Copyright 0 1994 by the Genetics Society of America
Structure, Expression and Phylogenetic Analysis of the Gene Encoding
Actin I in Pneumocystis carinii
Leah D. Fletcher,* John M. McDowell,t Richard R. Tidwell,” Richard B. Meaghert and
Christine C. Dykstra”
*Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599,
and +Department of Genetics, University of Georgia, Athens, Georgia, 30602
Manuscript received September 27, 1993
Accepted for publication March 23, 1994
ABSTRACT
Actin is a major component of the cytoskeleton and one of the most abundant proteins found in
eukaryotic cells. Comparativesequence analysis shows that this essentialgene has been highly conserved
throughout eukaryotic evolution makingit useful for phylogenetic analysis. Complete cDNA clones for
the actinencoding gene wereisolated and characterized from Pneumocystis carinii purified from
immunosuppressed rat lungs. The nucleotide sequence encodes a protein of 376 amino acids. The predicted actin protein of P. carinii shares a high degree of conservation to other known actins. Onlyone
major actin gene was found in P. carinii. The P. carinii actin sequence was compared with 30 other actin
sequences. Gene phylogenies constructed using both neighbor-joining and protein parsimony methods
places the P. carinii actin sequence closest tothe majority of the fungi. Since the phylogenetic relationship
of P. carinii to fungi and protists has been questioned, these data on the actin gene phylogeny support
the grouping of P. carinii with the fungi.
A
CTIN genes are descended by duplication and divergence from common ancestral genes and arose
early in eukaryotic evolution. Crucial cellular processes
influenced by actin include motility, regulation of cell
growth and differentiation, endocytosis, exocytosis and
structural stability (POLLARD 1990;
POLLARD
and COOPER
1986;STOSSEL1984). Approximately six different isoforms of actin are expressed in warm-blooded vertebrates and plants. Many of these actin isoforms appear
to be
developmentally regulatedand also exhibita cell
type-specific expression (MCLEAN
et al. 1990). Recent
studies have shown that several lower eukaryotes also
contain multiple actin isoforms or actin-related proteins ( i . e . , Plasmodiumfalciparum (WESSELING
et al.
1988a,b;1988), Schizosaccharomycespombe (LEES-MILLER
et al. 1992), and Saccharomyces cerevisiae (SCHWOB
and
MARTIN 1992)) .
P. carinii has been a challenge to taxonomists for
more than 70 years. It was originally classified as a protozoan based on its misidentification as a trypanosome
(CHAGAS
1909) and its susceptibility to anti-protozoal
agents ( i. e., pentamidine, bereniland primaquine) and
resistance to anti-fungal agents. Although some life cycle
stages of P. carinii are similar in appearance to those of
protozoans like Toxoplasma gondii,recent studies at the
molecular levelhave suggested that P.carinii may
bemore closely relatedto fungi in the Ascomycota
group (EDMANet al. 1988).Others have suggested a
closer association with the ‘‘F&izopoda/Myxomycota/
Zygomycota” group ( W A T A N et
~ Eal. 1989) than with
protozoa. Studies comparing the 18s rRNA sequence
Genetics 157: 743-750 uuly, 1994)
from P . cariniiwith eight other taxa showedthe greatest
amount of homology 18s rRNA sequences from fungi
such as S. cerevisiae and Neurospora crassa (CUSHION
et al. 1988). Additional evidence for a fungal classification is demonstrated by the fact that the dihydrofolate
reductase and thymidylate synthase genes of P. carinii
are separateand not encoded
by a bifunctional enzyme
0.C. EDMANet al. 1989;U. EDMANet al. 1989).Generally,
the fungi, unlike protozoans, have the two enzyme activities on separate polypeptides. Comparisons of gene
sequences could help resolve the debate over the classification of P. carinii. The ability to classify this organism has significant clinical relevance for the determination of new therapeutic agents.
We have completely sequenced cDNA clones containing themajor actin gene. Since the aminoacid sequence
and biochemical properties of this protein have been
highly conserved throughout evolution, actin is an ideal
gene for phylogenetic analysis. P. carinii appears to
have onlyone major actin gene. We have alsoidentified
a second actin-related gene with homology to mammalian centractin (L. D. FLETCHER
and C. C. DYKSTRA,
manuscript in preparation). This study describes the isolation
and characterization of the P. carinii actin I gene and
the phylogenetic analysis ofthe nucleotide and derived
amino acid sequences.
MATERTALSANDMETHODS
Source of P. carinii: P. carinii organisms were produced
in immunosuppressed male Sprague-Dawley rats as previously
et al. 1990).Organisms werepurified from
described (TIDWELL
rat lung tissue and DNA was isolated from P. carinii cysts and
h
744
D. Fletcher et al.
trophozoites by methods previously described (FLETCHER
et al.
