A sequence-specific protease cleaves a maternal, cortical protein during early embryogenesis in Sciara coprophila (Diptera).

One of the first events of egg activation in Sciara coprophila (Diptera) is the disappearance of an abundant maternal 38-kDa protein (p38) and the simultaneous emergence of an abundant 35-kDa protein (p35). Western blotting experiments using monoclonal antibodies directed against p38 reveal that p38 and p35 are serologically related and indicate that maternal p38 is transformed into p35 during early development. This transition is possibly accompanied by a conformational change in the part of the protein that is common to both protein species. The processing of p38 to p35 can be mimicked by trypsin treatment in vitro, suggesting that a trypsin-like protease is responsible for this conversion in vivo. Immunostaining indicates that the p38 class of antigens is evenly distributed in the periplasm of early cleavage embryos. After the arrival of nuclei in the periplasm, the antigens become associated with the infolding cellular membranes. A similar membrane association of actin can be observed with anti-actin antibodies. Nevertheless, p38 and actin are clearly distinct from each other. We presume that p38 is a product of a maternal effect gene necessary for early dipteran development.

Specific radioimmunoprecipitation of histone H2A antigens by protein A conjugated sepharose.

A modified radioimmunoprecipitation technique is described which allows the specific detection of histone H2A antigens. The technique circumvents unspecific binding of histones to the bacterial adsorbent.

Appearance of two maternally directed histone H2A variants precedes zygotic ubiquitination of H2A in early embryogenesis of Sciara coprophila (Diptera).

A monoclonal antibody raised against Drosophila histone H2A.l (15 kDa) recognizes two H2A isoforms, H2As (16kDa) and H2Af (15 kDa), and a modified ubiquitinated form, uH2A (25 kDa), in the fungus gnat Sciara coprophila. Both variants derive from different mRNAs as shown by in vitro translation, immunoprecipitation, and fluorography. Whereas H2Af is present throughout embryogenesis, the Sciara-specific variant H2As is preferentially expressed during cleavage. These developmental profiles reflect a program of histone H2A expression that is regulated in early embryogenesis by differential translation of maternal mRNAs. Later control is effected by zygotic transcription and degradation of maternal mRNA. Ubiquitination of H2A is first detected at the syncytial blastoderm stage: these results and data obtained in other dipteran systems suggest that this modification event is general for Diptera and shows a tight correlation with initiation of zygotic transcription. The first phases of the histone H2A expression program are maternally controlled and not dependent on fertilization. In sharp contrast, ubiquitination of H2A depends on fertilization-dependent processes rather than on egg activation alone. Expression and modification of histones H2A are therefore differently controlled. The significance of uH2A and H2As is discussed.

Regulation of phosphate incorporation into four brain phosphoproteins that are affected by experience.

Various regulators of protein kinase activities were tested for their effects on the in vitro transfer of phosphate from [gamma-32P]ATP to four proteins of rat brain synaptic particulate preparations. One protein, of apparent molecular weight 44,000, accepted 32P in the presence of 8 mM EDTA and no added Mg2+. It was the major phosphoprotein of brain mitochondria. Its phosphorylation was inhibited by pyruvate and stimulated by K+, and it comigrated in electrophoretic gels with authentic alpha-subunit of pyruvate: lipoamide oxidoreductase (decarboxylating) (EC 1.2.4.1) from bovine heart. The major kinase acting on three proteins of apparent molecular weights 24,000, 21,000, and 19,000 was stimulated by Ca2+, by preincubation with phospholipase C, and by 12-tetradecanoyl 4-beta-phorbol 13-acetate. Phosphorylation of these lower-molecular-weight proteins was inhibited by ACTH1-24, by cyclic 3',5'-adenosine monophosphate, and by 50 microM trifluoperazine. The stimulatory effect of Ca2+ was antagonized by calmodulin. The kinase in question appears to be B-50 protein kinase or protein kinase C.