The stringlike genes of the limpet Patella vulgata

Gene, 172(1996)261l265 0 1996 Elsevier Science B.V. All rights reserved.

GENE

261

03781119/96/$15.00

09761

The stringlike genes of the limpet Patella vulgata (Cloning; nucleotide sequence; genomic organization; cdc25 homolog; gene expression; development)

Annemieke van der Kooij, Alexander J. Nederbragt, Hans J. Goedemans and Andr6 E. van Loon Department

of Experimental Zoology, Utrecht University, Utrecht, The Netherlands

Received by J.L. Slightom:

3 August

1995; Revised/Accepted:

20 November/30

November

1995; Received at publishers:

20 February

1996

SUMMARY

As a first step in analyzing the function of a cdc25 homolog during the embryonic development of Patella vulgata (Pv), genomic clones encoding these stringlike proteins (Stl) were isolated and characterized. These clones belong to four groups which are derived from different regions of the Pv genome. As the sequences of Stl genes from two of these groups are almost identical, we suggest that these genes represent copies of the same gene. The St13 gene, which has been analyzed in detail, consists of four exons separated by three introns. Its sequence encodes a 250-amino-acid protein with a calculated weight of 28 kDa. The St1 protein contains regions conserved in all other cdc25 proteins. Stl messengers are not stored maternally in Pv oocytes and Stl transcription only starts after the first embryonic cleavages.

INTRODUCTION

In this report, we describe the isolation and characterization of Pv stringlike (Stl) genes. The St1 proteins are related to the cdc25 protein family, conserved predominantly in the C-terminal parts. The cdc25 protein acts as a dual phosphatase, capable of dephosphorylating Thr and Tyr residues. It activates the cyclin B-~34”~“~complex, essential for cells to enter mitosis, at the end of the GZphase (Millar and Russell, 1992). Expression of the Stl genes might function as a keyregulator of the cell division arrest in trochoblasts, which accompanies trochoblast differentiation during embryCorrespondence

to: Dr. A.E. van Loon,

Department

of Experimental

Zoology, Utrecht University, Padualaan 8, 3584 CH Uttecht, Netherlands. Tel. (31-30) 253 35 73; Fax (31-30) 253 28 37;

The

e-mail: [email protected] Abbreviations: cDNA, DNA

aa, amino acid(s); bp, base pair(s); cdc, cell division cycle; complementary to RNA; Ce, Caenorhabditis elegans;

Dm, Drosophila melanogaster; Hs. Homo sapiens; kb, kilobase or 1000 bp; nt, nucleotide(s); MPFSW, Millipore filtered sea-water; ORF, open reading

frame; PCR, polymetase

chain reaction;

Pu, Patella uul-

gata; rRNA, ribosomal RNA, SC, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Stl, stringlike protein; Stl, gene (DNA, RNA) encoding Stl; UTR, untranslated region(s); Xl, Xenopus laeois. PII SO378-1119(96)00164-3

onic development of the limpet Pv. Trochoblasts complete their final division during the sixth mitotic cycle when the embryonic cell cycle changes considerably and are arrested later in G2-phase. Whereas the first cleavage cycles occur synchronously and consist of alternating Sand M-phases only, cells divide asynchronous from this sixth mitotic cycle. Elongation of this cell cycle is caused by the appearance of a G2-phase (Van den Biggelaar, 1977; unpublished results). A similar transition in embryonic cell cycle has been described for the Drosophila embryo. The first thirteen cycles are rapid and synchronous. The fourteenth is asynchronous and elongation of this cycle is also based on the appearance of a G2-phase (Glover, 1991). In this fourteenth cycle, initiation of mitosis is controlled by zygotic expression of the string gene, encoding a protein related to the cdc25 family. Moreover, a mutation in this gene results in a cell cycle arrest in G2 during the fourteenth mitotic cycle (Edgar and O’Farrell, 1989; 1990). The resemblance of the fourteenth mitotic cycle in Drosophila to the sixth in Pu, in which trochoblasts complete their final division, and the cell cycle arrest of trochoblasts in G2 suggest that trochoblast arrest is controlled by expression of a Pv string homolog.