1993).
Oligonucleotideprimers: The primer sequences were
based on known conserved regions of actin proteins from several organisms and designed with the P. carinii A + T codon
bias described by FLETCHER
et al. (1993).The actin primer pairs
were 5'dGG GAT GAT ATG GAA AAA AT(T/A) TGG C and
5'd-GC ATA (A/T/C)CC CTC
ATA
(A/T)AT (A/T)GG
(A/T)AC, corresponding to nucleotides 239-262 and
490-512,respectively,of
the P. falciparum actin I gene
(WESSELING
et al. 1988a). Oligonucleotides were synthesizedby
the University of North Carolina, Department of Pathology
core facility.
Polymerase chain reaction and cloningof amplified products: The polymerase chain reactions (PCR) were performed
in a BioTherm thermal cycling oven. Thirty rounds of amplification were performed by denaturation at 92", annealing at
48", and elongation at 72" (the duration of each step was 1
min). Each amplification reaction contained 100 ng of
genomic P. carinii DNA.Rat testes and S. cerevisiae DNA
samples were used as negative controls. P. carinii PCR products wereisolated from a 1%low melting point agarose gel by
GlasPak (National ScientificSupply Company) procedures.
Purified PCR products were ligated directly into apBluescript
I1 K
S
' (Stratagene) T-tailed vector according to MARCHUK et al.
(1991). Ligated DNAwas transformed into Escherichia coli
(DH5-a) cells by electroporation at 25 pF, 200 ohms, and 25
kV (Bio-Rad Cell-Porator).Bacteriawere allowed to recover for
1 hr at 37" with shaking, before selection on LB plates containing ampicillin (50 pg/ml), withX-gal
isopropyl POthiogalactopyranoside, permitting blue us. white recombinant
selection. Plasmid DNA wasisolated from the recombinants by
the alkaline lysis method (SAMBROOK
et al. 1989). Recombinant
DNA fragments were digested from the vector with PstI/
Hind111 restriction endonucleases and isolated from 1% low
melting point agarose gels by the GlasPak (National Scientific
Supply Company) protocol. The recovered DNA was used for
random primed DNA labeling (Boehringer Mannheim) with
[32P]dCTP(Amersham).
Isolation of the actin I cDNAs: A hgtll P. carinii cDNA
library (kindly provided by J. A. FISHMAN,
Infectious Disease
Unit, Massachusetts General Hospital, 149 13th Street,
Charlestown, Massachusetts 02129), was screened with the 32Plabeled actin gene fragment described above. Three clones,
(41-1, 6-4-1 and El-1) were selected and purified by two
rounds of single plaque isolation and rescreening. The EcoRI
fragments from each of the cDNA clones was isolated, subcloned into the pBluescript I1 K
S
' (Stratagene) vector and
sequenced.
Nucleic acid hybridization:Restriction endonucleases were
used according to the manufacturers' instructions. Genomic
P. carinii, S. cerevisiae and rattestes DNAs were digested with
a variety of endonucleases, fractionated by agarose gel electrophoresis and blotted onto nitrocellulose membranes according to SOUTHERN
(1975). Hybridizations were performed
overnight at 55-60" in 6 X SSC, 1 X Denhardt's solution and
50 pg/ml salmon sperm DNA, after adding the '*P-labeled
actin probe. Following hybridization, filters were washedonce
in 2 X SSC/O.l% sodium dodecyl sulfate (SDS) for 5 min at
room temperature and three timesin0.1 X SSC/O.l% SDS for
10 mineach at 55-60". Filters were
exposed to Kodak XARfilm
(Rochester, New York).
Isolation and purification of P. carinii mRNk P. c a r i n i i
mRNA was isolated using a FastTrack mRNA isolation kit (Invitrogen Corp., San Diego, California). Two different sources
of organisms were utilized: (1) cysts and trophozoites purified
et al. (1993) and frozen in l-g quanas described by FLETCHER
+
TABLE I
Actin sequences used for phylogenetic analysis
Name
Abbreviation
Pneumocystis carinii
GenBank
accession no.
