. J. Mol.
Biol. 59:1-18

19. Sambrook, J., E.F. Fritsch and T. Maniatis 1989.
Molecular Cloning. second ed. Cold Spring Harbor Laboratory
press.

20. Sogin, M.L., A.Ingold, M. Karlok, H. Nielsen and J.
Engberg. 1986. Phylogenic evidence for acquisition of
ribosomal RNA introns subsequent to the divergence of some of
the major Igttghymgng groups. EMBO J. 5:3625-3630

21. Starner, W.T. and D.T. Sullivan 1989. A shift in a
third-codon-position nucleotide frequency in alcohol
dehydrogenase genes in the genus Dtgggphila. Mol. Biol.
Evol. 6(5):546-552

21

22. Sussman, D.J., E.Y. Lai and C. Fulton 1984. Rapid
disappearance of translatable actin mRNA during cell

differentiation in Nagglgtig. J. Biol. Chem. 259(11):7355-
7360

23. Taylor, D.L. and J.S. Condeelis 1979. Cytoplasmic
structure and contractility ih amoeboid cells. Int. Rev.
Cytol. 56:57—114

24. Wong, M.M., S.L. Karr Jr. and C.K. Chow, 1977. Changes
in the virulence of Naggleria foulgxi maintained in vitro.
J. Parasitol. 63:872-878 '

Axm1m2 m3m4

18$—
.“ —NfActin1

Figure 1. Autoradiograph of Northern blot of RNA from
axenically grown amoebae and amoebae from mouse brain. Total
RNA (lOug) was electrophoretically size separated on an
agarose gel containing formaldehyde. Lane Ax (axenic
amoebae); m1. m2, m3 and m4 amoebae after 1, 2, 3 and 4
passages of infection in mouse brain. The blot of the gel

was probed with and 32P-end labelled oligonucleotide
homologous to an actin mRNA sequence.

22

23

 

(kb)

23.1=4>
fin
.- e
9.4=> _’ ..
6.7={>

4.4={> at;

 

 

 

Figure 2. Autoradiograph of southern blot probed with the
act2 cDNA. Genomic DNA (long) digested with BamH1(B),
EcoR1(E), HindIII(H) and Pst(P) restriction enzymes was
electrophoretically size separated, blotted onto GeneScreen
and hybridized to 32P-oligolabelled act2 cDNA insert. Size
markers on the left were from lambda DNA digested with
HindI I I .

