The sexual inducer of Volvox carteri : purification, chemical characterization and identification of its gene

Abstract

The sexual inducer of Volvox carteri f. nagariensis is a glycoprotein and one of the most potent biological effector molecules known. It is synthesized by sperm cells and converts asexually growing males and females to the sexual pathway. Until now, large‐scale production of the inducer was made impossible by an inherent biological ‘switch’ mechanism, the spontaneous self‐induction of asexually growing males. Here we describe a method overcoming this problem for the first time. Large‐scale production and purification allowed a detailed chemical characterization of the inducer with respect to partial amino acid sequences and sugar composition. Chemically synthesized oligodeoxynucleotides corresponding to derived amino acid sequences were used to screen a genomic gene bank of V. carteri HK 10. A positive clone (Ind‐28) was shown to encode the inducer gene by subcloning and sequencing.

The sexual inducer of Volvox carteri Primary structure deduced from cDNA sequence

“…A cDNA library was constructed in the vector Agtll, using the Pharmacia cDNA synthesis kit. The library was screened either with a 2.7 kb fragment from the sexual inducer gene [8] or with the 25-mer oligonucleotide 5 ' -GAGATGTCAGCTGAGTGGCTGCCGA-3 ' , derived from the genomic sequence of the sexual inducer gene (exon 3 The hgtll cDNA library was screened with inducer-specific genomic DNA (2.7 kb fragment described in [8]). Five recombinant clones were obtained from 1 x lo5 transformants.…”

“…The inducer is one of the most potent biological effector molecules known as it exhibits full effectiveness at about lo-l6 M [2,7]. Only recently has it become possible to purify this inducer in sufficiently large amounts to obtain amino acid sequence data and to identify the inducer gene [8]. A rather complex exon-intron organization of this gene made it impossible to deduce the complete primary structure of the inducer.…”

The sexual inducer of Volvox carteri

“…The growth medium was diluted with 9 volumes of water. Subse¢uently, the indu~r was adsorbexi to SP-scphadex and purified as ~*reviously described [3]. In order to match the relative distances of the original phosphatase gene, the 5" non-coding region of the sexual inducer eDNA was trimmed by digestion with Bal3 !…”

“…Remarkably, the signal peptide of the Voh,ox gene is correctly recognized in yeast and causes secretion of the pheromone into the culture medium. Therefore a purification protocol worked out previously [3] using Volvox culture fluids as the starting material could be applied with only minor modifications (s~tion 2).…”

“…Only recently the inducer was isolated in amounts sufficient for obtaining amino acid sequence data [3] and both the gene and the eDNA of the inducer were cloned [3,4]. However, isolation of the giycoprotein from l/olvox cultures is still a difficult task and produces amounts hardly exceeding the range of 10-100/,tg.…”