Pea
L2183
Ad
Ani
Cal
Kla
Sba
See
M64729
M22869
X16377
M25826
Dl2534
LO0026
YO0447
S37672
S51076
X07463
Fungi:
Absidia glauca
Aspergillus nidulans
Candida albicans
Kluyveromyces lactis
Saccharomyces bayanus
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Phytophthora infestans
Phytophthoramegasperma
Thermomyces lanuginosus
Protists:
Acanthamoeba castellani
Achlya bisexualis
Cryptosporidium paruum
Dictyostelium discoideum
Entamoeba histolytica
Naegleria fowleri
Oxytrichia nova
Physarum polycephalum
Plasmodium falciparum
Tetrahymena thermophila
Trypanosomabrucei
Green algae:
Volvox carteria
Plant:
Oryza sativa
Invertebrate:
Piastrer ochraceus
Drosophila melanogaster
Caenorhabditis elegans
Vertebrate:
Gallus gallus pactin
Rattus rattus a-actin
Homo sabiens a-actin
SPO
Pin
Pme
Tla
Aca
Abi
CPa
Ddi
Ehi
Nfo
Ono
Pfa
Tth
Tbr
v00002
X59936
M86241
X03284
MI9871
M90311
M22480
M21501
M19146
M 13939
M20310
Vca
M33963
4
0
Osa
16280 X
POC
Dme
Cae
M26501
KO0670
X1 6796
Gga
Rra
Hsa
LO8 165
X06801
105192
~~
tities at -80" until needed and (2) P. carinii-infected rat lung
tissue excised, immediately frozen in liquid nitrogen (1-g
quantities) and stored at -80" until needed. P. carinii mRNA
was isolated from these two different sources using the FastTrack protocol. Contaminating DNA was removed with RQ1
RNase-freeDNase I(Promega). The mRNA samples were
stored in elution buffer at -20" after the addition of 20 units
RNasin (Promega).
Northern blot hybridization: P. c a r i n i i mRNA isolated as
described abovewas used for Northern blot hybridization.
Samples of mRNA (2.5 pl) were denatured in 12.5 pl electrophoresis sample buffer (0.75 ml deionized formamide/O. 15 ml
10 X MOPS/0.24 ml formaldehyde/O.l ml diethyl pyrocarbonate-treated dH,O/O.l ml glycero1/0.008 ml 10% (w/v)
bromophenol blue) and applied at 1-4 pg to a 1.5% agarose
formaldehyde gel in 1 X MOPS (20 mM morpholinopropanesulfonic acid/5.0 mM sodium acetate/l .O mM EDTA, pH 7.0).
After electrophoresis at 10 V/cm for 4 h, thegel was blotted
onto Hybond-N+ nitrocellulose by using the RNA alkali blotting procedure as described by the manufacturer (Amersham). RadioactiveDNA probes of full length actin I were used
for hybridization of the blots at 60-65" overnight in 5 X
SSPE/5 X Denhardt's solution/0.5% (w/v) SDS (SAMBROOK
et al. 1989). The filters were washed in 2 X SSPE/O.l% (w/v)
SDS at room temperature for
10 min and thenin 1 X SSPE/
0.1% SDS at 65" for 15 min. A high stringency wash in
0.1 X SSPE/O.l% SDS at 65" for 10 min was performed
P. carinii Actin I
745
1 2 3
kb
2.3 -
23.1
9.4
6.6
4.4
2.0
FIGURE
1.4outhern blot analysis of the P. carinii actin
gene. The actin I-specific DNA fragment was isolated from
from clone 41-1 and used for random primed DNA labeling
(Boehringer Mannheim) with [32P]dCTP (Amersham)
and hybridized to the filter as described in the MATERIAIS
AND METHODS.
Lanes 1 and 4 contain genomic P. carinii DNA digested with
HincII and EcoRV, respectively;lanes 2 and 5 contain genomic
S. cerevisiae DNA restriction digested with H i n d and EcoRV,
respectively;and lanes 3 and 6 contain genomic rat testes DNA
restriction digested with HincII and EcoRV, respectively.
twice. Filters were covered with Saran-Wrap and exposed to
Kodak XAR film.
DNA sequencing: The DNA sequence of the actin gene was
generated with a Sequenase kit (U.S. Biochemical Corp.) and
[a-%]dATP (Amersham) on selectedsubclones. The sequences were aligned and analyzed with the Macvector (IBI)
and GCG (DEVEREUX
et al. 1984) programs.
Species and GenBank accession numbers of actin genes
used used for phylogenetic analysis: Table 1 lists the 31 species, abbreviations,and GenBank accession numbers used in
the actin phylogenetic analysis. Examples from six kingdoms
were chosen. Every protozoan and most of the fungal actin
sequences available in GenBank were used for the analysis.
RESULTSANDDISCUSSION
Identification, isolation, and nucleotide sequenceof
P. carinii actingenes: Actinis
a ubiquitoussingle
polypeptide chain ofabout 375 residues.The amino acid
sequence and biochemical propertiesof actin have been
(VANDEKERCKHOVE
highly conserved throughout evolution
and WEBER
1984). Two criteria were employed to selectively identify the P. carinii actin gene(s). Previous studies have suggestedthat P. carinii has a strong preferred
codon usage that could be employed for P. carinii gene
identification (EDMANet al. 1989; FLETCHER
et al. 1993).
