On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.     

Chloroplast DNA base substitutions: an experimental assessment

An experimental assessment was carried out to determine directly the frequency and types of spontaneous base substitutions that occur in chloroplast DNA. A target site within the chloroplast 16S rRNA gene of the green alga Chlamydomonas reinhardtii was chosen for the assay. Mutations at this site were known to confer spectinomycin resistance and simultaneously result in the loss of an AatII cleavage site. In the experiments reported here, base substitutions at any individual base occurred at a frequency in the range of 0.9–11 per 10⁹ viable cells plated. Four new mutations that confer resistance to spectinomycin were identified at the target site in the Chlamydomonas chloroplast 16S rRNA gene. When the relative rates of transition and transversion mutations were quantified, a bias toward transversions was observed. The prominence of A/T C/G transversions in the observed mutation spectrum suggests that oxidative damage may be the major cause of base substitution mutations within the chloroplast.     

A unique type of eubacterial 5S rRNA in members of the order Planctomycetales

Summary Analysis of the 5S ribosomal RNA from members of the eubacterial order Planctomycetales, i.e.,Planctomyces, Pirella, Gemmata, andIsosphaera, reveals several unexpected features. Firstly, the primary structures are significantly shorter than those of the majority of eubacteria and vary in length between 109 and 111 nucleotides. Secondly, the lack of an insertion at position 66 is a feature not encountered before in prokaryotic 5S rRNAs. Thirdly, as compared to the proposed eubacterial minimal 5S rRNA structure (Erdmann and Wolters 1986) the secondary structure contains numerous basepair transversions. The isolated position of the planctomycetes as an individual eubacterial division and the phylogenetic position of its genera are in accord with the results obtained from 16S rRNA cataloguing.     

Comparative immunological and electrophoretic studies on ribosomal proteins of Bacillaceae

Summary The ribosomal proteins from several Bacillus species were compared by two-dimensional gel electrophoresis and immunological methods. The results revealed great heterogeneity among most Bacillus species. Comparison of ribosomal proteins from Bacilli with those of E. coli by two-dimensional gel electrophoresis showed little similarities, while structural homologies could be found by immunological methods. SDS two-dimensional gel electrophoresis revealed that the molecular weight of ribosomal proteins is conserved in all tested bacteria.Paper No. 68 on Ribosomal Proteins. Preceding paper is by Geisser et al., Molec. gen. Genet. 127, 111–128 (1973).     

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

Evolutionary relationship and secondary structure predictions in four transport proteins ofSaccharomyces cerevisiae

Summary The comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor. The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9–12 membrane-spanning -helices in each polypeptide chain. These results are concordant in that 90% of the -helices were determined by both methods to be at the same positions. In the aligned sequencesHIP1 andCAN1, the postulated membrane-spanning -helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30%. In the aligned DNA coding sequences ofCAN1 andHIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different. Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented.     

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. (Apicomplexa: Eimeriidae) from the black-capped bulbul,Pycnonotus xanthopygos

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. are described from the black-capped bulbul Pycnonotus xanthopygos from Lincoln Park Zoo, Chicago, Illinois. The bird came from southwestern Africa seven years earlier. I. ernsti oocysts are ellipsoidal to bluntly ovoid, 28–38 × 23–31m (mean 34 × 28 m) and have a single-layered oocyst wall. Micropyle, oocyst residuum and polar granules are absent. Sporocysts are elongate ovoid, 24–30 × 11–16 m (mean 27×13 m). Stieda and substiedal bodies and sporocyst residuum are present. I. blagburni oocysts are spherical to subspherical. 21–28 × 19–26 m (mean 25 × 23 m) and have a single oocyst wall. Sporocysts are ovoid and 17–23 × 10–13 m (mean 20 × 12 m). Stieda and substiedal bodies and sporocyst residuum are present.     

Evolution of proteins in mammalian cytoplasmic and mitochondrial ribosomes

Summary The proteins of cytoplasmic and mitochondrial ribosomes from the cow and the rat were analyzed by co-electrophoresis in two dimensional polyacrylamide gels to determine their relative evolutionary rates. In a pairwise comparison of individual ribosomal proteins (r-proteins) from the cow and the rat, over 85% of the cytoplasmic r-proteins have conserved electrophoretic properties in this system, while only 15% of the proteins of mitochondrial ribosomes from these animals fell into this category. These values predict that mammalian mitochondrial r-proteins are evolving about 13 times more rapidly than cytoplasmic r-proteins. Based on actual evolutionary rates for representative cytoplasmic r-proteins, this mitochondrial r-protein evolutionary rate corresponds to an amino acid substitution rate of 40×10^–10 per site per year, placing mitochondrial r-proteins in the category of rapidly evolving proteins. The mitochondrial r-proteins are apparently evolving at a rate comparable to that of the mitochondrial rRNA, suggesting that functional constraints act more or less equally on both kinds of molecules in the ribosome. It is significant that mammalian mitochondrial r-proteins are evolving more rapidly than cytoplasmic r-proteins in the same cell, since both sets of r-proteins are encoded by nuclear genes. Such a difference in evolutionary rates implies that the functional constraints operating on ribosomes are somewhat relaxed for mitochondrial ribosomes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

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     

The distribution of genetic parameter estimates and confidence intervals from small disconnected diallels

