cDNA encoding maternal cyclin E (HpCycE) has been cloned from the oocyte cDNA library of the sea urchin, Hernicentrotus pulcherrimus, by differential screening with a cDNA probe covering the total poly(A) RNAs of 16 cell-stage embryos and gastrulae. In this communication we describe similarity of amino acid sequences between HpCycE and those of cyclin E from other organisms and maternal origin of HpCycE. The amino acid sequence deduced from the nucleotide sequence of HpCycE cDNA is highly similar to those of human, rat, chicken, Xenopus, zebrafish and Drosophila, while its similarity to other cyclins is much lower. A gene for HpCycE exists as a single copy in the genome of H. pulcherrimus. Northern blotting revealed that the mRNA for HpCycE is maintained at a high level up to the morula stage and thereafter declines.
INTRODUCTION
Cyclins are a group of proteins required for the correct progression of the cell cycle in various organisms, Cyclin E was first isolated from human cells as the cDNA that can restore the yeast CLN mutation (Lew et al., 1991). The amount of human cyclin E is maximal in the late G1 and early S phases, and the cyclin E-cdk2 complex regulates the G1-S transition (Dulic et al., 1992; Koff et al., 1992; Jackson et al., 1995), and transforming-growth-factor-β (TGF-β) prevents the stable assemblage of the cyclin E-cdk2 complex (Koff et al., 1993). In sea urchins, from which cyclin was first detected as a protein that appears and disappears in a cell cycle-dependent manner (Evans et al., 1983), only cDNAs for G2 cyclins have been cloned thus far (Pines and Hunt, 1987), while none of the G1 cyclins such as cyclins C, D and E has been cloned yet.
As part of our efforts to isolate and classify the maternal cDNAs of the sea urchin, we screened the oocyte cDNA library of Hernicentrotus pulcherrimus with cDNA probes that cover the total poly(A)+ RNAs of 16 cell-stage and gastrula embryos, and isolated cDNA clones that are present in the 16 cell-stage embryo but absent from the gastrula. By the cloning and nucleotide sequencing of these cDNAs, we identified cDNAs encoding cyclins A, B and E.
Since little information is available regarding echinodermal cyclin E, we report here the nucleotide sequence of the HpCyclinE cDNA as well as the pattern of its ontogenic accumulation.
MATERIALS AND METHODS
Differential screening of oocyte cDNA library
The total RNA was extracted by the method of Chomczynski and Sacchi (1987) from the 16-ceII embryos and from gastrulae of the sea urchin Hernicentrotus pulcherrimus, and poly(A)+ RNA was isolated from the total RNA using Oligotex-dT30 (Roche, Tokyo, Japan). Digoxigenin (DlG)-labelted cDNA probes representing total poly(A)+ RNA of 16-cell embryos and gastrulae were prepared by reverse transcription with Superscript II (Gibco BRL, Rockville, MD, USA). Duplicate filters of the λgt11 oocyte cDNA library (a gift from Dr. I. Mabuchi of University of Tokyo) were probed with DIG-labelled total cDNA of 16-cell embryo or of gastrula, and 68 plaques with positive signals for the 16-cell probe but negative for the gastrula probe were selected by three rounds of screening. cDNAs isolated from these plaques were amplified by polymerase chain reaction (PCR) with the λgt11 primer (TaKaRa, Otsu, Japan). Amplified cDNA fragments were digested with EcoRI and inserted into pBluescript SK-(Stratagene, La Jolla, CA, USA). The nucleotide sequences of the subcloned fragments were determined by the chain termination method (ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin-Elmer, Foster City, CA, USA) with the pBluescript SK-reverse primer or with the M13-20 primer.
Cloning of HpCycE cDNA
Using as a probe the HpCycE fragment that was excised from HpCycE clone 1, the cDNA library of unfertilized eggs constructed in λgt10 vector was screened for a full-length HpCycE cDNA. Positive clones were rescued into pBluescript SK-, and the nucleotide sequences were determined by exonuclease III deletion followed by the chain termination procedure using the kit described above.
Genomic Southern hybridization
DNA isolated from the sea urchin sperm was digested by EcoRI, Sacl and Pstl. The digests were electrophoresed in 0.7% agarose gel, transferred to a nylon membrane (NY13N, Schleicher & Schuell, Dassel, Germany), and hybridized to the Sad-digested fragment of HpCycE clone 1.
Northern blottings
Five (μg of the total RNA extracted from embryos at various developmental stages was electrophoresed in a 1% agarose gel containing formaldehyde and transferred onto a nylon membrane (NY13N, Schleicher & Schuell). HpCycE mRNA was probed by digoxygenin-labeled antisense RNA prepared from the HpCycE clone 1 using a DIG-RNA labeling kit (Boehringer-Mannheim, Mannheim, Germany), and the hybridized bands were visualized with a DIG Luminescent Detection Kit (Boehringer-Mannheim). Probe hybridizations were performed according to the manufacturer's instructions.