Thus, we designed A + T-biased degenerate oligonucleotide primers to specifically amplifythe P. carinii actin
gene byPCR techniques. Secondly, the regions of the
actin consensussequence selected for primer design incorporated the amino acids that have shown the least
2.4
-.
1.3 "
FIGURE
2.-Northem analysis of P. carinii mRNA. Poly(A+)
RNA isolated fromeither purified cysts and trophozoites (lane
1) or infected rat lung tissue (lanes 2 and 3) was electrophe
resed and blotted onto nitrocellulose as described inMATERIALS
AND METHODS. The filters were hybridized
with DNA from actin
I clone (41-1). RNA sizemarker positions are indicated on the
left.
degeneracy intheir codon selection as much
as possible
(FLETCHER
et al. 1993). Thisapproach optimizes primer
hybridization to P. carinii actin sequencesand reduces
the chances ofhybridizationto
other actin-related
genes. The actin primers amplified two fragments from
genomic P. carinii DNA, one of 263 bp and the other
-350 bp (data not shown). Amplification of genomic
S.
cerevisiae DNA and rat testes DNA with the same primers, yieldedno amplified DNA, confirming the utility of
this approach. DNA sequence analysis of recombinants
containing the PCR-amplified products revealed that
the 263-bp fragment (actin I) had significant homology
to vertebrate cytoplasmic ( p ) actin while the -350-bp
fragment (actin 11) had less than 60% homology to vertebrate actin, but appeared tobeanactin-related
sequence (L. D. FLETCHER,
L. CHRISTOPHER
and C.C.
D w m , in preparation). The actin I PCRgenerated
fragment was radiolabeled and used to screen a hgtll
cDNAlibrary. DNA sequence analysis and restriction
mapping of subclones for three individual Agtl1 isolates
(41-1, 6-41, and El-1) indicated that they were identical tothe actin I PCRfragment.Actin I clones 41-1 and
64-1werefull
length cDNA clones and R-1-1 was a
shorter (-two-thirds the size) cDNA clone that mapped
to the 3' end of the gene. The complete P. carinii actin
I DNA sequence was obtained from these three clones
(GenBank accession no. L21183). Information on re-
746
Leah D. Fletcher et al.
Pileup start :1
1
MEDEIAALVIDNGSGMCKFAGDDAPRAVFPSIVGRPREIQGIMVGMGQKDSYVGDEAQS
MDDD-----V------------------_-------------V----------------MEEE------------------------------------H----------------_-MEEEV-----------------------------------H---I--------------MDSEV-------------------------------------------------------
DGEDVQ---------------------------_------T-V----------------MGEEWQ---V-----NV---V-------S---------KNP------EE--AF------T
61
121
KRGILTLKYPIEHGIVSNW~DMEKIWHHTFYNELRVAPE~HPALLTEAPLNPKSNREKMT
----------------T-------------------------V----------A---------------------N-------------------------C-----------------------R------V-T-------------------------V------I----------------R--------T-------------------------V------M-------------------------T-------------------------V----------A------------------.---T----------_-------A------V----------G---R-QIMFETFNTPAFWAIQAV~SLYASGRTTGIVLDSGDG~HTVPIYEGYALPHAILRLNL
-----------M--------------------M-------------------------D--I-----A----------------------------------------------M--D- -V-- - _-V--"-S
- _- - - _ _ - _ _ - _- - - - - - - - - "-V- _ _ _ _ -F- - - - - -S -VDM
FrGURE 3"comparison
Of p' carinii actin I
- _ _ _ _ _ -v
_ _ _ _ _ -s-- _ _ _ - - -s- - - - _ _ _ _- _ _ _ - - - --v- - --A-FS-- - - - _ _ ID- with actins from other species. The amino acid
- _ _ _ _ - _ _ - _"M- _ _ _ _ - _ _ _ - _ _ _ _ _ - _ _ "M _ _ _ _ _ _ s - - - - - _ _ _ _ _ - _ _ _ - - - -D- sequence ofP. carinii actin I was aligned with
-----S--V--M-----------S---------------S---------------M--D- the sequences of G . gallus cytoplasmic (p) ac-
__
181
tin (KOSTet al. 1983), S. pombe actin (MERTINS
AGRDLTDYLMKILTERGYNTTTAEREIVRDIKERLCYVLDFEQEIHTASSSSSLEKSY
and GALLwTZ1987), A . nidulans actin (FIDEL
-----------_------SF------_-------"-----------"-"-------_--------_-_
M_
_ _ _ _ TFS _ _ _ _ _ _ _ _ _ _ _ _ _ K- - - _ _ _ _ _ _ _ _ LQ--AQ _ _ _ _ _ _ _ _ et al. 1988), S. cerevisiae actin ( N G and
_ _ _ _ _ _ _ _ _ _ _ _ _ A_ _ _ _ TFS
ABELSON 1980), D. discoideum actin (ROWS
- _ _ _ _ _ _ _ _ _ _ _ _ _ K - - _ _ _ _ _ _ _ _ - _ o"-o _ _ _ _ _ _ _ _
..
