Cloning of a Locusta cDNA encoding a precursor peptide for two structurally related proteinase inhibitors.

Two peptides of respectively 35 and 36 residues were recently isolated from Locusta migratoria and their full structural characteristics were established by Edman degradation and mass spectrometry. These peptides were subsequently shown to have a proteinase inhibiting activity. We report here the cloning and characterization of a cDNA encoding a 92-residue precursor with three distinct domains: (I) a typical signal peptide of 19 residues; (II) the peptide sequence of the 35-residue inhibitor separated by a Lys-Arg dipeptide cleavage site from (III) the peptide sequence of the 36-residue inhibitor. We show by Northern blot analysis that the gene encoding this precursor is mainly transcribed in the cells of the fat body.

Isolation and structural determination of three peptides from the insect Locusta migratoria. Identification of a deoxyhexose-linked peptide.

We have isolated three novel peptides from the aqueous extract of the pars intercerebralis of male and female adults of the insect Locusta migratoria. After extensive HPLC purification, the peptides were characterised by automated Edman degradation and electrospray mass spectrometry: one is a 35-residue peptide (3752.3 +/- 1.1 Da) containing six cysteines involved in three intramolecular disulfide bridges, the second is a 36-residue peptide (3919.2 +/- 0.9 Da), also cross-linked by three intramolecular disulfide bridges. This second peptide is post-translationally modified by a single fucose moiety, which was identified by gas chromatography coupled to mass spectrometry. These two peptides show a strong sequence similarity. Additionally, they were also found in larger amounts in the fat body of Locusta; this finding raises the question of their exact site of synthesis. The third peptide (5776.3 +/- 0.9 Da), a 54-residue peptide cross-linked by six intramolecular disulfide bridges, is structurally related to the two other peptides, but to a lesser extent. Mass spectrometry has shown that all the cysteines within these three peptides are involved in intramolecular disulfide bridges; however, the location of these bridges is not yet established and is currently being investigated. A computer search of sequence data banks did not reveal any significant similarity of these three peptides with other known proteins.

Changes in the patterns of RNA synthesis in early embryogenesis of Xenopus laevis.

We analyzed the accumulation of newly-synthesized heterogeneous mRNA-like RNA, 4 S RNA, 5 S RNA, snRNAs and rRNA before and after the midblastula transition (MBT) in Xenopus laevis embryogenesis. Based on the kinetics of the labeling, we concluded that the pattern of RNA synthesis in Xenopus embryogenesis changes following at least three characteristically different phases. The first phase is the pre-MBT stage, which is characterized by the synthesis of heterogeneous mRNA-like RNA, accompanied by the synthesis of small amounts of 4 S RNA, 5 S RNA and snRNAs. The second phase is the MBT stage which is characterized by a large activation (about 50-fold increase on a per cell basis) of 4 S RNA synthesis. The third phase is the post-MBT stage which is characterized by the commencement and increase in rRNA synthesis. We assume that RNA polymerases II, III and I are activated in this order in early Xenopus embryogenesis.

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells.

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.

Synthesis of heterogeneous mRNA-like RNA and low-molecular-weight RNA before the midblastula transition in embryos of Xenopus laevis.

It has been proposed and is now widely accepted that in Xenopus laevis embryogenesis RNA synthesis starts only at and after 12 rounds of cleavage, at the time of the midblastula transition (MBT). In this report, however, we provide evidence that RNA synthesis takes place prior to the MBT stage in normally developing Xenopus embryos. In the present experiments, we cultured fertilized eggs in 80 mM phosphate buffer and loosened the adhesion between blastomeres, so that [3H]uridine could be incorporated into blastomeres from the surrounding medium. By this method and also by microinjection of [3H]GTP, we found that embryos synthesize heterogeneous, nonribosomal, high-molecular-weight RNAs and a relatively small amount of low-molecular-weight RNA as early as the sixth cleavage. RNAs synthesized were not of mitochondrial origin, and the synthesis was sensitive to actinomycin D and alpha-amanitin. From these results we conclude that mRNA-like RNA and low-molecular-weight RNA start to be synthesized during the cleavage stage.

Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis.

The fate of bacteriophage lambda-DNA was examined after injection into the fertilized eggs of Xenopus laevis. Injection of a large amount of lambda-DNA (ca. 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes. Southern blot analysis revealed the persistence of injected lambda-DNA until the blastula stage. The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.

Ammonium ion as a possible regulator of the commencement of rRNA synthesis in Xenopus laevis embryogenesis.

Recently, we have shown that ammonium salts and amines at an external concentration of 3 mM selectively inhibit rRNA synthesis in Xenopus disaggregated neurula cells. We studied here the change in the amount of ammonia within the embryo and its inhibitory action on the commencement of rRNA synthesis which normally occurs at the blastula stage of development. Ammonia exists at ca. 50 ng/egg (or ca. 3.0 mM at an intra-egg concentration) in the unfertilized egg. This level was maintained during cleavage and then sharply decreased during the blastula stage to the level of ca. 20 ng/embryo (or 1.2 mM) in postblastular stages. Ammonia was extracted from cleavage embryos in a form of ammonium chloride and confirmed to selectively inhibit rRNA synthesis in neurula cells. With authentic ammonium chloride, ammonia was found to be promptly incorporated into cells and to inhibit rRNA synthesis within 1 hr after treatment. In blastula cells, ammonium salts reversibly inhibited the commencement of the synthesis of rRNA, but not hnRNA, 5 S RNA and U1, U2, and U5 snRNAs. The inhibition was at the step of transcription of 40 S pre-rRNA but not the processing or degradation of the processed rRNA. Ammonium salts did not inhibit DNA synthesis, protein synthesis, cell division, and cellular reaggregation. These observations suggest that ammonium ion may be involved in the regulation of the commencement of rRNA synthesis in Xenopus embryogenesis, although it is not yet clear if the ammonium ion exerts its effect directly upon the rDNA transcription system.