Unique Telomeric Expression Site of Major-Surface-Glycoprotein ...

DNA RESEARCH 3, 55-64 (:.996)

Unique Telomeric Expression Site of Major-Surface-GlycoproteinGenes of Pneumocystis carinii

Miki WADA and Yoshikazu NAKAMURA*Department of Tumor Biology, The Institute of Medical Science, The University of Tokyo,

P.O. Takanawa, Tokyo 108, Japan

(Received 4 April 1996)

Abstract

Major cell surface glycoproteins (MSG) of Pneumocystis carinii play a crucial role in the host-parasiteinteractions involved in P. carinii pneumonia in AIDS patients. Genes encoding MSGs are repeated, highlypolymorphic, and distributed among all of the 14-15 chromosomes. Here we show, by BAL-31 exonucleasecleavage and DNA cloning experiments, that the unique expression site (previously termed UCS) of MSGgenes located in the 500-kb chromosome is telomeric. The 11-kb genomic UCS fragment isolated andsequenced in this study contained one MSG coding sequence (termed msglO5), subtelomeric repetitivesequences and telomere-specific tandem repeats of TTAGGG oriented 5' to 3' towards the DNA end.Despite the N-terminal polymorphism, the C-terminal one-third sequence of MSG105 was identical to oneof the known MSG-cDNAs, suggesting homologous recombination within the MSG coding sequences. Thesefeatures closely resemble the Variant Surface Glycoprotein system of the protozoan parasite Trypanosomabrucei, suggesting that the genetic heterogeneity of MSGs is generated by recombination between the UCSexpression site and multiple MSG genes by means of reciprocal exchange or gene conversion.

Key words: Pneumocystis carinii; major surface glycoprotein; expression site; telomere

1. Introduction

Pneumocystis carinii is an opportunistic pathogen thatoften causes fatal pneumonia in patients under immuno-suppressed or immunodeficient conditions such as AIDS,cancer chemotherapy or organ transplantation. Morethan 60% of AIDS patients suffer from P. carinii pneu-monia at some time during the course of the disease.1 P.carinii is a fungus2'3 that is coated by abundant het-erogeneous major surface glycoproteins (MSGs) calledMSG,4-5 P115,6-7 gpl208 or gpA.9'10 The MSGs arethought to play a crucial role in the host-parasite inter-action during P. carinii pneumonia because of the par-tial protection conferred by passive immunization withan anti-MSG monoclonal antibody against the progres-sion of P. carinii pneumonia,11 the attachment of the or-ganism via interaction between MSGs and fibronectin onalveolar epithelial cells and macrophages,12'13 a specificT-cell response to MSGs after immunization and natu-ral infection,14 and the uptake of P. carinii by alveolarmacrophages mediated by the mannose receptor that tar-

Communicated by Mituru Takanami

* To whom correspondence shculd be addressed. Tel. +81-3-5449-5307, Fax. +81-3-5449-5415, E-mail: [email protected]

f The nucleotide sequence data reported in this paper will ap-pear in the DDBJ, GenBank said EMBL nucleotide sequencedatabases with the accession number D82031.

gets the mannose moiety of MSG.15

MSG is highly glycosylated with mannose and itsmolecular mass has been reported to be from 100 to 125kDa.6'8'16 It accounts for three-quarters of the total cel-lular protein when isolated from rats.6 Genes encodingMSGs are repeated, highly polymorphic, and distributedamong all of the 14-15 chromosomes.4'5'7'9-10'17'18 Weshowed that MSG gene expression is mediated by aDNA element termed the upstream conserved sequence,UCS.19 The UCS element maps to a single chromosomeof about 500 kb, is attached to expressed MSG genes, andencodes the sequence found at the 5' ends of most MSGmRNAs. The UCS is not highly repeated, but P. cariniipopulations contain many different MSG genes attachedto the UCS, suggesting that the genetic heterogeneity ofMSGs is generated by UCS-MSG recombination.19 Herewe show that the UCS locus is telomeric, suggesting thatUCS-MSG recombination occurs by recombination be-tween the UCS expression site and multiple MSG genesby means of reciprocal exchange or gene conversion.

2. Materials and Methods

2.1. OrganismsPathogen-free athymic (rnu/rnu) nude rats were in-

fected with P. carinii as described,6 and P. carinii or-ganisms were prepared from bronchoalveolar lavage as

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56 Telomeric Expression Site of P. carinii [Vol. 3,

described.6'19 The purity of P. carinii organisms as as-sessed by the relative intensity of ribosomal RNA bandsafter agarose gel electrophoresis was about 95%.6'19

2.2. Genomic libraryGenomic DNA was prepared from P. carinii organ-

isms as described.6'19 P. carinii DNA samples (3 fig)were digested with 10 units of Mung Bean Nuclease(Takara Shuzo, Kyoto. Japan), followed by BamHI. Af-ter the fill-in reaction with the Klenow fragment, BamHIlinkers were added, followed by cleavage by BamHI. tofacilitate cloning into AEMBL3 DNA. The DNA wasthen ligated into the BamHI site of AEMBL3, pack-aged with Gigapack Gold Plus packaging extract (Strata-gene, La Jolla, CA), plated on Escherichia coli LE392cells,20 and screened by plaque hybridization with theUCS probe. The probe was a 505-bp UCS segment am-plified by the polymerase chain reaction (PCR) fromgenomic DNA using the UCS primers (sense primer,5'-TGTGCAATAATGACCATTGC-3'; antisense primer,5'-CCTCGATCAAATGACCTCCA-3'). Detection afterblot hybridization was performed using the ECL DirectNucleic Acid Labelling and Detection Systems (Amer-sham, Buckinghamshire, UK). Probe DNAs were labelledwith the enzyme horseradish peroxidase, hybridized toDNA blots and detected by enhanced chemiluminescenceusing the Hyperfihn™-ECL (Amersham) according tothe manufacturer's instructions. Two positive cloneswere selected from 9 x 104 recombinant plaques and thenucleotide sequence of one recombinant clone, AMW124,was determined.

