Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain.

Mechanisms controlling the transition of a neural precursor cell from proliferation to differentiation during brain development determine the distinct anatomical features of the brain. Nitric oxide (NO) may mediate such a transition, because it can suppress DNA synthesis and cell proliferation. We cloned the gene encoding the neuronal isoform of Xenopus NO synthase (XNOS) and found that in the developing brain of Xenopus tadpoles, a zone of XNOS-expressing cells lies adjacent to the zone of dividing neuronal precursors. Exogenous NO, supplied to the tadpole brain in vivo, decreased the number of proliferating cells and the total number of cells in the optic tectum. Conversely, inhibition of NOS activity in vivo increased the number of proliferating cells and the total number of cells in the optic tectum. NOS inhibition yielded larger brains with grossly perturbed organization. Our results indicate that NO is an essential negative regulator of neuronal precursor proliferation during vertebrate brain development.

Nitric oxide interacts with the retinoblastoma pathway to control eye development in Drosophila.

Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development [1] [2]. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway [3] [4], which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger [5], can reversibly suppress DNA synthesis and cell division [6] [7]. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly [8] [9] [10]. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however. We used transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors to address this issue. We found that manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. Our data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway.

Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression.

Structure/function relationships of acid beta-glucosidase, the enzyme deficient in Gaucher disease, were evaluated by characterizing the proteins expressed from cDNAs encoding normal and mutant enzymes. Twenty-two Gaucher disease mutations or created mutations were expressed in Spodoptera frugiperda (Sf9) cells and analyzed for catalytic properties, stability, inhibitor binding, and modifier interactions. Many Gaucher disease mutations encoded highly disruptive amino acid substitutions (e.g. P289L and D409V) and produced severely compromised proteins with very reduced activity (kcat < 1% of normal) and/or stability. Six mutant enzymes had sufficient catalytic activity (kcat approximately 5-30% of normal) for extensive studies. The highly conservative substitutions, i.e. F216Y or S364T and V394L, led to severe, but selective, abnormalities of enzyme stability or large decreases in catalytic activity, respectively. The T323I, N370S, and V394L enzymes interacted abnormally with active site-directed inhibitors and localized these residues to the glycon binding region. Selected mutant enzymes were poorly activated by phosphatidylserine (V394L, L444P, and R463C) or by saposin C (L444P and T323I), indicating that the enzyme sites for interaction with these activators were within the carboxyl one-third of the enzyme. Substitutions of Ser, Glu, and/or Gly at residues Asp-443 and/or Asp-445 demonstrated important steric roles for these residues in the active site, but neither is the catalytic nucleophile. Together with previous studies, the present analyses provide an insight into the pathogenesis of Gaucher disease and the functional organization of acid beta-glucosidase.

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.

A long terminal repeat-containing retrotransposon is mobilized during hybrid dysgenesis in Drosophila virilis.

A hybrid dysgenesis syndrome similar to those described in Drosophila melanogaster occurs in Drosophila virilis when a laboratory stock is crossed to a wild strain collected in the Batumi region of Georgia (U.S.S.R). Mutations in various loci obtained during these crosses are presumably induced by the insertion of DNA sequences. We have cloned an induced white mutation and characterized the insertion sequence responsible for the mutant phenotype. This sequence is a 10.6-kilobase (kb) transposable element we have named Ulysses. This element is flanked by unusually large 2.1-kb long terminal repeats. Ulysses also contains other landmarks characteristic of the retrotransposon family, such as a tRNA-binding site adjacent to the 5' long terminal repeat and open reading frames encoding putative products with homology to the reverse transcriptase, protease, and integrase domains typical of proteins encoded by vertebrate retroviruses. Some of the mutations obtained do not contain a copy of the Ulysses element at the mutant locus, suggesting that a different transposable element may be responsible for the mutation. Therefore, Ulysses may not be the primary cause of the entire dysgenic syndrome, and its mobilization may be the result of activation by an independent mobile element.

A hybrid dysgenesis syndrome in Drosophila virilis.