RESULTS AND DISCUSSION
Sixty-eight cDNA clones were isolated by the differential screening of the oocyte cDNA library of the sea urchin Hemicentrotus pulcherrimus, with cDNA probes covering the total poly(A)+ RNA population of the 16 cell-stage embryo and gastruia. One of them, Clone 1, consists of the nucleotide sequence with a significantly long ORF (open reading frame) containing a cyclin box. Since the ORF of Clonel is not completed and lacks a termination codon, we rescreened the cDNA library of unfertilized eggs with Clone 1 as a probe. Clone 2 thus isolated has a significant overlap with Clone 1 in its 5′ half but contains a poly(A)+ tail at its 3′ end. Figure 1 shows the nucleotide sequence constructed from Clone 1 and Clone 2 and the amino acid sequence deduced from it. It consists of 5,333 bp with an ORF of 1,272 bp encoding a polypeptide of 424 amino acids. Its calculated relative molecular mass is 48 kDa. A putative polyadenylation signal is located between +5,238 and +5,283. As Fig. 2 shows, the ORF of Clone 1 contains a stretch of the amino acid sequence highly homologous to the cyclin box of cyclin E of various other animals, with 70.6%, 67.0%, 70.6%, 65.1%, 67.0% and 68.8% sequence identity with human (Koff et al., 1991), rat (EMBL Access. No. P39949), chicken (EMBL Access. No. P49707), Xenopus(Rempel et al., 1995), zebrafish (EMBL Access. No. S52288) and Drosophila (EMBL Access. No. P54733), respectively.
Fig. 1
Nucleotide and amino acid sequence of HpCycE cDNA. The nucleotide sequence was determined from those of Clone 1 and Clone 2. The cyclin box is indicated by an open box and the polyadenylation signal is underlined.
Fig. 2
Comparison of the amino acid sequence of the cyclin box of HpCycE with those of other animals. Dots indicate identical amino acids between HpCycE and those of other animals.
Since the amino acid sequence of the cyclin box of the cloned ORF is very similar to the cyclin E of other animals and evolutionally well conserved, we concluded that the cDNA clones we isolated represent a sea urchin homologue of the cyclin E gene, and designated it HpCycE. The amino acid sequence similarity of HpCycE with other types of cyclins is low (45% with mouse cyclinA and 43% with chicken cyclinA).
To estimate the copy number of the HpCycE gene within the H. pulcherrimus genome, we Southern blotted the EcoRi-, Sad- and Psfl-digests of the sea urchin genomic DNA using DIG-labeled clonel cDNA as the probe. As shown in Fig. 3, only a single band was detected, suggesting that HpCycE is present as a single copy gene in the H. pulcherrimus genome.
Fig. 3
Southern blot of H. pulcherrimus genomic DNA. Two micrograms of sperm DNA was digested with EcoRI, Sad and PstI. HindIll-digested λ DNA was used as the DNA size marker.
Figure 4 shows the results of the Northern blotting of the total RNA extracted from various stages of H. pulcherrimus embryos with DIG-labeled Clone 1 RNA as the probe. Transcripts of HpCycE had accumulated in unfertilized eggs, as expected from their maternal origin. The level of HpCycE mRNA remained almost unchanged until the morula stage, but thereafter gradually declined, indicating the possible involvement of cyclin E with rapid cell proliferation during cleavage, while no obvious zygotic expression of the HpCycE gene during early embryogenesis was detectable. This is similar to the case of Drosophila Cyclin E (Richardson et al., 1993) in that cyclin E (DmcycE) mRNA is supplied maternally, but is rapidly degraded after the completion of cleavage division. Studies of mammalian tissue culture cells reveal that the level of cyclin E is maximal in the late G1 phase and in the early S phase regulating the entry of cells into the S phase (Dulic et al., 1992; Gong et al., 1995, Ohtsubo et al., 1995), while in Drosophila embryos, DmcycE is absent in the G1 phase but appears at the onset of the S phase (Richardson et al., 1995). The rapidly proliferating CNS cells that exhibit no obvious G1 stage express cyclin E constitutively (Duronio and O'Farrell, 1995). The ontogenic fluctuation of HpCycE mRNA observed in the present study agrees well with the results reported on Drosophila embryos and mammalian cells, suggesting that HpCycE is also related in a similar way to the cell cycle transition from G1 to S during cleavage. The constitutive presence of cyclin E in the sea urchin cleaving embryo may be related to the lack of G1 at this stage of development.
Fig. 4
Ontogenic changes in the accumulation of HpCycE mRNA. Total RNA populations were extracted from sea urchin embryos at various stages, and 10 (μg each was electrophoresed, transferred to a nylon membrane, and hybridized with the antisense RNA probe synthesized from Clone 1. The blank arrowhead indicates HpCycE mRNA, and the solid arrowheads indicate rRNAs. 0, unfertilized eggs; 16, 16 cell-stage embryos; Mo, morulae; HB, hatched blastulae; MB, mesenchyme blastulae; G, gastrulae; PI, pluteus larvae.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (08254212) and for Scientific Research (B) to H.S.(07458195), and for Scientific Research (C) to K.A. (08680795) from the Ministry of Education, Science, Sports and Culture, Japan. This research was also supported in part by a Grant Pioneering Research Project in Biotechnology given to H.S. by the Ministry of Agriculture, Forestry and Fisheries, Japan. Research support by a Grant from The Mitsubishi Foundation to H.S. is also acknowledged.