2.3. BAL-31 analysisThe P. carinii DNA (2.5 /j,g) was digested with 1.8

units of BAL-31 at 30°C for 0, 30 or 60 min in 200 filof BAL-31 buffer (20 mM Tris-HCl, pH 8.0, 600 mMNaCl, 12 mM CaCl2, 12 mM MgCl2, 1 mM EDTA).The reaction was stopped by adding ethyleneglycol-fris-(/3-aminoethyl)-AT,iV,./V',./V/-tetraacetic acid (EGTA) toa final concentration of 15 mM. The DNA was re-solved by agarose-gel electrophoresis after digestion withBamHI, blotted onto Hybond™-N+ membranes (Amer-sham), and hybridized to the UCS or the translationfactor EF3-coding21 probe DNA using the ECL DirectNucleic Acid Labelling and Detection Systems (Amer-sham). The EF3-coding sequence was amplified by PCRfrom genomic DNA using the probes (sense primers, 5'-CCGAATTCCCGGTTGAGCAACACATATG-3'; anti-sense primer, 5'-CCGAATTCCTATATGACATCTGGC-ACGG-3'), digested at the internal BamHI site and the3' fragment (377 bp) was used as the EF3 probe.

2.4- Karyotype blot hybridizationThe membrane blot of P. carinii chromosomes re-

solved by field inversion gel electrophoresis was pre-pared as described.19 The membrane was hybridized tothe (TTAGGG)5-oligonuclectide probe labeled with the3'ECL oligolabeling system (Amersham). After strippingthe Southern blots as described by Sambrook et al.,22 thesame karyotype blot was hybridized to subtelomeric re-gion II (330-bp Hindi, positions 10306-10636) and regionIV (650-bp EcoRl, positions 7604-8253) probes generatedfrom AMW124 DNA using t i e ECL Direct Nucleic AcidLabelling and Detection Systems (Amersham).

3. Results

We isolated three UCS-MSG clones of 2-6 kb from a P.carinii genomic library consisting of BamHI fragments.19

Since Southern blotting predominantly detected an 11- to13-kb segment hybridizable to the UCS probe when theP. carinii DNA was cleaved with BamHI,19 these threeisolates might have represented minor sets of UCS-MSGsequences among P. carinii populations that incidentallycontained BamHI site(s) within the polymorphic codingregion. Further trials to isolate a UCS clone having the11- to 13-kb fragment from the conventional library wereunsuccessful even after screening more than a half mil-lion plaques. This suggested that the 11- to 13-kb UCSfragment is telomeric and that it is not susceptible toconventional cloning using restriction endonucleases.

To determine whether the UCS is telomeric, P. cariniiDNA was digested with BAL-31 exonuclease for 0, 30 and60 min, then with BamHI. The samples were resolvedby agarose gel electrophoresis for Southern hybridizationto the UCS and EF3 probes. The EF3 gene fragmentwas not sensitive to BAL-31 (Fig. 1A, right). On theother hand, the size of the BamHI fragments that hy-bridized to the UCS probe slightly, though significantly,decreased depending on the time periods (Fig. 1A, left).Image analysis using the NIH Image 1.52 computer pro-gram confirmed the finding (Fig. IB). A minor UCS frag-ment that migrated faster than the others was sensitiveto BAL-31. These results suggest that most, if not all,of the UCS of the MSG genes are telomeric.

A P. carinii DNA library containing the telomeric re-gions was constructed by digesting with Mung Bean Nu-clease and BamHI, then ligating with BamHI linker af-ter the fill-in reaction with the Klenow fragment (seeMaterials and Methods). The recombinant phages werescreened by plaque hybridization using the UCS probe,and two A clones carrying an 11-kb fragment were iso-lated. Sequence analysis of one of them, AMW124,showed that it contained the UCS and one MSG-codingsequence, but no other significant open reading frames(Fig. 2). The MSG gene in AMW124, named msglO5, is


No. 21 M. Wada and Y. Nakamura m

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Figure 1. Sensitivity of the UCS and EF3 genes to BAL-31 digestion. (A) P. carinii DNA was digested with BAL-31 for 0, 30 and60 min, followed by BamHl, then fragments were resolved by 0.8% agarose-gel electrophoresis and Southern blotted with the UCS(left) and EF3 (right) probes. Experimental procedures and conditions are described in Materials and Methods. Hindlll digests ofA DNA served as size markers shown on the left. (B) Densitometry scanning of Southern blots using the NIH Image 1.52 program.The solid and dotted arrows (left panel) indicate the positions of the major and minor UCS bands, respectively. The solid line (rightpanel) indicates the position of the EF3 bands.

highly homologous, but not identical (except for the C-terminal part; see Discussion), to known MSG genes. Atthe 3' terminus, there were seven tandem hexanucleotiderepeats with the sequence TTAGGG oriented 5' to 3' tothe chromosomal end (Fig. 2, also see Table 1). The samehexamer repeats were also present at the 3' terminus ofthe other isolate (data not shown). TTAGGG is the se-quence of telomeric DNA in all vertebrates, the protozoanTrypanosoma, and several slime molds and fungi (for re-view, see ref. 23), revealing that the UCS is telomeric.

