Molecular characterization of a phosphoserine aminotransferase gene in Antheraea pernyi and assessment of its value for phylogenetic inference

A gene encoding phosphoserine aminotransferase from Antheraea pernyi (ApPSAT) was isolated and characterized, and its value for phylogenetic inference was assessed. The resulting 1309 bp cDNA sequence contains an open reading frame of 1095 bp encoding a polypeptide of 364 amino acids, with 58% sequence identity to that from Homo sapiens. RT-PCR analysis showed that the ApPSAT gene was transcribed at four developmental stages, and in all examined tissues with the most abundance in malpighian tubules. Sequence alignment suggested that ApPSAT protein sequence exhibited 41–87% homology with the known PSATs from bacteria, fungi, plants, invertebrates and vertebrates. Phylogenetic analysis revealed that 37 representative PSAT protein sequences were well divided into five groups corresponding to the known fungi, bacteria, plants, invertebrates and vertebrates. The phylogenetic relationships obtained were consistent with the traditional classification and other molecular data, suggesting the potential value of the PSAT protein in phylogenetic inference.     

Small,basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression,structure, function and potential application

Peptides and proteins with antimicrobial activity are produced throughout all kingdoms in nature, from prokaryotes to lower and higher eukaryotes, including fungi, plants, invertebrates and vertebrates. These proteins contribute to an important constitutive or induced defense mechanism of the producer against microorganisms. According to their variety in structure and function, these proteins are classified arbitrarily into groups that are based on their mechanism of action, their structure and their similarity to other known proteins. The present review focuses on a new group of antimicrobial proteins, namely small, basic and cysteine-rich antifungal proteins, which are secreted from filamentous fungi of the group Ascomycetes. These proteins are encoded by orthologous genes and exhibit both similarities and differences concerning their species-specificity, primary structure, protein activity and target sites. The properties of these proteins, their possible mode of action and their potential application for human benefits are discussed in comparison with other already well known antimicrobial proteins.     

Analysis of NADH dehydrogenase proteins, ATPase subunit 9, cytochrome b, and ribosomal protein L14 encoded in the mitochondrial DNA of Paramecium.

The mitochondrial (mt) encoded ndh1, ndh3, ndh4, ndh5, rpl14, cyt b and atp9 gene products were identified by sequence comparisons with known proteins. Amino acid sequence comparisons between predicted Paramecium mt gene products and proteins in current databases were quantitated approximately by the means of similarity scores for pairs of aligned sequences. The comparisons show that the Paramecium gene products are very divergent from all others with the exception of those from a closely related ciliate, Tetrahymena. The similarity scores of comparisons between a Paramecium mt DNA encoded protein, cytochrome b for example, and the homologous protein from a group of organisms as diverse as other protozoans, vertebrates, fungi, plants, and prokaryotes were all about the same. The Paramecium gene products appear to be equally divergent from proteins representing a number of different kingdoms and organelles.     

Mitochondrial alternative oxidase across the tree of life: Presence,absence, and putative cases of lateral gene transfer

The alternative oxidase (AOX) is a terminal oxidase in the electron transport system that plays a role in mitochondrial bioenergetics. The past 20 years of research shows AOX has a wide yet patchy distribution across the tree of life. AOX has been suggested to have a role in stress tolerance, growth, and development in plants, but less is known about its function in other groups, including animals. In this study, we analyzed the taxonomic distribution of AOX across >2800 species representatives from prokaryotes and eukaryotes and developed a standardized workflow for finding and verifying the authenticity of AOX sequences. We found that AOX is limited to proteobacteria among prokaryotes, but is widely distributed in eukaryotes, with the highest prevalence in plants, fungi, and protists. AOX is present in many invertebrates, but is absent in others including most arthropods, and is absent from vertebrates. We found aberrant AOX sequences associated with some animal groups. Some of these aberrant AOXs were contaminants, but we also found putative cases of lateral gene transfer of AOX from fungi and protists to nematodes, springtails, fungus gnats, and rotifers. Our findings provide a robust and detailed analysis of the distribution of AOX and a method for identifying and verifying putative AOX sequences, which will be useful as more sequence data becomes available on public repositories.     

