- Email: [email protected]
A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase a in the macronucleus of Oxytricha nova
Gene, 144 (1994) 1555161 0 1994 Elsevier Science B.V. All rights reserved.
0378-l 119/94/$07,tX~
155
GENE 07946
A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase a in the macronucleus of Oxytricha nova (Hypotrichous
ciliates; gene structure;
Sam J. Mansour,
DNA replication;
David C. Hoffman
molecular
evolution;
non-universal
genetic code)
and David M. Prescott
Department qfA4olecu/ar, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA Received by A.-M. Skalka: 20 December
1993; Revised/Accepted:
4 February/7
February
1994; Received at publishers:
10 March
1994
SUMMARY
We have isolated
a gene-sized
molecule
encoding
the catalytic
subunit
of DNA polymerase
c1 from a macronuclear
genomic library of Oxytricha nova, by using a 0.7-kb fragment of the corresponding human gene as a hybridization probe. Two different versions of the gene are present in the macronucleus, one with an EcoRI site (RI+) and one without an EcoRI site (RI-). The cloned RI- version has been characterized. It is 4938 bp in length, excluding telomeres. It consists of a 329-bp 5’ leader, a 4479-bp coding region and a 130-bp 3’ trailer. The deduced amino-acid sequence shares conserved regions with the yeast and human polypeptides. We also demonstrate by Southern analysis that gene-sized molecules of similar size, homologous to the isolated 0. noua gene are present in the mat genome of closely and distantly related hypotrichs.
INTRODUCTION
Hypotrichous ciliates contain two types of nuclei, a micronucleus (mic) and a macronucleus (mat). The mic genome contains typical chromosomes with hundreds of genes per chromosome. It serves as the germline genome. The mat genome contains millions of gene-sized molecules (0.4-15 kb), each with a single coding function (Klobutcher and Prescott, 1986). Seventy-four molecules have been characterized in various species of the hypotrichs Oxytricha, Stylonychia and Euplotes. These moleCorrespondence to: Dr. D.M. Prescott, University 492-8381;
of Colorado, Fax (l-303)
Boulder, 492-7744;
Department
CO 80309-0347,
of MCD
Biology,
USA. Tel. (l-303)
e-mail: [email protected]
Abbreviations: aa, amino acid(s); bp, base pair(s); E., Euplotes; EtdBr, ethidium bromide; kb. kilobase or 1000 bp; mat, macronucleus( mic, micronucleus( nt, nucleotide(s); O., Oxytricha; ORF, open reading frame; l?, Paramecium; polcc, DNA polymerase(s) a; RI’, version of polcc containing an EcoRI site; RI-, cloned version of polcl without an EcoRI site; S., Stylonychia; SC, Saccharomyces cerevisiae; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OlS M Naacitrate pH 7.6; T., Tetrahymena. SSDI 0378-l
119(94)00157-N
cules conform to one structural form with a 5’ leader, a coding segment and a 3’ trailer. All molecules are flanked by telomeric sequences (Klobutcher and Prescott, 1986). Because of the single coding function, macronuclear DNA molecules are referred to as gene-sized molecules. After cell mating, a mic genome is converted to a new mat genome in which various mic DNA sequences are cut, spliced, eliminated, reordered and amplified to produce gene-sized molecules to which are added telomeric repeats (Klobutcher and Prescott, 1986). In the mature mat of 0. nova, each different gene-sized molecule is present on average in 1000 copies. Characterization of the gene-sized molecule encoding the large catalytic subunit of DNA polymerase CL(pola) in the mat of 0. noua is the subject of this paper. This work is the foundation for a comparison of the mic and mat versions of the gene to be described in a later paper. We chose pola because it is twice the size of the largest scrambled gene identified to date, and it did prove to show a more complex scrambled pattern in the mic than the shorter genes studied. The gene encoding polcl has been cloned from six other eukaryotic species
156 (Braithwaite and Ito, 1993; White et al., 1993). The holoenzyme is composed of four subunits, the largest of which (studied here) is a 160-180-kDa protein that is directly involved in the catalysis of DNA synthesis (Wang, 1991; So and Downey, 1992). The aa sequence comparisons show the presence of several conserved regions dispersed along the polypeptide (Wang et al., 1989; White et al., 1993). In addition to sequencing the large catalytic subunitencoding gene and comparing its putative product to the subunit of other eukaryotes, we have compared the genesized molecules encoding the subunit in several hypotrich species.
RESULTS
1
b
2
1
a
6.5 -
6.5
4.3 -
4.3
2
2.3 2.0 -
AND DISCUSSION
(a) Cloning of the mat gene
The polcl of 0. nova shows a high degree of aa identity in twelve conserved regions (designated I-VII and A-E) associated with DNA polymerases in general and a-type polymerases in particular (see section e below, and Fig. 3). Using a 0.7-kb probe corresponding to the portion of the human pola-encoding gene for regions VI-IIII-C-VII-V (the HindIII-BamHI fragment from pcD-KB pola, a cDNA clone kindly provided by Teresa S.-F. Wang), we probed blots of native 0. now mat DNA by hybridization. ~acronuclear DNA ranges in size from 0.4 kb to 15 kb (Fig. la, lane 1). In Southern analysis, the human probe hybridizes to a 5.0-kb gene-sized molecule (Fig. la, lane 2). Hybridization of the human probe to EcoRI-cut mat DNA detected two molecules, one of 5 kb and one of 2.3 kb (data not shown). Analysis of the pattern indicates that two versions of the pola-encoding gene are present, one that lacks EcoRI sites (RI-) (5-kb molecule) and one with at least a single EcoRI site (RI ‘), which generated the 2.3-kb fragment. No hybridization to other fragments was observed for the RI+ version, indicating that either the probe does not span the EcoRI site, or that the probe does not share significant sequence identity with more than the single 2.3-kb fragment. Subsequent hybridization of the full-length cloned mat pola-encoding gene to EcoRI-cut mat DNA showed three bands, one at 5.0 kb (full-length gene-sized molecule without an EcoRI site) and two at 2.7 kb and 2.3 kb (Fig. lb, lane 2) (the two fragments produced by cutting at a single EcoRI site). (b) Characteristics product
of the mat gene and its encoded
Screening a mat genomic library of 0. nova yielded a single positive h clone designated DC8 Sequencing showed that the insert in the DC8 clone is 4978-bp long
Fig. 1. Southern
analysis
0.7-kb HindHI-BamHf EcoRI insert in DC8 stained
agarose
of 0. notru mat
DNA
probed
with (a) the
fragment of the human gene, and (b) the 5.0 kb Methods: (a) lanes: 1, Native 0. nooa mat (EtdBr
gel). 2, Native
0. nou~l mat (Southern
blot). Native 0.
noau mat DNA (3 ug) was electrophoresed in a 1% agarose gel and stained with EtdBr, after which the DNA was blotted onto Zetabind membrane (Life Science Products, Denver, CO, USA) (Ausubel et al., 1987). The blot was prehybridized at 42-C for 6 h in 20% formamide~3
x SSCj5 x Denhardt’s
solution/i
mM N~pyrophosphate/
1% SDSjlOtI ug/per ml of E. coli tRNA, and then incubated at 42°C for 19 h with the 32P-labeled human probe (Random primed DNA labeling
kit, US Biochemical,
Cleveland,
OH, USA) in the presence
of
5% dextran sulfate. The membrane was washed twice at room temperature for 15 min in 2 x SSC/O.l % SDS, followed by two similar washes at 60°C. Hybridization
signals were visualized
by autoradiography
with
a 21 h exposure period. (b) Lanes: 1, Native and 2, EcoRi digested. Macronuclear DNA (3 ug) was blotted onto Zetabind. prehybridized at 65°C in
I M NaCI/SO mM Tris 8.0/l mM EDTA/O.l%
SDS/ZOO pig
per ml sheared salmon sperm DNA, and then incubated at 65°C for 19 h with the 32P-labeled 5.0-kb insert in DC8. The blot was washed under stringent conditions at 65 ‘C in 0. I x SSC/O.l% SDS. The autoradiograph was developed after a 19-h exposure. Size markers are Hind111 digests of h phage DNA (US Biochemical).