In most organisms, the subtelomeric regions immedi-ately internal to the simple repieats consist of moderatelyrepetitive sequences, called telomere-associated DNA (for

review, see ref. 23). Between msglO5 and the TTAGGGrepeats, there are distinct regions (I-IV) that have im-perfect, but moderately repetitive sequences as revealedby the Macintosh software Dotty Plotter analysis (Fig.3). Most of these regions were composed of character-istic core sequences that appeared frequently within therespective regions (Table 1): region I has 23 repeats ofT3^4A5^s, region II has 17 repeats of TaA5(A/T), re-gion III has 18 repeats of GAx^GAGA, and region IVhas 8 repeats of TTCACAAAGTG. In addition to theT3A5(A/T) core sequence, region II has 12 tandem re-peats of a highly homologous 160-bp sequence in the re-gion proximal to the end. The nucleotide content in re-



Table 1. Sequence features of the telomere-associated DNA.

Region

III

IIIIVTelomere

Positions(nucleotide)5250-64006600-93007500-93009300-9900

10285-1095011322-11363

CharacteristicsCore sequence

T3~4A5~8T3A5(A/T)(160 bp)a)

GAi^2GAGATTCACAAAGTG

TTAGGG

Frequency

2317121887

GC content(%)191515315050

Shown in Fig. 2 by arrows.

gions III and IV is highly biased toward GC (Table 1),even though the AT content of the P. carinii chromosomeis high. These findings were consistent with the commonfeatures of subtelomeres.

The P. carinii chromosome blot resolved by pulse-fieldgel electrophoresis was probed with the (TTAGGG)soligonucleotide probe. As shown in Fig. 4, most of the14-15 chromosomes hybridized to the hexamer sequence,indicating that they retain the same simple repeats. Thekaryotype hybridization using region II or IV probesyielded essentially the same results, suggesting that most,if not all, of the P. carinii chromosomes share the telom-ere structure as well as the subtelomere structure.

4. Discussion

Several findings indicated that the UCS expressionsite of the MSG genes is telomeric. Firstly, the 11-kb UCS-MSG clone contained seven tandem repeats ofTTAGGG oriented 5' to 3' towards the DNA end, whichis characteristic of the telomere DNA repeats of all ver-tebrates, the protozoan Trypanosome, as well as severalslime molds and fungi.23 In most organisms, telomeresconsist of simple tandem repeats regardless of the differ-ent amounts of telomeric DNA at the chromosome end.Mice have as much as 150 kb of telomeric DNA per telom-ere, whereas telomeres on Oxytricha macronuclear DNAmolecules are only 20 bp in length (for review, see ref.23). Secondly, the sequence between the UCS-MSG geneand the TTAGGG repeats showed the characteristic sub-telomeric feature of middle repetitive sequences referredto as telomere-associated DNA.23'24 The region betweenthe UCS-MSG gene and the TTAGGG repeat is about

6 kb long and is composed of moderately repetitive se-quences classified into regions I-IV (see Fig. 3), whichseemed to be present in most of the 14-15 chromosomes ofP. carinii. Thirdly, the UCS segment in the chromosomewas sensitive to BAL-31 exonuclease prior to endonucle-ase digestion. Fourthly, the major, full-size UCS-MSGfragment detected by Southern blotting was almost im-possible to clone from the standard BarriHl DNA librarybut was cloned from a genomic library prepared by di-gestion with Mung Bean Nuclease for blunting the chro-mosomal end.

We isolated several restriction fragments carrying theUCS sequence and the truncated MSG genes.19 Thereare more MSG genes connected to the UCS than canbe accommodated by the copy-number (perhaps onlyone) of the UCS in the genome, suggesting that theP. carinii population is genetically heterogeneous at theUCS locus.19 A sequence comparison of the telomericUCS-MSG clone (this work) with known UCS clones re-vealed that they were identical in the UCS and the up-stream sequences. These findings suggest that all of theUCS-MSG clones originated from the unique, telomericexpression site of the P. carinii chromosome.

In many organisms including yeast and protozoa,telomere-associated DNA is repeated, can extend for tensor hundreds of kb, can include genes, and can be a sourceof chromosome polymorphism with different yeast strainsor Plasmodium lines containing different combinations ofrepeated sequence elements at their telomeres.25~28 Thepresence of these repeats and their polymorphic distribu-tion is thought to reflect the exchange of DNA sequencesbetween nonhomologous telomeres. As this manuscriptwas near completion, we learned of recent studies that

Figure 2 Sequence of the P. carinii chromosome encoding the telomere and the UCS-MSG gene. The number of the nucleotideposition is indicated from the 5' end of the clone AMW124. Boxed regions represent UCS and subtelomeric regions I-IV. Aminoacids of MSG are shown by single-letter code and the nucleotides that are not translated to amino acids represent the positionof the intron encoded in UCS. CRJE sequence, a potential MSG recombination site (see text), is double underlined. The arrowsin region II denote tandem repeats of the 160-bp sequence. Seven tandem TTAGGG repeats characteristic of telomere ends arelocated at the 3' terminus.


No. 2] M. Wada and Y. Nakamura 59

GGATCCGTTTTTATTATAAAATTTCATGrTTTATGTATAAAACTTTCTTTTTTTTATTTTTACTTGGTCCCTTTCTTTCCCTCATCATACTTGGAATTTTCTGATATATTACAATAGATG 120

GTTCATTCCTCTACTCACTTTTAAAATCrTATTTAACTGACCATTCATGCTATTTTTCCCCTATTTTTTATAAAAAGACTCGTCGCTTATATATAGAATGGCGTATACGTAAAAATAGAA 240

ACGTGTATGCGTGAAATAGAATGGTGCTrTTAAATTTAGAAACTTTATTCAAATTAGGAAATCTTTTTTTCCTTTTTAGTTTTGGAATCCATTATTCTTCTATACAAAGTATTTCTTTTT 360

TTCCTAACATGACTATTTATACTTTATArTTGGAAACCTATGCATATACTCGATATCACTCTGACTTTGCATATTCTACTCCCTCCTTTCCTTTCACCCCCTCTGGGACACTCCCCTGTC 480

