Biosynthesis of acyclic methyl branched polyunsaturated hydrocarbons inPseudomonas maltophilia

Summary The hydrocarbon composition ofPseudomonas maltophilia was determined by gas chromatography-mass spectrometry. Mono-, di- and tri-unsaturated alkenes were identified with a predominance of polyunsaturated components. The carbon chains of the alkenes contained single methyl branches iniso andanteiso position and double methyl branches in theiso-iso andanteiso-anteiso configurations. The composition of the hydrocarbons from cells grown in synthetic media enriched with amino acids or volatile fatty acids demonstrated that the probable precursors incorporated into individual hydrocarbons were branched and normal fatty acid chains in the range from C₃ to C₁₆. The probable fatty acid precursors which were connected together to form the major triunsaturated hydrocarbon chains were two monounsaturated chains, whereas the major liunsaturated chains resulted from condensation of saturated and monounsaturated chains. The probable precursors for the major monounsaturated hydrocarbons were C₁₄ (C₁₅) and C₁₆ (C₁₅) fatty acids. The accumulation of hydrocarbons was not detected until the cells were in the late exponential phase of growth; the maximal levels were reached at the mid-stationary phase of growth.     

Globin evolution in the genusXenopus: Comparative analysis of cDNAs coding for adult globin polypeptides ofXenopus borealis andXenopus tropicalis

Summary Globin mRNAs ofXenopus borealis andXenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data fromXenopus laevis. This analysis rendered clear evidence that the common ancestor ofX. laevis andX. borealis, but not ofX. tropicalis, had lost one amino acid of the -globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between theX. laevis and theX. tropicalis groups occurring approximately 110–120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and₁₂ contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genusXenopus.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

Comparative Genomics Reveals Long, Evolutionarily Conserved, Low-Complexity Islands in Yeast Proteins

Eukaryotic proteomes abound in low-complexity sequences, including tandem repeats and regions with significantly biased amino acid compositions. We assessed the functional importance of compositionally biased sequences in the yeast proteome using an evolutionary analysis of 2838 orthologous open reading frame (ORF) families from three Saccharomyces species (S. cerevisiae, S. bayanus, and S. paradoxus). Sequence conservation was measured by the amino acid sequence variability and by the ratio of nonsynonymous-to-synonymous nucleotide substitutions (K _a /K _s ) between pairs of orthologous ORFs. A total of 1033 ORF families contained one or more long (at least 45 residues), low-complexity islands as defined by a measure based on the Shannon information index. Low-complexity islands were generally less conserved than ORFs as a whole; on average they were 50% more variable in amino acid sequences and 50% higher in K _a /K _s ratios. Fast-evolving low-complexity sequences outnumbered conserved low-complexity sequences by a ratio of 10 to 1. Sequence differences between orthologous ORFs fit well to a selectively neutral Poisson model of sequence divergence. We therefore used the Poisson model to identify conserved low-complexity sequences. ORFs containing the 33 most conserved low-complexity sequences were overrepresented by those encoding nucleic acid binding proteins, cytoskeleton components, and intracellular transporters. While a few conserved low-complexity islands were known functional domains (e.g., DNA/RNA-binding domains), most were uncharacterized. We discuss how comparative genomics of closely related species can be employed further to distinguish functionally important, shorter, low-complexity sequences from the vast majority of such sequences likely maintained by neutral processes. [Reviewing Editor: Dr. Stuart Newfeld]     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Vitellogenin motifs conserved in nematodes and vertebrates

Summary Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrogram, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies betweenThermotoga and the other bacterial lineages.     

