Primary structure of a gene-sized DNA encoding calmodulin from the hypotrichous ciliate Stylonychia lemnae

Gene. 119 (1992) 191-198 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

191

0378-l 119/92/$05.00

06613

Primary structure of a gene-sized DNA hypotrichous ciliate Stylonychia lemnae

encoding

(Recombinant

expression;

Christine

DNA;

Gaunitz,

calcium-binding

proteins;

S 1 nuclease

by J.R. Kinghorn:

macronucleus;

from

copy number;

the

protozoa)

Hubert Witte and Frank Gaunitz

Abteilung Zellbiologie, Zoologisches Institut, Eberhard-Karls-Universitiit, Received

analysis;

calmodulin

15 August

1991; Revised/Accepted:

Tiibingen, Germany

10 April/21

April 1992; Received

at publishers:

25 May 1992

SUMMARY

We have isolated and characterized a gene-sized DNA encoding calmodulin (Clm) from macronuclear (MA) DNA of the hypotrichous ciliate, Stylonychiu lemnae. The gene has 3500 copies per macronucleus. The length of the gene was deduced by agarose-gel electrophoresis of MA DNA and Southern blot analysis using a Clm cDNA probe from chicken. We then isolated the gene from a MA library. The overall length of the gene is 821 bp with a 450-bp intronless coding region. The deduced amino acid (aa) sequence of ciliate Clm has 149 aa and an M,. of 16819. Both ends of the cloned gene have the hypotrichous telomeric C,A, repeat. The coding region is flanked by a 158-bp 5’-leader sequence and a 3’-trailer sequence of 213 bp. Sl analysis was used to locate the transcription start point (tsp) 49 bp upstream from the start codon. No common eukaryotic transcription signals were found upstream from the tsp. A second gene-sized DNA, detected by its cross-hybridization with the Clm DNA, predicts the existence of a second Ca2+ -binding protein with only one Ca2 + binding site. It’s function and biological significance is yet unknown.

INTRODUCTION

Calmodulin Ca* + -binding and Hunziker, Ca2 + -binding

(Clm) is one of the many members of the protein family (for review, see Heizmann 1991). It is a monomeric protein with four sites. In high-resolution crystal structure

Correspondence to: Dr. F. Gaunitz, Institut,

Abteilung

Germany.

Tel.

Zellbiologie, (49-7071)

Universitat

Tttbingen,

Auf der Morgenstelle

296949;

Fax

Zoologisches

28, 7400 Tubingen,

(49-7071)

294634;

e-mail:

[email protected]. Abbreviations:

aa, amino acid(s);

bp, base pair(s);

C/EBP,

CCAAT/en-

hancer-binding protein; cDNA, DNA complementary to mRNA; Clm, calmodulin; Clm, gene (DNA) encoding Clm; E., Escherikhia; EtdBr, ethidium bromide; leotide(

kb, kilobase

or 1000 bp; MA, macronuclear/us;

oligo, oligodeoxyribonucleotide;

ment of E. coli DNA

polymerase

I; SDS,

PolIk, sodium

Klenow dodecyl

staining density; S.I., Stylonychia lemnae; SSC, 0.15 M NaCljlS citrate pH 7.6; tsp, transcription

start point(s);

nt, nu-

(large) sulfate;

fragsd,

mM Na,.

u, unit(s); UV, ultraviolet.

analysis, Clm appears to be a dumbbell-shaped protein (Babu et al., 1985; 1988): A long central helix separates two domains (Strynadka and James, 1989; Van Eldik et al., 1982; Klee and Vanaman, 1982). Each domain has two EF-hand Ca2 + -binding loop s with a very high affinity for Ca2 + . On binding of Ca2 + , Clm changes its conformation and transmits the Ca2’ -second messenger signal by activating enzymes that are central to cellular regulatory processes. Clm, as well as enzymes that are activated by Ca2+ Clm, have been found in every eukaryotic organism so far examined. Clm DNA has been isolated from various organisms such as filamentous fungi (L&John, 1989), yeast (Davis et al., 1986), green algae (Zimmer et al., 1988), trypanosome (Tschudi et al., 1985), fruit fly (Smith et al., 1987; Yamanaka et al., 1987; Doyle et al., 1990), electric eel (Lagace et al., 1983), chicken (Putkey et al., 1983), Xenopus (Chien and Dawid, 1984), and potato (Jena et al., 1989). Additionally, there is growing evidence that Clm can perform its essential function without the ability to bind Ca2+

192 ,lOObp,

Calmodulin 3008ac1

321bp

579EcoRl

607 EcoRV

Clone719 719

775XbaI

527Sal1 903Xbn I

bp

I

w

4 I

Telomeres m

non-coding

regions

coding regions

Fig. 1. Organization binding proteins.

of the two gene-sized

S.I. DNAs

Two clones were isolated

encoding

Ca’ +

below. Methods.

as described

MA DNA was resolved by electrophoresis on a 1.5:/, agarose gel, transferred to a nylon membrane (Biodyne B, Pall) and fixed by baking at 80°C for 2 h. The chicken Clm cDNA probe (0.29-kb EcoRI-PstI the vector pGEM-GM, was labeled according with [a-32P]dCTP

that was kindly provided to the method

fragment

from

by Dr. C. Rasmussen)

of Feinberg

and Vogelstein

(spec. act. > 110 TBq/mmol,

Amersham,

(1983)