CGGACACACCCCGGACAGCTCTTGGCTGrCCCTCCCTCGACCCTTCTATACCCATCTATCCATCTATCCATCTATATATACACCGTTTTTACTCATTTCTATCCTATAAAAATTGACTTG 600

TAATTTTGGATATATTCCTTCTATATGCATTTTTAGGACTGGCTAATATTTTAAGGTCTAGCTCTTTTTATTTCCGGCTAGTCATCCCCCCATCCGGAAATTGGCTTTTCCTTTTTCAGC 720

GTTTTCTTCGATTCATCCGTAAATTTCC3TCAATATTTAAGGGTCTTTGCATATTAAGGGGACTGTTTCTGGACTGTATTTTTACCATACCCGTTGCTTCCTTTAGCGCTTGGAAAATAT 840

ATTTTTTCTi|GATATCCGTCTCGTCTTCAGTTTGTTTGTGCAATAATGAGGATTGCATTTTTTGCGCTTTTTGCGCAACTTAGTTGTATTTTAGrTTATTCAATAGCAGAAAGGGATTTC | 960

M R I A F F A L F A Q L S C I L V Y S I A E R D F

ATGTCArTAGATGAAATATATGGTGAGTrTTTrTTCCTTAATTTCCTGrTGTATCATTTTrTACACGTCTTTTTTTACrCGrCTTTrACTCATCTTTTTACrCATCTTTCTATTCTCTCC

M S L D E I Y

TCTTCTTTTGGAAATCTCTTTTTCCTTTTATGGTTTTACTCATGTTTTCTAGAAGGAGGCGATATAAGTTTTGATCATGAAAAACTCGAATTTAACGAATATAATCAAGTTTTACAAATG

E G G D I S F D H E K L E F N E Y N Q V L Q M

CTTGAAAAGGCAAAAAAATTGGGAACCGGCTTTGTTGATAGAACCAAAGATTTTTCTAATAGACGATATGAAGGGAGAATTGAGTTAAATCATrTGGGGAGACGCCCAGGAGTCGACTAT

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

TTTAGGAAAGGTGGGGATGTTTTTJ '

F R K G G D V F T D G Y P R G G H L I E D E L S E E V A M A R P V K R Q

3CACCACAAGCA

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GCACCAGCAGCGGATGGGATTAAGGAGG.\ACACCTTTTGGCTTTCATTCTGAAGGAGAAATATAAAAACGGATGCAAAGATGAGCTTGAAAAATATTGTAAAGAGTTGAAGGAAGCAGAT

A P A A D G I K E E H L L A F I L K E K Y K N G C K D E L E K Y C K E L K E A D

AAAAATCTAGAGAAAGTGAATGATAAAGTTAAAGGATTCTGTGAAGATGGAAAACAAGACGAAAAATGCAAAGGCCTGAAAGAGAAAGTTGAACAAGAATTGGACACTTTTGATGCAGAA

K N L E K V N D K V K G F C E D G K Q D E K C K G L K E K V E Q E L D T F D A E

CTTCCAGATGATTTAGGAAATCTAAAAG.MGAAGAATGTGAAAAACATGAAGAAAAATGTATCCTTTTAGAAGAGACGGGTTATAATGATGATATTAAGAAGAAGTGTGTCAACTTGAGG

L P D D L G N L K D E E C E K H E E K C I L L E E T G Y N D D I K K K C V N L R

GAAGGATGTTACAAATTGAAGCGTGAAA,^GGTGGCAGAGGAGCTCCTTTTGAGGGCGCTCGGAAAGGAAGCTAAAGAAAAAAACACATGTGAACTAAAGATGAAAACTGTTTGCCCAGTG

E G C Y K L K R E K V A E E L L L R A L G K E A K E K N T C E L K M K T V C P V

TTAAGCCGAGAAAGCGACGAATTGATGTTTTTCTGCCTTGATCCGACTAAAACATGTGGAGATATGAAAACAAAATTGGAAGAAAATTGCAAGCCTTTAAAAGAAAAGCTTAAAGATAAC

L S R E S D E L M F F C L D P T K T C G D M K T K L E E N C K P L K E K L K D N

GAATTAGAGGGAAAGTGTCATGAAAGACTTGAGAAATGTCATTrTTACGGAGAAGCGTGTGCTGATGCGAAATGTAAGGAGGATAAGACGAAATGCGAGGGAAAAGGATTCACATATAAA

E L E G K C H E R L E K C H F Y G E A C A D A K C K E D K T K C E G K G F T Y K

ACGCCAGAATCCGATTTTAGTCCGGTGAJÂCCGAAGGCGTCGTTGTTGAGAAGTATTGGGTTGGATGATGTGTATAAAAACGCAGAAAAAGATGGAATTATTATTGGAAAAGCAGGAGTG

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

GATCTACCAAGGAAGTCGGGTACAAAATTTCTGCAAGATCTCTTGCTAGTGTTGAGCAGAGATGAGAATGATAAGAATACAGATAAAAAGTGCAAAAAAGCGTTAGAAAAATGTGGTGCT

D L P R K S G T K F L Q D L L L V L S R D E N D K N T D K K C K K A L E K C G A

TCTGAGTATTTGGATGCTGATTTGAAAA^^GTTATGCAAAGATGGTGATAAACAAGAGAAATGCAAAGCATTACTAGATGTAAATGTAAAAGAAAGATGTACAAATCTCAAATTAAAACTT