A new example of "hybrid dysgenesis" has been demonstrated in the F1 progeny of crosses between two different strains of Drosophila virilis. The dysgenic traits were observed only in hybrids obtained when wild-type females (of the Batumi strain 9 from Georgia, USSR) were crossed to males from a marker strain (the long-established laboratory strain, strain 160, carrying recessive markers on all its autosomes). The phenomena observed include high frequencies of male and female sterility, male recombination, chromosomal nondisjunction, transmission ratio distortion and the appearance of numerous visible mutations at different loci in the progeny of dysgenic crosses. The sterility demonstrated in the present study is similar to that of P-M dysgenesis in Drosophila melanogaster and apparently results from underdevelopment of the gonads in both sexes, this phenomenon being sensitive to developmental temperature. However, in contrast to the P-M and I-R dysgenic systems in D. melanogaster, in D. virilis the highest level of sterility (95-98%) occurs at 23-25 degrees. Several of the mutations isolated from the progeny of dysgenic crosses (e.g., singed) proved to be unstable and reverted to wild type. We hypothesize that a mobile element ("Ulysses") which we have recently isolated from a dysgenically induced white eye mutation may be responsible for the phenomena observed.

Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction.

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter. The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close. The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease. The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem. The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase.

A rapid method for mapping exposed cytosines in polyribonucleotides. Application to tRNATrp (yeast, beef liver).

A rapid method for mapping exposed cytosine residues in 5'-[32P]-labeled RNA molecules is suggested. The exposed cytosines (C's) are converted into uracyls (U's) by bisulphite treatment at pH 5.8 in the presence of Mg2+, followed by complete modification of the residual (non-exposed) C's by a methoxyamine and bisulphite mixture at pH 5.0. The control RNA is modified only by methoxyamine and bisulphite without the preliminary C leads to U conversion. The location of the exposed C's is determined by comparing the products of partial T1, T2, A and U2 ribonuclease digestions of the C leads to U converted and control RNAs after slab gel polyacrylamide electrophoresis and autoradiography. The method has been applied for mapping exposed cytosine bases in tRNATrp (yeast) which have been found in the anti-codon loop and at the 3'-end of the molecule. In tRNATrp (beef liver), in addition to the same exposed bases, C in the diHU-loop is exposed. The data obtained are in full agreement with what is known about exposed C's for other tRNAs.

The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.

Immunochemical studies of beef pancreas tryptophanyl-tRNA synthetase and its fragments. Determination of the number of antigenic determinants and a comparison with tryptophanyl- tRNA synthetases from other sources and with reverse transcriptase from avian myeloblastosis virus.

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl-tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl-tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl-tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl-tRNA synthetases. The interaction of antibodies with the fragments of beef tryptophanyl-tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. On third of the antiserum antibodies interacting with the C-terminal fragment of the enzyme (Mr approximately equal to 40000) inhibits its activity whereas the antibodies to the N-terminal fragment (Mr approximately equal to 20000) have no effect on the enzyme activity. The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl-tRNA synthetase. A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl-tRNA synthetase was found to bind 9 (+/- 1) molecules of Fab fragments. Antibodies against tryptophanyl-tRNA snythetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl-tRNA synthetase by radioimmunochemical assays.

Tritium labelling of nucleic acids accompanied by conversion of cytosine to uracil.

A new method of incorporation of tritium into nucleic acids with an accompanying conversion of cytosine to uracil is proposed. The method is based on the reaction of nucleic acids with bisulfite in the presence of 3H2O. Under certain conditions poly(C) is quantitatively converted to a radioactive poly(U), whereas similar bisulfite treatment of poly(U) does not result in any tritium incorporation. Specificity of the reaction is confirmed by the results of analysis of modified tRNA and rRNA. Incubation of tRNA with bisulfite and 3H2O does not lead to cleavage of the polynucleotide chain. Similar treatment of the denatured DNA results in tritium incorporation into DNA which is accompanied by a conversion of cytosine to uracil. There is virtually no reaction between native DNA and bisulfite. Only certain cytosone residues in yeast tRNAVal/2a interact with bisulfate providing that reaction is carried out under sufficiently mild conditions.