UK) to a

specific activity of 2 x 10’ cpm/pg DNA (10 ng/ml). Hybridization of the labeled probe to the filters with the transferred MA DNA was carried out at 60°C for 14 to 16 h in 6 x SSC/Denhardt’s

solution in the presence

salmon sperm DNA (150 pg/ml). The filter was then extensively in 2 x SSCjO. I “; SDS at 60°C and autoradiographed at -70°C intensifying

screen. Hybridization

signals were detected

of

bp and of 650-750

signals detected

BAL 31 (BRL, Eggenstein) stopped mediately

bp, respectively,

by hybridization

extracted

to standard

el et al., 1991) the DNA was then blunt-filled pBluescriptKS

+ vector

(Stratagene)

formed into E. co/i JM83 according hybridization

using the 0.29-kb

mixture was immethods

(Ausub-

which was cut with EcoRI The plasmids

to the method of Hanahan

PstI-EcoRI

were

with PolIk and ligated into

hringer) and treated with alkaline phosphatase.

Clm cDNA

(Boe-

were trans(1983). By

probe from the

chicken, approx. lo4 colonies were screened from the library originating from the 750-850-bp MA DNA and approx. 5 x lo4 colonies originating from the MA DNA of 650-750 bp. One clone from each library was isolated. They were subcloned into pBluescriptKS + vector for sequencing with [c(-“S]dCTP

(specific

using the chain-termination shows the organization entation

activity

technique

> 37 TBq/mmol; (Sanger

of the two isolated

and regions determined

(Geiser et al., 1991). It would be ize the Clm gene and Clm protein such as the hypotrichous ciliates to how the Clm-binding sites and

Amersham),

et al., 1977). The figure

clones.

by separate

Arrows

sequencing

as described

DNA corresponding depending

clones (see Fig. 1) to MA DNA

electrophoresis.

Hybridization

in Fig. 1. Each

and washhybridized

to the length it originated

on hybridization

to the length of the other clone, The size of the bands

to

from and

temperature

and washing

respectively.

L1857Sam7

DNA cut with EcoRI + Hind111 and labeled with used as a size marker.

clone

[ r-32P]dCTP

is indicated

has been

in kb.

to the two

at 0°C for I,2 and 3 min. The reactions

with phenol. According

macronuclear conditions,

with 0.07 u exonuclease

with l/20 vol. EDTA pH 8.0, and the reaction

ing were performed cross-hybridized,

800 bp

of the isolated

in 1.5 :, agarose-gel

with an

at approx.

corresponding

were digested

separated

washed

and at approx. 700 bp. The corresponding clones were isolated from macronuclear libraries constructed as follows. MA DNA (1 pg) DNA of 750-850

Fig. 2. Rehybridization

indicate

ori-

reactions.

interesting to characterof unicellular eukaryotes to obtain information as other sequence elements

have evolved in organisms that branched early in the evolution to higher eukaryotes (Schlegel, 1991). The hypotrichous ciliates show a very special genetic apparatus in having two different nuclei: a generative micronucleus, containing chromosome-sized DNA that seems to be transcriptionally inactive, and a larger and transcriptionally active macronucleus (Steinbriick, 1990; Ammermann, 1990). The MA DNA is organized as gene-sized DNA: small DNA molecules with mostly intronless coding regions flanked by 5 ‘- and 3’ -noncoding sequences. At both ends of such genes are inverted telomeric repeats. Each MA gene has a specific copy number. It is assumed that all c®ulatory signals for replication, as well as for expression, are in the 5’- and 3’-noncoding regions. Therefore, the gene-sized DNA offers a good opportunity for targetting questions of gene regulation. The aim of the present study was to clone and sequence the Clm gene of the hypotrichous ciliate, S. lemnae, and to compare the deduced aa sequence with other Clm genes. We also analyzed the transcript and tsp, as well as regula-

193 Clm probe. Autoradiography

tory signals. Another aim of our study was to describe a second gene with some similarity to Clm, but of unknown significance.

RESULTS

AND

showed a hybridization

signal

corresponding to a length of approx. 820 bp and two weaker signals at lengths of 4.2 and 0.7 kb. MA DNA of the length that gave the main hybridization signal and DNA corresponding to the signal at 0.7 kb were extracted from preparative agarose gels and used to construct partial genomic plasmid libraries. From one library, we isolated a clone with a 821-bp insert that strongly hybridized with the chicken Cfm probe. From the other library, we isolated a clone with a 719-bp insert that on hybridization with the same Clm probe gives a much weaker signal. Both clones were analyzed by restriction endonuclease analysis, sub-

DISCUSSION

(a) Identification, cloning and nt sequence of the Clmencoding gene-sized DNA from the macronucleus In order to identify gene-sized DNA encoding Clm, total MA DNA was electrophoresed in agarose gels and, after transfer to nylon membranes, hybridized with a chicken