Primary structure of chain I of the heterodimeric hemoglobin from the blood clamBarbatia virescens

The blood clamBarbatia virescens has a heterodimeric hemoglobin in erythrocytes. Interestingly, the congeneric clamsB. reeveana andB. lima contain quite different hemoglobins: tetramer and polymeric hemoglobin consisting of unusual didomain chain. The complete amino acid sequence of chain I ofB. virescens has been determined. The sequence was mainly determined from CNBr peptides and their subpeptides, and the alignment of the peptides was confirmed by sequencing of PCR-amplified cDNA forB. virescens chain I. The cDNA-derived amino acid sequence matched completely with the sequence proposed from protein sequencing.B. virescens chain I is composed of 156 amino acid residues, and the molecular mass was calculated to be 18,387 D, including a heme group. The sequence ofB. virescens chain I showed 35–42% sequence identity with those of the related clamAnadara trapezia and the congeneric clamB. reeveana. An evolutionary tree forAnadara andBarbatia chains clearly indicates that all of the chains are evolved from one ancestral globin gene, and that the divergence of chains has occurred in each clam after the speciation. The evolutionary rate for clam hemoglobins was estimated to be about four times faster than that of vertebrate hemoglobin. We suggest that blood clam hemoglobin is a physiologically less important molecule when compared with vertebrate hemoglobins, and so it evolved rapidly and resulted in a remarkable diversity in quaternary and subunit structure within a relatively short period.     

Variation in the primary structure ofwaxy proteins (granule-bound starch synthase) in diploid cereals

The molecular weights ofwaxy proteins, by SDS-PAGE, and the N-terminal amino acid sequences of mature protein and of V8 protease-induced fragments were determined in diploid cereals. The homology of the primary structure was relatively high among cereals examined here, and there appeared to be a common sequence, V-F-V-G-A-E-M-A, in the vicinity of the N terminus. Based on the amino acid sequences, these cereals could be divided into two groups, including corn and rice in one and diploid wheat, fourAegilops species, rye, and barley in the other. In diploid wheat andAegilops species there were substitutions of amino acids in the primary structure. Variations of this sort suggest that the primary structure ofwaxy proteins would provide clues to the phylogenetic relations in the wheat group.     

Nucleotide sequence ofatpB,rbcL,trnR,dedB andpsaI chloroplast genes from a fernAngiopteris lygodiifolia: a possible emergence of Spermatophyta lineage before the separation of Bryophyta and Pteridophyta

To elucidate the evolutionary relationship between the Spermatophyta, Pteridophyta and Bryophyta, we cloned a fragment of chloroplast DNA from the fernAngiopteris lygodiifolia (Pteridophyta) and determined its nucleotide sequence. The fragment contained theatpB,rbcL,trnR-CCG,dedB andpsaI genes. Comparisons of the deduced amino acid and nucleotide sequences of these genes from the three plant groups indicate thatAngiopteris sequences are more closely related to those of Bryophyta species (85% identity on average) than to those of seed plants (76% identity on average), supporting a hypothesis that the Bryophyta and Pteridophyta diverged more recently from one another than their common progenitor diverged from that of the Spermatophyta.     

Conserved amino acid networks involved in antibody variable domain interactions

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody V_H and V_L function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which V_H and V_L domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (?‐values). We provide rosters of the key conserved amino acid pairs in antibody V_H and V_L domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in V_H and V_L are at or adjacent to the V_H–V_L interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the V_H domain, but not the V_L domain, residue pairs at the variable‐constant domain interface (V_H–C_H1 interface) are also strongly conserved. The same network of conserved V_H positions involved in interactions with both the V_L and C_H1 domains is found in camelid V_HH domains, which have evolved to lack interactions with V_L and C_H1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A computer system for the analysis of molecular evolution modes of protein-encoding genes (SAMEM): The relationship between molecular evolution and phenotypic traits

An online computer system for the analysis of the molecular evolution modes of genes and proteins has been developed (SAMEM: ). SAMEM computations are based on the ratio of radical to conservative amino acid substitutions (K _R /K _C ), the rate of amino acid substitutions in the course of protein evolution (V _P ), and statistical relationships between the evolutionary changes of all known amino acid properties and particular features of an organism. The system facilitates the interpretation of the results of K _R /K _C and V _P analyses.     