.~
"
-------------s----sFS----__-----K-"K-----------MQ--AQ-_-I----
--------M---------SF--------------K-A---------~--A---A----------E------H----GFS-S--K--------K---I--N-DE-MK-SEQ--DI---241
ELPDGQVITIGNERFRAPEALFQPSIVGMETCGIHETTF~SIMKCDVDIRKDLYSNIVMS
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ c _ _ _ _ _ _ _ - FL"-S - _ _ _ - _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ A-T-L-------------------------AL-L-NA----A-~---------------G-~--------------------K------VL-L-SG---V------I-----V-----G-----
and FIRTEL
1985), and P. falciparum actin I
(WESSELING
et al. 1988a). Residues which are
identical to the P. carinii protein are indicated
by dashes
while differences are shown by
the amino acidwhichoccurs
in the other
proteins.
(-)7
----------------------H--vL"-sA"DQ--DQ--y-_-------V--E--G-----
----------------C--------FL---SA------Y---------------G-V-L-----NI--V------C--------FL-K-AA---T------K-----------G---L-
301
GGTTMYPGIADRMQKEITA~APSSMKIKIVAPPERKYS~IGGSILASLSTFQQ~ISKQ
-----------_-----------T-----I----------------------------------------------Q--------V---------------------------------
-""-"-s""""""""v"I"""""""""""""""
-----F----E---------------V--I-------------------T--------------F-------N--L------T-----I--------------------_--------E
------E-TGE-LTRD--T----T----V-----------------S----------T-E
361
EYDENGPSIVYRKCF*
"--s""-H""-
"--S--G"""--
-"-s""-H""-"-S-""HH""
-"-s""-H""""S""-H""-
P.carinii
G. gallus (p)
S. pombe
A. nidulans
S. cerevisiae
D. discoideum
P. falciparum
striction mapping and sequencing strategy will be provided upon request.
To confirm that the recombinantswere derived from
P. carinii DNA, the actin I gene coding region was hybridized toaSouthernblot
of restriction digested
genomic P. carinii, S.cerevisiae and rat testes DNA (Figure 1). Specific hybridization to genomic P. carinii DNA
was observed for the actin I gene. The second, fainter
band in the hybridization to the EcoRV digest was o b
served due to the presenceof an EcoRV site in an intron
at the 5' end of the genomic DNA. No specific hybridization was detected in the yeast or rat DNA controls.
The hybridization results confirm that the clones originated from P. carinii DNA.
Detection of actin I m
R
N
k Northern blotting was
performed to determine thetranscript size ofthe P. ca-
rinii actin gene. Since any purification of P. carinii has
the potentialto remove yet unidentified forms thatmay
contain lifecyclestage-specificactin-like mRNAs,we
isolated mRNA directly from whole, quick frozen,
P. carinii-infected rat lungs aswell as from purified
cysts and trophozoites.
Northern hybridization analysis was performed on
both types of mRNAsamples (Figure 2).When the complete P. carinii actin I cDNAwas used as a hybridization
probe, asingle mRNA species of 1.3 kb was identified in
the mRNA ofboth purified and non-purified organisms.
Some hybridization to degradedmRNAwas also evident.
This sequence did not hybridize to rat or S. cerevisiae
RNA under theconditions used for this experiment (see
Figure 1).In addition, PCR analysis ofcDNAs from rat
or S. cerevisiaewith P. carinii-specific actin primers,
747
P. carinii Actin I
Tbr
on0
\
\
Ddi
113
Osa
A.
Tbr
58
Trh
211
Osa
32
I I3
Nfo
32
25
-
pfa
26
l3
CPa
Abi
34
13
,
Pi"
Pme
810
Hsa
Ca;&
II
"
Dme 3l
Ehi
16
4 0
19
lo
7
FIGURE
4.-Gene trees for actin sequences. The branches are drawn to
scale withthe distance values placed
in each node for both trees. In addition, for both trees the protists are
generally grouped at the top andthe
fungi are grouped towards the bottom. (A) A gene tree relating 28 actin sequences from six eukaryotic
kingdoms was constructed by the
neighborjoining method (SAITOU
and NEI 1987). The neighborjoining distances correlate approximatelywith
the fraction of RNS
between sequences. This is an unrooted tree. The members of the
Ascomycetes have been underlined.