Calmodulin A

CCCZAAACCCCGTGATAZAAGAATTATTCAM

ccccAAAAccccAAAAc

GAAAUAAGATTAATTCTGATTGTTTTGTG

79

ACAAAACAAGAAATCATTATTTAAGAGCGCTTAGTTAAAT

158

ATG

GCT

GAT

AAT

CTA

ACT

GAA

GAA

CAA

ATT

GCT

GAG

TTC

AAG

GAA

GCC

TTC

TCC

CTC

TTC

1

Met

Ala

Asp

Asn

Leu

Thr

Glu

Glu

Gin

Ile

Ala

Glu

Phe

Lys

Glu

Ala

Phe

Ser

Leu

Phe

GAT

A&G

GAC

GGT

GA!? GGA

ACC

ATT

ACC

ACC

AZ&

GAG

CTT

GGT

ACT

GTC

ATG

AGA

TCA

CTC

21

Asp

Lys

Asp

Gly

Asp

Gly

Thr

Ile

The

Thr

LyS

Glu

Leu

Gly

Thr

Val

Met

Arg

Ser

Leu

GGA

CAA

AAC

CCA

ACT

GAA

GCT

GAG

CTC

CAA

GAT

ATG

ATC

AAC

GA?+ GTT

GAT

GCT

GAC

GGT

338

Gly

Gln

Asn

Pro

Thr

Glu

Ala

Glu

Leu

Gin

Asp

Met

Ila

Asn

Glu

Val IAS 398

41

AAC

GGC

ACC

ATT

GAT

TTT

CCA

GAG

TTC

CTT

TCC

CTC

ATG

GCA

AGA

A&G

ATG

AAG

GAC

ACT

61

Asn

Gly

Thr

Ile

Asp

Phe

Pro

Glu

Phe

Leu

Ser

Leu

Met

Ala

Ax-g Lys

Met

Lys

Asp

Thr

GAT

ACC

GAG

GAG

GA&

CTG

GTA

WLA

GCC

TTC

AR0

GTC

TTC

GAT

AGA

GAT

GGA

AAC

GGT

CTC

81

Asp

Thr

Glu

Glu

Glu

Le"

Vs.1 Glu

Ala

Phe

LyS

Val

Phe

Asp

Arg

Asp

Gly

Asn

Gly

Leu

ATC

TCA

GCT

GCT

GA

CTT

AGA

CAC

GTC

ATG

ACA

AAT

CTC

GGA

GAA

AAG

TTG

ACC

GAC

GAG

Se+

Ala

Ala

Glul Leu

Arg

Eis

Val

Met

The

Asn

Leu

Gly

Glu

Lys

Leu

Thr

Asp

Glu

lOl(Ile GAG

GTC

GAT

GAG

ATG

ATT

AGA

GAG

GCT

GAT

GTT

GAC

GGT

GAT

GGT

CAC

ATT

A&C

TAC

GAG

121

Glu

Val

Asp

Glu

Met

Ile

Arg

Glu

Ala

Asp

Val

Asp

Gly

Asp

Gly

His

Ile

Asn

Tyr

Glu

GAA

0

TTC

GTC

AGA

ATG

ATG

AT0

GCT

AAG

TGA

141

Glu

Phe

Val

Arg

Met

Met

Met

Ala

Lys

OPA

TCTCCCAAATGTTAAATATACATATTCACTATTTTGTAA

218

270

458

518

578

647

CCTAAGAGCCATTCAATCTTGACTTTTTTTTCTTT~TTTGTCT~TAGTCTT~TCTTTATTAGTTCTT~TATT

726

ATTTACCACCCATAATTCTTTATATTCTCCGTACTA~~~CTCT~T~TTCTATTGTTA~C~CGGGG

805

TTTTGGGGTTTTGGGG

Clone

719

B

21

ATG

GAA

GAA

TAA

TTA

AGT

GAG

GAT

CAG

ATT

MT

GAA

TGT

AGA

GAA

ACA

TTT

AAA

ATG

TTT

Met

Glu

Glu

Gin

Leu

Ser

Glu

Asp

Gln

Ile

Asn

Glu

Cys

Arg

Glu

Thr

Phe

Lys

Met

Phe

GAC

AAG

GAT

GGT

GAT

GGG

ACT

ATT

ACA

GCC

AAG

GAG

CTT

GGT

ATA

GTG

ATG

AGG

TAA

CTT

Asp

Ly5

Asp

Gly

Asp

Gly

Thr

Ile

Thr

Ala

Lys

Glu

Leu

Gly

Ile

Val

Met

Arg

Gin

Leu

GGA

CTC

AAC

CCC

ACT

GAG

GAC

GAG

TTA

TTG

GAA

ATG

ATT

CAA

GAG

GTA

GAC

GAG

GAT

GGT

41

Gly

Leu

Am,

Pro

Thr

Glu

Asp

Glu

Leu

Leu

Glu

Met

Ile

Gln

Glu

Val

Asp

Glu

Asp

Gly

ATG

GGG

AAA

TCA

ATT

TCA

CAG

AGT

TTC

TGA

61

Met

Gly

Lys

Ser

Ile

Sar

Gln

Ser

Phe

OPA

CTATTATGGCTCATAAAATGAAGTAAATAATTGATCCAA

TAACATTAMTTATCAGAGATGCTGATACTGAATTGGGAACTTTAGAAGCATTTAGAGTTTTT

205

265

334

GATTTATATCGAAAACTGTTAAGAATATTATTAT~T-TCTTG~~~C~T~CT~G~~CT~G-CT

413 492

TCTATAGGAAGCAGAA

571

GTAMTCCAGATGGCAATGTCGACTACATGAGTTTTGTTCCTTTG

ATAGCTAAWLTGCCATCAAATTTACGARCATTCTAGATAT

GACAAAGAAAGGACTG

145

650

ATTAATTTCATAATGTTATTTTACTAAATAATCTCTCT~T~T~TTGCCGGGGTTTTG~GTTTT~GG

Fig. 3. Complete

nt sequences

of the two gene-sized

DNAs

and deduced

of the deduced product of Clone71Y have been boxed and shaded. Nos. M76407 (Clm) and M90073 (Clor~7lY).

aa sequences.

