Structure, expression and phylogenetic analysis of the gene encoding actin I in Pneumocystis carinii.

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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. Comparative sequence analysis shows that this essential gene has been highly conserved throughout eukaryotic evolution making it useful for phylogenetic analysis. Complete cDNA clones for the actinencoding gene were isolated 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. Only one 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 to the 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 di- vergence 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; STOSSEL 1984). Approximately six different isoforms of actin are expressed in warm-blooded verte- brates and plants. Many of these actin isoforms appear to be developmentally regulated and also exhibit a 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 . , Plasmodium falciparum (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, berenil and 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 level have suggested that P. carinii may be more closely related to fungi in the Ascomycota group (EDMAN et al. 1988). Others have suggested a closer association with the ‘‘F&izopoda/Myxomycota/ Zygomycota” group ( W A T A N ~ E et al. 1989) than with protozoa. Studies comparing the 18s rRNA sequence

Genetics 157: 743-750 uuly, 1994)

from P . cariniiwith eight other taxa showed the 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 separate and not encoded by a bifunctional enzyme

0.

C. EDMAN et al. 1989; U. EDMAN et a l. 1989). Generally, the fungi, unlike protozoans, have the two enzyme ac- tivities on separate polypeptides. Comparisons of gene sequences could help resolve the debate over the classification of P. carinii. The ability to classify this organ- ism has significant clinical relevance for the determina- tion of new therapeutic agents.

We have completely sequenced cDNA clones containing the major actin gene. Since the amino acid 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 only one major actin gene. We have also identified 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 of the nucleotide and derived amino acid sequences.

MATERTALS AND METHODS

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744 Leah D. Fletcher et al.

trophozoites by methods previously described (FLETCHER et al.

1993).

Oligonucleotide primers: 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

(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 synthesized by the University of North Carolina, Department of Pathology core facility.

Polymerase chain reaction and cloning of amplified prod- ucts: 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 were isolated from a 1% low melting point agarose gel by GlasPak (National Scientific Supply Company) procedures. Purified PCR products were ligated directly into a pBluescript I1

K

S

'

(Stratagene) T-tailed vector according to MARCHUK et al.

(1991). Ligated DNA was 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), with X-gal

+

isopropyl PO- thiogalactopyranoside, permitting blue us. white recombinant selection. Plasmid DNA was isolated 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 32P- labeled 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, sub- cloned 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 rat testes 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 washed once in 2 X SSC/O.l% sodium dodecyl sulfate (SDS) for 5 min at room temperature and three timesin 0.1 X SSC/O.l% SDS for 10 min each 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 (In- vitrogen Corp., San Diego, California). Two different sources of organisms were utilized: (1) cysts and trophozoites purified as described by FLETCHER et al. (1993) and frozen in l-g quan- 5'd-GC ATA (A/T/C)CC CTC ATA (A/T)AT (A/T)GG

TABLE I

Actin sequences used for phylogenetic analysis

Name

GenBank Abbreviation accession no.

Pneumocystis carinii

Absidia glauca Aspergillus nidulans Candida albicans Kluyveromyces lactis Saccharomyces bayanus Saccharomyces cerevisiae Schizosaccharomyces pombe Phytophthora infestans Phytophthora megasperma

Thermomyces lanuginosus

Acanthamoeba castellani Achlya bisexualis Cryptosporidium paruum Dictyostelium discoideum Entamoeba histolytica Naegleria fowleri Oxytrichia nova Physarum polycephalum Plasmodium falciparum Tetrahymena thermophila Trypanosoma brucei

Volvox carteria

Oryza sativa

Piastrer ochraceus Drosophila melanogaster

Caenorhabditis elegans

Gallus gallus pactin Rattus rattus a-actin Homo sabiens a-actin Fungi: Protists: Green algae: Plant: Invertebrate: Vertebrate:

Pea L2183

A d M64729

A n i M22869

Cal X16377

Kla M25826

Sba Dl2534

See LO0026

P i n S37672

Pme S5 1076

T l a X07463

Aca v00002

Abi X59936

Ddi X03284

Ehi MI9871

Ono M22480

S P O YO0447

CPa M86241

Nfo M90311

4 0 M21501

Pfa M19146

T t h M 13939

Tbr M20310

Vca M33963

Osa X 16280

POC M26501

Dme KO0670

Cae X1 6796

Gga LO8 165

Rra X06801

Hsa 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 Fast- Track protocol. Contaminating DNA was removed with RQ1 RNase-free DNase I (Promega). The mRNA samples were stored in elution buffer at -20" after the addition of 20 units RNasin (Promega).