(B) A consensus gene tree relating
30 actin protein sequences from six
kingdoms to that of P. carinii was
constructed by the PAUP method
(SWOFFORD1991)utilizing
the heuristic and MULPARS options with
bootstrapping.
Aco
680
POC
I1
E H a l
Rra
23
Pca
19
22
15
spO
23
Ani
T , ~
B.
with its high sensitivity,
did not amplify any bands(data
not shown).
Predicted amino acid sequence
comparisons: A
comparison of the deduced amino
acid sequence with
the GenBank sequence data revealed several actin
proteins with high conservation to the P. carinii actin
I including; Rattusrattus
p-actin (93%identity),
S. pombe (91%identity), Aspergillus nidulans (95%
identity), S. cermisiae (94.7% identity), Dictyostelium
discoideum (89% identity) and Plasmodium falciparum
actin I (88.8% identity). These sequences are shown
in Figure 3, lined up to illustrate their sequence
conservation.
Phylogenetic analysis of P. carinii actin I: Since the
taxonomic assignment of P. carinii has been controversial and actin is commonlyused for phylogenetic
analysis, a major goal of this study
was to determine the
evolutionary relationship of P. carinii actin I to actins
from other eukaryotes with particular
attention devoted
to members of the fungi and protozoa.Thirty sequences, listed in Table 1, from organismsrepresenting
six eukaryotic kingdoms were included in the analysis
with P. carinii. Only the coding sequence of each organism was used. To optimize the alignment, each sequence alignment began with the codon homologous to
alanine 7 of P. carinii actin I and a one-codon gapwas
introduced at nucleotides 812-814 to accommodate
the
extra codon found in Trypanosoma brucei and
Oxytrichia nova.A distance matrixof corrected replacement nucleotide substitution (RNS) rates was calculated
according tothe method of LI et al. (1985) (Table2) for
28 of the sequences. RNS is a measure of the nucleotide
changes that result in amino acid changes.
The distance matrixwas used to construct a gene genealogy usingthe neighborjoining method (SAITOU
and
NEI1987). This method determines neighboring pairs
748
Leah D. Fletcher et al.
TABLE 11
Matrix of replacement nucleotide substitution data between pairs of actin genes
Pfa
Cpa
Pfa
Ehi
Ddi
Pin
Pme
Agl
SPO
Sce
Sba
Kla
Cal
Nfo
Abi
Rra
Hsa
Ani
Tla
Ppo
Aca
Gga
Pi0
Vca
Osa
Tth
Tbr
Ono
Pca
Cpa
EhiPin
0.0981
0.1597
0.1487
0.1818
0.1850
0.1593
0.1555
0.1849
0.1662
0.1671
0.1584
0.1792
0.1642
0.1651
0.0177
0.1446
0.1661
0.1437
0.1411
0.1445
0.1332
0.1590
0.1701
0.1429
0.1612
0.1496
0.1399
0.0712
0.1390 0.1493 0.1356
0.1397 0.1472 0.1363
0.1405 0.1521 0.1391
0.1408 0.1589 0.1569
0.1197
0.1223
0.1384
0.1668
0.1580
0.1469
0.1400
0.1391
0.1226
0.1199
0.1226
0.1324
0.1196 0.1508 0.1549
0.1350 0.1622 0.1221
0.1329 0.1586 0.1241
0.1626 0.1820 0.1422
0.1473 0.1765 0.1424
0.1282 0.1573 0.0897
0.1329 0.1565 0.0979
0.1147 0.1391 0.0832
0.1308 0.1536 0.0941
0.1349 0.1579 0.1432
0.1616 0.1801 0.1372
0.1702 0.1880 0.1983
0.2226 0.2126 0.2314
0.2894 0.2758 0.2573
0.1499 0.1825 0.1397
Ddi
Pme
Agl
Spo
Sce
SbaCal
Nfo
Kla
Abi
-
-
-
-
0.1489
0.1475
0.1517
0.1702
0.1450
0.1437
0.1578
0.1646
0.1424
0.1410
0.1516
0.1633
0.0829
0.0816
0.0906
0.1082
0.0879
0.0879
0.1064
0.1149
0.0000
0.0244
0.0256
0.0429
0.0417
0.0435
-
0.1525
0.1393
0.1393
0.1607
0.1560
0.0957
0.1013
0.1230
0.1303
0.1533
0.1718
0.2375
0.2727
0.3342
0.1488
0.0800
0.1551
0.1565
0.1548
0.1459
0.1259
0.1243
0.1448
0.1375
0.1383
0.1724
0.2105
0.2263
0.3147
0.1539
0.0880
0.1505
0.1491
0.1485
0.1475
0.1239
0.1228
0.1442
0.1371
0.1350
0.1769
0.2176
0.2336
0.3124
0.1512
0.1109
0.0907
0.0933
0.0710
0.0712
0.0766
0.0655
0.0863
0.0906
0.1003
0.1413
0.2088
0.2348
0.3165
0.0762
0.1288
0.0912
0.0918
0.0791
0.0715
0.0809