The nt sequence

The four Ca’ * -binding domains of the deduced data have been submitted to GenBank and assigned

Clm and that the accession

194 cloned and sequenced in both orientations (Fig. 1). Rehybridization of the clones to MA DNA that was sizefractionated in agarose-gel electrophoresis gave two different signals corresponding to two different gene-sized chromosomes (Fig. 2). Sequence comparison clearly showed that the 821-bp insert is a Cfm gene. It has an open reading frame of 450 bp starting with “ATG’ and terminating with the stop-codon, “TGA’. This corresponds to a 149-aa protein. The coding region is preceded by a 130-bp 5’-noncoding sequence and followed by a 193-bp 3’noncoding sequence each ending with telomeric sequences of 28 bp and 20 bp, respectively. Despite some similarity to Clm, the 719-bp gene-sized DNA (Clone719) could not be related to any known gene product. Its open reading frame predicts a protein of 69 aa with one Ca*+ -binding motif from aa 21 to aa 32 that has a very high homology to the first Ca2 + -binding site of Clm. The complete nt sequences of both genes and the predicted aa sequences are shown in Fig. 3. (b) Determination of the tsp and identification of putative ens-regu~tory elements In order to identify the corresponding transcripts, total RNA and poly(A)’ RNA were separated on denaturing gels and hybridized with the cloned genes. On hybridization with Clm gene autoradiography revealed a transcript of about 640 nt {Fig. 4). Even under less stringent conditions or with long exposure times, hyb~dization with the 719-bp gene-sized DNA does not reveal any signal aside from the signal at nt 640. We can not rule out the possibility that Clone719 is not transcribed at all, but it may also be that it is transcribed at a very low level and has just the same length as the transcript of Cfnl. The tsp of Cfm was then identified by Sl mapping (Favaloro et al., 1980) (Fig. 5). The mRNA has a 5’-untranslated leader of 49 nt and starts with a T-residue. The region located upstream from the tsp was searched for potential &-regulatory elements: There is no TATA-box-like sequence in the correct position that matches those considered as optimal for strong TATA-boxes (Efstratiadis et al., 1980; Bucher and Trifonov, 1986). There are TATA-box-like sequences 28, 36 and also 45 bp upstream from the translational start codon, but because they are downstream from the determined tsp, they are unlikely to be functions. Sequences considered to be typical TATA-boxes in other ciliate genes, like the consensus ‘T A/T T/A A/T A A A’ (Gaunitz, 1991) cannot be found either. On the other hand, the occurrence of TATA-box-like sequences downstream from t.sp has also been reported for the actin genes of the two hypot~chous ciliates, Oxytricha nova and O~yt~chafffl~a~ (Greslin, 1988). Clm also lacks a typical CCAAT box, originally found to bind the nuclear factor, C/EBP (Graves et al., 1986; Johnson et al., 1987). Inverted repeats believed to be in-

kb

4.4 2.4

-

O-24*

4h Fig. 4. Northern-blot

analysis

of total RNA and of poly(A) + RNA iso-

lated from S.1. for the presence of transcripts gene. Total

RNA

Lhiocy~ate

method

(10 gg) (lanes of Chirgwin

(lanes B) extracted go(dT)cellu

columns

methods

from the Clm-encoding

A) isolated

et al. (1979) and 2 pg of poly(A) + RNA

according

to standard

in denaturing

(Hames and Higgins,

protocols

formaldehyde

on oli-

(Ausubel

of Feinberg

> 110 TBq/mmol, dpm/pg

DNA. Hybridization

5 x Denhardt’s

to

1987). The RNA was then trans-

and Vogelstein ( f 983) with [ r-32P]dCTP Amersham,

et al.,

gels according

ferred to nylon membrane (Biodyne B, Pall) by capillary transfer. hybridized with the Clm probe from S. lernnae labeled according method

MA

using to the guanidinium

from total RNA by affinity chromatography

1991) were electrophoresed standard

3d

It was to the

(spec. act.

UK) to a spec. act. of approx.

5 x 10”

was carried out at 54 o C for IO h in 6 x S SC/

in the presence

of salmon

sperm DNA (100 pgjml).

The

nylon filter was then extensively washed in 0.2 x SSCjO. 17; SDS at 54°C and autoradiographed

at -70°C

for 3 days. As a size-marker

with an intensifying

(lane M), a BRL-iadder

was used. After electrophoresis,

the marker

was cut from the rest of the

gel, stained with EtdBr and the bands were detected size of the marker

bands

screen for 4 h and (BRL, Eggenstein) under UV light. The

is given in kb. The size of the RNA signal was

determined by kqarithmic regression to be approx. 640 bp. Even with a 3 day exposure or with less stringent washing (2 x SSC, 42°C; not shown) no other signat could be detected.

volved in the replicative processes of the gene-sized DNA (Wegner et al., 1989) do occur in the 5’-leader and the 3’-trailer, but their functional significance cannot be assessed yet. We also searched the 3’-trailer for processing

195 -130

-120

I

I

CCCCAAAACCCCAAAACCCCAMACCCC -70

-60

I

I

GAAATCATTA

GTGATAAAAG

-50

-40

AATTATTCAA

TCa$l%TA+T

-60

I

ATTAATTCTG

-20

I

ATTGTTTTGT

GACAAAACAA

-10

I

I

ACAAACACAA

~Q%A~X~~GATACCCTA

4

I

-90

I

W

I..