262

primers, when compared on the aa level. Several Pv cDNA libraries of different developmental stages were screened with this PCR probe. We did not succeed in isolation of a Pv cdc25 cDNA clone, although its mRNA should be present at these developmental stages according to the PCR results and Northern blot analysis (section c). However, screening the Pv genomic library with the PCR fragment enabled us to isolate ten clones. One clone was analyzed in detail. A restriction map of this hSt13 clone is shown in Fig. 1A. The PCR fragment hybridizes to three fragments after Sal1 + XhoI digestion. Sequence analysis of these fragments revealed two ORFs. The first ORF contains a complete St1 gene (Fig. 1B). This St13 gene consists of four exons separated by three introns. A region with repeating sequences was found about 1 kb upstream from the ORF. This might suggest that the 1-kb sequence between these two features contains the 5’ UTR of the gene as well as the promoter sequence. The aa sequence was deduced from the nt sequence based on comparison with known cdc25 aa

As a first step to analyze the involvement of such a homolog in the trochoblast arrest, we have isolated and characterized genomic clones encoding these St1 genes. We report here the sequence of the St13 gene, the results on the genomic organization of StZ genes and the expression during early embryonic stages.

EXPERIMENTAL

AND DISCUSSION

(a) Cloning, mapping and sequencing of a StZ gene The C-terminal part of the cdc25 protein is conserved between all known homologs. Degenerate primers were designed to fit two of the conserved parts in this region, namely the IIDCRYPYE - and FHCEFSSER sequences (see Fig. 2). We have isolated a 180-bp fragment from PCR reactions performed with first-strand cDNAs produced from total RNA of embryonic stages, but not from oocyte RNA. Sequence determination revealed additional sequence identity in the region between the degenerate

A AStl3 clone

s

Xh

I

I

S

Xh

Xh

S

S

I

1

B X

S

St!3 gene

I

I E B I

III EBE

I I

I

E

B

ORFl

I

C St/

X

6

B

. I

Ba

I

S I

pseudogene

c

EX

n

B

I

X 2

1

ORFZ

’

I I

EaX

Fig. 1. Genomic organization of the St13 gene. (A) Restriction map of the 14.6-kb 1St13 clone, showing the Sal1 (S) and XhoI (Xh) restriction sites. The 3.2-kb, 1.9-kb and 1.3-kb fragments that hybridized to the PCR probe are marked by the striped box. The open boxes indicate the two ORFs, which represent fragment

the St/3 gene and a St[ pseudogene

and the S-terminal

respectively.

700 bp of the 1.9-kb XhoI-XkoI

(B) Restriction

fragment.

map of the StI3 gene. The map consists

The BamHI

of the 3.2-kb SalI-XhoI

(Ba), BglII (B), EcoRI (E) and XbaI (X) restriction

sites are

indicated as well as the SaiI and XkoI restriction sites. The gene exists of four exons (filled rectangles) and three introns (open rectangles). A putative polyadenylation signal is present 180 bp downstream from the stop codon (filled square). The thick line marks the area that contains repeating sequences. XkoI-Sal1 C-terminal

(C) Restriction map of the Stl pseudogene. The map consists of the 3’-terminal 1.2 kb of the 1.9-kb Xkol-XkoI fragment and the 1.3-kb fragment. Restriction sites, exons and introns are indicated as in (B). Compared to the St/3 gene, a 112-bp fragment, containing the 16 aa and the first part of the 3’ UTR, is deleted (filled triangle).

A mutated

polyadenylation

signal is present

115 bp downstream

from the

ORF (open square). A 250-bp fragment that is nearly identical to the 3’ UTR of the St/3 gene (from nt 3258 to nt 3525, see Fig. 2) is marked by thick line 1. Thick line 2 indicates a 485-bp fragment that is also nearly identical to the 3’ UTR of the Sr/3 gene (from nt 3356 to nt 3827, see Fig. 2). The 485-bp fragment

contains

an intact polyadenylation

signal. Methods: Degenerate

primers

were designed,

based on sequence

similarities

between

the cdc25 protein homologs of SC, Sp, Dm and Hs. The St11 primer set consists of 5’-ATGGATCCAT(YA)AT(YA)GAYTGYMGNTAYCCNTAYGAR and the St13 primer set of T-ATGTCGACNCGYTCNGANGARAAYTCRCARTGRAA (M, A/C; N, A/C/G/T, R, A/G, Y, C/R). cDNA was synthesized using random hexanucleotide primers and total RNA of immature and mature oocytes as well as of 4, 8 and 16 h old embryos (GeneAmp RNA PCR kit, Perkin Elmer Cetus). PCR reactions were performed, consisting of 40 cycles: 2 min 94°C 3 min 40°C and 3 min 72°C. A 1X0-bp fragment was isolated from these reactions, digested with SalI+BamHI and cloned into the pGEM3 sequenced, which proved to be identical (T7 sequencing kit, Pharmacia).

