Bürglin TR et al. (1987), Cloning of nucleoplasmin from Xenopus laevis oo...

XB-ART-28246

Genes Dev 1987 Mar 01;11:97-107. doi: 10.1101/gad.1.1.97.

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Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression.

Bürglin TR , Mattaj IW , Newmeyer DD , Zeller R , De Robertis EM .

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Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes. We cloned a cDNA coding for nucleoplasmin from an expression library of immature Xenopus laevis oocytes. The deduced amino acid sequence shows that the carboxyl terminus is very hydrophilic and contains an unusual stretch of 12 glutamic acid residues, which is consistent with one of the proposed functions of nucleoplasmin--that of promoting chromatin assembly. The last 50 amino acids are lysine- and alanine-rich and contain short stretches of homology to histone H1. These regions could be involved in interactions with nucleosomes. The levels of nucleoplasmin mRNA and protein during oogenesis and embryogenesis were investigated using Northern blots, Western blots, and in situ hybridization to oocyte sections. The mRNA is detected during oogenesis but not during embryogenesis, suggesting that nucleoplasmin may be an exclusively maternally expressed gene. However, the protein is present throughout embryogenesis and undergoes pronounced changes in its level of phosphorylation during maturation of the oocyte and just after midblastula transition. These results support the notion that nucleoplasmin is not only important in oocytes but also plays a major role during the rapid cleavages of early embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: npm1 npm2 tbx2

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	Figure 1. Immunolocalization of nucleoplasmin in the nucleus of X. laevis oocytes. Paraffin sections of stage 5 oocytes (Dumont 1972} were incubated with anti-nucleoplasmin serum and the antibodies were detected with Protein A-gold complexes. The gold staining was then enhanced using a photochemical silver method. Bar, 200 um.
	Figure 2. Characterization of the clone using affinity-purified antibodies. Fusion proteins from hgtl 1 and clone N7.2 were expressed in E. coli Y1089. The cells were sonicated and the protein extracts were separated on 7.5% SDS-polyacrylamide gels. Gels were then either stained with Coomassie Blue {lanes a-d) or transferred to nitrocellulose and stained with anti-nucleoplasmin serum (lanes e-g) or stained with affinity-purified antibodies against the fusion protein (lane h). Xenopus oocyte extracts were separated on 15% SDS-polyacrylamide gels, transferred to nitrocellulose, and stained with affinity-purified antibody {lane i)or crude anti-nucleoplasmin serum {lane j). {Lane a) Protein molecular weight markers m first three markers from the top are: myosin, 200 kD; 13-galactosidase, 116 kD; phosphorylase B, 92.5 kD. (Lane b)Crude hgtl 1 protein extract from a culture not induced with IPTG. (Lane c) Same as lane b, but induced with IPTG. (Lane d) Crude protein extract of the N7.2 clone from a culture induced with IPTG. The extract was precipitated with 35% ammonium sulfate prior to gel separation. Note the difference in size of the fusion protein {arrow} compared with B-galactosidase. {Lanes e-g) Same samples as lanes b, c, and d, respectively, after transfer to nitrocellulose and staining with anti-nucleoplasmin serum. Note the staining of the fusion protein. (Lane h) Same as lane g, but stained with anti-nucleoplasmin serum which had been affinity- purified against N7.2 fusion {see text and Materials and methods). Only the fusion protein stains now. (Lane i) Same affinity-purified antibody as in lane h stains only nucleoplasmin on Xenopus oocyte extracts. {Lane j) Crude anti-nucleoplasmin serum shows some other cross-reacting bands on Xenopus oocyte extracts.
	Figure 3. Sequence of nucleoplasmin A. The deduced amino acid sequence of the clone is in frame with ~-galactosidase. The first two amino acids are coded by the EcoRI linker used for constructing the cDNA library. At the end of the clone a possible polyadenylation signal is found (underlined). The last four bases are EcoRI linker sequence. The numbering of the sequence is provisional, since the 5' end is missing.
	Figure 4. Homologies of the carboxyl terminus to histones. Gonadal histone H1 of sea urchin (Parechinus angulosus) and histone H1.3 of rabbit show short stretches of high homology to the lysine-rich parts of the carboxyl terminus of nucleoplasmin.
	Figure 5. Secondary structure prediction of the amino acid sequence of N7.2. The bars indicate the regions where a-helixes, turns, and B-sheets might form. The hatched box indicates the polyglutamic acid stretch. The open box indicates the stretch of four lysines, which is most likely involved in nuclear transport. The tail region starts upstream of the glutamic acid stretch.
	Figure 6. Dark-field micrograph of paraffin sections of oocytes hybridized with the clone N7.2 in situ. (a) Immature ovary of a young frog. Note the silver grains over the cytoplasm. Bar, 50 ~m. (b) Ovary of an adult frog with oocytes of different stages. Smallest oocytes are previtellogenic. Bar, 100 ~m. This method is specific, since hybridization using U2 snRNAs was shown to be nuclear (Mattaj et al. 1985).
	Figure 7. Developmental profile of nucleoplasmin message and protein during oogenesis. Stages of oogenesis are according to Dumont (1972). (a) Northern blot of stages 1-6 and in vitromatured (M) oocytes probed with clone N7.2. Total RNA from 2.5 oocytes was loaded per lane. Molecular weight markers (pBR322 digested with RsaI)are indicated on the left. (b) Western blot of stages 1-6, and in vitro-matured (M) oocytes. Extracts of five oocytes per lane were separated on a 15% SDS gel, transferred to nitrocellulose, and immunostained with anti-nucleoplasmin serum. Note the shift in mobility between stage 6 and matured oocytes, and that the cross-reacting bands do not shift. The gel is somewhat overexposed so as to show the fainter bands in the early stages. Arrows indicate the nucleoplasmin bands.
	Figure 8. Developmental profile of nucleoplasmin message and protein during embryogenesis. Stages are according to Nieuwkoop and Faber {1967). {a) Northern blot of different stages of embryogenesis probed with clone N7.2. Poly(A) + RNA from 40 embryos was loaded in each lane. The same blot had previously been used by Carrasco et al. (1984} and Miiller et al. {1984}, showing that poly(A) + RNA is present in all stages. (b) Western blot of different stages of embryogenesis. Two stage 5 oocytes (oo), eggs, or embryos of the indicated stages were separated on a 10% SDS gel and analyzed as in Fig. 7b. {Lane 3) Four-cell stage; {lane 5) 16-cell stage; {lanes 8 and 9a) blastula; {lane 9b) late blastula; {lanes 10 and 11) gastrula; (lane 27)tail bud; (lane 41) swimming tadpole. Note the reduced electrophoretic mobility of nucleoplasmin protein during the period from egg to midblastula transition, when compared with oocytes and late embryos. [The impression of a gradual increase in electrophoretic mobility during early embryogenesis (egg to stage 9) is merely due to the curvature artifact {"smile" of the gel).] Arrows indicate the shifts in electrophoretic mobility.