Lengths
are indicated
in kb.
(Fig. 2b). Telomeric repeat sequences (20 bp each) occur at the ends of the insert, indicating the h clone contained a complete gene-sized molecule. The gene-sized molecule contains a 5’ leader of 329 bp extending from the inner end of the 5’ telomere to the ATG start codon (nt 350), and a 3’ trailer of 130 bp extending from the TGA stop codon (nt 4828) to the inner end of the 3’ telomere. The assigned ATG start codon is the fourth ATG triplet from the 5’ end, and is the only one followed by a long ORF. It defines an ORF that begins with an aa region that is conserved at the N terminus in the large subunit of four polcl proteins (Fig. 3, region A) (White et al., 1993). The 5’ leader lacks TATAA, CCAAT and GC boxes.
157
a) sense strand
anti-sense strand
b)
. . . . . ccccaaaaccccaaaaccc~tctagtt=~t=~~=g~g==~t=g=~~tt~=t~g~tt==~g=tttt~~ .
.
.
.
.
.
120
cattttgaacttaaaatagagtatgagcgacaactatactatactactgccc~tgcagagaagtaaagaaaggtggtaaaggagaagttgttaaaaagggattagcagctttgagagcagccaga gaaggtggag~caag~gaaCtg~ct~gtac~C~gta~gtat~~~gcattaatgCtaat~atctttctga~tggCttttc~aaaat~ttgcaacatcatcctcctttctcATGA~~CA MIKHl
240 360
CATTTTATAGGTIGAAGAAAC
480
AAAMAGA’ICTITGAAGAGATAGA~ACMCGAATA~AMAAA
rrTAAGATTrCCGCAAAAA~~A~~~A~~A~~A~ATA
ILQVEETKKIFEEIDDNEYEKIQDSRKNDDFIVDDDGYGY44 TCGCGATCACGGAGGAGAAA~~CAGAGA'PGOAGCC RDHGGEIWDRDGDVEEVGKKKKKKQNNHDPNENIMNYMMP84
600
‘PGCAAAAGATGTAGA~AC~M~~~M~~GC
840
mAGAGTCAAGAGAAGAGCAAGAGCGCGCCGTCGAGA LESREEQERRRQSEQLKQQANIGQNQSDVNPFSKKRKLDE204
960
GlTCTAGCAAGTTCAAGCT~~CATAACT FQQVQANSYQSKQNSHSVSKSKPGDHEMANHADGVDLNLL244
1080
TGCTA?TGACGACACTAAGATGACAGATAGTCACCCTAGC AIDDTKMTDSHPSEIITQNQRASAVSSVNQQIMESTNGKN284
1200
CCAGCTCGAGAAAAA CGATACCGAATOGCMTAGATGAAGGAG~~~C~~~~A~AC~~G~~C~T~~A~~~TM~~C~GACTA~C~~C QLEKNDTEWQQMKEKNAVFNQDLRHNDNALMSNQQDYPLP324
1320
~~GA~AACTCTTTCrrPCTA~~TAGAT LNEDGTLSFYWIDAHEENNGADLFVFGKIYQHEERKFVSC364
1440
CTCTATCAAAGTGAATGGTATGCAAAGAGAOCTATTPGTTA ELFVLPKMSGKSRAAMTTEEEKEQARKVMME404 SIKVNGMQR
1560
ACTTGAAGGAGTAGAAAGAGATTCCAGCTATCACTA LEGVRKRFPAITKWRCKPVTRKYAFELPIQHGEHQFVKIK444
1680
CAAAAACA~PG?TCG~'CAAGAAATGAACAGACCAGTI(;GTCAC AKDVDELEDVNEAKNMFVQEMNRPVAFNKEEDFNNRYSVT164
ATACGA~CAACCTTCCCTTCrrrrCCTAGCACAGTACAG~C YDATFPSLPSTVQGNTFECIFGA
NQSMLESFI
L
K
R
K
I
R
G
1800 P484
A~C~ATGACTATTAGAAACCCTCAAAAGGTAAC? CWMTIRNPQKVTDFRRTWCKQEILVSNPKDIEITLDDLNK524
1920
AACTGAGCTACCTCCTTTGACCTCTGTCACATPTGCCATAG TELPPLTSVTFAIKTCRSSQNTNEIAHLSCIVNENISQEG564
2040 GCCTATGCCAATIGAATAlGAGAGAGCTTl'CAGAGATAAGAAAGAT'I'XT'TCATTTAG~CTT PIEYERAFRD KK DSFIQFF604
TCC~CAAAAATPGA~'PCCACAAA?rA?TCACmACTC PSKIDVHKSFTLLRKLDGKPM CTAACATGAGAGACAACTTAT?GAAGCC~G~CTAAA QHERQLIEAFVAKIYQLDPDLMVAHNLCGGMFD
L
L
L
A
R
I
2160
2280 Q644
GA~CTCAAAATTAGTCAC~G~CAG~~~GCCT~G~~CT~~CCT~C~~~AGA~~GC~AGCC~~A~AGG~~~~~~CC~~T~GT PNKKSDQSGANYGGSQWIPRQV684 HLKISHWSRIGRLKKNQI
2400
TACTPGTGGAAGATPGTTAGTTGATACTPTCTPGACTGCC~~GC~A~A~~~CT~CTA~C~ACCCA~TAGCT~~TA~C~T~G~TA~~AC~A RETNYDLTHLAKVQLQKDRIDFD724 TCGRLLVDTFLTAKELI
2520
TGA~ACCmTGCCAACTTA~TCC~~C~C~~~C~~~ACAC~AG~GACGCCTATCTCAC~~T~~~~~ACC~TAGG~A~C~T LFQLIDHTEKDAYLTLQLMNHLQVIPL764 DDLLPTFYVQMAK
2640
GACTAAGCMCTCACACA~T~~~~A~~~~~CGCCA~GC~~G~~~C~TAC~CAC~G~~G~~G~~~ACC TKQLTNIAGNLWFRSLQNARAERNEHLLLHEFKKKKFVLP804
2760
AGACAAAAAGCAACTTAACGCCAAAGA~~G~G~CA~~CA~~AGTACGM~~G~~~~~~G~~~GA~G~GCAGCGTA~CAG~~~T EYEEGDGKTKGGKRKKAAYAGG DKKQLNAKDLKKNMFAD TGTGA~CCCAAffiCTGG'ITTrTATGATAATATCATC VIEPKAGFYDNII
2880 L 844 3000
IQEYNLCFTTVNRRP884
LLLDFNSLYPSI
TACCAAGAACTTCG4~GC~~~~G~CTAGTAC~G~~~~~~~GAGGTAGATA~~~GC~~~CC~AC~~~T~C~G~~C ENGEEEVDIEEADLPDKNVNLKDA924 TKNFDGSEMKNQYKKG AG'PXlTCCCATGGT-lT%AGAGACCTCGTCTiUAAGAGAAAAGC~l-CAAGGACAAGAnXAAAC VKDKMKTEKDHVKLSQLE VLPMVLRDLVQKRKA
lWdAAAGA%!ACGTCAAGTTATCGCAATTAGAAA!FZAGATAAAAGGCTATTAA I R Q K A I
3120 3240 K964
158 3360 1004 tGCAGAAAAT~GCTTGGATPCAATGTAGTGTATGGn;ATACTT AENKLGFNVVYGDTDSIMINTGSNQLQQALEMGKRLKGEV
3480 1044
~GAAGTATGCAGCTCTG?diGTAl'JAAAACTICCTAAGTCCAGCTGAAGT AAACTGC'lTGTACAAATGCCTlGAAATAGAUiTAGATGGAGTTTIl'AAGAGlT'FXTCTTA NCLYKCLEIEIDGVFKSLLLLKKKKYAALKYENFLSPAEV
3600 1084 3720 1124 3840 1164
3960 RLRDQGKSENQLVNNFIPYVICQQTYGDT1204 IKGQPHVAVAK * . . . . . . . . . 0 . TACCAAATCAAGCACAGCACTCTCAGATAAGGC~ACCACCCAGAXiAAGTGATrAGCTCAAGAGGAAAAGTGACTATAGATAGl?.3AC'lWZTATG?GTCTACCTAATItXTlK!CTCCCAT 4080 TKSSTALSDKAYHPDEVISSRGKVTIDSDWYVSTQLLPPIl244 4200 1284 4320 1324 AGGMTCC~~CCCAGCrrAAACAATACA~GCrrACA
GILVPSSNNTELTGLACVKCNQR
TCTlT'XTXAAGCAACT IPDAYMLNRLNLFLKQL
4440 1364 4560 1404 4680 1444
GCCTM~CMTCATTCAAGC~~~GG~~~~A~~GM~~ATAC~C~G~GA~AGGCMTA~GG~A~AGC~G~AGCT~~C PNFQSFKELQKKVESFMIRSGYNKVDLGNIFGFMSKGANP
4800 1484
TCACTAAC~~GCA~~Aatggcggcattttgatcagagttaatttattttactcaaattccttaagaacaagataaacctcgcgccttttaagaggttatctgcattta H Q Q K M S I F"*
4920 1492
atgcattcatttagtttataaatttacacaaaaaacttggggttttggggttttgggg-3'
4978