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P. carinii Actin I 745

23.1

-

9.4

-

6.6

-

4.4

-

2.3

-

2.0

-

FIGURE 1 . 4 o u t h e r n 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 X A R 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 selected subclones. The se- quences 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.

RESULTS AND DISCUSSION

Identification, isolation, and nucleotide sequence of P. carinii actin genes: Actin is a ubiquitous single polypeptide chain of about 375 residues. The amino acid sequence and biochemical properties of actin have been highly conserved throughout evolution (VANDEKERCKHOVE

and WEBER 1984). Two criteria were employed to selec- tively identify the P. carinii actin gene(s). Previous studies have suggested that P. carinii has a strong preferred codon usage that could be employed for P . carinii gene identification (EDMAN et al. 1989; FLETCHER et al. 1993). Thus, we designed A

+

T-biased degenerate oligonucleotide primers to specifically amplify the P. carinii actin gene by PCR techniques. Secondly, the regions of the actin consensus sequence selected for primer design in- corporated the amino acids that have shown the least

1 2 3

kb

2.4 -.

1.3 "

FIGURE 2.-Northem analysis of P. carinii mRNA. Poly(A+) RNA isolated from either purified cysts and trophozoites (lane 1) or infected rat lung tissue (lanes 2 and 3) was electrophe resed and blotted onto nitrocellulose as described in MATERIALS AND METHODS. The filters were hybridized with DNA from actin

I clone (41-1). RNA size marker positions are indicated on the left.

degeneracy in their codon selection as much as possible (FLETCHER et al. 1993). This approach optimizes primer hybridization to

P.

carinii actin sequences and reduces the chances of hybridization to 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 prim-

ers, yielded no 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 to be an actin-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 cDNA library. DNA sequence analysis and restriction mapping of subclones for three individual Agtl 1 isolates (41-1, 6-41, and El-1) indicated that they were identical to the actin I PCRfragment. Actin

I

clones 41-1 and 64-1 were full 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

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746 Leah D. Fletcher et al.

Pileup start

:1

1 MEDEIAALVIDNGSGMCKFAGDDAPRAVFPSIVGRPREIQGIMVGMGQKDSYVGDEAQS MDDD---V---_---V--- MEEE---H---_-- MEEEV---H---I--- MDSEV---

MGEEWQ---V---NV---V---S---KNP---EE--AF---T

DGEDVQ---_---T-V---

61 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--

121 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 of P. carinii actin I was aligned with

181

AGRDLTDYLMKILTERGYNTTTAEREIVRDIKERLCYVLDFEQEIHTASSSSSLEKSY

and tin (KOST GALLwTZ et al. 1987), 1983), A . S. nidulans actin pombe actin (MERTINS (FIDEL

_ - - - _ - _ _ M

_ _ _ _

TFS

_ _ _ _ _ _ _ _ _ _ _ _ _

K- - - _ _ _ _ _ _ _ _ LQ--AQ

_ _ _ _ _ _ _ _

et al. 1988), S. cerevisiae actin ( N G and

_ _ _ _ _ _ _ _ _ _ _ _ _ A

_ _ _ _

TFS

_ _ _ _ _ _ _ _ _ _ _ _ _

K - - _ _ _ _ _ _ _ _ - _ o"-o

_ _ _ _ _ _ _ _

ABELSON 1980), D. discoideum actin (ROWS

---S--V--M---S---S---M--D- the sequences of G . gallus cytoplasmic (p) ac-

---_---SF---_---"---"-"---

..

---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----

.~ - "

and FIRTEL 1985), and P . falciparum actin I

(WESSELING et al. 1988a). Residues which are identical to the P. carinii protein are indicated 2 4 1 ELPDGQVITIGNERFRAPEALFQPSIVGMETCGIHETTF~SIMKCDVDIRKDLYSNIVMS by dashes (-)7 while differences are shown by

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

c

_ _ _ _ _ _ _ -

FL"-S

- _ _ _ - _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _

A-T-L- the amino acid which occurs in the other ---AL-L-NA----A-~---G-~--- proteins.