-24

Actl TTCCTCTCCAACAAGAACAACAAA

MetCysAspAspVal
ATGTGTGACGACGTT
********Act2

PheAlaGlyAspAsp
TTCGCTGGTGATGAT

LysSerIleMetVal
AAGTCCATCATGGTT

LysArgGlyIleLeu
AAGAGAGGTATTTTG

AspMetGluLysIle
GATATGGAAAAGATC

HisProValLeuLeu
CATCCAGTCTTGTTG

GlnIleMetPheGlu
CAAATCATGTTTGAA

SerLeuTyrAlaSer
TCTTTGTATGCTTCT

HisThrValProIle
CACACTGTTCCAATT
AlaGlyArgAspLeu

GCTGGTAGAGATTTG

AsnThrThrAlaGlu
AATACCACTGCTGAG

30
GlnAlaLeuValVal
CAAGCACTCGTAGTT

a
Glu

90
AlaProArgAlaVal
GCACCAAGAGCTGTC

150
GlyMetGlyAsnLys
GGTATGGGTAACAAG

210
ThrLeuLysTerro
ACTTTGAAGTATCCA

270
TrpHisHisThrPhe
TGGCATCACACCTTC

330
ThrGluAlaProLeu
ACTGAAGCTCCATTG

390
ThrPheSerValPro
ACCTTCTCTGTTCCA

450
GlyArgThrThrGly
GGTCGTACCACTGGT

510
TyrGluGlyTyrAla
TATGAAGGTTATGCT

570
ThrAspTereuIle
ACTGATTACTTGATC

630
ArgGluIleValArg
AGAGAAATTGTCAGA

tg caa
CysGly

24

AspAsnGlySerGly
GATAACGGATCTGGT

PheProSerIleIle
TTCCCTTCCATCATT

AspAlaTeralGly
GATGCCTATGTTGGT

IleGluHisGlyIle
ATTGAACACGGTATT

TyrAsnGluLeuArg
TACAATGAATTGAGA

AsnProLysAlaAsn
AATCCAAAGGCTAAC

AlaMetTyrvalAla
GCCATGTATGTTGCC

IlevalLeuAspSer
ATTGTTTTGGACTCT

LeuProHisAlaIle
TTGCCTCATGCTAGG
c
Ala

GluAspSerHisGly
GAAGATTCTCATGGA

AsleeGluGlyLys
GATATCGAAGGAAAA
a
Glu

60
MetCysLysAlaGly
ATGTGTAAGGCTGGT

120
GlyArgProLysGln
GGTAGACCAAAGCAA

180
AspGluValGlnSer
GATGAAGTCCAATCC

240
valThrAsnTrpAsp
GTCACCAATTGGGAT

300
valAlaProGluGlu
GTTGCTCCAGAGGAA

360
ArgGluLysMetThr
AGAGAAAAGATGACT

420
IleGlnAlaValLeu
ATTCAAGCTGTCTTG

480
GlyAspGlyValSer
GGTGATGGTGTCTCT

540
LeuArgLeuAspLeu
TTGAGATTGGATTTG

600
ThrCysTyrSerPhe
ACGTGTTACTCATTC

660
AlaLeuLeuTerys
GCTCTGTTATATTGC

PheAspPheGluGln
TTTGACTTTGAACAA

GluLeuProAspGly

GAATTGCCAGACGGT

LeuPheGlnProAsn
TTGTTCCAACCAAAC

SerIleGlyLysCys
TCGATTGGAAAGTGT

GlyGlyThrThrMet
GGTGGTACTACCATG

AlaProAlaSerMet

GCTCCTGCTTCCATG

IleGlyGlySerIle

ATTGGAGGTTCCATC

GluTyrGluAspAla
GAATATGAGGATGCC

690
GluMetLysIleAla
GAAATGAAGATTGCT

750
AsnvalIleThrVal
AACGTGATTACTGTT

810
PheIleGlyMetGlu
TTCATTGGTATGGAA

870
AsleeAsleeArg
GATATTGATATCAGA

930
PheGluGlyIleAla
TTTGAAGGTATTGCT

990
LysIleLysValVal
AAGATTAAGGTTGTG

1050
LeuAlaSerLeuSer
TTGGCTTCATTGTCC

1110
GlyProGlyIleVal
GGTCCAGGTATTGTC

25

AlaGluSerSerThr

GCTGAATCATCCACC
t
Ser

GlyAsnGluArgPhe
GGAAATGAAAGATTC

AlaAlaGlyValHis
GCTGCTGGTGTCCAT

LysAspLeuTyrGly
AAGGATTTGTATGGT

GluArgMetThrLys
GAGAGAATGACCAAG

AlaProProGluArg

GCTCCACCAGAAAGA

ThrPheGlnGlnMet

ACCTTCCAACAAATG

HisArgLysSerPhe
CACAGAAAGAGCTTC

720
ValGluLysSerTyr
GTTGAAAAGTCTTAT

ctc
Leu

780
ArgCysProGluval
AGATGTCCAGAAGTT

840
GluThrThrPheAsn
GAAACTACTTTCAAC

900
Asnva1ValLeuSer
AACGTTGTCTTGTCT

'960
GluLeuThrAsnMet
GAATTGACCAACATG

1020
LysTyrSerValTrp
AAGTACTCGGTCTGG

a
Lys

1080
TrleeThrLysGlu
TGGATCACCAAGGAA

1128
stop
TAA

ATTGACCTTGGATGCACATTATCAAATTCCAATGTABIABAACATAAAAATCTATGT

AAAAAAAAAAA 1196

attggatgcacattatcaaattccaataaatccaataattgtaatacttcaaaaaaaaaaa 1189

Figure 3.