Fig. 2. Nucleotide sequence of the polcc-encoding gene and the deduced aa sequence. (a) The RI- version of the gene-sized molecule. Black boxes at the ends of the DNA molecule denote telomeric sequences; the single-stranded extension of telomeres was removed during cloning. The extent of sequence generated from each strand is indicated with arrows. Lengths are indicated in kb. The hatched box denotes the 0.7-kb fragment of the human pola-encoding gene, and is depicted along its target hybridization region in both the RI- and RI+ versions, The EcoRI site (E) in RI+ is shown. The RI’ version has not been isolated. (b) Complete nt sequence of the RI version, and the deduced aa sequence. The ORF is in upper case; the leader and trailer are in lower case. The sequence has been submitted to the GenBank database under the accession No. UO2001. A mat genomic library of 0. nova was constructed by inserting the gene-sized molecules into the unique EcoRl site of hgt 10 as described elsewhere (Klobutcher et al., 1984). About 1.2 x 10’ plaques representing five times the sequence complexity of mat DNA were screened with the 0.7-kb human probe. The screen yielded one positive h clone, DC8. The 5.0-kb insert in this clone was recovered after EcoRl digestion, indicating that it represents the RIversion. The 5.0-kb insert was subcloned in both orientations into the unique EuoRI site of pTZI8R (US Biochemical). Procedures for subcloning and for the generation of nested deletions were according to established protocois (Ausubei et al., 1987). Sequence determination was performed on double-stranded DNA templates with a Sequenase version 2.0 kit (US Biochemical) (Hsiao, 1991).
There are four TATAA-like boxes located in the region 52-279 bp upstream from the translation start codon; however, since the leader is A +T-rich (66% A+T), TATAA-like sequences are expected to occur by chance. Comparison of the leader sequences of different genes within a hypotrich species or between the same gene in different hypotrichs shows little or no sequence similarity among them with the exception of a histone H4-encoding gene in S. herniae and 0. rz~~a(Herrick, 1992). This suggests that hypotrichs may define promoters in other ways. The TGA stop codon at nt 48264828 is followed in the same reading frame by another TGA codon at nt 4841-4843. The 130-bp trailer sequence does not contain the poly(A) addition signal, 5’-AATAAA. The sequence S-ATAAA occurs 109 bp downstream from the translation stop codon, but this may be only by chance in the 71% A i-T 3’ trailer. Of the 47 hypotrich mat sequences deposited in the GenBank database, 17 sequences lack
the poly(A)-addition signal (Prescott, 1994). Thus it is not unexpected that this signal sequence is not present in the pola gene-sized molecule. It is not clear what guides poly(A) addition in hypotrich genes lacking the conventional signal. The ORF is 4479-bp long, including the stop codon, and is 62% A +T. It is not interrupted by A+ T-rich regions that suggest the presence of introns, which average 80% ACT in hypotrichs. The ORF specifies a putative polypeptide of 1492 aa (approx. 172 kDa). It contains 23 TAA and 18 TAG codons. In the ciliate genera Oxytricha, Stylonychia, Tetrahymena and Paramecium, TAA and TAG codons specify Glu rather than signalling translation termination as in most other organisms (Helftenbein, 1985; Preer et al., 1985; Hanyu et al., 1986; Herrick et al., 1987; Gray et al., 1991). Comparison of the sequence of the 0.7-kb human probe with the target sequence reveals an overall identity of
159 Region
A
Region
E
Region
D
10
Region
IV
Region
B
Region
Region
VI
Region
III
Region
Fig. 3. Conserved
II
aa sequence
10
20
10
20
I 5
motifs shared
Region
30
40
30
C
40
Region
VII
Region
V
10
between
the catalytic
subunits
10
of polcc from 0. nova (On), human
(h) and SC. Regions
are presented
in
the order they appear along the polymerase polypeptide (from N to C terminus): A-E-D-IV-B-II-VI-III-I-C-VII-V. Identical aa in all three polypeptides are shown in shaded boxes, while aa shared between any two of the polypeptides are shown in open boxes. The regions encompass the following aa: A: aa 33-48 (On), 76-91 (h), 56-71 (SC); E: aa 349-353
(On), 361-365
(h), 368-372
(SC); D: aa 483-487
(On), 495-499
(h), 493-497
(SC); IV: aa 601-642
(On), 609-650 (h), 610-651 (SC); B: aa 772-807 (On), 772-807 (h), 781-816 (SC); II: aa 840-880 (On), 839-879 (h), 843-883 (SC); VI: aa 924-940 909-925 (h), 904-920 (SC); III: aa 9577998 (On), 943-984 (h), 937-978 (SC); I: aa 1013P1020 (On), 998-1005 (h), 992-999 (SC); C: aa 1051-1064 1036-1049
(h). 1030-1043
(SC); VII: aa 1067P1071
(On), 1052-1056
(h), 1046-1050
56%, with a 185-bp region exhibiting 70% sequence identity. The homologous region maps to the 3’ half of the sense strand (Fig. 2a), spanning the portion of the gene between nt 3110 and 3820. The EcoRI site in the RI+ version is further upstream in the vicinity of nt 2700
(Fig. 2a). (c) Evolutionary conservation of aa sequence motifs The deduced aa sequence of the 0. nova protein was aligned with sequences of the human and Saccharomyces cerevisiae (SC) pola proteins (Fig. 3). Sequence comparisons show the presence of several conserved motifs associ-
(SC); V: aa 1087-1101
(On), 1071-1085
(h), 1065-1079
(On), (On),
(SC).