S E Y L D A D L K K L C K D G D K Q E K C K A L L D V N V K E R C T N L K L K L

TATCTGAAAGGATTGTCTACGGAATATD^TGATCAAGAATCAGATCCTTTATCGTGGGGACAGCRTCCAACATTATTTACGAAGGGAGAGTGRGCAGAACTTGAGTCGGAATGTTTCTAT

Y L K G L S T E Y D D Q E S D P L S W G Q L P T L F T K G E C A E L E S E C F Y

TTAGAAAATGCGTGTAAGGATAATAAGATTGATGAAGCATGTCAAAATGCAAGAGCAGCGTGCTATAAAAAGGGACAAGACAGGATGTTGAATAAGTTCTTTCAAAAGGAATTGAGGGGA

L E N A C K D N K I D E A C Q N A R A A C Y K K G Q D R M L N K F F Q K E L R G

AATCTTGGTCTTGTAAGATTTTATAGCG/1TCCTGAAGAATGCAAAAAATCTGTGGTAGGAAACTGTACAAAACTTAAAGAAGATAGTAGATACCTTTCAAAATGTCTTTATCCTAAAGAA

N L G L V R F Y S D P E E C K K S V V G N C T K L K E D S R Y L S K C L Y P K E

TTATGTTATGCGCTTTCAAATGATATTTTTCTTCAATCCAAAGAGTTAAGTTCGCTTTTGGATGATCAAAGGGATTTTCCATTAGAAAAGGATTGTCTTGAATTGGTGGAGAAGTGTGAT

L C Y A L S N D I F L Q S K E L S S L L D D Q R D F P L E K D C L E L V E K C D

GAACTTAGTAGTGATTCATTATTGAATTTAGAAAAGTGTATAACATTGAAAAGACGCTGTGAATACTTTAAGGTTACAGAGGGATTTAGAAACGTATTTTTAGAAAAAAAGGATGATTCG

E L S S D S L L N L E K C I T L K R R C E Y F K V T E G F R N V F L E K K D D S

TTAATGACTCAGGAAAACTGTACAAAGGCATTGCATGAGAAATGTAATGCTTTATCTAGGAGGAGAAAGAATTCATTTAGTGTRTCATGTGCTTTACCAGAAGAAACATGTAGTTATATG

L M T Q E N C T K A L H E K C N A L S R R R K N S F S V S C A L P E E T C S Y M

GTATTCCACACAAGTCAAGATTGTAAATATTTAAAAGAAAACATCAAGAATGGAGGAATTGTAGGAAAAATTGGGGGAGCAAATGGAAATGAAGCAACACTCGAAGAACTCTGCACAACA

V F H T S Q D C K Y L K E N I K N G G I V G K I G G A N G N E A T L E E L C T T

TGGGGCCAACATTGTCACCAACTTGTGG;\GAATTGTCCAGATCAGTTGAAAAAAGAGAATGGCAATAARCATAACTGRGATGAACTCGATGAAAAATGCAGTGATACCRTTAAAAGGTTG

W G Q H C H Q L V E N C P D Q L K K E N G N N H N C D E L D E K C S D T F K R L

AAATTAGAGGAGGAGCTGACTCATCTGT"GAAAGGCAGTTTAAAGAAGGATGATCAATGTACAGAAGCATTAGGAAAGAATTGCACTGACTTGCAAAAGAATGAAACATTCAAAATTCTG

K L E E E L T H L L K G S L K K D D Q C T E A L G K N C T D L Q K N E T F K I L

CTTAGTGATTGTGAAAAAGATAAGCAAA;VTGTTTGCACAAAATTAGTTAAAAAAGTACAAGAGAGATGTCCTACTTTAAAAGCTGATCTGGATAAAGCGAAAGATGAGTTGACAAAGATG

L S D C E K D K Q N V C T K L V K K V Q E R C P T L K A D L D K A K D E L T K M

AAGAATGAGTACGAAAAGGCAAAAGAGG;\GGCAGAAAAATCTACAAATAAAGCTAGCTTATTGCTATCAAAGTCTGGAAAAGCCGCAATGCCAAGTGGACAGGATAGCAATGGCTCTGTA

K N E Y E K A K E E A E K S T N K A S L L L S K S G K A A M P S G Q D S N G S V

P A P P A E P A P G T L P P P P S T D G T V N T P A G G S G T S A G G S G T P S

AGTGGAACGACAGACTCTGCAAAACTTGGACTCGTTAAAAGAGCGTARGTAGAAGGAGGAGTATCAGAAGTAGAGGTAAAAGCATTTGATGCAACGACGGTAGCATTGGAATTGTATTTG

S G T T D S A K L G L V K R A Y V E G G V S E V E V K A F D A T T V A L E L Y L

GAATTGAAAGAGGAATGCAATGCTTTASlACTAGATTGCGGTTTTAAAGAGGATTGTTCAGATCTTCAAGTTTGCGGAGAAATAGACACGTTATGCGGAGGAATAAAACCATTAGAAATT

E L K E E C N A L E L D C G F K E D C S D L Q V C G E I D T L C G G I K P L E I

AAGCCTCATCATACAGAGACAATAACAAJiTAAGGTGACGGAAACGAAGACGGAAACAAAGACAGAAACAAAGACTGATGACAAGGCTGATGAGAAGACCGGTACGAAAACTGTTACAGAA