Nucleotide and deduced amino acid sequence of

N.fgnlgti is shown in capital letters in full length cDNA
(Actl), while the partial cDNA (Ath) with small letters.
Nucleotides are numbered relatively to the A of the ATG
initiation codon in Actl, negative numbers indicate 5'

flanking sequence.

The deduced amino acid sequence of Actl
is shown above the nucleotide sequence.
sequence is shown below the nucleotide sequence.

In Act2,Amino acid

The

underline indicates a potential poly A additional site

AAIWJUX.

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Dictyostelium
Human
Saccharomyces
Acanthamoeba
Plasmodium
Entamoeba
Tetrahymena

Trypanosoma

Naegleria
Dictyostelium
Human
Saccharomyces
Acanthamoeba
Plasmodium
Entamoeba
Tetrahymena

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Figure 4. Amino acid sequence alignment and comparison of the
actin sequences in Nimrleri— (actl), Dicticstelinm discoideum.
human b-actin, Sacchammcea sen-insides, Acanthamoeba
castellanii, Elasmcdium falninafnm, Entamoeba bismlxtica.
Tetrahymena thermophila, W Emmi. In the species
that have isoforms of actin, the most homologous amino acid
sequence is chosen for the comparison. Amino acid identities
are boxed only when shared by all nine members. The residues
in actin that interact with the nucleotide (n), calcium (c),
and myosin (m) and that involved in actin-actin interactions
(a) are in small letters above the actl sequemce.

TABLES

Table 1. Codon usage in N, fgnlgri actin gene. All 375
codons from the initiator ATG to the st0p TAA are
represented. The numbers are total number of occurrences of
each codon.p

 

 

 

 

 

 

 

 

 

 

 

 

ARG CGT 1 LED TTA 1 SER TCT 9
CGC 0 TTG 22 TCC 7
CGA 0 CTT 0 TCA 3
CGG 0 CTC 1 TCG 2
AGA 15 ,CTA 0 AGT 0
AGG 0 CTG 1 AGC 1
ALA GCT 22 GLY GGT 25 PRO CCT 3
GCC 4 GGC ~ 0 CCC 0
GCA 2 GGA 6 CCA 15
GCG 0 GGG 0 CCG 0
VAL GTT 14 THR ACT 11 ILE ATT 18
GTC 11 ACC 11 ATC 9
GTA 1 ACA 0 ATA O
GTG 2 ACG 1
ASN AAT 5 ASP GAT 17 CYS TGT 5
AAC 8 GAC 5 TGC 1
GLN CAA 9 GLU GAA 25 HIS CAT 5
CAG 0 GAG 5 CAC 4
LYS AAA 1 PHE TTT 4 TYR TAT 10
AAG 19 TTC 11 TAC 4
MET ATG 15 TRP TGG 4

 

 

 

35

36

Table 2. Amino acid comparison of Nggglgzia‘fignlgti with
other species (Ac; Acanthamoeba castellanii,
Dd;D.im'.¥Qs.t.elinmdis.cnideum Hsrncmcsaniens. Eh;Entamceba
111.319.111.11“, Pf; Blasmcdium falcinarum, Sc; Sacchammxces
cemisidae Tt Tetrahymena W Th; Trypanosoma
hrnggi). Matching column shows the number of identical amino
acids between the two species. Similar replacement is the
amino acid changes within the same side chain polarity while
different replacement shows different polarity. H-value is
calculated as Ham/(m+u+g/2), m is the number of sequence
positions with matching amino acids in the two positions, u
is with unmatching (replacements) positions, and g is the
number of positions that have a gap in one sequence opposite
an amino acid in the other. The greater H-value represents
closer relationships between the two species.

 

 

 

replacement
comparison matching H-value
- similar different

N-Ac 318 46 11 0.848
N-Dd 316 47 12 0.845
N-Hs 312 50 13 0.832
N-Eh 307 56 ' 12 0.817
N-Pf 302 61 12 0.805
N-Sc 296 68 11 0.789
N-Tt 276 72 27 0.736
N-Tb 272 82 21 0.725

 

 

 

 

 

 

 

 

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