A mathematical model for the vertical distribution of chlorophyllA in estuarine intertidal sediments

Coastal and estuarine intertidal sediments are commonly colonized by dense populations of microphytobenthos. Due to wind and tides, important fractions of microphytobenthic populations may be buried. A mathematical model describing the depth variation of chlorophyll a in intertidal sediments was developed and experimentally tested. The model assumed first-order chlorophylla degradation and a constant mean burial velocity which resulted in a negative exponential variationC _Z =C _O exp{-k/vz} (C _Z andC _O =chlorophylla concentration at depth zand at the surface;k=specific degradation rate of chlorophyll a to pheopigments;V=mean burial velocity). Chlorophylla concentration depth profiles in different sediment types measured at the Tagus estuary and Ria Formosa (Portugal) were used to validate the model. The model was adjusted to field data. The chlorophyll a degradation rate was measured in a microcosm experiment under total darkness and no tidal action, and sampled during three months. This rate was shown to be independent of time and depth for the upper 0–15 mm depth interval. This result allowed the estimation ofV for each sampling site. Comparison of predicted and observed temporal data further confirmed the validity of the model andk andV values. Despite its simplicity, the proposed model adequately described the depth distribution of chlorophylla in different types of intertidal sediments. The model allowed the quantitative characterization of the buried microphytobenthic biomass (depth-integrated biomass) and the assessment of its importance as potentially productive stock of cells.     

Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Irish Government under the National Development Plan 2000–2006     

Chromosomal location of seed storage protein genes in the genome ofDasypyrum villosum (L.) Candargy

Summary Genes coding for glutenin-like subunits and for several prolamin subunits with electrophoretic mobilities (lactate-PAGE) corresponding to those of omega- and gamma-gliadins of wheat were located inDasypyrum villosum chromosome1V. Genes controlling four gliadinlike subunits with electrophoretic mobilities corresponding to those of alpha- and gamma-gliadins were located on the short arm of chromosome6V and on the long arm of chromosome4V. N-terminal amino acid sequences of these four components were also determined and homology with alpha-type gliadins was demonstrated. The presence of genes coding for glutenin- and gliadin-like subunits on chromosomes1V and6V demonstrates homoeology between theD. villosum chromosomes1V and6V and the chromosomes of homoeologous groups 1 and 6 in wheat. It is likely that the additional locusGli-V3 on chromosome4V originated by translocation from theGli-V2 locus.     

Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences

Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a K_m value of 0.6 mM for arginine and a V_max value of 13 μmole Pi min⁻¹ mg protein⁻¹ for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1608 and 1239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66–73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63–71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55–57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis     

Origin and evolution of the sulawesi macaques 2. complete amino acid sequences of seven β chains of three molecular types

Seven β chains were identified as the typical molecular types carried by the seven species of Sulawesi macaques based on isoelectric focusing and urea starch gel electrophoresis. These β chains include the β3 chains ofmaura, tonkeana, nigra, andbrunnescens, β1 chains ofhecki andochreata and β5 chain ofnigra. The results of chromatography on cation-exchange and reversed phase columns and the amino acid compositions of the tryptic peptides suggested substitutions at the 9th and 13th amino acids from the N-terminal. Sequence analyses of these seven β chains from the N-terminal to the 18th amino acid and those of purified tryptic peptides from βT3 to βT15 by Edman degradation revealed the following facts: (1) the amino acid sequences of the β3 chains carried by the four species coincided with each other and as did those of the β1 chains of the two named species; and (2) the 9th and 13th amino acids were Lys and Thr in β3, Asn and Asn in β1, and Asp and Thr in the β5 chain, respectively. These three β chains are related with each other by at least two-base changes. The evolution of the β chains of the Sulawesi macaques was inferred to be as follows. (1) The β3 chain might have been dominant β chain in the past among Sulawesi macaques, since peripheral species separately carried this chain; (2) the β1 and β5 chains might have derived from a “missing link” because of more than two-base substitutions between β3 and β1 and between β3 and β5; (3) eight other macaque species, including the lion-tailed macaque (M. silenus), bear Asn and Thr at these two positions, while the Barbary macaque (M. sylvanus) has Thr and Thr; and (4) thus, if the parsimonious rule is followed, the type with Asn-Thr is the most plausible “missing link,” since only the Asn-Thr type can combine these five β chains by minimum one-base change. Two genetic events are postulated in the evolutionary process of the Sulawesi β chains: the first Lys-Thr type (β3) was distributed over the whole island, and next Asn-Thr, the common type in other macaques, produced Asn-Asn (β1) and Asp-Thr (β5).