Molecular characterization of a novel nucleolar protein in starfish oocytes which is phosphorylated before and during oocyte maturation

Abstract

In response to 1-methyladenine, a maturation-inducing substance, starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown through activation of p34 cdc2 -cyclin B, which results in the dispersal of the nucleolus. Little information has been elucidated thus far on nucleolar proteins that are phosphorylated by p34 cdc2 -cyclin B during meiotic maturation. Here, we describe a novel nucleolar protein of the starfish Asterina pectinifera oocyte, which is designated ANO39 and which is phosphorylated during meiotic maturation. A fulllength ANO39 cDNA of 2106 base pairs encodes a polypeptide of 346 amino acids having a calculated M r of 39 005. The amount of ANO39 is kept nearly constant during oocyte maturation and embryogenesis up to the midgastrula stage. The transcript encoding ANO39 was present in growing oocytes but not in full-grown ones, as evidenced by Northern blot hybridization. Ser145 is specifically phosphorylated when ANO39 is incubated in vitro with purified starfish p34 cdc2 -cyclin B. This phosphorylation site corresponds to that is phosphorylated during meiotic maturation in vivo. Immunoblot analysis using phosphoserine145-specific antibody as a probe revealed that some populations of ANO39 of the immature oocytes at the G 2 stage have been already phosphorylated on Ser145 and Ser145 is maximally phosphorylated during meiotic maturation.

Mapping the Functional Domains of Nucleolar Protein B23

“…In the Cterminal end of this sequence there are five aromatic residues: FINYVKNCFRMTDQEAIQDLWQWRKSL. The placement of the aromatic residues, especially the two tryptophans is highly conserved in analogous proteins including starfish nucleolar protein ANO39 (37), sea urchin mitosis apparatus protein p62 (38), Xenopus NO38 (4), and B23 from chickens (39) and humans (3). The requirement for the two tryptophans is reinforced by experiments in which they were replaced by leucines; the resulting mutant protein did not bind DNA.…”

Molecular characterization of a novel nuclear transglutaminase that is expressed during starfish embryogenesis

“…Samples of poly(A) + RNA (0.5 lg) were prepared from staged embryos as described previously [22]. They were denatured and separated by formaldehyde gel electrophoresis and transferred to nylon filters (Amersham Pharmacia Biotech).…”

“…Following centrifugation and removal of the supernatant, the pellets were washed twice with IP buffer, and resuspended with 400 lL of IP buffer (total volume, 500 lL). Control experiments were performed using the affinity-purified anti-ANOC Ig, which was raised against the C-terminal portion of ANO 39, a starfish protein unrelated to nTG [22].…”

“…Twenty picoliters of the solution, along with a small amount of KF96 silicone oil (Shin-Etsu Chemical, Tokyo, Japan), were then microinjected into the germinal vesicle of an oocyte as described previously [22]. Three to four hours later, the injected oocytes were examined for localization of fluorescent proteins under a fluorescence microscope equipped with differential interphase and epifluorescence optics.…”

“…Early starfish development may be directed by two sources of mRNA: (a) a pool stored in the immature oocyte transcribed from the maternal genome during oogenesis such as ANO39 mRNA [22], and (b) newly synthesized mRNA transcribed from the embryonic genome. Northern blot hybridization on poly(A) + RNA from blastulae and gastrulae showed a gradual increase in the signal at 5.0-kb during the progression of embryonic development (Fig.…”