CTTAGTTAAA

-100

I

-30

I

TTTMGKGCG

-110

ATG..................

I

Fig. 5. Determination

of the tsp of Clm. A 345bp

at the 5’-end with polynucleotide annealed

to total RNA from S.I.. After digestion

M urea/6%

polyacrylamide)

5’-nontranslated downstream

mRNA

from the

and

leader

Sac1 fragment

kinase (Boehringer)

with Sl nuclease

sized by comparison

starting

covering

[ y-32P]dATP

and

the shortened

with a dideoxy

with a thymidine

residue.

the 5’-leader

according

of the MA Clm gene and part of its coding region was labeled

to standard fragment

sequencing

ladder

protocols

(Ausubel

was analyzed run alongside.

The rsp and start codon

et al., 1991). The fragment

by gel electrophoresis The detected

have been marked

fragment

by arrows.

was then

on a sequencing contained

TATA-box-like

gel (8 a 49-nt

sequences

tspare boxed and shaded.

signals, known to be responsible for transcription termination and polyadenylation in other eukaryotes. Despite poly(T) clusters, known to be able to induce transcription termination in genes that are transcribed by eukaryotic RNA polymerase III, no typical sequence element is found. Even the well-conserved polyadenylation signal ‘AATAAA’ (Proudfoot and Brownlee, 1976; Wickens and Stephenson, 1984) does not occur. The consensus sequence, ‘TAAAC’,

supposed to be a ciliate-specific polyadenylation signal (Helftenbein et al., 1989), is also missing. Probably there may be no need for a transcription termination signal, because of the shortness of 3’-trailer regions in gene-sized DNA. Whether or not a processing signal exists relative to polyadenylation must now be assessed. (c) Determination of the copy number of the Clm gene in the macronucleus The copy number of Clm in the MA was determined by hybridization experiments and densitometer scanning of X-ray films after autoradiography of slot-blots (Fig. 6). A number of 3500 copies per MA was estimated. Compared to, for example, 150000 and 30000 copies for the ai/&tubulinand cc,/&tubulin-encoding genes, respectively (Helftenbein, 1985; Conzelmann and Helftenbein, 1988), or to 6 x lo6 copies for highly amplified genes (unpublished), it is the smallest copy number in S.1. yet determined. Fig. 6. Determination of the copy number of Clm in the MA of S.I.. Methods. Different concentrations of cloned Cfm DNA were applied to a nylon membrane

alongside

MA DNA of different concentrations.

was done by the use of a micro-sample Schuell, Dassel, Germany) were hybridized hybridization

and washed

231pg

“&@5bF,

4.95

6.6

23.1 pg

8.25

as described

(Ultrascan

This

(Schleicher protocols.

the film was scanned

XL, LKB) and the staining density,

in the following way. First, we calculated

and

Filters

in Fig. 2 legend to avoid cross-

for each slot. The gene copy number

the relative staining density,

pg

46.1

to manufacturer’s

with Clone719. After autoradiography,

by laser densitometry was measured

according

filtration manifold

sd,

was then determined

the relation (sd,,,/[m])

sd,,,, and the applied amount

between

of cloned Clm

DNA, [ml,, (values along left side of filter) and the corresponding relation for the amount of MA DNA, [m] MA,applied alongside (values along right side of filter). A mean value was calculated from the values for the

11.5 pg

conditions. We calculated for sd,,,/ sd,,, measured under nonsaturating [ml,, = 2.0 x lo-*/pg and for sd,,,/[m],, = 1.6 x lo-‘/pg. From these

5.9 Pg

relations, we calculated the percentage of Clm DNA in the MA DNA to be 8 x 10 “%. Referring to a value of 780 pg DNA/MA of a single S. lemnae cell (Ammermann

et al., 1974), we calculated

the amount

2-8Pg

MA DNA to be 6.24 x 10 _ 3 pg/cell. Taking into account

l*4Pg

n x 660 x 3.32 x 10 34 g, we calculated

stranded

DNA molecule of n bp length has a molecular

should have a copy number S. lemnae.

of approx.

of Clm

that a double-

weight of approx.

for the 821-bp Clm DNA that it 3500 molecules

in a single MA of

196 10 II s.l. an S.l. Clone719 Tetrahymena Electrophorus xenopus Drosophila Trypanosom Saccharomyces

20 ll

90

u

an

100

u

11

(Yazawa

(Yamanaka

120

QL.SN.S.Q..L

aa sequences

The Ca”-binding

130

..,... .TM..K .TM... .TM... .L....

.A. .S. 140

. .. . .. . .

.Q..T.. .Q_.T.. .T. .TS. .K...S. -AALLS.

:: ............ ,...I........ . . . .D.L. .K..L.SI .... .A..

......

of Clo~e7lY (S.I. CIone719) and of the Clm (S.I. Clm) gene, with aa sequences

domains

are framed.