ated with eukaryotic, viral, and some prokaryotic replicative DNA polymerases, including domains I through VII, and motifs associated specifically with cl-type DNA polymerases, including domains A through E (Wong et al, 1988; Damagnez et al., 1991; White et al., 1993). The overall arrangement of the sequence blocks (from N to C terminus of the protein) is A-E-D-IV-B-IIVI-III-I-C-VII-V (White et al., 1993), a pattern that is also observed in the 0. nova polypeptide (Fig. 3). Regions I through VI are present in seven eukaryotic pola, and in the replicative DNA polymerases of herpes simplex virus, human cytomegalovirus, Epstein-Barr
160 virus and vaccinia
1993). The prokaryotic merases
examined
III, or regions Region
Based
and mitochondrial
contain
VII is also present viral
DNA
suggested
pola, as 6 and in
polymerases
in most
single aa substitutions sensitivity analogs,
(Spicer Wang
have
a set of core
polymerases.
Indeed,
in regions II and III confer altered
to a variety suggesting
DNA
et al., 1988).
et al. (1989)
of nucleotide
these regions
play an important
role
in DNA pblymerization (Wang et al., 1989). Regions A through E are conserved only in the seven eukaryotic region
pola examined
A, which
is missing
to date, with the exception from the T. brucei
and
of P.
fulciparum
polypeptides (White et al., 1993; and this paper), and are thus thought to be associated only with a-type DNA polymerases (Damagnes et al., 1991). A single aa substitution in region D alters the stability of the DNA polymerase-primase complex in SC, implicating this region in protein-protein interactions with other
components of the et al., 1988). Functions determined. Another conserved tides is the Cys-rich nova polypeptide also
replication machinery (Pizzagalli of A, B, C, and E remain to be
Fig. 4. Southern probed
aa 13 14 to 1398, where the spacing between Cys (C) residues is CX,CX,,CX2CX,,CX,CX,,cX,C, where X represents any aa other than Cys. This arrangement closely matches the spacing in the SC (CX,CX,,CX,CX,, CX,CX,JX,C) and the human (CX,CX,,CX,CX,, CX,CX,,CX,C) polypeptides. The Cys residues may form DNA-binding zing-fingers (Wang et al., 1989). (d) Homologous gene-sized molecules coding for pola in other hypotrichs Using the 0. nova mat gene as probe, Southern analysis of native mat DNA from ten closely and distantly related ciliates identified homologous gene-sized molecules ranging from 4.5 kb to 5.2 kb in every species tested (Fig. 4). The size variation observed is not remarkable because DNA polymerases are generally composed of multiple conserved regions separated by variable length spacers (Wang et al., 1989); it may also result from differences in the length of leader or trailer sequences. Lynn and Sogin (1988) have compiled a phylogenetic tree for the ciliates based upon alignments of 16s rRNA sequences that indicates the genera Oxytricha and Stylonychia are closely related, while the genus Euplotes is more distantly related to both. Our Southern analysis confirms this relationship;
4
5
6
7
8
9
10
analysis
of native
mat
DNA from ten ciliate species
insert of DC8. Species examined are 0. trifallax (lane I), 0. sp. (WR) (I ane 2), S. lemnae (lanes 3 and 5), S. mytilus (lane 4), Urost$u grundis (lane 6), Paraurostylu weissii (lane 7),
with the 5.0-kb EcoRl
sp. (lane 8), E.
Holosticha
10). Methods:
Cultures
temperature
Prescott, extracted
octocarinatus (lane 9) and E. aediculutus (lane
of the various
hypotrich
species were grown
at
using the algae Chlorogonium as food. Preparation
of mat was performed
according
to established
procedure
(Zahler
and
1988). The mat suspension was lysed in 0.3% SDS and phenolat 23°C in order to eliminate or inactivate nucleases. The
aqueous phase was subsequently treated with proteinase K (Sigma, St. Louis, MO, USA) and extracted twice with phenol and once with chloroform/isoamyl
alcohol
(24:l).
Macronuclear
DNA
was subse-
quently ethanol precipitated and resuspended in TE ( 10 mM Tris/l EDTA pH 7.5). The native mat DNA was then electrophoresed
mM in a
1% agarose
was
gel and stained
with EtdBr,
after which
the DNA
blotted onto a Zetabind membrane (Life Science Products) (Ausubel et al., 1987). The blot was hybridized at 65’C for 18 h in NES (0.5 M NaH,PO, 1989)
pH 7.2/l mM EDTA
with
the
probe
labeled
pH 8.0/7% with
SDS) (Sambrook
32P using
random
et al.,
hexamers
(Pharmacia, Cambridge, UK) (Sambrook et al., 1989). Lanes 1 through 8 were washed to a final stringency of 0.1 x SSC/O.l% SDS at 53 C for 20 min, and visualized 9 and
feature among the pola polypepregion at the C terminus. The 0. contains a Cys-rich region between
3
n”-~ nl, _‘ Gs .n:*: :”
room
and pyrophosphate
2
5.0 4.3 -
et al., 1989).
in seven eukaryotic
that regions I, II and III comprise present
poly-
I, 11 and
T4, SC DNA polymerase
on these comparisons,
sequences
DNA
only either regions
I, II, III, IV and V (Wang
well as bacteriophage several
1
virus (Wang et al., 1989; White et al.,
by autoradiography
10 were washed
to a final stringency
room temperature, and visualized posure. Background hybridization presence washing
after a 20-h exposure.
Lanes
of 0.1 x SSCjO.1 %SDS
at
by autoradiography after a 17-h exin lanes 9 and 10 resulted from the
of telomeres on the probe fragment and the low-stringency conditions. Size markers are the 0. notrcl gene and Hind111
digest of h phage DNA (US Biochemical).
Lengths
are indicated
in kb.
the bands in both E. octocarinatus and E. aediculatus (Fig. 4, lanes 9 and 10) are visible only after lowstringency washes (0.1 x SSC/O.l% SDS at room temperature). In contrast, bands in the remaining lanes are visible after washing at much higher stringency (0.1 x SSC/O.l% SDS at 53°C). The catalytic subunit of pola in 0. nova contains multiple, highly conserved regions associated with DNA polymerases in general and with cl-type DNA polymerases in particular (Fig. 3). Because of the unique structure and organization of hypotrich genes, studies in 0. nova may yield useful information about transcriptional regulation of the pola-encoding gene.
ACKNOWLEDGEMENTS
This work was supported by NIGMS Grant No. GM19199 to D.M.P. The authors wish to thank Gayle Prescott for assistance with manuscript preparation.
161 among
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Smith, J.A. and Struhl,
K. (Eds.): Current
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R.E., Moore,
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(1991) 807-814. Hanyu, N., Kuchino, Y. and Nishimura, S.: Dramatic evolution: alteration of UAA and UAG termination mine
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lemnae. Special codon. Nucleic Kotter,
K.P.:
Multiple sequence versions of the Oxytricha ,fa/lax Sl-MAC ternate processing family. J. Protozool. 34 (1987) 4299434. Herrick,
G.: Non-coding
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G.: DNA poly-
cerevisiae: nucleotide mutation, and protein
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B.M. and Barnett,
A.J.: Deviation
from the universal code shown by the gene for surface protein in Paramecium. Nature 314 (1985) 188-190. Prescott, D.M.: The DNA (1994) in press. J., Fritsch,
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So, A.G. and Downey, K.M.: Eukaryotic DNA replication. Biochem. Mol. Biol. 2 (1992) 1299155. Konigsberg,
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Preer Jr., J.R., Preer, L.B., Rudman,
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W.H.: Primary relatedness
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Wang, TS-F., Wong, S.W. and Kern, D.: Human DNA polymerase CL: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 3 (1989) 14-21. Wang, T.S.-F.: Eukaryotic DNA polymerases.