Follow the leader: preference for specific amino acids directly following the initial methionine in proteins of different organisms

It is well established that the vast majority of proteins of all taxonomical groups and species are initiated by an AUG codon, translated into the amino acid methionine (Met). Many attempts were made to evaluate the importance of the sequences surrounding the initiation codon, mostly focusing on the RNA sequence. However, the role and importance of the amino acids following the initiating Met residue were rarely investigated, mostly in bacteria and fungi. Herein, we computationally examined the protein sequences of all major taxonomical groups represented in the Swiss-Prot database, and evaluated the preference of each group to specific amino acids at the positions directly following the initial Met. The results indicate that there is a species-specific preference for the second amino acid of the majority of protein sequences. Interestingly, the preference for a certain amino acid at the second position changes throughout evolution from lysine in prokaryotes, through serine in lower eukaryotes, to alanine in higher plants and animals.     

Nucleobase transporters (review)

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

A novel cyclophilin from parasitic and free-living nematodes with a unique substrate- and drug-binding domain

A highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus (OvCYP-16). This 25-kDa cyclophilin shares 43-46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans (CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu(124) (C. elegans) or Asp(123) (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A. CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism.     

Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.     

The evolutionary position of the rhodophytePorphyra umbilicalis and the basidiomyceteLeucosporidium scottii among other eukaryotes as deduced from complete sequences of small ribosomal subunit RNA

Summary The complete small ribosomal subunit RNA (srRNA) sequence was determined for the red algaPorphyra umbilicalis and the basidiomyceteLeucosporidium scottii, representing two taxa for which no srRNA sequences were hitherto known. These sequences were aligned with other published complete srRNA sequences of 58 eukaryotes. Evolutionary trees were reconstructed by a matrix optimization method from a dissimilarity matrix based on sections of the alignment that correspond to structurally conservative areas of the molecule that can be aligned unambiguously. The overall topology of the eukaryotic tree thus constructed is as follows: first there is a succession of early diverging branches, leading to a diplomonad, a microsporidian, a euglenoid plus kinetoplastids, an amoeba, and slime molds. Later, a nearly simultaneous radiation seems to occur into a number of taxa comprising the metazoa, the red alga, the sporozoa, the higher fungi, the ciliates, the green plants, plus some other less numerous groups. Because the red alga diverges late in the evolutionary tree, it does not seem to represent a very primitive organism as proposed on the basis of morphological and 5S rRNA sequence data. Asco- and basidiomycetes do not share a common ancestor in our tree as is generally accepted on the basis of conventional criteria. In contrast, when all alignment positions, rather than the more conservative ones, are used to construct the evolutionary tree, higher fungi do form a monophyletic cluster. The hypothesis that higher fungi and red algae might have shared a common origin has been put forward. Although the red alga and fungi seem to diverge at nearly the same time, no such relationship can be detected. The newly determined sequences can be fitted into a secondary structure model for srRNA, which is now relatively well established with the exception of uncertainties in a number of eukaryote-specific expansion areas. A specific structural model featuring a pseudoknot is proposed for one of these areas.     

Nucleobase transporters

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members fromarchea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

Divergent evolution of the chloroplast small heat shock protein gene in the genera Rhododendron (Ericaceae) and Machilus (Lauraceae)