The distributions of genetic variance components and their ratios (heritability and type-B genetic correlation) from 105 pairs of six-parent disconnected half-diallels of a breeding population of loblolly pine (Pinus taeda L.) were examined. A series of simulations based on these estimates were carried out to study the coverage accuracy of confidence intervals based on the usual t-method and several other alternative methods. Genetic variance estimates fluctuated greatly from one experiment to another. Both general combining ability variance (²_g) and specific combining ability variance (²_s) had a large positive skewness. For ²_g and ²_s, a skewness-adjusted t-method proposed by Boos and Hughes-Oliver (Am Stat 54:121–128, 2000) provided better upper endpoint confidence intervals than t-intervals, whereas they were similar for the lower endpoint. Bootstrap BCa-intervals (Efron and Tibshirani, An introduction to the bootstrap. Chapman & Hall, London 436 p, 1993) and Halls transformation methods (Zhou and Gao, Am Stat 54:100–104, 2000) had poor coverages. Coverage accuracy of Fiellers interval endpoint(J R Stat Soc Ser B 16:175–185, 1954) and t-interval endpoint were similar for both h² and r_B for sample sizes n10, but for n=30 the Fiellers method is much better.     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Adsorption of small biological molecules on silica from diluted aqueous solutions: Quantitative characterization and implications to the Bernal's hypothesis

To describe quantitatively the adsorption of prebiotically important compounds of low molecular weight (amino acids, short linear peptides, cyclic dipeptides, the Krebs's cycle and other carboxylic acids, nucleosides and related phosphates) on silica surface from diluted neutral aqueous solutions, equilibrium constants (K) and free energies (–G) of adsorption were determined from the retention values measured by means of high-performance liquid chromatography on a silica gel column and from the isotherms measured under static conditions. For most carboxylic acids (including amino acids and linear peptides) –G values were negative and K<1, thus showing very weak adsorption. Cyclic dipeptides (2,5-piperazinediones) exhibited higher adsorbability; –G>0 and K>1 were found for most of them. Influence of the structure of -substituent on the adsorbability is analyzed. A linear dependence of –G on the number of aliphatic carbon atoms in a sorbate molecule was found for the series of aliphatic bifunctional amino acids, related dipeptides and 2,5-piperazinediones, as well as for the row from glycine to triglycyl glycine. The adsorption of nucleosides and their phosphates is characterized by much higherK and –G values (of the order of 10² and 10⁴, respectively). The adsorption data available from our work and literature are summarized and discussed with implications to the Bernal's hypothesis on the roles of solid surfaces in the prebiotic formation of biopolymers from monomeric building blocks.corresponding author: on leave of absence from Institute of Surface Chemistry, Academy of Sciences of the Ukraine, Kiev, Ukraine.     

DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class I introns of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal RNA (rRNA) fromPodospora anserina is interrupted by two class I introns. The coding region for the large subunit rRNA itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length. Secondary structure models for the large subunit rRNA were constructed and compared with the equivalent structure fromEscherichia coli 23S rRNA. The two structures were remarkably similar despite an 800-base difference in length. The additional bases in theP. anserina rRNA appear to be mostly in unstructured regions in the 3 part of the RNA. Secondary structure models for the two introns show striking similarities with each other as well as with the intron models from the equivalent introns inSaccharomyces cerevisiae, Neurospora crassa, andAspergillus nidulans. The long open reading frames in each intron are different from each other, however, and the nucleotide sequence similarity diverges as it proceeds away from the core structure. Each intron is located within regions of the large subunit rRNA gene that are highly conserved in both sequence and structure. Computer analysis showed that the open reading frame for intron r1 contained a common maturase-like polypeptide. The open reading frames of intron r2 apeared to be chimeric, displaying high sequence similarity with the open reading frames in the r1 and ATPase 6 introns ofN. crassa.     

MucAB but not UmuDC proteins enhance ?2 frameshift mutagenesis induced by N-2-acetylaminofluorene at alternating GC sequences

N-2-acetylaminofluorene has been shown efficiently to induce both –1 and –2 frameshift mutations in Escherichia coli as well as in mammalian cells. In E. coli, the genetic characteristics of –1 and –2 frameshift mutations were found to be distinct. The –1 frameshift mutation pathway occurs at monotonous runs of G residues (i.e. GGGGG). This pathway exhibits the same genetic requirements as UV light-induced base substitution mutagenesis. Indeed, optimal mutagenesis requires the expression of both UmuDC and the activated form of RecA. The –2 frameshift mutation pathway operates at short alternating GpC sequences, such as the NarI sequence (i.e. GGCGCCGGCC). In contrast to the –1 frameshift mutation pathway, optimal induction does not require the UmuDC and RecA proteins. This pathway involves a LexA-repressed function tentatively called Npf (for NarI processing factor). In this paper, we show that MucAB efficiently stimulates the –2 frameshift mutation pathway. However, unlike the Npf pathway, MucAB-mediated stimulation requires expression of the RecA protein.     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

Amino acid composition and the evolutionary rates of protein-coding genes

Summary Based on the rates of amino acid substitution for 60 mammalian genes of 50 codons or more, it is shown that the rate of amino acid substitution of a protein is correlated with its amino acid composition. In particular, the content of glycine residues is negatively correlated with the rate of amino acid substitution, and this content alone explains about 38% of the total variation in amino acid substitution rates among different protein families. The propensity of a polypeptide to evolve fast or slowly may be predicted from an index or indices of protein mutability directly derivable from the amino acid composition. The propensity of an amino acid to remain conserved during evolutionary times depends not so much on its being features prominently in active sites, but on its stability index, defined as the mean chemical distance [R. Grantham (1974) Science 185862–864] between the amino acid and its mutational derivatives produced by single-nucleotide substitutions. Functional constraints related to active and binding sites of proteins play only a minor role in determining the overall rate of amino acid substitution. The importance of amino acid composition in determining rates of substitution is illustrated with examples involving cytochrome c, cytochrome b₅,ras-related genes, the calmodulin protein family, and fibrinopeptides.