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(1991) 5133552. White, J.H., Kilbey, B.J., de Vries, E., Goman, M., Alano, P., Cheesman, S., McAIeese, S. and Ridley, R.G.: The gene encoding DNA polymerase c1 from Plasmodium falciparum. 364333646. Wong, SW., Wahl, A.F., Yuan, K.-I.,
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tic replicative DNA polymerases. EMBO J. 7 (1988) 37-47. Zahler, A.M. and Prescott, D.M.: Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha now and a model for replication of the ends of linear DNA molecules. Nucleic Acids Res. 16 (1988) 695336972.
0378-l 119/94/$07,tX~
155
GENE 07946
A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase a in the macronucleus of Oxytricha nova (Hypotrichous
ciliates; gene structure;
Sam J. Mansour,
DNA replication;
David C. Hoffman
molecular
evolution;
non-universal
genetic code)
and David M. Prescott
Department qfA4olecu/ar, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA Received by A.-M. Skalka: 20 December
1993; Revised/Accepted:
4 February/7
February
1994; Received at publishers:
10 March
1994
SUMMARY
We have isolated
a gene-sized
molecule
encoding
the catalytic
subunit
of DNA polymerase
c1 from a macronuclear
genomic library of Oxytricha nova, by using a 0.7-kb fragment of the corresponding human gene as a hybridization probe. Two different versions of the gene are present in the macronucleus, one with an EcoRI site (RI+) and one without an EcoRI site (RI-). The cloned RI- version has been characterized. It is 4938 bp in length, excluding telomeres. It consists of a 329-bp 5’ leader, a 4479-bp coding region and a 130-bp 3’ trailer. The deduced amino-acid sequence shares conserved regions with the yeast and human polypeptides. We also demonstrate by Southern analysis that gene-sized molecules of similar size, homologous to the isolated 0. noua gene are present in the mat genome of closely and distantly related hypotrichs.
INTRODUCTION
Hypotrichous ciliates contain two types of nuclei, a micronucleus (mic) and a macronucleus (mat). The mic genome contains typical chromosomes with hundreds of genes per chromosome. It serves as the germline genome. The mat genome contains millions of gene-sized molecules (0.4-15 kb), each with a single coding function (Klobutcher and Prescott, 1986). Seventy-four molecules have been characterized in various species of the hypotrichs Oxytricha, Stylonychia and Euplotes. These moleCorrespondence to: Dr. D.M. Prescott, University 492-8381;
of Colorado, Fax (l-303)
Boulder, 492-7744;
Department
CO 80309-0347,
of MCD
Biology,
USA. Tel. (l-303)
e-mail: [email protected]
Abbreviations: aa, amino acid(s); bp, base pair(s); E., Euplotes; EtdBr, ethidium bromide; kb. kilobase or 1000 bp; mat, macronucleus( mic, micronucleus( nt, nucleotide(s); O., Oxytricha; ORF, open reading frame; l?, Paramecium; polcc, DNA polymerase(s) a; RI’, version of polcc containing an EcoRI site; RI-, cloned version of polcl without an EcoRI site; S., Stylonychia; SC, Saccharomyces cerevisiae; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OlS M Naacitrate pH 7.6; T., Tetrahymena. SSDI 0378-l
119(94)00157-N
cules conform to one structural form with a 5’ leader, a coding segment and a 3’ trailer. All molecules are flanked by telomeric sequences (Klobutcher and Prescott, 1986). Because of the single coding function, macronuclear DNA molecules are referred to as gene-sized molecules. After cell mating, a mic genome is converted to a new mat genome in which various mic DNA sequences are cut, spliced, eliminated, reordered and amplified to produce gene-sized molecules to which are added telomeric repeats (Klobutcher and Prescott, 1986). In the mature mat of 0. nova, each different gene-sized molecule is present on average in 1000 copies. Characterization of the gene-sized molecule encoding the large catalytic subunit of DNA polymerase CL(pola) in the mat of 0. noua is the subject of this paper. This work is the foundation for a comparison of the mic and mat versions of the gene to be described in a later paper. We chose pola because it is twice the size of the largest scrambled gene identified to date, and it did prove to show a more complex scrambled pattern in the mic than the shorter genes studied. The gene encoding polcl has been cloned from six other eukaryotic species
156 (Braithwaite and Ito, 1993; White et al., 1993). The holoenzyme is composed of four subunits, the largest of which (studied here) is a 160-180-kDa protein that is directly involved in the catalysis of DNA synthesis (Wang, 1991; So and Downey, 1992). The aa sequence comparisons show the presence of several conserved regions dispersed along the polypeptide (Wang et al., 1989; White et al., 1993). In addition to sequencing the large catalytic subunitencoding gene and comparing its putative product to the subunit of other eukaryotes, we have compared the genesized molecules encoding the subunit in several hypotrich species.
RESULTS
1
b
2
1
a
6.5 -
6.5
4.3 -
4.3
2
2.3 2.0 -
AND DISCUSSION
(a) Cloning of the mat gene
The polcl of 0. nova shows a high degree of aa identity in twelve conserved regions (designated I-VII and A-E) associated with DNA polymerases in general and a-type polymerases in particular (see section e below, and Fig. 3). Using a 0.7-kb probe corresponding to the portion of the human pola-encoding gene for regions VI-IIII-C-VII-V (the HindIII-BamHI fragment from pcD-KB pola, a cDNA clone kindly provided by Teresa S.-F. Wang), we probed blots of native 0. now mat DNA by hybridization. ~acronuclear DNA ranges in size from 0.4 kb to 15 kb (Fig. la, lane 1). In Southern analysis, the human probe hybridizes to a 5.0-kb gene-sized molecule (Fig. la, lane 2). Hybridization of the human probe to EcoRI-cut mat DNA detected two molecules, one of 5 kb and one of 2.3 kb (data not shown). Analysis of the pattern indicates that two versions of the pola-encoding gene are present, one that lacks EcoRI sites (RI-) (5-kb molecule) and one with at least a single EcoRI site (RI ‘), which generated the 2.3-kb fragment. No hybridization to other fragments was observed for the RI+ version, indicating that either the probe does not span the EcoRI site, or that the probe does not share significant sequence identity with more than the single 2.3-kb fragment. Subsequent hybridization of the full-length cloned mat pola-encoding gene to EcoRI-cut mat DNA showed three bands, one at 5.0 kb (full-length gene-sized molecule without an EcoRI site) and two at 2.7 kb and 2.3 kb (Fig. lb, lane 2) (the two fragments produced by cutting at a single EcoRI site). (b) Characteristics product
of the mat gene and its encoded
Screening a mat genomic library of 0. nova yielded a single positive h clone designated DC8 Sequencing showed that the insert in the DC8 clone is 4978-bp long
Fig. 1. Southern
analysis
0.7-kb HindHI-BamHf EcoRI insert in DC8 stained
agarose
of 0. notru mat
DNA
probed
with (a) the
fragment of the human gene, and (b) the 5.0 kb Methods: (a) lanes: 1, Native 0. nooa mat (EtdBr
gel). 2, Native
0. nou~l mat (Southern
blot). Native 0.