Numbering

et al., 1981); Electrophorus, Electrophphorus electricus (Lagact

meiunogaster

70 11

1)

.

Saccharomyces of the deduced

60 11

!DEEN'DEMIA .. .

ZrypdIlOS~a

Fig. 7. Comparison

110

KMKDTDTEEELVEZF

Tetrahymena Electrophorus Xenopus Drosophila

from other organisms.

50 11

40 u

MADNLTEEQIAEFKFJWS TVMR%GQNPT~AELQDMI .EEQ.S.D..N.CR.T .KM .I...Q..L....D..LK .. -..Q ....................................... ...Q ....................................... . ..Q ....................................... -..Q ............... ..j :........................ ...Q.SN...S.........~ 2 ;"....................... ..SS.............A..~ 2 :.A.......LS.S...VN.L?.l..I

80 s.l.

30 11

et al., 1987); Trypanosoma,

is according

to the S.1. Clm sequence.

et al., 1983); Xenopus, Xerzopus luevis (Chien and Dawid,

Tr~~punosoma hrucei gumhiense (Tschudi

deduced

from CIm gents

(TerrahJann2enu, Tetruhymerru pyrl~wmis

et al., 1985); Sacchurom~res,

1984). Drosophila,

Drosophilu

Saccharon?,ce.s c~erevisiae (Davis

et al.. 1986); see section d for details.

(d) Amino acid sequences The aa sequence of S. lemnae Clm and the possible aa sequence of the product of Clone719 have been deduced from the nt sequences of the cloned genes, and were compared among Clms from other invertebrates and vertebrates (Fig. 7). The aa sequence of S. lemnae Clm shows high homology to aa sequences of Clms from the representatives of other phyla, apart from the sequence from Saccharomyces cerevisiae which also differs from all other eukaryotic Clms. Among the Clms, the first 70 aa spanning the two Ca2 + binding domains I and II, and the spacer region between binding sites III and IV, are highly conserved compared to the binding sites III and IV, and the spacer region between the first two binding sites and the second two binding sites. The N-terminal domain of Clm is believed to be responsible for the interaction of Clm with enzymes (Klee and Vanaman, 1989). Its high conservation among vertebrates and invertebrates (except yeast), thus, may reflect the universal nature of regulatory processes mediated by Ca2+ -Clm in different organisms. The most conserved region is the first Ca2 + -binding site. Surprisingly, this side shares 11 of 12 aa with the only obvious Ca2 + -binding site of Clone719. According to the theory of Baba et al. (1984) Clm with its four Ca2+ -binding domains was produced by ancient gene duplications originating from a one-domain polypeptide about 36-40 aa residues long with one central 12-residue Ca2 + binding site. Two tandem duplications should have occurred to produce a four-domain Clm-like protein with domains I and III descended from the N-terminal half of the earlier two-domain protein, and domains II and IV descended from the C-terminal half. It is tempting to imagine that the protein that could be encoded by Clone719 may resemble the ancestral Ca’+ -binding protein which had

only one Ca’ + -binding site, but we do not know if this gene still encodes a functional protein. Another aspect of speculating on the role of the predicted product of Clorze71Y is that the corresponding gene also contains many silent mutations in the Ca2+ -binding domain, indicating that Clone719 and Cbn diverged from each other early in cvolution, but because of high evolutionary pressure on the Ca’+ -binding domain, the accumulating mutations did not result in a drastic change of the protein domain responsible for Ca2 + -binding. The conservation of the Ca” binding site also argues against the consideration that there may be no functional product of Clone71Y. Possibly our inability to find a gene transcript results from very low expression of Clone719 in the cells we usually use for RNA isolation (vegetative, nonconjugating cells, slightly starved). Considering the high similiarity of the S.I. Clm with the Clms of higher eukaryotes, the low similiarity of the Clm of Succharomyces cerevisiae with that of other organisms is surprising, because it is believed that the phylogenetic rclationship of S. cerevisiae to the multicellular eukaryotes is closer than that of S. lemnae (Schlegel, 1991). One possible explanation is to interprete the many substitutions observed in the Clm as a derived character of S. cerevisitre. Thus, the interpretation of the yeast Clm in terms of function and evolution must be reconsidered.

(e) Conclusions (I) Two cloned and sequenced gene-sized DNAs from the MA of S.I. that crosshybridized with a Clm cDNA from the chicken predict a 149-aa Clm and a Ca” -binding protein of 69 aa.

197 (2) The highly A+T-rich

5’- and 3’-regions

of the gene-

sized DNAs show none of the common eukaryotic processing signals similiar to other ciliate genes (Helftenbein et al., 1989). The only signals that can be found are TATAbox-like sequences, but the functionality of these elements remains to be assessed. (3) The predicted aa sequence encoded by Clm shows similiarity to Clms from other vertebrates and invertebrates. Highest similiarity is observed in the N-terminal half of the predicted protein including the first two Ca2 + -binding sites. Because this region is known to be responsible for the interaction of Clm with enzymes, this similiarity indicates the conserved role of Clm function in a group of organisms that branched early in evolution from the tree that led to higher eukaryotes. (4) The aa sequence predicted from the second genesized DNA, referred to as Clone719, shows only one calcium-binding site which is very similiar to the first Ca* + binding domain of Clm. The evolutionary significance and the function of this putative Ca2+ -binding protein is yet unknown.