vector (Promega,

Madison,

WI, USA). Five clones were

263 sequences and on consensus sequences for splice donor and acceptor sites. The putative start codon at nt 1995 matches the Kozak consensus sequence, as it has an A at the -3 position (Kozak, 1986). Both in frame ATGs, at nt 1992 and at nt 2121, do not satisfy this requirement. A putative polyadenylation signal (AATAAA) is present at nt 3364,180 bp downstream from the stop codon. The sequence of the St13 gene encodes a 250-aa protein with a calculated molecular weight of 28 kDa (Fig. 2). Another ORF was found 730 bp downstream from the St13 stop codon. The sequence of this ORF is almost identical to the St13 gene. It consists of the same alternation of exons and introns (Fig. 1C). However, several mutations causing frame-shift were found in the protein encoding parts, which would prevent synthesis of a functional protein. Furthermore, sixteen C-terminal aa and the first part of the 3’ UTR are deleted in comparison to the St13 gene. The AATAAA polyadenylation signal in the remaining part of the 3’ UTR has been changed into AACAAA. Part of the 3’ UTR is repeated further downstream. The features of this ORF imply it to be a pseudogene. The ten genomic clones were subdivided in four groups based on restriction analysis. A clear overlap in fragments between these groups was not found. Each group had its characteristic set of internal EcoRI, BglII or XhoI fragments, which hybridized to the 1.9-kb XhoI fragment of the hStl3 clone. These sets were subsets of the hybridizing

exons are snown with encoded putative primers,

aa smgle letter codes. An in frame upstream

EcoRI, BglII or XhoI fragments found after Southern blotting of Pu genomic DNA, implying the four groups to be derived from different regions of the Pu genome. Thus, we assume that there are at least four stringlike genes in the Pv genome. Based on our preliminary sequence data of a 3.2-kb XhoI fragment subcloned from the hStl12 clone, these could prove to be very similar to the St13 gene. The sequence of this 3.2-kb XhoI fragment, which also hybridized to the PCR fragment, is nearly identical to the 5’part of the St13 gene (99% nt identity), and its deduced aa sequence is indistinguishable from st13. (b) Similarity with other cdc25 proteins The C-terminal part of the St1 protein is similar to the C-terminal parts of other cdc25 proteins (Fig. 2). Conservation is concentrated in a stretch of 150 aa starting from aa 61. This region shows the greatest sequence identity with the Dm string protein (51%, Edgar and O’Farrell, 1989), and the least with SC MIHl protein (33%, Russell et al., 1989). Apart from the Ce cdc25 protein, the St1 protein has the lowest molecular weight of all known cdc25 proteins (Sulston et al., 1992). In both cases, this is caused by the limited length of the nonconserved N-terminal part of the proteins. Combinations of the aa SP or TP preceded or followed by polar or basic aa are potential sites for phosphorylation by the ~34”~~~ -cyclin B kinase, which activates cdc25

stop codon

and a putative

polyadenylation

signal are underlined.

The

phosphorylation sites for the ~34~“- cyclin B complex are shown in bold (SP). The aa sequences, which are homologous to the degenerate are shown in italic. The aa consensus based on identity between six of nine cdc25 sequences from different species is indicated by asterisks

below the aa sequence

(Edgar

and O’Farrell,

1989; Kakizuka

et al., 1992; Kumagai

and Dunphy,

1992; Galaktionov

and Beach,

1991; Russell and

Nurse, 1986; Sulston et al., 1992; O’Connell et al., 1992; Russell et al., 1989). Methods: All fragments of the hSt13 clone, hybridizing with the PCR probe, were cloned bluntended in pGEM5 (Promega). Sequence analysis (T7 sequencing kit, Pharmacia) was performed using pGEM3 or pGEM5 subclones containing inserts produced by restriction enzymes. GenBank accession No. X89982.