0.0748
0.0867
0.0821
0.1115
0.1418
0.1991
0.2448
0.2999
0.0729
0.1360
0.1141
0.1120
0.0804
0.0803
0.0989
0.0933
0.0964
0.1055
0.1186
0.1653
0.1899
0.2377
0.2764
0.1024
0.1330
0.1134
0.1100
0.0797
0.0808
0.0995
0.0945
0.0964
0.1056
0.1172
0.1641
0.1876
0.2362
0.2798
0.1005
0.1510
0.1189
0.1175
0.0913
0.0955
0.1076
0.0980
0.1117
0.1185
0.1316
0.1648
0.1927
0.2482
0.2841
0.1097
0.1577
0.1348
0.1300
0.1014
0.0996
0.1247
0.1137
0.1166
0.1320
0.1458
0.1881
0.1929
0.2483
0.2874
0.1126
0.1273
0.1275
0.1259
0.1602
0.1562
0.1246
0.1224
0.1224
0.1252
0.1299
0.1532
0.1862
0.2169
0.2930
0.1454
0.1263
0.1256
0.1171
0.1203
0.1014
0.0929
0.1170
0.1217
0.1100
0.1523
0.1929
0.2157
0.3283
0.1210
closest fungal actin to that fromP. cariniiis the actin of
of taxa and iteratively clusters them into nodes which
A. nidulans.
minimizes the total branch length, producing a “miniIt is also interesting to note that two oomycetous spemum evolution” tree. This tree is shown in Figure 4A.
cies
actins (Phytophthora megaspermaand Phytophthora
The fungal, protist, green algal, plant and animal actin
infestins)
and onezygomycete actin (Achlya bisexualis)
sequences generally form distinct groups in the tree.
were
found
in the middleof the protist region of both
The P. cariniiactin is most closely related to most of the
the
neighbor-joining
and protein parsimony trees.
fungal actins, but away from most of the ascomycetous
These
three
species
are
poorly described fungi, so more
actins. To confirm these results, theamino acid seinformation
is
required
to determine whether this is a
quences of each of the 28 organisms were aligned by the
case
for
convergent
evolution
or misplacement of these
GCG Pileup program (DEVEREUX
et al. 1984) and used
organisms
with
the
fungi.
in a “Protpars” (Phylip 2.9) protein parsimony program.
Twelve trees were produced (data not shown), all of
CONCLUSION
which were very similar in topology to the neighborDesigning probes or primers based on known conjoining tree. The only difference observed from thetwo
served regions of actin proteins from other organisms,
methods was that in 3 of the 12 trees, the order of
coupled with codon preference information facilitated
S . pombe and Absidia glaucawere switched.Each of the
the isolation of the P. carinii actin I gene. Complete
12 trees indicated that P. carinii actin was most closely
cDNA
clones forthe
actin I gene have been serelated to the fungal actin sequences. As a further test,
quenced.
The
P.
carinii
actin I is highly conserved
the protein parsimony method described by SWOFFORD
with
other
known
actins
and
phylogenetic analysis of
(1991) was utilized to perform a second protein parsithis
gene
by
two
independent
methods places the P .
mony analysis on theoriginal 28 sequences with two adcarinii actin with fungal actins, closest to that of S.
ditional sequences added (Drosophila melanogasterand
pombe. Several recent studies have suggested that P .
Caenorhabditis elegans) . This analysis was performed
carinii may be more closely related to the fungi than
heuristically with the bootstrapping option The results
to its original classification with the protozoa (EDMAN
are shown in Figure 4B as a cladistic tree. Thus three
et al. 1988; STRINGER
et al. 1989; WAKEFIELD
et al. 1992;
different methods of phylogenetic analysis placed the
W A T A N ~etEal. 1989) andourdata
are consistent
P. carinii actin with the fungal actins. While it has been
with this observation. The taxonomic placement of P .
suggested that P. carinii should be placed among the
carinii within the fungi continues to be controversial
ascomycetous fungi, S. pombe is the only ascomycete ac(TAILORand BOWMAN1993;WAKEFIELD
et al. 1992).