noau mat DNA (3 ug) was electrophoresed in a 1% agarose gel and stained with EtdBr, after which the DNA was blotted onto Zetabind membrane (Life Science Products, Denver, CO, USA) (Ausubel et al., 1987). The blot was prehybridized at 42-C for 6 h in 20% formamide~3
x SSCj5 x Denhardt’s
solution/i
mM N~pyrophosphate/
1% SDSjlOtI ug/per ml of E. coli tRNA, and then incubated at 42°C for 19 h with the 32P-labeled human probe (Random primed DNA labeling
kit, US Biochemical,
Cleveland,
OH, USA) in the presence
of
5% dextran sulfate. The membrane was washed twice at room temperature for 15 min in 2 x SSC/O.l % SDS, followed by two similar washes at 60°C. Hybridization
signals were visualized
by autoradiography
with
a 21 h exposure period. (b) Lanes: 1, Native and 2, EcoRi digested. Macronuclear DNA (3 ug) was blotted onto Zetabind. prehybridized at 65°C in
I M NaCI/SO mM Tris 8.0/l mM EDTA/O.l%
SDS/ZOO pig
per ml sheared salmon sperm DNA, and then incubated at 65°C for 19 h with the 32P-labeled 5.0-kb insert in DC8. The blot was washed under stringent conditions at 65 ‘C in 0. I x SSC/O.l% SDS. The autoradiograph was developed after a 19-h exposure. Size markers are Hind111 digests of h phage DNA (US Biochemical).
Lengths
are indicated
in kb.
(Fig. 2b). Telomeric repeat sequences (20 bp each) occur at the ends of the insert, indicating the h clone contained a complete gene-sized molecule. The gene-sized molecule contains a 5’ leader of 329 bp extending from the inner end of the 5’ telomere to the ATG start codon (nt 350), and a 3’ trailer of 130 bp extending from the TGA stop codon (nt 4828) to the inner end of the 3’ telomere. The assigned ATG start codon is the fourth ATG triplet from the 5’ end, and is the only one followed by a long ORF. It defines an ORF that begins with an aa region that is conserved at the N terminus in the large subunit of four polcl proteins (Fig. 3, region A) (White et al., 1993). The 5’ leader lacks TATAA, CCAAT and GC boxes.
157
a) sense strand
anti-sense strand
b)
. . . . . ccccaaaaccccaaaaccc~tctagtt=~t=~~=g~g==~t=g=~~tt~=t~g~tt==~g=tttt~~ .
.
.
.
.
.
120
cattttgaacttaaaatagagtatgagcgacaactatactatactactgccc~tgcagagaagtaaagaaaggtggtaaaggagaagttgttaaaaagggattagcagctttgagagcagccaga gaaggtggag~caag~gaaCtg~ct~gtac~C~gta~gtat~~~gcattaatgCtaat~atctttctga~tggCttttc~aaaat~ttgcaacatcatcctcctttctcATGA~~CA MIKHl
240 360
CATTTTATAGGTIGAAGAAAC
480
AAAMAGA’ICTITGAAGAGATAGA~ACMCGAATA~AMAAA
rrTAAGATTrCCGCAAAAA~~A~~~A~~A~~A~ATA
ILQVEETKKIFEEIDDNEYEKIQDSRKNDDFIVDDDGYGY44 TCGCGATCACGGAGGAGAAA~~CAGAGA'PGOAGCC RDHGGEIWDRDGDVEEVGKKKKKKQNNHDPNENIMNYMMP84
600
‘PGCAAAAGATGTAGA~AC~M~~~M~~GC
840
mAGAGTCAAGAGAAGAGCAAGAGCGCGCCGTCGAGA LESREEQERRRQSEQLKQQANIGQNQSDVNPFSKKRKLDE204
960
GlTCTAGCAAGTTCAAGCT~~CATAACT FQQVQANSYQSKQNSHSVSKSKPGDHEMANHADGVDLNLL244
1080
TGCTA?TGACGACACTAAGATGACAGATAGTCACCCTAGC AIDDTKMTDSHPSEIITQNQRASAVSSVNQQIMESTNGKN284
1200
CCAGCTCGAGAAAAA CGATACCGAATOGCMTAGATGAAGGAG~~~C~~~~A~AC~~G~~C~T~~A~~~TM~~C~GACTA~C~~C QLEKNDTEWQQMKEKNAVFNQDLRHNDNALMSNQQDYPLP324
1320
~~GA~AACTCTTTCrrPCTA~~TAGAT LNEDGTLSFYWIDAHEENNGADLFVFGKIYQHEERKFVSC364
1440
CTCTATCAAAGTGAATGGTATGCAAAGAGAOCTATTPGTTA ELFVLPKMSGKSRAAMTTEEEKEQARKVMME404 SIKVNGMQR
1560
ACTTGAAGGAGTAGAAAGAGATTCCAGCTATCACTA LEGVRKRFPAITKWRCKPVTRKYAFELPIQHGEHQFVKIK444
1680
CAAAAACA~PG?TCG~'CAAGAAATGAACAGACCAGTI(;GTCAC AKDVDELEDVNEAKNMFVQEMNRPVAFNKEEDFNNRYSVT164
ATACGA~CAACCTTCCCTTCrrrrCCTAGCACAGTACAG~C YDATFPSLPSTVQGNTFECIFGA
NQSMLESFI
L
K
R
K
I
R
G
1800 P484
A~C~ATGACTATTAGAAACCCTCAAAAGGTAAC? CWMTIRNPQKVTDFRRTWCKQEILVSNPKDIEITLDDLNK524
1920
AACTGAGCTACCTCCTTTGACCTCTGTCACATPTGCCATAG TELPPLTSVTFAIKTCRSSQNTNEIAHLSCIVNENISQEG564
2040 GCCTATGCCAATIGAATAlGAGAGAGCTTl'CAGAGATAAGAAAGAT'I'XT'TCATTTAG~CTT PIEYERAFRD KK DSFIQFF604
TCC~CAAAAATPGA~'PCCACAAA?rA?TCACmACTC PSKIDVHKSFTLLRKLDGKPM CTAACATGAGAGACAACTTAT?GAAGCC~G~CTAAA QHERQLIEAFVAKIYQLDPDLMVAHNLCGGMFD
L
L
L
A
R
I
2160
2280 Q644
GA~CTCAAAATTAGTCAC~G~CAG~~~GCCT~G~~CT~~CCT~C~~~AGA~~GC~AGCC~~A~AGG~~~~~~CC~~T~GT PNKKSDQSGANYGGSQWIPRQV684 HLKISHWSRIGRLKKNQI
2400
TACTPGTGGAAGATPGTTAGTTGATACTPTCTPGACTGCC~~GC~A~A~~~CT~CTA~C~ACCCA~TAGCT~~TA~C~T~G~TA~~AC~A RETNYDLTHLAKVQLQKDRIDFD724 TCGRLLVDTFLTAKELI
2520
TGA~ACCmTGCCAACTTA~TCC~~C~C~~~C~~~ACAC~AG~GACGCCTATCTCAC~~T~~~~~ACC~TAGG~A~C~T LFQLIDHTEKDAYLTLQLMNHLQVIPL764 DDLLPTFYVQMAK
2640
GACTAAGCMCTCACACA~T~~~~A~~~~~CGCCA~GC~~G~~~C~TAC~CAC~G~~G~~G~~~ACC TKQLTNIAGNLWFRSLQNARAERNEHLLLHEFKKKKFVLP804
2760
AGACAAAAAGCAACTTAACGCCAAAGA~~G~G~CA~~CA~~AGTACGM~~G~~~~~~G~~~GA~G~GCAGCGTA~CAG~~~T EYEEGDGKTKGGKRKKAAYAGG DKKQLNAKDLKKNMFAD TGTGA~CCCAAffiCTGG'ITTrTATGATAATATCATC VIEPKAGFYDNII
2880 L 844 3000
IQEYNLCFTTVNRRP884
LLLDFNSLYPSI
TACCAAGAACTTCG4~GC~~~~G~CTAGTAC~G~~~~~~~GAGGTAGATA~~~GC~~~CC~AC~~~T~C~G~~C ENGEEEVDIEEADLPDKNVNLKDA924 TKNFDGSEMKNQYKKG AG'PXlTCCCATGGT-lT%AGAGACCTCGTCTiUAAGAGAAAAGC~l-CAAGGACAAGAnXAAAC VKDKMKTEKDHVKLSQLE VLPMVLRDLVQKRKA
lWdAAAGA%!ACGTCAAGTTATCGCAATTAGAAA!FZAGATAAAAGGCTATTAA I R Q K A I
3120 3240 K964
158 3360 1004 tGCAGAAAAT~GCTTGGATPCAATGTAGTGTATGGn;ATACTT AENKLGFNVVYGDTDSIMINTGSNQLQQALEMGKRLKGEV
3480 1044
~GAAGTATGCAGCTCTG?diGTAl'JAAAACTICCTAAGTCCAGCTGAAGT AAACTGC'lTGTACAAATGCCTlGAAATAGAUiTAGATGGAGTTTIl'AAGAGlT'FXTCTTA NCLYKCLEIEIDGVFKSLLLLKKKKYAALKYENFLSPAEV
3600 1084 3720 1124 3840 1164
3960 RLRDQGKSENQLVNNFIPYVICQQTYGDT1204 IKGQPHVAVAK * . . . . . . . . . 0 . TACCAAATCAAGCACAGCACTCTCAGATAAGGC~ACCACCCAGAXiAAGTGATrAGCTCAAGAGGAAAAGTGACTATAGATAGl?.3AC'lWZTATG?GTCTACCTAATItXTlK!CTCCCAT 4080 TKSSTALSDKAYHPDEVISSRGKVTIDSDWYVSTQLLPPIl244 4200 1284 4320 1324 AGGMTCC~~CCCAGCrrAAACAATACA~GCrrACA
GILVPSSNNTELTGLACVKCNQR
TCTlT'XTXAAGCAACT IPDAYMLNRLNLFLKQL
4440 1364 4560 1404 4680 1444