K P H H T E T I T N K V T E T K T E T K T E T K T D D K A D E K T G T K T V T E

ucs


60 Telomeric Expression Site of P. carinii [Vol. 3.

ACCAAGACAATAGGTGGAGGAAAAGTAACAGAAGAGTGTACAATGATACAAACAACAGATACATGGGTAACACGTACATCATTGCATACGAGTACGACAACGAGTACGTCAACGGTGACG

T K T I G G G K V T E E C T M I Q T T D T W V T R T S L H T S T T T S T S T V T

TCGACAGTGACGTTGACGTCGATGCGCAAGTGCAAGCCTACCAAATGTACTACTGATTCAAGCAGAGAGACAGATAAAGGAGAAGAAGGAAAAGAAGGAGAAGAACATGTAAAACCGAAT

S T V T L T S M R K C K P T K C T T D S S R E T D K G E E G K E G E E D V K P N

GATGGGATGAAAATAAGAGTCCCTGATATGATTAAAATAATATTGTTGGGAGTGATTGTTATGGGGATGATGTAAATGAATGAAAAAAATGTTAATAGATTGAAAJTGTGCATATATCCA

D G M K I R V P D M I K I I L L G V I V M G M M

TTGTTTATATATAATAGAAATCTAAATGAATGAAATGAAGTTTTAATTATTTTAACACACCATCTTAGTTCGAAACTATGCTTTATCGGGTAAAGTTTAAGCTTATCGAAGAACTAAAGT

TTATGATAAGAAGATGTTTAAATTTGTTGTAAAAGCACTTGAATGAATGGATTATCGTGAAAAAAAGAGAGTTCTGCGTATCTTAAAGAATTAGTAGGGGTTATaiTTATTTTAAAAAAA

AATCCCACTAACCAGAGAAAGAAGTTArCTTGAACCCATTAAAAGACGATTTGAAArCGAGAGCTTTATGATCATCAAAAAGAGAAACATTAGArCGATTGAAAATGGAATGATCATGAA

AAGTTGTAACCCTAACCCTAACCCTAAAGTGACTAACCAGATCACCCGACTCTTCTAAAACCACTAGAATCTCATTGCAAAATCAGTCAkcrGAATGTCCATTrwAAAGAAGTACrACG

CTTTATGATTATTTTAAAAAAACTAGTGAAATCTATTGTTTATTTAAAAAAATTAGTAAAATCCAAGTTTGTTGTAAAAAAATTAACTCTACGCTAATTACAAAA/AACTAGGAAATCTA

TGTAAAAAACTAGTGAAATCCAAGTTTGTTATAAAAAACCACTAGAATGATTGTAAAAGAACTAATAAACTTrATGATTACTTTAAAAAAAATAAAATCTGTTGA"TATTTCAAAAAAAC

ATCTAAArTTTATGArTACTCCAAAGAArTAGTAGAATCTATrGTTTArTTTAAAAAAATCAGTAAACrCTACGCTAATTACAAAAGAACTAGTGAAATCAATTG"TTATTTAAAAAAAA

TTACTAAAATCCAAGTTTGTTGTAAAAAACTAGGAAATCTATTGATTATTTCAAAAAAATTAATAAACTCTAAAATCATTGAAAAAAACCAGTGAAATCCAAGTT"ATTGTAAAAACGAT

TAGAATAATTATAAAAGAAGTAATAAACTTTATGATTATTTAAAAAAAAAGAGAAAAGTTTATGATTACTTTAAAAAAAATAGTCAAATCCAAGTTTGTTGTAAA/jmAATATTTAACTC

ATITCTTATTTAAAAAAATAGTCAAATCCAAGTTTGTTGltlAAAAACCAGTAAAATGATTGAAAAAAAAGTAGTGAAATCTATTGTTTATCAAAAAGAGTCTATaTTATGATAAAAAAA

GAGTTAGACATTGGTTATCGTGAAAAAAAACGAATCCArCTGTrTAGCAAAAATTAGTCTGTCGTTAGGGCGAAGCTAGTGGTGATTTAATAAAAGTAAGCTATTCAATAAGGTTGAAlf

TAAAAAAAATTGTTAATCATTAATCAAATAAAAAAGAATTGAATTTTCATTGATAAATATAAGAAATGAAAGCATAGACTAGGTTTTATTAAAAAAAAGATGTTTATTTAAAAATATCTT

TAAAAAAATTTGAAGAGATTTTAAAATTAAAAATGAAAATCAAAAATAAAAATGATATCTAGAAAAATTTATTGAATAGATCAAGTAAGATTGAATTAATTTTTG/vTAGTAAAAGAAAAG

ATTAGAAGGAGGAGTrTTATTAAAAAGAAGAAAAAAAGAGATTGATTTGAGAGATATTTTTATTTAAAAATAACTTGAAAAGAAAGGATAGAGAATAGGTTTTAT"AAAAAGAAGAAGAG

ATTGATTTAAGAGATATTTTTATTTAAAAATAATTTAAAAAAAAAGAAAAGATTTTAAAATGAAATTTAAAAATGAAAATAAGAAAAAAAAAATGAATAAATTGGACAAGTTGAGTTGAA

TTTTCATTGATAAATATAAGAAATGAAAGGATAAAGTAGGTTTTATTAAAAAAAAGATGTCCATTTAAAAATATCTTTAATTTAAAAAAAAAAAGATTTTAAAAT"AAAAATGAAAATGA

AAAATAAAAATGAAATCTAAAAAAATTTGTTAAATAGATTAAATAAGATTGAATTGATTTTTGATAGTAAAGATGAGAAATGAAGGGTTAGAGTAGGTTTTATTACyiAGTACAATTGAAT

GAAATTTAAAAGGATTTTAAAAATGAAGATGAAATTAAAAGTGAAATTGAAATGTAAAAAAAATTGTTCACACTTGATCAAATGAAATAGAATTGATTTTTTATTCATTTAAATAAGAAT

TTAGAAATATTTTTGTTAAAAGTACAATTTTTTGTTAAAAGTAGAATTCAATGAAATTTAAAATTAAAAATGAAATCTAAAAAAAAAATCAAATAIAAAAAAAAT-'GTTAACAGTTGATC