264

(Moreno and Nurse, 1990). Such sites are also present in the N-terminal part of the St1 protein (2 x SP, Fig. 2). Six of the seven putative phosphorylation sites in the human cdc25C protein were phosphorylated by the p34”dc2cyclin B kinase in vitro (Strausfeld et al., 1994). (c) St1 messenger level during early embryonic

tion of the level (Fig. 3A and B). The increase of St1 messenger level is cell cycle-independent during these stages. In situ hybridization experiments confirmed that Stl messengers are present during the whole cell cycle (our unpublished data). It should be noted that the St! expression can not be contributed to only one of the St1 genes, as the St1 genes known so far show 99% nt identity.

development

After Northern blot analysis, Stl messengers could not be detected in the RNA extracts of immature and mature oocytes. The absence of the messengers in oocytes explains why we did not succeed in isolating a fragment from oocyte RNA extracts via the PCR method. St1 messengers were detected not earlier than the 4-cell stage (Fig. 3). A faint band was visible at that point only after long exposure of the film. The St1 messengers were determined to be 1.7 kb of length, and their level rises from the S-cell stage, and reaches a peak at 10 min after the fifth cleavage, when the embryo has reached the 32-cell stage. The level decreases again at the time the first divisions of the asynchronous sixth mitotic cycle occur (at 60 min after the 32-cell stage). A short and a longer exposure of the film are shown to allow a correct interpreta-

abcdefgh

C

Fig. 3. The level of Stl messengers Lanes: (a) 4-cell stage, (third cleavage);

during

embryonic

20 min after second

(c) 20 min after third cleavage;

development.

cleavage;

(d) Conclusions (I) We suggest that the Pv St/3 and St112 genes represent two copies of the same gene, as these are almost identical throughout their sequences, including introns and the immediate upstream sequences, while more distant upstream sequences differ obviously. Only two of the four hSt1 groups, isolated after screening of the genomic library, have been analyzed in more detail. It remains possible that the St1 genes present in the hSt1 clones, other than hStl3 and hStll2, represent homologs to the St13 gene. The existence of homologs to the St13 gene in Pv is not unlikely, as different cdc25 homologs are known to exist within one organism, which have distinct functions (Sadhu et al., 1990; Galaktionov and Beach, 1991; Nagata et al., 1991; Alphey et al., 1992; Jinno et al., 1994; Hoffmann et al., 1994). (2) The presence of Pv St1 messengers is not crucial for oocyte maturation or the first mitotic divisions, as St1 messengers were absent during these developmental stages. However, in Dm, maternal messengers of the cdc25 homologs string and twine are stored during oogenesis, and are essential for meiosis and early embryonic development (O’Farrell et al., 1989, Alphey et al., 1992, WhiteCooper et al., 1993). As involvement of a cdc25 homolog in these divisions in Pv seems inevitable, we suggest that, instead of messengers, the protein could be stored maternally, and its activity regulated depending on the cell cycle phase. Such a mechanism operates during maturation and early embryonic cleavage cycles in Xl (Jessus and Beach, 1992; Izumi et al., 1992; Kumagai and Dunphy, 1992).

(A)

(b) S-cell stage

REFERENCES

(d) 16-cell stage (fourth

cleavage); (e) 20 min after fourth cleavage; (f) 32-cell stage (10 min after fifth cleavage); (g) 1 h after fifth cleavage; (II) 2 h after fifth cleavage.

Alphey, L., Jimenez, J., White-Cooper, H., Dawson, I., Nurse, P. and Glover, D.M.: Twine, a cdc25 homolog that functions in the male

The decrease of the level at the 16-cell stage is due to a lower amount of total RNA loaded on the gel, as is visible after methylene blue staining

Edgar, B.A. and O’Farrell,

of the blot. (B) The same as in A, but two times longer exposure. (C) Methylene blue staining of 18s rRNA in samples analyzed above. Methods Total RNA was isolated at various developmental stages as

in the Drosophila embryo. Cell 57 (1989) 177-187. Edgar, B.A. and O’Farrell, P.H.: The three postblastoderm cell cycles of Drosophila embryogenesis are regulated in G2 by string. Cell 62

described in Rosenthal and Wilt (1986). Total RNA (10 pg per sample, 6.6% formaldehyde/0.8% agarose gel) was analyzed by Northern blot-

(1990) 469-480. Galaktionov, K. and Beach, D.: Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic

ting (Sambrook XhoI fragment

et al., 1989). The filter was probed of the hStl3 clone.

with the 3.2-kb SalI-

and female germline

of Drosophila.

Cell 69 (1992) 977-988.

P.H.: Genetic control

cyclins. Cell 67 (1991) 1181-1194.

of cell division patterns

265 Glover, D.M.: Mitosis in the Drosophila embryo - in and out of control. Hoffmann,

I., Draetta,

tase activity

G. and Karsenti,

of human

E.: Activation

cdc25A by a cdk2-cyclin

E dependent

phos-

of the Xenopus cdc25 phosphatase

regulate

its activity.

Mol.

