Organization of ribosomal RNA genes in Mycoplasma capricolum

Summary DNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5)16S-23S-5S(3). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes. Present address: Division of Hematology and Immunology of Internal Medicine, Kanazawa Medical University, Uchinada-Cho, Kahoku-Gun Ishikawa Pref. 920-02, Japan     

A soluble calcium-binding protein from the terrestrial annelidLumbricus terrestris L.

Summary Soluble calcium-binding proteins (SCBP) considerably different from calmodulin were purified from the body wall muscle of the earthwormLumbricus terrestris. Three isoforms were obtained with similar UV absorption spectra and amino acid compositions and an apparent molecular weight close to 20 kDa. They can be distinguished by their histidine and proline content and by their peptide maps. The tissue content, as determined by quantitative ELISA varies individually from 0.1 to 0.3 mmol kg^–1. The calcium-binding property can be demonstrated by Ca²⁺-dependent electrophoretic mobility shift and⁴⁵Ca²⁺ autoradiography on nitrocellulose sheets. The apparentK _D values for the SCBP-Ca²⁺ complex is approximately 10^–7 mol l^–1 as revealed by euquilibrium and flow dialysis experiments. In the presence of 1 mmol l^–1 MgCl₂ the maximum binding capacity of SCBP was determined to be either 2 mol Ca²⁺ mol^–1 protein (SCBP₂) or 3 mol Ca²⁺ mol^–1 protein (SCBP₃). Preliminary studies concerning the functional role of SCBP indicate that it facilitates the diffusion of Ca²⁺ ions by a factor of 2 and is capable of inhibiting the ATPase of isolated body wall muscle actomyosin. The results reveal that earthworm SCBP are similar to vertebrate parvalbumin and to SCBP characterized from aquatic invertebrates.Abbreviations ABTS 2,2-azino-di-(3-ethyl)-benzothiazolinsulfonate - CN-PDE 3:5-cyclic nucleotide-phosphodiesterase - DEAE diethylaminoethyl - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ELISA enzyme linked immuno sorbent assay - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - HRP horseradish peroxydase - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - P _i inorganic phosphate - PMSF phenylmethylsulfonyl fluoride - SCBP soluble calcium-binding protein - SDS sodium dodecyl sulphate - SPDP N-succininydyl-3-(2-pyridyldithio)propionate - SR sarcoplasmic reticulum - Tris tris(hydroxymethyl)-aminomethane - UV ultraviolet     

Differential coupling efficiency of chemically activated amino acid to tRNA

Summary Interaction based on possible chemical affinity of an amino acid for tRNA was examined as a model for the aminoacylation of primitive tRNA without aid of an enzyme system. Two types of reaction were carried out and compared. One was the acyl linkage of amino acid to the 5-terminal phosphate of a tRNA activated as an imidazolide. The other was the incorporation of an amino acid activated as an imidazolide into 2(3)-hydroxyl groups of intact tRNA. Both types of reaction indicated that none of the amino acids tested had any selectivity for the tRNAs examined. However, the rates of reaction with a given tRNA were different among amino acids. In the second type of reaction, amino acids were found mainly at loop-out regions of tRNA, but not at either its 5- or 3-terminal sitesOneA ₂₆₀ unit is defined as an amount of material which gives an absorption of 1.0 at 260 nm when dissolved in 1 ml water and measured with a 1-cm light path     

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

Summary The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5-and 3-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5- and 3-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.     

Uncharged tRNA inhibits guanosine 3'',5''-bis (diphosphate) 3''-pyrophosphohydrolase [ppGppase], the spoT gene product, from Escherichia coli

Summary The spoT gene product from Escherichia coli, the guanosine 3,5-bis(diphosphate) 3-pyrophosphohydrolase [ppGppase] catalyzes the specific release of pyrophosphate from the 3-position of guanosine 3,5-bis(diphosphate) [ppGpp]; this reaction is significantly inhibited in the presence of uncharged tRNA _yeast ^Phe . Little or no inhibition is observed with Phe-tRNA^Phe, tRNA^Phe-CpCpA_oxi-red or ribosomal RNA (16S and 23S).     