Bucher,

P. and Trifonov,

PolII promoter

Chien, Y. and Dawid, Chirgwin,

I.B.: Isolation

J.M., Przybyla,

of eukaryotic

and characterization

A.E., MacDonald,

lation of biologically ribonuclease. Conzelmann,

of calmodulin

active ribonucleic

Biochemistry

R.J. and Rutter, W.J.: Isoacid from sources

enriched

in

18 (1979) 5294-5299.

K.K. and Helftenbein,

E.: Nucleotide

sequence and expres-

sion of two B-tubulin genes in Scylonychia lemnae. J. Mol. Biol. 198 (1987) 643-653. Davis, T.N., Urdea, M.S., Masiarz, yeast calmodulin

F.R. and Thorner,

gene: calmodulin

J.: Isolation

is an essential

protein.

of the Cell 47

(1986) 423-431. Doyle,

K.E.,

Kovalick,

G.E.,

meiunogaster contains expression Efstratiadis,

K.: Drosophila

Lee, E. and Beckingham,

a single calmodulin

gene. Further

structure

and

studies. J. Mol. Biol. 213 (1990) 599-605. A., Posakony,

J.W., Maniatis,

T., Lawn, R.M., O’Connell,

C., Spritz, R.A., DeRiel, J.K., Forget, B.G., Weissman, tom, J.L., Blechl, A.E., Smithies, and Proudfoot,

O., Baralle,

N.J.: The structure

S.M., Sligh-

F.E., Shoulders,

and evolution

C.C.

of the human

b-

globin gene family. Cell 21 (1980) 653-658. Favaloro,

J., Treisman,

oma virus-specific mapping. Feinberg,

Methods

R. and Kamen,

them.

R.: Transcription

RNA: analysis by two-dimensional Enzymol.

A.P. and Vogelstein, endonuclease

maps of polynuclease

S I gel

65 (1980) 718-749. B.: A technique

fragments

for radiolabeling

DNA

to high specific activity. Anal. Bio-

132 (1983) 6-13.

Gaunitz,

F.: Charakterisierung

verschiedener

The excellent technical assistance of Mrs. M. Kneer and Mrs. C. Handwerker is gratefully acknowledged. We thank Dr. Colin Rasmussen, Department of Cell Biology, Baylor College of Medicine, Houston, USA for the gift of the plasmid pGEM-CaM containing the chicken Clm probe, and we thank Dr. W. Seyffert, Universitat Tubingen, Germany, for help with the computer programs used for the analysis. This work was supported by the Deutsche Forschungsgemeinschaft, grant AM 26114-9.

and analysis

Nucleic Acids Res. 14 (1986) 10009-10026.

genes from Xenopus laevis. Mol. Cell. Biol. 4 (1984) 507-513.

restriction ACKNOWLEDGEMENTS

E.N.: Compilation

sequences.

von r-Tubulingenen

hypotricher

Ciliaten.

ologie der Eberhard-Karls-Universitat, Geiser, J.R., Van Tuinen, T.N.:

function

der Fakultat

Tubingen,

D., Brockerhoff.

Can calmodulin

aus den Makronuclei

Dissertation

fur Bi-

1990.

S.E., Neff, M.M. and Davis,

without

binding

calcium?

Cell 65

(1991) 949-959. Graves,

B., Johnson,

a promoter

domain

P.F., McKnight, common

S.L.: Homologous

to the MSVLTR

recognition

of

and HSV tk gene. Cell

44 (1986) 565-576. Greslin,

A.F., Loukin,

the macronuclear Hames,

S.H., Oka, Y. and Prescott,

B.D. and Higgins,

Practical Hanahan,

D.M.: An analysis

of

actin genes of Oxytricha. DNA 7 (1988) 529-536.

Approach.

S.J. (Eds.): Transcription

IRL Press, Oxford,

and Translation.

A

1987.

of Eschericha coli with plasmids.

D.: Studies on transformation

J. Mol. Biol. 166 (1983) 557-580. Helftenbein, REFERENCES Ammermann, standing

D.: The contribution of molecular

biology

of hypotrichous and evolution

ciliates to our underof ciliates.

Zoo.

Sci. 7

(1990) 13-22. Ammermann,

sequence

of a macronuclear

DNA molecule

G., v.Berger,

of the macronucleus

mytilus. Chromosoma

L. and Henning,

in the ciliated protozoan

W.: The Srylonychiu

(Berl.) 45 (1974) 401-429.

E., Conzelmann,

Green Publishing Medical

Associates,

School,

Massachusetts

Cambridge,

General

Hos-

MA, 1991.

Baba, M.L., Goodman, M., Berger-Cohn, J., Demaille, J.G. and Matsuds, G.: The early adaptive evolution of calmodulin. Mol. Biol. Evol. 1 (1984) 442-455. T.J., Bugg, C.E., Means, A.R. and structure of calmodulin. Nature 3 15

(1985) 37-40. Babu, Y.S., Bugg, C.E. and Cook, W.J.: Structure at 2.2 A resolution.

of calmodulin

J. Mol. Biol. 204 (1988) 191-204.

refined

K.K., Becker, K.F. and Fritzenschaf, of gene

expression,

C.W. and Hunziker,

DNA-replication

H.: and

genes of Slylonychia lemnae, a

in macronuclear

ciliate. Eur. J. Protistol.

25 (1989) 158-167.