GCCTM~CMTCATTCAAGC~~~GG~~~~A~~GM~~ATAC~C~G~GA~AGGCMTA~GG~A~AGC~G~AGCT~~C PNFQSFKELQKKVESFMIRSGYNKVDLGNIFGFMSKGANP
4800 1484
TCACTAAC~~GCA~~Aatggcggcattttgatcagagttaatttattttactcaaattccttaagaacaagataaacctcgcgccttttaagaggttatctgcattta H Q Q K M S I F"*
4920 1492
atgcattcatttagtttataaatttacacaaaaaacttggggttttggggttttgggg-3'
4978
Fig. 2. Nucleotide sequence of the polcc-encoding gene and the deduced aa sequence. (a) The RI- version of the gene-sized molecule. Black boxes at the ends of the DNA molecule denote telomeric sequences; the single-stranded extension of telomeres was removed during cloning. The extent of sequence generated from each strand is indicated with arrows. Lengths are indicated in kb. The hatched box denotes the 0.7-kb fragment of the human pola-encoding gene, and is depicted along its target hybridization region in both the RI- and RI+ versions, The EcoRI site (E) in RI+ is shown. The RI’ version has not been isolated. (b) Complete nt sequence of the RI version, and the deduced aa sequence. The ORF is in upper case; the leader and trailer are in lower case. The sequence has been submitted to the GenBank database under the accession No. UO2001. A mat genomic library of 0. nova was constructed by inserting the gene-sized molecules into the unique EcoRl site of hgt 10 as described elsewhere (Klobutcher et al., 1984). About 1.2 x 10’ plaques representing five times the sequence complexity of mat DNA were screened with the 0.7-kb human probe. The screen yielded one positive h clone, DC8. The 5.0-kb insert in this clone was recovered after EcoRl digestion, indicating that it represents the RIversion. The 5.0-kb insert was subcloned in both orientations into the unique EuoRI site of pTZI8R (US Biochemical). Procedures for subcloning and for the generation of nested deletions were according to established protocois (Ausubei et al., 1987). Sequence determination was performed on double-stranded DNA templates with a Sequenase version 2.0 kit (US Biochemical) (Hsiao, 1991).
There are four TATAA-like boxes located in the region 52-279 bp upstream from the translation start codon; however, since the leader is A +T-rich (66% A+T), TATAA-like sequences are expected to occur by chance. Comparison of the leader sequences of different genes within a hypotrich species or between the same gene in different hypotrichs shows little or no sequence similarity among them with the exception of a histone H4-encoding gene in S. herniae and 0. rz~~a(Herrick, 1992). This suggests that hypotrichs may define promoters in other ways. The TGA stop codon at nt 48264828 is followed in the same reading frame by another TGA codon at nt 4841-4843. The 130-bp trailer sequence does not contain the poly(A) addition signal, 5’-AATAAA. The sequence S-ATAAA occurs 109 bp downstream from the translation stop codon, but this may be only by chance in the 71% A i-T 3’ trailer. Of the 47 hypotrich mat sequences deposited in the GenBank database, 17 sequences lack
the poly(A)-addition signal (Prescott, 1994). Thus it is not unexpected that this signal sequence is not present in the pola gene-sized molecule. It is not clear what guides poly(A) addition in hypotrich genes lacking the conventional signal. The ORF is 4479-bp long, including the stop codon, and is 62% A +T. It is not interrupted by A+ T-rich regions that suggest the presence of introns, which average 80% ACT in hypotrichs. The ORF specifies a putative polypeptide of 1492 aa (approx. 172 kDa). It contains 23 TAA and 18 TAG codons. In the ciliate genera Oxytricha, Stylonychia, Tetrahymena and Paramecium, TAA and TAG codons specify Glu rather than signalling translation termination as in most other organisms (Helftenbein, 1985; Preer et al., 1985; Hanyu et al., 1986; Herrick et al., 1987; Gray et al., 1991). Comparison of the sequence of the 0.7-kb human probe with the target sequence reveals an overall identity of
159 Region
A
Region
E
Region
D
10
Region
IV
Region
B
Region
Region
VI
Region
III
Region
Fig. 3. Conserved
II
aa sequence
10
20
10
20
I 5
motifs shared
Region
30
40
30
C
40
Region
VII
Region
V
10
between
the catalytic
subunits
10
of polcc from 0. nova (On), human
(h) and SC. Regions
are presented
in
the order they appear along the polymerase polypeptide (from N to C terminus): A-E-D-IV-B-II-VI-III-I-C-VII-V. Identical aa in all three polypeptides are shown in shaded boxes, while aa shared between any two of the polypeptides are shown in open boxes. The regions encompass the following aa: A: aa 33-48 (On), 76-91 (h), 56-71 (SC); E: aa 349-353
(On), 361-365
(h), 368-372
(SC); D: aa 483-487
(On), 495-499
(h), 493-497
(SC); IV: aa 601-642
(On), 609-650 (h), 610-651 (SC); B: aa 772-807 (On), 772-807 (h), 781-816 (SC); II: aa 840-880 (On), 839-879 (h), 843-883 (SC); VI: aa 924-940 909-925 (h), 904-920 (SC); III: aa 9577998 (On), 943-984 (h), 937-978 (SC); I: aa 1013P1020 (On), 998-1005 (h), 992-999 (SC); C: aa 1051-1064 1036-1049
(h). 1030-1043
(SC); VII: aa 1067P1071
(On), 1052-1056
(h), 1046-1050
56%, with a 185-bp region exhibiting 70% sequence identity. The homologous region maps to the 3’ half of the sense strand (Fig. 2a), spanning the portion of the gene between nt 3110 and 3820. The EcoRI site in the RI+ version is further upstream in the vicinity of nt 2700
(Fig. 2a). (c) Evolutionary conservation of aa sequence motifs The deduced aa sequence of the 0. nova protein was aligned with sequences of the human and Saccharomyces cerevisiae (SC) pola proteins (Fig. 3). Sequence comparisons show the presence of several conserved motifs associ-
(SC); V: aa 1087-1101
(On), 1071-1085
(h), 1065-1079
(On), (On),
(SC).