Biol. Cell 3 (1992) 9277939. Jessus, C. and Beach, D.: Oscillation odic association

between

of MPF is accompanied

cdc25 and cdc2-cyclin

by peri-

B. Cell 68 (1992)

S., Suto, K., Nagata,

and Okayama,

A., Igarashi,

A., Sebastian,

Bolada,

M., Kanaoka,

H.: Cdc25A is a novel phosphatase

in the cell cycle. EMBO Kakizuka,

J., Hunter,

Y., Nojima, functioning

H.

early

J. 13 (1994) 154991556. B., Borgmeyer,

T., Hoekstra,

M.: Point mutations

tor codon that modulates

and

I.,

R.M.: A mouse

developmentally

expressed.

translation

flanking

by eukaryotic

A. and

W.G.:

Regulation

the cell cycle in Xenopus extracts.

ventional

protein

phosphatase.

S. and Nurse,

protein

Cell 70 (1992) 1399151. inducer:

A., Igarashi,

additional humans

Russell, P., Moreno,

homolog

M., Jinno,

of fission

Sambrook,

Cell

yeast

cdc25+

H.: An

gene occurs

in

in some cancer cells. New Biologist

in the mitotic

H. and Russell, P.L.: Human

cdc25

mitotic

inducer

homo-

is predominantly

in G2. Proc. Natl. Acad. Sci. USA 87 (1990) 5139-5143.

Laboratory Strausfeld,

of mitotic controls

yeast. Cell 57 (1989) 2955303.

yeast

J., Fritsch,

E.F. and Maniatis,

Manual.

T.: Molecular

CSHL Press, Cold Spring

U., Fernandez,

A., Capony,

J.P., Girard,

kinase by microinjection

J., Du, Z., Thomas,

Halloran,

Cloning.

Harbor,

of human

K., Wilson,

N., Green, P., Thierrymieg,

Ainscough,

M., Durbin,

F., Lautredou,

Nature

Van den Biggelaar,

N.,

of ~34~~~

cdc25C into mammalian

R., Hillier,

L., Staden,

R.,

J., Qui, L., Dear, S., Coulson,

R., Berks, M., Metzstein,

R. and Waterston,

A

NY, 1989.

J., Labbe, J.C. and Lamb, N.J.C.: Activation

~ a beginning.

for p34 cdc2: in vivo veritas?

S., Suto, K. and Okayama,

of the fission

as an inducer

S. and Reed, S.I.: Conservation

K., Reed, S.1, Richardson,

logue

mesentoblast

and is highly expressed

3 (1991) 959-968.

Sadhu,

RNA accumula-

of fission yeast. Cell 45 (1986) 145-153.

A., Craxton,

an uncon-

61 (1990) 549-551. Nagata,

control

protein

of the cdc25

J. 11 (1992)

in Urechis caupo. Dev. Biol.

cells. J. Biol. Chem. 269 (1994) 598996000.

Cell 68 (1992) 407-410.

P.: Substrates

of maternal

oogenesis

117 (1986) 55-63. Russell, P. and Nurse, P.: cdc25+ functions

Sulston, Dunphy,

Millar, J.B.A. and Russell, P.: The cdc25 M-phase Moreno,

during

ribosomes.

44(1986)283-292. Kumagai, during

E.T. and Wilt, F.H.: Patterns

tion and adenylation

the AUG initiaCell

suppresses

nidulans. EMBO

2139-2149. Rosenthal,

Derancourt,

define a sequence

S.A.: An extra

levels and partially

of nimTCdcz5 in Aspergillus

expressed

U., Hermans-Borgmeyer,

M. and Evans,

cdc25 homolog is differentially Genes Dev. 6 (1992) 5788590. Kozak,

A.H., Morris, N.R. and Osmani,

in fission and budding

323-332. Jinno,

M., Osmani,

mutation

of the phospha-

phorylation at the GljS transition. EMBO J. 13 (1994) 430224310. Izumi, T., Walker, D.H. and Maller, J.L.: Periodic changes in phosphorylation

O’Connell,

copy of nimE cyc’i”Belevates pre-MPF

TIG 7 (1991) 1255132.

M., Hawkins,

R.: The C. elegans sequencing

T.,

project

356 (1992) 37-41.

J.A.M.: Development determination

in

of dorsoventral

Pctellu

polarity

and

tlulgcrtn. J. Morph.

154

(1977) 1577186. White-Cooper,

H., Alphey, L. and Glover,

D.M.: The cdc25 homologue

twine is required for only some aspects of the entry into meiosis in Drosophila. J. Cell Sci. 106 (1993) 103551044.