Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1995 Nov 1; 131(3): 563–570.
PMCID: PMC2120627

A zinc-binding domain is required for targeting the maternal nuclear protein PwA33 to lampbrush chromosome loops


In oocytes of the newt Pleurodeles waltl, the maternal nuclear protein PwA33 occurs on the lampbrush chromosomes and in some nucleoplasmic particles of the germinal vesicle. PwA33 is a modular protein and we used site-directed mutagenesis to alter the sequences encoding two metal-binding regions, the C3HC4 (or RING finger) and B-box motifs. Several mutant clones were generated and their synthetic transcripts were injected into Pleurodeles oocytes for in vivo analysis. In the oocyte, all translation products localized in the germinal vesicle. Proteins encoded by RING finger mutant clones were distributed in a pattern identical to that of the wild type protein, but when His266 of the B-box was mutated, PwA33 failed to localize in the lampbrush chromosomes and the nucleoplasmic particles. Using an in vitro colorimetric assay, we demonstrated that PwA33 is a zinc-binding protein and that mutations in the RING finger and B-Box altered its metal-binding properties. The RING finger motif bound two Zn2+ ions and the binding ratios of several mutants were consistent with the tertiary structure recently proposed for this motif. The B-box coordinated one Zn2+ and this binding was inhibited by the His266 mutation. The failure of the His266 mutation to bind zinc and to localize properly within the germinal vesicle suggests that an intact B-box is required for normal functioning of the PwA33 protein in the oocyte.