BACKGROUND AND AIMS: Evolutionary and ecological roles of the chloroplast small heat shock protein (CPsHSP) have been emphasized based on variations in protein contents; however, DNA sequence variations related to the evolutionary and ecological roles of this gene have not been investigated. In the present study, a basal angiosperm, Machilus, together with the eudicot Rhododendron were used to illustrate the evolutionary dynamics of gene divergence in CPsHSPs. METHODS: Degenerate primers were used to amplify CPsHSP-related sequences from 16 Rhododendron and eight Machilus species that occur in Taiwan. Manual DNA sequence alignment was carried out according to the deduced amino acid sequence alignment performed by CLUSTAL X. A neighbour-joining tree was generated in MEGA using conceptual translated amino acid sequences from consensus sequences of cloned CPsHSP genes from eight Machilus and 16 Rhododendron species as well as amino acid sequences of CPsHSPs from five monocots and seven other eudicots acquired from GenBank. CPsHSP amino acid sequences of Funaria hygrometrica were used as the outgroups. The aligned DNA and amino acid sequences were used to estimate several parameters of sequence divergence using the MEGA program. Separate Bayesian inference of DNA sequences of Rhododendron and Machilus species was analysed and the resulting gene trees were used for detection of putative positively selected amino acid sites by the Codeml program implemented in the PAML package. Mean hydrophobicity profile analysis was performed with representative amino acid sequences for both Rhododendron and Machilus species by the Bioedit program. The computer program SplitTester was used to examine whether CPsHSPs of Rhododendron lineages and duplicate copies of the Machilus CPsHSPs have evolved functional divergence based on the hydrophobicity distance matrix. KEY RESULTS: Only one copy of the CPsHSP was found in Rhododendron. However, a higher evolutionary rate of amino acid substitutions in the Hymenanthes lineage of Rhododendron was inferred. Two positively selected amino acid sites may have resulted in higher hydrophobicity in the region of the alpha-crystallin domain (ACD) of the CPsHSP. By contrast, the basal angiosperm, Machilus, possessed duplicate copies of the CPsHSP, which also differed in their evolutionary rates of amino acid substitutions. However, no apparent relationship of ecological relevance toward the positively selected amino acid sites was found in Machilus. CONCLUSIONS: Divergent evolution was found for both Rhododendron lineages and the paralogues of CPsHSP in Machilus that were directed to the shift in hydrophobicity in the ACD and/or methionine-rich region, which might have played important roles in molecular chaperone activity.     

Molecular cloning,expression pattern and phylogenetic analysis of the <Emphasis Type="Italic">will die slowly</Emphasis> gene from the Chinese oak silkworm, <Emphasis Type="Italic">Antheraea pernyi</Emphasis>

The will die slowly (wds) gene coding for a WD-repeat protein with seven repeats has been characterized in Drosophila melanogaster. In this paper, the wds gene was isolated and characterized from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae). The obtained 1733 bp cDNA sequence contains an open reading frame of 1041 bp encoding a polypeptide of 346 amino acids, with 85% sequence identity to that from D. melanogaster. RT-PCR analysis showed that the wds gene was transcribed during four developmental stages and in all the tissues tested, consistent with the result observed in Bombyx mori based on EST resources and genome-wide microarray information. The mRNA expression level of the A. pernyi wds gene was not significantly down- or up- regulated under temperature stress compared to the control, indicating that it may be not involved in temperature stress tolerance. In search of database, the wds protein homologues were found in various kinds of eukaryotes, including fungi, plants, invertebrates and vertebrates, with 50–93% amino acid sequence identities between them, suggesting that they are highly conserved during the evolution of eukaryotes. Phylogenetic analysis based on the wds protein homologue sequences clearly separated the known fungi, plants, invertebrates and vertebrates, consistent with the topology tree on the classical systematics, suggesting the potential value of wds protein in eukaryotic phylogenetic inference. In vertebrates, two apparent types of the wds proteins were also defined by sequence alignment and phylogenetic analysis.     

Evolution of RNA-dependent RNA polymerases of positive riboviruses

A comparative analysis is presented of 24 known amino acid sequences of RNA-dependent RNA polymerases of positive strand RNA viruses infecting animals, plants and bacteria. Using a newly proposed methodology of group alignment for weakly similar sequences, evolutionary conserved fragments of all these proteins were unambiguously aligned. A unique pattern (consensus) of 7 invariant amino acid residues was revealed which is absent from the sequences of other RNA and DNA polymerases and is thought to unequivocally identify the RNA-dependent RNA polymerases of positive strand RNA viruses. Based on the obtained alignment a tentative phylogenetic tree of viral RNA polymerases was constructed for the first time. The RNA-dependent RNA polymerases of positive strand RNA viruses are concluded to comprise a distinct family of evolutionary related proteins.     

Patterns of temperature adaptation in proteins from Methanococcus and Bacillus

It has long been known that amino acid substitutions in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are not all symmetrical; for example, more aligned sites have lysine in mesophiles and arginine in thermophiles than have the opposite pattern. This is generally taken to indicate that certain amino acids are favored over others by selection at different temperatures. Previous comparisons of protein sequences from mesophiles and thermophiles have used relatively small numbers of sequences from a diverse array of species, meaning that only the most common amino acid substitutions could be examined and any taxon-specific patterns would be obscured. Here, we compare a large number of proteins between mesophiles and thermophiles in the archaeal genus Methanococcus and the bacterial genus Bacillus. Each genus exhibits dramatically asymmetrical substitution patterns for many pairs of amino acids. There are several pairs of amino acids for which one amino acid is favored in thermophilic Bacillus and the other is favored in thermophilic Methanococcus; this appears to result from the higher G + C content of the DNA of thermophilic Bacillus, a complication not seen in Methanococcus.     