ated with eukaryotic, viral, and some prokaryotic replicative DNA polymerases, including domains I through VII, and motifs associated specifically with cl-type DNA polymerases, including domains A through E (Wong et al, 1988; Damagnez et al., 1991; White et al., 1993). The overall arrangement of the sequence blocks (from N to C terminus of the protein) is A-E-D-IV-B-IIVI-III-I-C-VII-V (White et al., 1993), a pattern that is also observed in the 0. nova polypeptide (Fig. 3). Regions I through VI are present in seven eukaryotic pola, and in the replicative DNA polymerases of herpes simplex virus, human cytomegalovirus, Epstein-Barr
160 virus and vaccinia
1993). The prokaryotic merases
examined
III, or regions Region
Based
and mitochondrial
contain
VII is also present viral
DNA
suggested
pola, as 6 and in
polymerases
in most
single aa substitutions sensitivity analogs,
(Spicer Wang
have
a set of core
polymerases.
Indeed,
in regions II and III confer altered
to a variety suggesting
DNA
et al., 1988).
et al. (1989)
of nucleotide
these regions
play an important
role
in DNA pblymerization (Wang et al., 1989). Regions A through E are conserved only in the seven eukaryotic region
pola examined
A, which
is missing
to date, with the exception from the T. brucei
and
of P.
fulciparum
polypeptides (White et al., 1993; and this paper), and are thus thought to be associated only with a-type DNA polymerases (Damagnes et al., 1991). A single aa substitution in region D alters the stability of the DNA polymerase-primase complex in SC, implicating this region in protein-protein interactions with other
components of the et al., 1988). Functions determined. Another conserved tides is the Cys-rich nova polypeptide also
replication machinery (Pizzagalli of A, B, C, and E remain to be
Fig. 4. Southern probed
aa 13 14 to 1398, where the spacing between Cys (C) residues is CX,CX,,CX2CX,,CX,CX,,cX,C, where X represents any aa other than Cys. This arrangement closely matches the spacing in the SC (CX,CX,,CX,CX,, CX,CX,JX,C) and the human (CX,CX,,CX,CX,, CX,CX,,CX,C) polypeptides. The Cys residues may form DNA-binding zing-fingers (Wang et al., 1989). (d) Homologous gene-sized molecules coding for pola in other hypotrichs Using the 0. nova mat gene as probe, Southern analysis of native mat DNA from ten closely and distantly related ciliates identified homologous gene-sized molecules ranging from 4.5 kb to 5.2 kb in every species tested (Fig. 4). The size variation observed is not remarkable because DNA polymerases are generally composed of multiple conserved regions separated by variable length spacers (Wang et al., 1989); it may also result from differences in the length of leader or trailer sequences. Lynn and Sogin (1988) have compiled a phylogenetic tree for the ciliates based upon alignments of 16s rRNA sequences that indicates the genera Oxytricha and Stylonychia are closely related, while the genus Euplotes is more distantly related to both. Our Southern analysis confirms this relationship;
4
5
6
7
8
9
10
analysis
of native
mat
DNA from ten ciliate species
insert of DC8. Species examined are 0. trifallax (lane I), 0. sp. (WR) (I ane 2), S. lemnae (lanes 3 and 5), S. mytilus (lane 4), Urost$u grundis (lane 6), Paraurostylu weissii (lane 7),
with the 5.0-kb EcoRl
sp. (lane 8), E.
Holosticha
10). Methods:
Cultures
temperature
Prescott, extracted
octocarinatus (lane 9) and E. aediculutus (lane
of the various
hypotrich
species were grown
at
using the algae Chlorogonium as food. Preparation
of mat was performed
according
to established
procedure
(Zahler
and
1988). The mat suspension was lysed in 0.3% SDS and phenolat 23°C in order to eliminate or inactivate nucleases. The
aqueous phase was subsequently treated with proteinase K (Sigma, St. Louis, MO, USA) and extracted twice with phenol and once with chloroform/isoamyl
alcohol
(24:l).
Macronuclear
DNA
was subse-
quently ethanol precipitated and resuspended in TE ( 10 mM Tris/l EDTA pH 7.5). The native mat DNA was then electrophoresed
mM in a
1% agarose
was
gel and stained
with EtdBr,
after which
the DNA
blotted onto a Zetabind membrane (Life Science Products) (Ausubel et al., 1987). The blot was hybridized at 65’C for 18 h in NES (0.5 M NaH,PO, 1989)
pH 7.2/l mM EDTA
with
the
probe
labeled
pH 8.0/7% with
SDS) (Sambrook
32P using
random
et al.,
hexamers
(Pharmacia, Cambridge, UK) (Sambrook et al., 1989). Lanes 1 through 8 were washed to a final stringency of 0.1 x SSC/O.l% SDS at 53 C for 20 min, and visualized 9 and
feature among the pola polypepregion at the C terminus. The 0. contains a Cys-rich region between
3
n”-~ nl, _‘ Gs .n:*: :”
room
and pyrophosphate
2
5.0 4.3 -
et al., 1989).
in seven eukaryotic
that regions I, II and III comprise present
poly-
I, 11 and
T4, SC DNA polymerase
on these comparisons,
sequences
DNA
only either regions
I, II, III, IV and V (Wang
well as bacteriophage several
1
virus (Wang et al., 1989; White et al.,
by autoradiography
10 were washed
to a final stringency
room temperature, and visualized posure. Background hybridization presence washing
after a 20-h exposure.
Lanes
of 0.1 x SSCjO.1 %SDS
at
by autoradiography after a 17-h exin lanes 9 and 10 resulted from the
of telomeres on the probe fragment and the low-stringency conditions. Size markers are the 0. notrcl gene and Hind111
digest of h phage DNA (US Biochemical).
Lengths
are indicated
in kb.
the bands in both E. octocarinatus and E. aediculatus (Fig. 4, lanes 9 and 10) are visible only after lowstringency washes (0.1 x SSC/O.l% SDS at room temperature). In contrast, bands in the remaining lanes are visible after washing at much higher stringency (0.1 x SSC/O.l% SDS at 53°C). The catalytic subunit of pola in 0. nova contains multiple, highly conserved regions associated with DNA polymerases in general and with cl-type DNA polymerases in particular (Fig. 3). Because of the unique structure and organization of hypotrich genes, studies in 0. nova may yield useful information about transcriptional regulation of the pola-encoding gene.
ACKNOWLEDGEMENTS
This work was supported by NIGMS Grant No. GM19199 to D.M.P. The authors wish to thank Gayle Prescott for assistance with manuscript preparation.
161 among
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