Full Text

The Full Text of this article is available as a PDF (1.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bandziulis RJ, Swanson MS, Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. [PubMed]
  • Barlow PN, Luisi B, Milner A, Elliott M, Everett R. Structure of the C3HC4 domain by 1H-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger. J Mol Biol. 1994 Mar 25;237(2):201–211. [PubMed]
  • Bellini M, Lacroix JC, Gall JG. A putative zinc-binding protein on lampbrush chromosome loops. EMBO J. 1993 Jan;12(1):107–114. [PMC free article] [PubMed]
  • Berg JM. Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. Proc Natl Acad Sci U S A. 1988 Jan;85(1):99–102. [PMC free article] [PubMed]
  • Berg JM. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. J Biol Chem. 1990 Apr 25;265(12):6513–6516. [PubMed]
  • Borden KL, Martin SR, O'Reilly NJ, Lally JM, Reddy BA, Etkin LD, Freemont PS. Characterisation of a novel cysteine/histidine-rich metal binding domain from Xenopus nuclear factor XNF7. FEBS Lett. 1993 Dec 6;335(2):255–260. [PubMed]
  • Briggs R. Further studies on the maternal effect of the o gene in the Mexican Axolotl. J Exp Zool. 1972 Aug;181(2):271–280. [PubMed]
  • Briggs R, Cassens G. Accumulation in the oöcyte nucleus of a gene product essential for embryonic development beyond gastrulation. Proc Natl Acad Sci U S A. 1966 May;55(5):1103–1109. [PMC free article] [PubMed]
  • Briggs R, Justus JT. Partial characterization of the component from normal eggs which corrects the maternal effect of gene o in the Mexican axolotl (Ambystoma mexicanum). J Exp Zool. 1968 Jan;167(1):105–116. [PubMed]
  • Brothers AJ. Stable nuclear activation dependent on a protein synthesised during oogenesis. Nature. 1976 Mar 11;260(5547):112–115. [PubMed]
  • Chan EK, Hamel JC, Buyon JP, Tan EM. Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen. J Clin Invest. 1991 Jan;87(1):68–76. [PMC free article] [PubMed]
  • de Thé H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991 Aug 23;66(4):675–684. [PubMed]
  • Evan GI, Lewis GK, Ramsay G, Bishop JM. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. [PMC free article] [PubMed]
  • Everett RD, Barlow P, Milner A, Luisi B, Orr A, Hope G, Lyon D. A novel arrangement of zinc-binding residues and secondary structure in the C3HC4 motif of an alpha herpes virus protein family. J Mol Biol. 1993 Dec 20;234(4):1038–1047. [PubMed]
  • Freemont PS. The RING finger. A novel protein sequence motif related to the zinc finger. Ann N Y Acad Sci. 1993 Jun 11;684:174–192. [PubMed]
  • Freemont PS, Hanson IM, Trowsdale J. A novel cysteine-rich sequence motif. Cell. 1991 Feb 8;64(3):483–484. [PubMed]
  • Gall JG, Stephenson EC, Erba HP, Diaz MO, Barsacchi-Pilone G. Histone genes are located at the sphere loci of newt lampbrush chromosomes. Chromosoma. 1981;84(2):159–171. [PubMed]
  • Hunt JB, Neece SH, Ginsburg A. The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase. Anal Biochem. 1985 Apr;146(1):150–157. [PubMed]
  • Inoue S, Orimo A, Hosoi T, Kondo S, Toyoshima H, Kondo T, Ikegami A, Ouchi Y, Orimo H, Muramatsu M. Genomic binding-site cloning reveals an estrogen-responsive gene that encodes a RING finger protein. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11117–11121. [PMC free article] [PubMed]
  • Itoh K, Itoh Y, Frank MB. Protein heterogeneity in the human Ro/SSA ribonucleoproteins. The 52- and 60-kD Ro/SSA autoantigens are encoded by separate genes. J Clin Invest. 1991 Jan;87(1):177–186. [PMC free article] [PubMed]
  • Jack LJ, Mather IH. Cloning and analysis of cDNA encoding bovine butyrophilin, an apical glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule membrane during lactation. J Biol Chem. 1990 Aug 25;265(24):14481–14486. [PubMed]
  • Kakizuka A, Miller WH, Jr, Umesono K, Warrell RP, Jr, Frankel SR, Murty VV, Dmitrovsky E, Evans RM. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell. 1991 Aug 23;66(4):663–674. [PubMed]
  • Kastner P, Perez A, Lutz Y, Rochette-Egly C, Gaub MP, Durand B, Lanotte M, Berger R, Chambon P. Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins. EMBO J. 1992 Feb;11(2):629–642. [PMC free article] [PubMed]
  • Lacroix JC, Azzouz R, Boucher D, Abbadie C, Pyne CK, Charlemagne J. Monoclonal antibodies to lampbrush chromosome antigens of Pleurodeles waltlii. Chromosoma. 1985;92(1):69–80. [PubMed]
  • Le Douarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat P, Heery D, Gronemeyer H, Chambon P, Losson R. The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO J. 1995 May 1;14(9):2020–2033. [PMC free article] [PubMed]
  • Li X, Shou W, Kloc M, Reddy BA, Etkin LD. Cytoplasmic retention of Xenopus nuclear factor 7 before the mid blastula transition uses a unique anchoring mechanism involving a retention domain and several phosphorylation sites. J Cell Biol. 1994 Jan;124(1-2):7–17. [PMC free article] [PubMed]
  • Lovering R, Hanson IM, Borden KL, Martin S, O'Reilly NJ, Evan GI, Rahman D, Pappin DJ, Trowsdale J, Freemont PS. Identification and preliminary characterization of a protein motif related to the zinc finger. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2112–2116. [PMC free article] [PubMed]
  • Mattaj IW. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. [PubMed]
  • Miki T, Fleming TP, Crescenzi M, Molloy CJ, Blam SB, Reynolds SH, Aaronson SA. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5167–5171. [PMC free article] [PubMed]
  • Patarca R, Freeman GJ, Schwartz J, Singh RP, Kong QT, Murphy E, Anderson Y, Sheng FY, Singh P, Johnson KA, et al. rpt-1, an intracellular protein from helper/inducer T cells that regulates gene expression of interleukin 2 receptor and human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2733–2737. [PMC free article] [PubMed]
  • Piñol-Roma S, Choi YD, Matunis MJ, Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 1988 Feb;2(2):215–227. [PubMed]
  • Reddy BA, Kloc M, Etkin L. The cloning and characterization of a maternally expressed novel zinc finger nuclear phosphoprotein (xnf7) in Xenopus laevis. Dev Biol. 1991 Nov;148(1):107–116. [PubMed]
  • Reddy BA, Etkin LD. A unique bipartite cysteine-histidine motif defines a subfamily of potential zinc-finger proteins. Nucleic Acids Res. 1991 Nov 25;19(22):6330–6330. [PMC free article] [PubMed]
  • Reddy BA, Etkin LD, Freemont PS. A novel zinc finger coiled-coil domain in a family of nuclear proteins. Trends Biochem Sci. 1992 Sep;17(9):344–345. [PubMed]
  • Roth MB, Gall JG. Monoclonal antibodies that recognize transcription unit proteins on newt lampbrush chromosomes. J Cell Biol. 1987 Sep;105(3):1047–1054. [PMC free article] [PubMed]
  • St Johnston D, Brown NH, Gall JG, Jantsch M. A conserved double-stranded RNA-binding domain. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10979–10983. [PMC free article] [PubMed]
  • Tafuri SR, Wolffe AP. Dual roles for transcription and translation factors in the RNA storage particles of Xenopus oocytes. Trends Cell Biol. 1993 Mar;3(3):94–98. [PubMed]
  • Takahashi M, Cooper GM. ret transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol Cell Biol. 1987 Apr;7(4):1378–1385. [PMC free article] [PubMed]
  • Takahashi M, Inaguma Y, Hiai H, Hirose F. Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene. Mol Cell Biol. 1988 Apr;8(4):1853–1856. [PMC free article] [PubMed]
  • Wu ZA, Murphy C, Callan HG, Gall JG. Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes. J Cell Biol. 1991 May;113(3):465–483. [PMC free article] [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • MedGen
    Related information in MedGen
  • Protein
    Protein translation features of primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.
  • Taxonomy
    Taxonomy records associated with the current articles through taxonomic information on related molecular database records (Nucleotide, Protein, Gene, SNP, Structure).
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...