Ultrastructural heterogeneity of the mesocoxal muscles of Periplaneta americana

Summary Ultrastructural studies have been performed upon the posterior coxal depressor muscle (136) and a coxal branch of the main depressor group (135d) from the mesocoxa of the cockroach, Periplaneta americana. The quantitative stereometric analyses performed have shown the latter muscle to consist of a dorsal band of fibers having 25.5% mitochondria and 13.6% sarcoplasmic reticulum (SR) and T-tubules (TTS), and a ventral group of fibers with only 4.4% mitochondria and 26.6% SR/TTS. The volume fractions characteristic of the ventral fibers of muscle 135 d are also typical of muscle 136.This work was supported by a grant from the McCandless Foundation, Atlanta, Georgia, to D.R. Stokes     

DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

Nucleotide sequence of the 16S-23S spacer region in the rrnA operon from a blue-green alga, Anacystis nidulans

Summary The nucleotide sequence of an entire spacer region between the 16S and 23S rRNA genes of the rrnA operon from a blue-green alga, Anacystis nidulans, has been determined. The spacer region is 545 base pairs long and encodes tRNA^fle and tRNA^Ala in the order of 16S rRNA-tRNA^fle-tRNA^Ala-23S rRNA. A striking feature is that the A. nidulans tRNA^fle gene contains no 3-CCA sequence while the tRNA^Ala gene does. These spacer tRNA genes show strong sequence homology with those of chloroplasts and bacteria.     

Phagostimulation of the mediterranean fruit fly, Ceratitis capitata by ribonucleotides and related compounds

Females of the medfly, Ceratitis capitata, prefer sucrose solutions containing ribonucleotides to sucrose solutions without them. The order of preference for the nucleotides was: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP=2 & 3GMP=RP>3AMP.2AMP, guanosine, inosine, adenine and 5TMP produced no significant stimulation. Females sterilized by irradiation showed reduced attraction to 5GMP as compared to non-irradiated females.Optimal molecular configuration for phagostimulation includes: phosphorylation at the 5 position of the ribose, free hydroxyl groups at 2 and 3 on the ribose, and an NH₂ group at the 2 position of the aromatic ring of purine.It is proposed that the 5GMP in yeast hydrolyzate can be used as a measure of the suitability of the hydrolyzate as a bait.

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Résumé La femelle de la mouche méditerranéenne des fruits, Ceratitis capitata, préfère les solutions de sucrose contenant des ribonucléotides aux simples solutions de sucrose. Lórdre de préférence pour les nucléotides est le suivant: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP =2 & 3GMP=RP>3AMP.Le 2AMP, la guanosine, l'inosine, l'adénine et le 5TMP provoquent une stimulation significative. Les femelles montrent aprés stérilisation par irradiation une attirance réduite pour le 5GMP par comparaison avec les femelles non-irradiées.La configuration moléculaire optimale pour la phagostimulation comprend: la phosphorylation en position 5 du ribose; des groupes hydroxyles libres en 2 et 3 sur le ribose; et un groupe NH₂ en position 2 sur le noyau aromatique.Nous proposons que le 5GMP dans l'hydrolysat de levure puisse être utilisé pour mesurer la capacité de l'hydrolysat comme appât.