---K---VL-L-SG---V---I---V---G---

---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* P. carinii "--s""-H""- G. gallus (p)

"--S--G"""-- S. pombe -"-s""-H""- A. nidulans

-"-S-""HH"" S. cerevisiae

""S""-H""- _{P. falciparum}

-"-s""-H""- D. discoideum

striction mapping and sequencing strategy will be pro- rinii actin gene. Since any purification of P. carinii has

vided upon request. the potential to remove yet unidentified forms that may

To confirm that the recombinants were derived from contain life cycle stage-specific actin-like mRNAs, we

P. carinii DNA, the actin I gene coding region was hy- isolated mRNA directly from whole, quick frozen, bridized to a Southern blot of restriction digested P. carinii-infected rat lungs as well as from purified genomic P. carinii,

S.

cerevisiae and rat testes DNA (Fig-

ure 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 presence of 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 origi- nated from P. carinii DNA.

Detection of actin I mRNk Northern blotting was performed to determine the transcript size of the P. ca-

cysts and trophozoites.

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P . carinii Actin I

on0 Tbr

\

Ddi

747

113

A.

Osa

58

32

Tbr

211 Trh

I I3 ₂₅ Osa

32 Nfo

-

₂₆ pfa

CPa

l 3

Abi

13 34

,

810 Pi"

Hsa

Pme

I I Ca;&

" Dme 3l

16 Ehi

4 0

19 l o Aco

7 680

I 1 E H a l

-

POC

Rra 23

19 Pca

22 spO

15

23 T , ~

Ani

FIGURE 4.-Gene trees for actin sequences. The branches are drawn to scale with the distance values placed in each node for both trees. In addition, for both trees the protists are generally grouped at the top and the fungi are grouped towards the bot- tom. (A) A gene tree relating 28 actin sequences from six eukaryotic kingdoms was constructed by the neighborjoining method (SAITOU and NEI 1987). The neighbor- joining distances correlate approximately with the fraction of RNS between sequences. This is an un- rooted 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 (SWOFFORD 1991) utilizing the heu- ristic and MULPARS options with

bootstrapping.

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; Rattus rattus 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 commonly used 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 organisms representing six eukaryotic kingdoms were included in the analysis with P. carinii. Only the coding sequence of each or- ganism 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 gap was introduced at nucleotides 812-814 to accommodate the

extra codon found in Trypanosoma brucei and

Oxytrichia nova. A distance matrix of corrected replacement nucleotide substitution (RNS) rates was calculated according to the method of LI et al. (1985) (Table

2)

for 28 of the sequences. RNS is a measure of the nucleotide changes that result in amino acid changes.

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748 Leah D. Fletcher et al.

TABLE 11

Matrix of replacement nucleotide substitution data between pairs of actin genes

Cpa Pfa Ehi Ddi Pin Pme Agl Spo Sce Sba Kla Cal Nfo Abi

Cpa -

Pfa 0.0981 -

Ehi 0.1487 0.1597 -

Ddi 0.1818 0.1850 0.1593 -

Pin 0.1555 0.1849 0.1662 0.1671 -

Pme 0.1584 0.1792 0.1642 0.1651 0.0177 -

Agl 0.1446 0.1661 0.1437 0.1411 0.1445 0.1332

-

Sce 0.1390 0.1493 0.1356 0.1489 0.1450 0.1424 0.0829 0.0879

-

Sba 0.1397 0.1472 0.1363 0.1475 0.1437 0.1410 0.0816 0.0879 0.0000 -

Kla 0.1405 0.1521 0.1391 0.1517 0.1578 0.1516 0.0906 0.1064 0.0256 0.0244 -

Cal 0.1408 0.1589 0.1569 0.1702 0.1646 0.1633 0.1082 0.1149 0.0429 0.0417 0.0435 -

Abi 0.1196 0.1508 0.1549 0.1525 0.0800 0.0880 0.1109 0.1288 0.1360 0.1330 0.1510 0.1577 0.1273

-

Rra 0.1350 0.1622 0.1221 0.1393 0.1551 0.1505 0.0907 0.0912 0.1141 0.1134 0.1189 0.1348 0.1275 0.1263

