Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.

Regulation of tissue-specific expression of the esterase S gene in Drosophila virilis.

The esterase S gene (estS) of Drosophila virilis is specifically expressed in the ejaculatory bulbs of males. Its sequencing shows similarities between estS product and other esterases of different origin. The transcription of estS in ejaculatory bulbs is at least 2 orders of magnitude higher than in other tissues of males. Two promoters, P1 (distal) and P2 (proximal), and two different transcripts were identified. The promoter P2 is used much more efficiently, and in a stringent, tissue-specific manner. The transcription from P1 takes place in different tissues and stages of development of D. virilis. However, the mRNA transcribed from P1 seems to be inactive in translation as there are three open-reading frames (ORF) between P1 and P2, which may block the translation in P1 initiated mRNA. Insertion of sequence containing the three ORFs into the 5' untranslated region of the CAT gene strongly inhibited expression of CAT. Point mutations destroying the three ORFs completely eliminate the inhibitory effect. Hence tissue-specific expression of the estS gene may depend on control at the level of transcription, promoter selection and translation.

[Primary structure of the esterase s gene from Drosophila virilis]. / Pervichnaia struktura gena ésterazy S Drosophila virilis.

Complete nucleotide sequence of the esterase S gene of Drosophila virilis has been determined. The length of the gene from the transcription initiation site down to the polyadenylation signal is about 1850 bp. Conceptual translation of the DNA sequence reveals a protein 549 amino acid residues long, its presumptive active site being highly similar to active sites of the known esterases of insects and mammals.

Transposition of mobile genetic elements in interspecific hybrids of Drosophila.

In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using 32P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions ("jumping") of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with "synthetic" karyotoypes characterized by different combinations of D. virilis homologous chromosomes and "hybrid" chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition ("jumping") took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such "synthetic" stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.

[Structure of nuclear pre-mRNA. XIII. Hybridization properties of triphosphorylated 5'-end fragments]. / Struktura iadernoi pre-mRNK. XII. Gibridizatsionnye svoistva trifosforilirovannykh 5'-kontsevykh fragmentov.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method. These fragments as well as fragments of total pre-mRNA were polyadenylated by ATP:RNA adenyl-transferase and used as templates for the synthesis of [32P]cDNA by reverse transcriptase in the presence of an oligo(dT)-primer, cDNA transcribed from total fragments of pre-mRNA and from 5'-end fragments (5'-cDNA) were hybridized with excess of nuclear pre-mRNA. The extent of hybridization was 65-70 and 80-85% in different experiments. 18% of total cDNA and 35% of 5'-cDNA hybridized with mRNA from polysomes. A high homology between mRNA and triphosphorylated 5'-ends of pre-mRNA may be explained in the terms of splicing. The sequences adjacent to the triphosphorylated 5'-ends of pre-mRNA represent a specific class with complexity about 2.10(5) nucleotides. Less than 30% of 5'-cDNA hybridized with intermediately repetitive DNA, while the main portion hybridized with unique DNA sequences. About 15% of 5'-cDNA contain oligo (dA) sequences, originated from oligo (U) in the pre-mRNA.

Hybridization properties of sequences adjacent to triphosphorylated 5'-ends of nuclear pre-mRNA from mouse Ehrlich carcinoma.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method /1/. These fragments as well as fragments of total pre-mRNA of the same size were polyadenylated in vitro by ATP:RNA adenyltransferase and used as templates for the synthesis of [32P] cDNA by reverse transcriptase in the presence of an oligo(dT) primer. The use of cDNA transcribed from the triphosphorylated 5'-end fragments of pre-mRNA (5'-cDNA) and from the total pre-mRNA fragments allows one to calculate the complexity of the 5'-end fraction pre-mRNA and to detect these sequences in polysomal mRNA. Sequences adjacent to 5'-phosphorylated ends of pre-mRNA represent a specific class of sequences with a complexity of about 200 kb. It was also found that about 25% of total pre-mRNA and about a half of sequences adjacent to triphosphorylated 5'-ends are present in polysomal mRNA. A high homology between triphosphorylated 5'-end fragments of pre-mRNA and mRNA sequences may be explained in terms of splicing. Less than 30% of 5'-cDNA hybridized to moderately repetitive DNA while most of them are represented by unique DNA sequences. About 15% of 5'-cDNA contained oligo(dA) sequences originated from oligo(U) in pre-mRNA from which it was transcribed.

[Structure of nuclear pre-mRNA. XI. Triphosphorylated and blocked 5'-ends in the pre-mRNA]. / Struktura iadernoi pro-mRNK. XI. Trifosforilirovannye i blokirovannye 5'-kontsy v pro-mRNK.

The nature of the 5'-termini in pre-mRNA isolated from Ehrlich carcinoma cells has been investigated. To discriminate between triphosphorylated 5'-ends and capped structures different methods were used including treatment by alkaline phosphatase and several chromatographic methods. It was shown that heavey pre-mRNA contains a significant number of non-blocked triphosphorylated nucleotides at the 5'-end termini. However, phosphatase resistent, blocked 5'-termini were also found. 5'-terminal nucleotides in triphosphorylated pre-mRNA are G in a 3 : 2 ratio. In contrast to nuclear pre-mRNA cytoplasmic poly(A)+mRNA does not contain triphosphorylated 5'-ends but does contain the "cap" structure only. To elucidate the pre-mRNA topography the localization of homopolymeric regions of pre-mRNA, poly(A) and oligo(U), in relation to 5'terminal structures has been investigated. The experiments showed that the distance between 3'-terminal poly(A) sequences and 5'-end triphosphates is longer than 1500--2000 nucleotides. At the same time the distance between the latter and oligo(U) in pre-mRNA is much shorter.