AGATGAAAATGGATTGATTTTTTATTGATTTAAATAAGAATTTAGAAATAATTTTGTTAGAAGTACAATTAAATCAAATTTAAAAGGATTTTTAAAATAAAAATTAAAATAAAAAAATAA

AAATAAAATAT;'AA»AAA.TTGTTAACAGTTGATCAAATGAAATAGAATTGATTTTTTATCAACTAAAAAGAGATAAGAATTTAGAAATCTCTTTGTTAAAAGTAGAATTAGATGAAATAT

GAAAGGATTTTGArTAAAATAAAAAATGAAAATGAAAATTAAAATGAAAGTGAAATTCAAAATGAAATGTAAAAAAAAA"TTGTTGACAGTTGATCAAATGAAGTA<»ATTGATTTTGTAT

TAAGTAAAAAAGATAAGAATTTAGAAATAATTTTGTTAGAAGTAAAATTAGATGAAATATAAAAGGATTTTAAAAATGAAGATGAAATTAAAAATGAAATAT,AAAAAAA T̂TTGTTGACAG

TTGATTATATGAAGTAGAATTGATTTTTTATTGATTTAAATAATTTAGAAATATTTTTGTTAAAAGTACAATTTTTTGTTAAAAGTAGAATTCAATGAAATTTAAAATTAAAAATAAAAT

TAAAATAAAAAATSAAATGTiAAAAAAAT

AATTTAAAAGGATtTTTAAAATAAAAATrAAAATAAAAAAArAAAAArAAAATATi•AAAAAJiA'•TTGTTAACAGTTGATCAAATGAAATAGAATTGATTTTTTATCAACTAAAAAGAGATAA

GAATTTAGAAATATTTTTGTTAGAAGTAGAATTTTTTATTAAAAGTAGAATTCAATGAAATTTAAAAGGATTTTAAAAATGAAAATTAATGAAATATi-AAAAAAAA1.rTGTTAACAGTTGAT

CAAATAAAGTAGAATTGATTTTGTATTAAGTAAAAAGGATAAGAATTTAGAGATAATTTTGTTAAAAGTAGAATTAGATGAAATTTAAAAGGATTTTTATTTAAWlATTAGAAAATGAAA

TTAAAATTAAAATGAAATATiGAAAAAA'L T T A T T T A G C A G T C A A T T A T A T A A A A A T G A A T T G A T T T T T A T T A G T A G A A A T G A G A A T T T A G A A A T A A T T C T G T T A G A A G T A G A A T T T T T T G T T

AAAACTAGAATTAGATGAAATTTTAAAATAAAAATTAAAATAAAAAAATAAAAATGAAATCTi•AAAAAAA'.TTTGTTCACAGTTGATCAAATGAAGTAGAATTGATT'-TTTATTGACTTAAT

AAGAATTTAGAAATAATTTTGTTAAAAGTAAAATTAGATGAAATTTAAAAGGATTTTTATTTAAAAATTAGAAAGTGAAATTAAAATTAAAATGAAATATAAAAAiJiTTATTTAGCACTC

AATTATATGAAAATGAATTGATTTTTCATTAGTAAGATGAAAATTTAGAAAJATTTTTAACAATTGATCAAATGAAGTAGAATTAAITTTTTATTGACTAAAAAAAATAAGAATTTAAAA

ATAATTTTGTTAAAAGTAGATTTTTTTGTTAAAAGTAGAATTAGATGAAAATTAAAGT^JÂAGAAGAGAGAAGAGAGTAGAGTGAGAGATATTTTTATTAATAAAAGGAAGAGAGTAG

AGAAACGTCAATTGAATAGATCTTCTGTTCGTGAGAAGAAGAAGAGAGTAGGATGAGAAGAAGAGAGTAGAGGGAGCTTTAAAAGAAGAGATTAGAGTGAGTTTT/WAAAGAAGAGATTA

TAGGAGATACTTTTGTTTTAAATAAAAAGGAATGAATTATTTTAAAAGAGAAGAGAGTAA[:GTCAATTGAATAGTTTTTTTCTTCGTAAAAAGGAGGCAGAGTAG(^TGTGAGATCATTT

GTTTTAAAAAGGGGCAGCGTCGAATGGAATGGCACTACCGAGGAATGCGACTACCGAGGAATGGCACTTCCGAGGAATGGTGCGTCCGAGGAATGTGCGTAGGAGTATAAATAGGAGGGA

AGGAAATAGGGTGGGTGGGGCGGTGGTTGGTGGTGGTTAGAAAGGGTTAGGAAGAAGTAGGGGGGGTGGGGGAGAGAGGAGAGGGGGGGGTCGAAAGTCACGTGACGAAGGACGAGGTCA

AGÂCCGGCTTAACGTCTGTTCA

TAAAGTGCTAGTACTTGGTGGACGTAAAAAGAAGTGCTAGAAAGAGGTGAACGTCAATTCACGAAGTGCTACTTCTGGGTGAACGTAAGTAGGAAAGGTGAGCGTMGTAGGAAAGGTGG

GCGTAAGTAGrACTCGGTGGGCGTAAGTAGTACTCGGTGGACGTCTATTCACAAAGTGCTCCGGGTAGGTGAACGCAGAATGTGAGTCGGTCGAGGGACGCAGTAGTCGGTCGGGGAAGT

GCTCCGGGTAGGCGGACGTAAGTAGGAGTTAGGTGGGTGTAGAAGTACTAGGTGGGCGCAGAAGTACTGGGTTAACGTCAATTCACAAAGTGCTCCGGGTAGGTGIÎCGCAAGTAGAGAA

GGGTTAACGTCTATTCACAAAGTGCTCCGGGTAGGCGGGCGTAGAAGTACTAGGTGAACGCCAATTCACAAAGTGCTCCGGGTAGGTGGGCGTAAGTAGTAGTACTAGGTGGACGTCTAT

TCACAAAGTGCTAGrGGTAGGTGGACGTCTGTTCACAAAGTGCTAGTACTGGGTGAGGTAAGTAAGAAAAGGTGGGCGTAAGTAGGGTTAGGTGACGTCTGTTCACAAAGTGCTCGGGGT

AGGTAACGCAAAGTGAAGTGCTATGAAGGTAAGG1JCAAAGTCACGTGACCAGTAAAAGTCGAGTAAGAGAGTTGTAGTACGTAGGTTCGAGTCGTAGTAGGTGAG"TCGAGTCTTCAGGT

TCACCTAGTCAAACGGTTAGGGGGGTTAGGTGGTTAGCACGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 11363

4800

4 920

5040

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636.0

6480

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III

Figure 2. Continued.


