Nucleotide sequence of the gene for the mitochondrial 15S ribosomal RNA of yeast

We have determined the nucleotide sequence of a DNA segment carrying the entire 15S ribosomal RNA gene of yeast mitochondrial genome. Many stretches of sequence are present which are homologous to the E. coli 16S ribosomal RNA gene. The gene sequence can be folded into a secondary structure according to the [1] model on bacterial ribosomal RNAs. The structure reveals a striking similarity between the two RNAs despite the large difference in their base compositions. In the middle of the gene, we found a guanine-cytosine rich sequence that is also present in several other regions of the mitochondrial genome.     

Vacuolar H+-ATPase: From mammals to yeast and back

Vacuolar H⁺-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V₁) and membrane (V₀) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPases that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by drinking the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.     

tRNase Z: the end is not in sight

Although the enzyme tRNase Z has only recently been isolated, a plethora of data has already been acquired concerning the enzyme. tRNase Z is the endonuclease that catalyzes the removal of the tRNA 3′ trailer, yielding the mature tRNA 3′ end ready for CCA addition and aminoacylation. Another substrate cleaved by tRNase Z is the small chromogenic phosphodiester bis(p-nitrophenyl)phosphate (bpNPP), which is the smallest tRNase Z substrate known so far. Hitherto the biological function as tRNA 3′-end processing enzyme has been shown only in one prokaryotic and one eukaryotic organism, respectively. This review summarizes the present information concerning the two tRNase Z substrates pre-tRNA and bpNPP, as well as the metal requirements of tRNase Z enzymes. Received 29 March 2007; received after revision 15 May 2007; accepted 21 May 2007     

Carboxypeptidases from A to Z: implications in embryonic development and Wnt binding

Carboxypeptidases perform many diverse functions in the body. The well-studied pancreatic enzymes (carboxypeptidases A1, A2 and B) are involved in the digestion of food, whereas a related enzyme (mast-cell carboxypeptidase A) functions in the degradation of other proteins. Several members of the metallocarboxypeptidase gene family (carboxypeptidases D, E, M and N) are more selective enzymes and are thought to play a role in the processing of intercellular peptide messengers. Three other members of the metallocarboxypeptidase gene family do not appear to encode active enzymes; these members have been designated CPX-1, CPX-2 and AEBP1/ACLP. In this review, we focus on the recently discovered carboxypeptidase Z (CPZ). This enzyme removes C-terminal Arg residues from synthetic substrates, as do many of the other members of the gene family. However, CPZ differs from the other enzymes in that CPZ is enriched in the extracellular matrix and is broadly distributed during early embryogenesis. In addition to containing a metallocarboxypeptidase domain, CPZ also contains a Cys-rich domain that has homology to Wnt-binding proteins; Wnts are important signaling molecules during development. Although the exact function of CPZ is not yet known, it is likely that this protein plays a role in development by one of several possible mechanisms.     

Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?

Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap₄A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap₄A hydrolases also efficiently remove pyrophosphate from the 5′ termini of mRNAs, suggesting a potential role for Ap₄A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.     

Gastric lysozyme as a digestive enzyme in the hoatzin (Opisthocomus hoazin), a ruminant-like folivorous bird

The hoatzin is the only bird known to have pregastric fermentation in the crop. This digestive strategy is supported by morphological and microbiological adaptations analogous to those present in ruminants and ruminant-like mammals. The hoatzin expresses a lysozyme-like bacteriolytic activity in its foregut. The enzyme has a high activity, and its low pH optimum, pepsin resistance and localization to the proventriculus allow it to be active for digestion in the stomach. The hoatzin enzyme and the ruminant gastric lysozyme present similar biochemical characteristics. The lysis of bacterial cells may be of significance for the nutrition of the hoatzin. We propose that the hoatzin expresses a lysozyme which has been recruited to function as a digestive enzyme, representing a unique case of evolutionary convergence of digestive adaptations in this bird and foregut fermenter mammals.     

Yeast prions and human prion-like proteins: sequence features and prediction methods

Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.     