W.: Intracellular

teins: more sites than insights.

Trends

calcium-binding

Biochem.

pro-

Sci. 16 (1991) 98-

103. Jena, P.K., Reddy, A.S.N.

and Poovaiah,

B.W.: Molecular

cloning

and

sequencing of a cDNA for plant calmodulin: signal-induced changes in theexpression ofcalmodulin. Proc. Natl. Acad. Sci. USA 86 (1989) 3644-3648. Johnson,

Babu, Y.S., Sack, J.S., Greenhough, Cook, W.J.: Three-dimensional

structures

DNA-rearrangement hypotrichous Heizmann,

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.M., Seidman. J.G., Smith, J.A. and Struhl, K. (Eds.), Current Protocols in Molecular pital, Harvard

(1985) 415-433. Helftenbein, Regulatory

D., Steinbrtick,

development

Biology.

E.: Nucleotide

for z-tubulin from the ciliate Stylonychia lemnae. Special codon usage: TAA is not a translation termination codon. Nucleic Acids Res. 13

P.F.,

Identification

Landschulz,

W.H.,

Graves,

B.J. and McKnight,

S.L.:

of a rat liver nuclear protein that binds to the enhancer

core element of three animal viruses. 146. Klee, C.B. and Vanaman, 213-321.

Genes

T.C.: Calmodulin.

Develop.

1 (1987) l33-

Adv. Prot. Chem. 35 (1982)

198 Lagace,

L., Chandra,

multiple

T., Woo, S.L.C. and Means, A.R.: Identification

species

of calmodulin

complementary LeJohn,

H.B.:

DNA.J.

Structure

messenger

of

RNA using a full length

Biol. Chem. 258 (1983) 1684-1688.

and expression

of fungal

N.J. and Brownlee,

eukaryotic Putkey,

messenger

G.G.:

and Means, A.A.; Chicken (1983) 11864-11870. Sambrook,

J., Fritsch,

Laboratory

E.F.

Manual,

gene. J.

region sequences

in

calmodulin

L., Stein, J.P., Lai, E.C.

genes.

J. Biol. Chem.

258

and Maniatis,

T.: Molecular

Cloning.

Laboratory

A

Press,

Calcium-binding Wegner,

with a common J.G.,

proteins

M., Helftenbein,

Grummt,

evolution

and phylogeny

RNA sequence

as discerned

comparisons.

from small

Eur. J. Protistol.

27

K.: Structure

J.F., Munjaal,

R.P. and Beckingham,

and sequence of the Drosophila melanoguster calmodulin

G.: Recent

Protistol. Strynadka,

in the study of ciliate genes.

Eur. J.

26 (1990) 2-14. N.C.J.

loop-helix 951-998.

advances

and Hames,

calcium-binding

M.N.G.: proteins.

Proc. Natl.

Marshak,

D.R. and Waterson,

D.M.:

basis of calcium

action.

of an amplification

M., Mnller, promoting

S. and

DNA

se-

ciliate Stylonychiu lemnae. Nucleic Acids

M. and Stephenson,

P.: Role of conserved

point mutants

AAUAAA

prevent messenger

sequence:

RNA 3’end forma-

tion. Science 226 (1984) 1045- 105 1. Yamanaka,

M.K.,

Saugstad,

J.A., Hanson-Painton, and expression

O., McCarthy,

B.J.

of the Drosophila calmod-

ulin gene. Nuclei Acids Res. 15 (1987) 3335-3348. Yazawa,

M., Yagi, K., Toda, H., Kondo,

sequence

S., Nagao,

K., Narita,

S. and Nozawa,

of the Tetrahymena calmodulin

with guanylate

cyclase.

Biochem.

Biophys.

K., Yamazaki,

R.,

Y.: The amino acid

which specifically

interacts

Res. Commun.

99 (1981)

1051-1057.

gene. J. Mol. Biol. 196 (1987) 471-485. Steinbrttck,

sequence.

F.F.:

and pro-

Res. 17 (1989) 8783-8802. Wickens,

Sobue, K., Kakiuchi,

(1991) 207-219. Smith, V.L., Doyle, K.E., Maune,

repeated

E., Mnller, F., Meinecke,

and Tobin, D.L.: Structure M.: Protist

5’leader

and the molecular

F.: Identification

four AAUAAA

NY, 1989.

5461. subunit ribosomal

C.L. and Richards,

are tandemly

Sci. USA 82 (1985) 3998-4002.

quence from the hypotrichous

Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463Schlegel,

Acad.

L., Patton,

Int. Rev. Cytol. 77 (1982) l-60.

263 (1976) 211-214.

T., Lagace,

2nd ed. Cold Spring Harbor

Cold Spring Harbor,

A.S., Ruben,

genes in trypanosomes

Van Eldik, L.J., Zendegui,

3’non-coding

RNA. Nature

J.A., Ts’ui, K.F., Tanaka,

C., Young,

Calmodulin

duce multiple mRNAs

calmoduhn

Biol. Chem. 264 (1989) 19366-19372. Proudfoot,

Tschudi,

Crystal

structures

of the helix-

Annu. Rev. Biochem.

58 (1989)

Zimmer, W.E., Schloss, J.A., Silflow, C.D., Youngblom, J. and Watterson, D.M.: Structural organization, DNA sequence and expression of the calmodulin

gene. J. Biol. Chem. 263 (1988) 19370-19383.