No. 2 M. Wada and Y. Nakamura 61

ucs(TTAGGG)7

5000

4632 /5250 64006600

8000 9000

9300 9900 1095010285

10000 11000. . . I I . .

(bp)11363

I V

wm. /'/•/.

"t ' •|.rV rf'V'V ^"• _ ; _ - : ' . / . / ; . - • '

, .s1. .::f jt- .->•'

n

-11000

-10000

- 9000

- 8000

- 7000

- 6000

5000

Figure 3. Schematic diagram of the UCS-MSG and telomere-associated sequences. The moderately repetitive sequences of regions I-IVwere defined using Macintosh software Dotty Plotter version 1.0 under the following conditions: match window 25 and stringency18.

warrant mention. Underwood et al.29 have reported thatone telomeric fragment of P. carinii contains an MSGgene. Given that the majority of MSGs genes are lo-cated at the chromosomal ends, our discovery of a UCSexpression site within the telomere region suggests thatMSG genes, or batteries of them, are translocated tothe UCS expression site by means of reciprocal recom-bination or gene conversion, as is thought to be trueof DNA rearrangement of the Variant Surface Glycopro-tein (VSG) genes of the protozoan parasite Trypanosoma

brucei.30 32 Trypanosome VSG undergoes antigenic vari-ation to evade the host immune system. There are manyVSG genes on the chromosomes and minichromosomes,but the VSG expression sites are restricted to telomericregions and silent VSG genes can be translocated to theexpression site by DNA recombination.30^32 The sametelomere-associated recombination model for P. cariniiMSGs has also been proposed.33

We assume that recombination in P. carinii MSGs ismediated by sequence homology, named CRJE for con-


•62 Telomeric Expression Site of P. carinii [Vol. 3,

i . o -

IV II(kb)700-

400-

300 -

m «•

Figure 4. Electrophoretic karyotype hybridization. Rat P. cariniichromosomes were separated by pulse-field gel electrophoresis.The blot was hybridized using probes specific to (TTAGGG)s(left), region IV (middle) or region II (right). A A ladder(48.5-kb increments) served as size markers (left).

served recombination junction element.19 The CRJE is a28-bp sequence that is shared by the 3' terminus of theUCS including that sequenced here (see Fig. 2) and the5' termini of silent msg coding sequences. Site-specificrecombination at this site is suggested in all the UCS-MSG junctions so far sequenced.19 The recombinationcould also occur between homologous regions within thecoding sequences other than the CRJE. In fact, whenthe full-length sequences of the known genomic or cDNAMSGs were compared using the PlotSimilarity programof the GCG software package, the C-terminal one-thirdsequence of MSG105 perfectly matched that of the MSG-cDNAs {msgl) cloned in AMW4.5 Except for the UCS,no other MSGs shared identical sequences of significantlength (Fig. 5). These findings provide the first geneticevidence for homologous recombination within the MSGcoding sequences. The similarity score plot revealed thatthe region between nucleotide positions 2905-3205, where

! o . s -

B <D 1 . 0 -

0 . 5 -

0 . 0 -

1454 3454 3845Position

Figure 5. The average similarity plot of the polymorphic se-quences of MSGs. Comparison scores (expressed in standarddeviations) between nucleotide positions 1454 (downstream ofUCS) and 3845 (upstream of C-terminus) in AMW124 (msglOS;see Fig. 2) were calculated using the PlotSimilarity programfrom the GCG software package (version 7.3.1). The averagesimilarity score along the entire sequence is indicated by thehorizontal line, (A) The similarity plot of three genomic MSGgenes (msglOO,34 msglO2,19 r.isglOS) and two MSG-cDNAs(msgl,5 msg57). The arrow indicates the region highly con-served in MSGs. (B) The similarity plot of msglO5 and msgl.

the recombination point of msg 105 and msgl (AMW4)maps (see Fig. 5B), is highly conserved in MSGs (seeFig. 5A) and may serve as a hot spot of homologous re-combination. Another hot spot of recombination may bethe 3'-flanking 200-bp sequence of the UCS-MSG genein AMW124 since it is highly homologous to the spacerregion of tandem linked MSG genes.34

P. carinii is the only fungus that uses the genetic sys-tem to switch cell surface determinants. The complexityof MSG gene expression implies that antigenic polymor-phism and variability play an important role in P. cariniipathobiology. Antigenic variation may contribute to theprevalence of P. carinii pneumonia in AIDS patients witha lower number of CD4+ T cells compared with other mi-crobes. Alternatively, we speculate that antigenic vari-ability is used to maintain opportunistic infection in asingle animal host, rather than for relapses of this or-ganism, because of moderate alterations (about 70% se-quence conservation) in the MSG structures. Much workremains to uncover the mechanism of UCS-MSG switch-ing as well as its pathobiological relevance.

Acknowledgments: This work was supported in partby grants from the Ministry of Education, Science, Sportsand Culture, Japan, and the Human Science Foundation.


No. 2] M. Wada and Y. Nakamura 63

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