庆大霉素产生菌GntB基因的克隆与转化

根据小单孢菌产生庆大霉素的生物合成机理,利用基因克隆方法从棘孢小单孢菌（Micromonospora echinospora）基因组中扩增出庆大霉素生物合成的关键酶基因—2-脱氧青蟹肌糖合成酶基因（GntB）,并将其通过大肠杆菌／链霉菌穿梭质粒pIJ699转化原菌株,采用硫链丝菌素抗性基因启动子带动2-脱氧青蟹肌糖合成酶基因在棘孢小单孢菌细胞中实现了转化。     

New insights into copper monooxygenases and peptide amidation: structure, mechanism and function

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.     

Genetic localization of a mutation rendering the growth of E coli K12 insensitive to illumination at 365 nm]

The genotype of the Nop mutant recently isolated from the E. coli K 12 strain AB 1157 has been characterized. This mutant lacks 4-thiouridine in its tRNA and is much less susceptible to near ultraviolet-induced growth delay than wild type cells. This phenotype results from a single mutation called nuv which has been localized on the E. coli genetic map. nuv is found by conjugation to lie between the origins of injection of Hfr P4X and Hfr cavalli in the vicinity of the lac gene. Cotrans-duction with bacteriophage P1 more precisely maps nuv at 0.3 min. clockwise from tsx.     

Chemical analysis of the pheromone blends produced by males and females of the neotropical moth,Mocis megas (Guénée) (Lepidoptera,Noctuidae, Catocalinae)

Summary Pheromonal secretions produced by females and males of the noctuid moth,Mocis megas (Guénée) have been analyzed by gas chromatography and mass spectrometry (EI (electron impact) and CI (chemical ionization)). The female sex pheromone was a blend of (Z,Z,Z) 3,6,9 heneicosatriene (55%) and (Z,Z) 3,6-cis-9S, 10R-epoxyheneicosadiene (45%). Male secretion produced at the level of a prothoracic organ was a blend of two unsaturated major hydrocarbons: (Z,Z) 6,9 heneicosadiene, (64%) and (Z,Z,Z) 3,6,9 heneicosatriene (24%) and C₁₉, C₂₀ and C₂₂ homologues (total ratio 12%), as minor components. The trienic hydrocarbon was present in both sexes. The behavioral role of this male secretion has not yet been elucidated.     

Recent observations on the structure and the properties of yeast NMN adenylyltransferase

Summary A homogeneous preparation of yeast NMN adenylyltransferase (EC 2.7.7.1) showed microheterogeneity, which was revealed by FPLC (Fast Protein Liquid Chromatography) ion exchange chromatography. The resolved components have been characterized with respect to electrophoretic behavior and adenine content. The results led to a hypothesis about a possible role of poly(ADP-ribosylation) in modulating the enzyme activity.     

Chitin in the epidermal cuticle of a vertebrate (Paralipophrys trigloides,Blenniidae, Teleostei)

Lectin binding, endo-chitinase binding and enzymatic degradation studies show that the epidermal cuticle of the bony fishParalipophrys trigloides (Blenniidae) is chitinous. This is the first evidence that a vertebrate species possesses a chitinous tissue. Recently aXenopus gene has been identified which has significant sequence similarity to the catalytic domain of yeast chitin synthase III, a chitin producing enzyme^1,2. Taken together these two findings imply that chitin synthesis capability may be a basic vertebrate feature.     

Recent observations on the structure and the properties of yeast NMN adenylyltransferase

A homogeneous preparation of yeast NMN adenylyltransferase (EC 2.7.7.1) showed microheterogeneity, which was revealed by FPLC (Fast Protein Liquid Chromatography) ion exchange chromatography. The resolved components have been characterized with respect to electrophoretic behavior and adenine content. The results led to a hypothesis about a possible role of poly(ADP-ribosylation) in modulating the enzyme activity.     

Methoxymercuration-demercuration and mass spectrometry in the identification of the sex pheromones ofPanolis flammea,the pine beauty moth

Summary The major components of the sex pheromone system ofPanolis flammea, pine beauty moth have been identified as (Z)-9-tetradecenyl acetate, (Z)-11-hexadecenyl acetate and (Z)-11-tetradecenyl acetate in the ratio 10051; the double bond position of these derivatives was established by microscale application of a methoxymercuration-demercuration technique and GC-MS followed by multiple ion monitoring.We thank the Science Research Council and Forestry Commission for financial support. We are grateful for the collaboration of Messrs Stoakley and Bevan, Dr C. Longhurst and Mrs J. Edwards.     

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.