Hsa 0.1329 0.1586 0.1241 0.1393 0.1565 0.1491 0.0933 0.0918 0.1120 0.1100 0.1175 0.1300 0.1259 0.1256

Ani 0.1626 0.1820 0.1422 0.1607 0.1548 0.1485 0.0710 0.0791 0.0804 0.0797 0.0913 0.1014 0.1602 0.1171

Tla 0.1473 0.1765 0.1424 0.1560 0.1459 0.1475 0.0712 0.0715 0.0803 0.0808 0.0955 0.0996 0.1562 0.1203

Ppo 0.1282 0.1573 0.0897 0.0957 0.1259 0.1239 0.0766 0.0809 0.0989 0.0995 0.1076 0.1247 0.1246 0.1014

Aca 0.1329 0.1565 0.0979 0.1013 0.1243 0.1228 0.0655 0.0748 0.0933 0.0945 0.0980 0.1137 0.1224 0.0929

Gga 0.1147 0.1391 0.0832 0.1230 0.1448 0.1442 0.0863 0.0867 0.0964 0.0964 0.1117 0.1166 0.1224 0.1170

Pi0 0.1308 0.1536 0.0941 0.1303 0.1375 0.1371 0.0906 0.0821 0.1055 0.1056 0.1185 0.1320 0.1252 0.1217

Vca 0.1349 0.1579 0.1432 0.1533 0.1383 0.1350 0.1003 0.1115 0.1186 0.1172 0.1316 0.1458 0.1299 0.1100

Osa 0.1616 0.1801 0.1372 0.1718 0.1724 0.1769 0.1413 0.1418 0.1653 0.1641 0.1648 0.1881 0.1532 0.1523

Tth 0.1702 0.1880 0.1983 0.2375 0.2105 0.2176 0.2088 0.1991 0.1899 0.1876 0.1927 0.1929 0.1862 0.1929

Tbr 0.2226 0.2126 0.2314 0.2727 0.2263 0.2336 0.2348 0.2448 0.2377 0.2362 0.2482 0.2483 0.2169 0.2157

Ono 0.2894 0.2758 0.2573 0.3342 0.3147 0.3124 0.3165 0.2999 0.2764 0.2798 0.2841 0.2874 0.2930 0.3283

Pca 0.1499 0.1825 0.1397 0.1488 0.1539 0.1512 0.0762 0.0729 0.1024 0.1005 0.1097 0.1126 0.1454 0.1210

SPO 0.1590 0.1701 0.1429 0.1612 0.1496 0.1399 0.0712

-

Nfo 0.1197 0.1223 0.1384 0.1668 0.1580 0.1469 0.1400 0.1391 0.1226 0.1199 0.1226 0.1324

-

of taxa and iteratively clusters them into nodes which minimizes the total branch length, producing a “mini- mum evolution” tree. This tree is shown in Figure 4 A .

The fungal, protist, green algal, plant and animal actin sequences generally form distinct groups in the tree. The P. carinii actin is most closely related to most of the fungal actins, but away from most of the ascomycetous actins. To confirm these results, the amino acid sequences of each of the

28

organisms were aligned by the GCG Pileup program (DEVEREUX et al. 1984) and used in a “Protpars” (Phylip 2.9) protein parsimony program. Twelve trees were produced (data not shown), all of which were very similar in topology to the neighbor- joining tree. The only difference observed from the two

methods was that in 3 of the 12 trees, the order of S . pombe and Absidia glauca were switched. Each of the 12 trees indicated that P. carinii actin was most closely related to the fungal actin sequences.

As

a further test, the protein parsimony method described by SWOFFORD (1991) was utilized to perform a second protein parsimony analysis on the original 28 sequences with two additional sequences added (Drosophila melanogaster and

Caenorhabditis elegans)

.

This analysis was performed

heuristically with the bootstrapping option The results are shown in Figure 4B as a cladistic tree. Thus three different methods of phylogenetic analysis placed the

P. carinii actin with the fungal actins. While it has been suggested that P. carinii should be placed among the ascomycetous fungi, S. pombe is the only ascomycete actin to be closely related to the P. carinii actin. The next

closest fungal actin to that from P. carinii is the actin of A. nidulans.

