Purification, primary structure, bacterial expression and subcellular distribution of an oocyte-specific protein in Xenopus.
This study defines a novel Xenopus laevis protein (P100) that has recently been shown to be recognized by scleroderma patient sera. Using a combination of differential solubility in detergents, hydroxyapatite chromatography and one-dimensional PAGE, P100 was purified to apparent homogeneity and the amino acid sequence was obtained. An oligonucleotide derived from this sequence was used to clone P100 cDNA through a polymerase-chain-reaction cloning strategy. The entire P100 cDNA sequence was determined, identifying a novel 83,000-Da protein. Two alleles for P100 were transcribed in the oocyte, with only one predicted amino acid change between them. Bacterial expression of a clone containing the entire P100 coding region produced a protein that migrated at a mass 15% greater than that predicted from the amino acid sequence, indicating an aberrant electrophoretic mobility. The mRNA transcript for P100 was only expressed during the previtellogenic stages of oogenesis (stages I and II) and was absent from other Xenopus tissues. Similarly, the P100 protein was found only in Xenopus oocytes and was localized to the cytoplasm of these cells. P100 irreversibly bound single-stranded-DNA--cellulose but not double-stranded-DNA--cellulose. These data demonstrate the presence of a novel oocyte-specific protein in Xenopus.
Rother
R P
RP
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City.
Frank
M B
MB
Thomas
P S
PS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002845:Chromatography; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000737:chemistry; D014018:Tissue Distribution; D014169:Transformation, Bacterial; D029867:Xenopus Proteins; D014982:Xenopus laevis
D011506:Proteins; D029867:Xenopus Proteins; C075198:PATL2 protein, Xenopus; D004247:DNA