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Abbreviations 5AMP Adenosine 5-monophosphate - 3AMP Adenosine 3-monophosphate - 2AMP Adenosine 2-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - ADP Adenosine 5-diphosphate - ATP Adenosine 5-triphosphate - 5GMP Guanosine 5-monophosphate - 2GMP Guanosine 2-monophosphate - 3GMP Guanosine 3-monophosphate - dGMP 2-deoxyguanosine 5-monophosphate - GDP Guanosine 5-diphosphate - GTP Guanosine 5-triphosphate - 5IMP Inosine 5-monophosphate - IDP Inosine 5-diphosphate - ITP Inosine 5-triphosphate - 5XMP Xanthosine 5-monophosphate - 5CMP Cytidine 5-monophosphate - dCMP 2 deoxycytidine 5-monophosphate - CTP Cytidine 5-triphosphate - 5UMP Uridine 5-monophosphate - 5TMP Thymidine 5-monophosphate - RP Ribose 5 monophosphate     

Measurement of one-bond 15N-13C′ dipolar couplings in medium sized proteins

A simple and accurate method is described for measurement of ¹ J _CN splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the ¹ J _CN splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with ¹ J _CN dephasing intervals of 1/(2¹ J _CN) (reference intensity) and 1/¹ J _CN (residual intensity). If the two spectra are recorded under identical conditions and with the same number of scans, the random error in the ¹ J _CN value extracted in this manner is inversely related to the signal-to-noise (S/N) in the reference spectrum. A S/N of 30:1 in the reference spectrum yields random errors of less than 0.2 Hz in the extracted ¹ J _CN value. Dipolar couplings obtained from the difference in ¹ J _CN splitting in the isotropic and liquid crystalline phase for the C-terminal domain of calmodulin are in excellent agreement with its 1.68-Å crystal structure, but agree considerably less with the 2.2-Å structure.     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.     

An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer

Summary We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA^Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5 and 3 flanking sequences, which bear similarity with promoter and termination signals in eubacteria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed.     

Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector

The pharmaceutically important plant, licorice (Glycyrrhiza uralenesis Fisher), was transformed with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. The transgenic state of transformed roots was confirmed by detection of agropine and mannopine and by Southern blot hybridization with T-DNA of pGSGluc1. One to three copies of T-DNA of pGSGluc1 was integrated into the genomic DNA of G. uralensis. The expression of chimeric neo and gus genes driven by TR 1 and 2 promoters, respectively, was demonstrated by enzymatic assays. Histochemical analysis showed that the chimeric TR2-gus gene was expressed specifically in phloem and pericycle tissues of the transformed licorice roots.Abbreviations NPT-II neomycin phosphotransferase II - neo NPT-II gene from Tn5 - GUS ß-glucuronidase - gus GUS gene from Escherichia coli - TR 1–2 genes 1 and 2 of TR-DNA of pTiAch5 - Rif rifampicin     

Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine

Conformational and dynamic properties of the anticodon loop of yeast tRNA^Phe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3 side. The influence of Mg²⁺, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s²UUC of E. coli tRNA ₂ ^Glu were investigated.Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes () and rotational relaxation times ( _R ) we conclude that wybutine occurs in various structural states: (i) one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (=6 ns, _R =19 ns), (ii) an unstacked conformation where the base can freely rotate (=100 ps, _R = 370 ps) and (iii) an intermediary state (=2 ns, _R = 1.6 ns).Under biological conditions, i. e. in the presence of Mg²⁺ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA ₂ ^Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3–5 stack.     

Postembryonic development ofParalabidocera antarctica (I. C. Thompson) (Copepoda,Calanoida) from the fast ice near Syowa Station,Antarctica

Six nauplius and five copepodid stages as well as adults ofParalabidocera antarctica (I. C. Thompson, 1898) (Copepoda:Calanoida) are described based on specimens obtained from fast ice and collected by a plankton net near Syowa Station (69°00 S, 39° 35 E), Antarctica. The adult male and female are redescribed in detail. Nauplius stages ofP. antarctica are very similar to the previously describedAcartia species. Sexual dimorphism becomes apparent from copepodid IV onwards in the morphology of antennule and leg 5. The copepodid stages of this species retain certain characteristics not only of Acartiidae but also of Pontellidae and Parapontellidae.     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.