tin to be closely related to theP. carinii actin. The next
749
P. carinii Actin I
Eme Hsa
Rra
Tla
Phy
0.0867
0.0715
0.0988
0.0977
0.1075
0.1563
0.1949
0.2300
0.2914
0.0766
0.0207
0.0503
0.0428
0.0790
0.0932
0.1957
0.2229
0.3012
0.0816
Aca
Gga
Pi0
Vca
OnoOsaTbr
0.0568
0.0536
0.0748
0.0969
0.1883
0.2114
0.2964
0.0609
0.0349
0.0894
0.1071
0.1848
0.2200
0.2720
0.0701
0.0873
0.1049
0.1964
0.2307
0.2791
0.0821
0.0935
0.2017
0.2114
0.2974
0.0962
0.2153
0.2462
0.3113
0.1424
Tth
Pca
CPa
Pfa
Ehi
Ddi
Pin
Pme
4 1
SPO
Sce
Sba
Kla
Gal
Nfo
Abi
Rra
Hsa
Ani
Tla
PPo
Aca
Gga
Pi0
Vca
Osa
Tth
Tbr
Ono
Pca
0.0017
0.1116
0.1235
0.0678
0.0649
0.0484
0.0527
0.0906
0.1136
0.1879
0.2298
0.2966
0.0867
0.1129
0.1242
0.0709
0.0623
0.0540
0.0558
0.0893
0.1163
0.1870
0.2305
0.2948
0.0892
0.0351
0.0879
0.0739
0.0977
0.0969
0.1040
0.1526
0.2087
0.2296
0.2917
0.0746
-
TAYLORand BOWMAN(1993) suggest that the closest
known relatives of P. carinii are most likely the ascomycete fungi rather than the basidiomycetous red yeasts as
suggested by WAKEHELD
et aL (1992).
An analysis of many more actins (and similar studies
with other genes) is required to fully resolve this question. For example, P. carinii could easily be classified in
the Mycetozoan class based on cytological features of
encysted cells or spores (S. H. HUTNER
in LEEet al. 1985),
but among the
Mycetozoans, onlyactins from the orders
Dictyosteliida and Physarida within this class have been
cloned. P. carinii shares some physical characteristics
with the cyst and trophic stages ofsome members of the
orders Protosteliida, the Guttulinala and thePlasmodiophoridae. The taxonomy of the Mycetozoans is even
more controversial as the protostelids, plasmodiophorids, cellular slime molds and myxomycetes have been
grouped together merely for convenience ( O L1975;
~
LEVINE
et al. 1980). Their unifying characteristics are
the presence of stalked fruiting bodies (with the exception of the plasmodiophorids) and
plasmodial vegetative stages in all groups except the cellular slime
molds, which form pseudoplasmodia. While P. cam’nii
has not been described as containing a “plasmodial”
stage, its habit of forming aggregates of trophozoites
that appear to be aggregated and enmeshed in polysaccharide appears similar to the pseudoplasmodial
stage in dictyostelids.
In addition, characteristics of some members of the
phylum Myxozoa (WEISER
1955;J. WEISER,
in LEEet al.
1985) can also be compared to P. carinii. Members of
0.2510
0.2910
0.1877
0.3433
0.2439
0.3024
-
this phylum are generally parasites of fish and have organized polar capsules and orfilaments, but some members appear similar to P. carinii cytologically with its
diffuse, multisized trophozoite form and highly organized cyst (for example Chloromyxum leydigi and TetNothing has been deractinomyxonintermedium).
scribed at the molecular level for these organisms.
Figure 4, A and B, is ordered so that the protists are
grouped on the top. It is worth noting that some of the
protist actingenes are as diverged from each
other as much
as they are from plants or animals or fungi as determined
by measuring the distance lines of the tree. In addition,
actins from three species classified inthe fungi fell within
the protist grouping. P. carinii’s actin, while grouped with
the fungal actins in this study,
is more closely related to the
vertebrate actins (asdetermined by measuring the distance
lines on the tree) than to two other ascomycetous actins
(S. c
e
r
&
a
e and C . albicans). P. carinii’s long parasitic association with mammalian hosts haspossibly made an impact on its genome, adding constraints to its evolutionary
rate. It is clear that more fungal and protist actins, especially those from organisms withstructural similaritiesto P.
can’nii and histories of host-parasite relationships,need to
be examined before definitive conclusionscan be made.
Finally, an analysis ofadditional P. carinii genes should be
performed. If every P. c a ~ n igene
i
falls out into a similar
tree, a classification withthe fungi for this parasiteshould
be contemplated.
We are gratefulto LOUISE
GORTON, WYNELLA
BRAKEand SUSAN
JONES
for assistance with the production of P. carinii organisms. We also
750
Leah D. Fletcher et al.
thank MICHAELDYKSTRA
for helpful discussions on protist taxonomy.
This work wassupported by National Institutes of Health grant 1 UO1
AI33363.
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Communicating editor: S. L. ALLEN