A method of detecting conserved and variable segments in the amino acid sequences of homologous proteins

A set of aligned homologous protein sequences is divided into two groups consisting of m and n sequences. Each group contains sequences from the most related organisms. Value of the position dissimilarity of proteins from different groups of m and n sequences is defined as a number of mismatches in comparison of all possible m X n pairs of amino acid residues in the position (each from different group) divided by m X n. Ten position average of dissimilarity values is plotted vs. the first position number. Area of the figure between the profile of dissimilarity values and its mean value line characterizes the overall irregularity of amino acid substitutions along the protein sequences. If the area is greater than the average area for 1000 random profiles by more than two standard deviation units, the profile extrema containing the "surplus" of area are cut off. The cut-off stretches are likely to be variable and constant regions. If necessary, each of stretches may be separately tested and statistically estimated using a standard size sample of artificial protein families. Intergroup comparison of protein sequences reveals high overall irregularity of amino acid substitutions and identifies variable and conservative regions for all considered families of proteins: phospholipases A2, aspartate aminotransferases, alpha-subunits of Na+, K(+)-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre, human rhodopsins.     

Graphic identification of conservative and variable segments in the amino acid sequences of homologous proteins

An algorithm is presented for localizing variable and constant regions in homologous protein sequences. A set of aligned protein sequences is divided into two groups consisting of m and n sequences. Each group contains sequences of most related species. Value of the position dissimilarity of proteins from different groups of m and n sequences is defined as a number of failures to coincide in comparison with all possible mXn pairs of amino acid residues in the position (each from different group) divided by mXn. The position dissimilarity value of m protein sequences within a group is defined as the number of failures to coincide in comparison with all possible mX X(m-1)/2 pairs of amino acid residues divided by mX(m-1)/2. Ten position average of dissimilarity values is plotted vs. the first position number. Area of the figure included between the profile of dissimilarity values and its mean value line characterizes the overall irregularity of amino acid substitutions along the protein sequences. If the area value is greater than the average area for 1000 random profile by more than two standard deviation units, the profile extrema containing the "surplus" of area are cut off. The cut off stretches are likely to be variable and constant regions. In case of "between groups" comparisons it is found that the overall irregularity of amino acid substitutions is very high for all considered families of proteins; phospholipases A2, aspartate aminotransferases, alpha-subunits of Na+,K(+)-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre, human rhodopsins.     

The Molecular Evolution of the Small Heat-Shock Proteins in Plants

The small heat-shock proteins have undergone a tremendous diversification in plants; whereas only a single small heat-shock protein is found in fungi and many animals, over 20 different small heat-shock proteins are found in higher plants. The small heat-shock proteins in plants have diversified in both sequence and cellular localization and are encoded by at least five gene families. In this study, 44 small heat-shock protein DNA and amino acid sequences were examined, using both phylogenetic analysis and analysis of nucleotide substitution patterns to elucidate the evolutionary history of the small heat-shock proteins. The phylogenetic relationships of the small heat-shock proteins, estimated using parsimony and distance methods, reveal that gene duplication, sequence divergence and gene conversion have all played a role in the evolution of the small heat-shock proteins. Analysis of nonsynonymous substitutions and conservative and radical replacement substitutions (in relation to hydrophobicity) indicates that the small heat-shock protein gene families are evolving at different rates. This suggests that the small heat-shock proteins may have diversified in function as well as in sequence and cellular localization.     

MCALIGN2: Faster, accurate global pairwise alignment of non-coding DNA sequences based on explicit models of indel evolution

Background  

Non-coding DNA sequences comprise a very large proportion of the total genomic content of mammals, most other vertebrates, many invertebrates, and most plants. Unraveling the functional significance of non-coding DNA depends on how well we are able to align non-coding DNA sequences. However, the alignment of non-coding DNA sequences is more difficult than aligning protein-coding sequences.