It is also interesting to note that two oomycetous species actins (Phytophthora megasperma and Phytophthora

infestins) and one zygomycete actin (Achlya bisexualis)

were found in the middle of the protist region of both the neighbor-joining and protein parsimony trees. These three species are poorly described fungi, so more information is required to determine whether this is a case for convergent evolution or misplacement of these organisms with the fungi.

CONCLUSION

Designing probes or primers based on known conserved regions of actin proteins from other organisms, coupled with codon preference information facilitated the isolation of the P. carinii actin I gene. Complete cDNA clones for the actin I gene have been sequenced. The P. carinii actin I is highly conserved with other known actins and phylogenetic analysis of this gene by two independent methods places the P . carinii actin with fungal actins, closest to that of

S.

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P. carinii Actin I

Rra Hsa Eme Tla Phy Aca Gga Pi0 Vca Osa Tth Tbr Ono Pca

CPa Pfa Ehi Ddi Pin Pme 4 1

SPO

Sce Sba Kla

Gal

Abi

Rra -

Hsa 0.0017 -

A n i 0.1116 0.1129 -

Tla 0.1235 0.1242 0.0351 -

Aca 0.0649 0.0623 0.0739 0.0715 0.0207 -

Gga 0.0484 0.0540 0.0977 0.0988 0.0503 0.0568 -

Vca 0.0906 0.0893 0.1040 0.1075 0.0790 0.0748 0.0894 0.0873 -

Osa 0.1136 0.1163 0.1526 0.1563 0.0932 0.0969 0.1071 0.1049 0.0935

-

Tbr 0.2298 0.2305 0.2296 0.2300 0.2229 0.2114 0.2200 0.2307 0.2114 0.2462 0.2510 -

Ono 0.2966 0.2948 0.2917 0.2914 0.3012 0.2964 0.2720 0.2791 0.2974 0.3113 0.2910 0.3433 -

Pca 0.0867 0.0892 0.0746 0.0766 0.0816 0.0609 0.0701 0.0821 0.0962 0.1424 0.1877 0.2439 0.3024

-

Nfo

PPo 0.0678 0.0709 0.0879 0.0867

-

Pi0 0.0527 0.0558 0.0969 0.0977 0.0428 0.0536 0.0349 -

Tth 0.1879 0.1870 0.2087 0.1949 0.1957 0.1883 0.1848 0.1964 0.2017 0.2153 -

TAYLOR and 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 ques- tion. 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 LEE et al. 1985), but among the Mycetozoans, only actins from the orders Dictyosteliida and Physarida within this class have been cloned. P. carinii shares some physical characteristics with the cyst and trophic stages of some members of the orders Protosteliida, the Guttulinala and the Plasmodio- phoridae. 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 L ~ 1975; LEVINE et al. 1980). Their unifying characteristics are the presence of stalked fruiting bodies (with the ex- ception of the plasmodiophorids) and plasmodial veg- etative 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 po- lysaccharide appears similar to the pseudoplasmodial stage in dictyostelids.

In addition, characteristics of some members of the phylum Myxozoa (WEISER 1955; J. WEISER, in LEE et al.

1985) can also be compared to P. carinii. Members of

this phylum are generally parasites of fish and have or- ganized polar capsules and or filaments, but some members appear similar to P. carinii cytologically with its diffuse, multisized trophozoite form and highly orga- nized cyst (for example Chloromyxum leydigi and Tet- ractinomyxon intermedium). Nothing has been described 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 actin genes 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 in the 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

(as

determined by measuring the distance lines on the tree) than to two other ascomycetous actins (S. cer&aeand C . albicans)

.

P. carinii’s long parasitic as-

sociation with mammalian hosts has possibly made an im- pact on its genome, adding constraints to its evolutionary rate. It is clear that more fungal and protist actins, espe- cially those from organisms with structural similarities to P. can’nii and histories of host-parasite relationships, need to be examined before definitive conclusions can be made. Finally, an analysis of additional P. carinii genes should be performed. If every P. c a ~ n i i gene falls out into a similar tree, a classification with the fungi for this parasite should be contemplated.

We are grateful to LOUISE GORTON, WYNELLA BRAKE and SUSAN JONES

(8)

750 Leah D. Fletcher et al.

thank MICHAEL DYKSTRA for helpful discussions on protist taxonomy. This work was supported by National Institutes of Health grant 1 UO1 AI33363.

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