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PubMed1991

Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA.

The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serratia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants.

Bacterial Outer Membrane Proteins↗
PubMed1995

Structural classification of plant chitinases: two subclasses in class I and class II chitinases.

For the classification of plant chitinases, phylogenetic relationships were analyzed besides the established classification of domain structure and C-terminal extension sequences. Two genetically different subclasses (high and low molecular weight) were found for the main structure of class I and class II chitinases in their phylogenetic trees. The genetic distance of these subclasses showed that the high molecular weight subclass may be an ancestral molecule.

Amino Acid Sequence↗
PubMed2006

Identification of forces shaping the commensal Escherichia coli genetic structure by comparing animal and human isolates.

To identify forces shaping the Escherichia coli intraspecies ecological structure, we have characterized in terms of phylogenetic group (A, B1, D and B2) belonging, presence/absence of extraintestinal virulence genes (pap, sfa, hly and aer) and intra-host phylotype diversity a collection of 1898 commensal isolates originating from 387 animals (birds and mammals) sampled in the 1980s and the 2000s. These data have been compared with 760 human commensal isolates, sampled from 152 healthy subjects in the 2000s, and analysed with the same approach. The prevalence of the E. coli phylogenetic groups in birds, non-human mammals and humans is clearly different with a predominance of D/B1, A/B1 and A/B2 strains respectively. A major force shaping the ecological structure is the environment with a strong effect of domestication and the year of sampling followed by the climate. Host characteristics, as the diet and body mass, also influence the ecological structure. Human microbiota are characterized by a higher prevalence of virulence genes and a lower intra-host diversity than the non-human mammal ones. This work identifies for the first time a group of strains specific to the animals, the B1 phylogenetic group strains exhibiting the hly gene. In conclusion, a complex network of factors seems to shape the ecological structure of commensal E. coli, with anthropogenic factors playing a major role and perturbing natural niche equilibrium.

Animals↗
PubMed1998

The cox2 locus of the primitive angiosperm plant Acorus calamus: molecular structure, transcript processing and RNA editing.

Acorus calamus, or sweet flag, is a semiaquatic plant of uncertain taxonomic position. Molecular phylogenetic analysis using plastid rbcL sequences have suggested that Acorus calamus might be the most ancient surviving representative of the ancestral monocotyledonous plants. In order to provide molecular and phylogenetic data for the mitochondrial genetic system of Acorus, we have determined the structure of a mitochondrial locus, the cytochrome oxidase subunit II gene cox2. The Acorus cox2 gene harbors an unusually small group II intron, the smallest plant mitochondrial intron known to date. The transcript undergoes C-to-U RNA editing at eight sites. One of these sites is likely to play a dual functional role in both intron splicing and protein function. The 3' end of the mature transcript folds into a characteristic stem-loop structure that is presumably required for mitochondrial mRNA stability. Phylogenetic analysis of the cox2 sequence data, as well as the unusual intron structure, all support an evolutionarily isolated position for Acorus calamus.

Base Sequence↗
PubMed2004

Saccharomyces SRP RNA secondary structures: a conserved S-domain and extended Alu-domain.

The contribution made by the RNA component of signal recognition particle (SRP) to its function in protein targeting is poorly understood. We have generated a complete secondary structure for Saccharomyces cerevisiae SRP RNA, scR1. The structure conforms to that of other eukaryotic SRP RNAs. It is rod-shaped with, at opposite ends, binding sites for proteins required for the SRP functions of signal sequence recognition (S-domain) and translational elongation arrest (Alu-domain). Micrococcal nuclease digestion of purified S. cerevisiae SRP separated the S-domain of the RNA from the Alu-domain as a discrete fragment. The Alu-domain resolved into several stable fragments indicating a compact structure. Comparison of scR1 with SRP RNAs of five yeast species related to S. cerevisiae revealed the S-domain to be the most conserved region of the RNA. Extending data from nuclease digestion with phylogenetic comparison, we built the secondary structure model for scR1. The Alu-domain contains large extensions, including a sequence with hallmarks of an expansion segment. Evolutionarily conserved bases are placed in the Alu- and S-domains as in other SRP RNAs, the exception being an unusual GU(4)A loop closing the helix onto which the signal sequence binding Srp54p assembles (domain IV). Surprisingly, several mutations within the predicted Srp54p binding site failed to disrupt SRP function in vivo. However, the strength of the Srp54p-scR1 and, to a lesser extent, Sec65p-scR1 interaction was decreased in these mutant particles. The availability of a secondary structure for scR1 will facilitate interpretation of data from genetic analysis of the RNA.

Alu Elements↗
PubMed1999

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.

Animals↗
PubMed1998

Trematode and monogenean rRNA ITS2 secondary structures support a four-domain model.

The secondary structure of rRNA internal transcribed spacer 2 is important in the process of ribosomal biogenesis. Trematode ITS sequences are poorly conserved and difficult to align for phylogenetic comparisons above a family level. If a conserved secondary structure can be identified, it can be used to guide primary sequence alignments. ITS2 sequences from 39 species were compared. These species span four orders of trematodes (Echinostomiformes, Plagiorchiformes, Strigeiformes, and Paramphistomiformes) and one monogenean (Gyrodactyliformes). The sequences vary in length from 251 to 431 bases, with an average GC content of 48%. The monogenean sequence could not be aligned with confidence to the trematodes. Above the family level trematode sequences were alignable from the 5' end for 139 bases. Secondary structure foldings predicted a four-domain model. Three folding patterns were required for the apex of domain B. The folding pattern of domains C and D varies for each family. The structures display a high GC content within stems. Bases A and U are favored in unpaired regions and variable sites cluster. This produces a mosaic of conserved and variable regions with a structural conformation resistant to change. Two conserved strings were identified, one in domain B and the other in domain C. The first site can be aligned to a processing site identified in yeast and rat. The second site has been found in plants, and structural location appears to be important. A phylogenetic tree of the trematode sequences, aligned with the aid of secondary structures, distinguishes the four recognized orders.

Animals↗
PubMed2002

Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata.

The dorsal skin of the crawfish frog, Rana areolata, is associated with numerous prominent granular glands. Proteomic analysis of electrically stimulated skin secretions from these glands enabled the identification and characterization of eight peptides with antimicrobial and hemolytic activity belonging to the previously identified brevinin-1, temporin-1, palustrin-2, palustrin-3, esculentin-1 (two peptides), and ranatuerin-2 (two peptides) families. The primary structures of the peptides were consistent with a close phylogenetic relationship between R. areolata and the pickerel frog, Rana palustris. Three structurally related cationic, cysteine-containing peptides were identified that show sequence similarity to peptide Leucine-Arginine, a peptide with immunomodulatory and histamine-releasing properties from the skin of the northern leopard frog, Rana pipiens. The skin secretions contained a 61-amino-acid-residue peptide that inhibited porcine trypsin and possessed a 10-cysteine-residue motif that is characteristic of a protease inhibitor previously isolated from the parasitic nematode, Ascaris suum. A 48-amino-acid-residue protein containing eight cysteine residues in the whey acidic protein (WAP) motif, characteristic of elafin (skin-derived antileukoproteinase) and secretory leukocyte protease inhibitor, was also isolated. The data suggest that protease inhibitors in skin secretions may play a role complementary to cationic, amphipathic alpha-helical peptides in protecting anurans from invasions by microorganisms.

Amino Acid Sequence↗
PubMed1991

Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs.

Base Sequence↗
PubMed2004

Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure, and phylogeny of the species.

A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed. Type strains of the two most closely related species (P. balearica, former genomovar 6, and P. mendocina), together with P. aeruginosa, as the type species of the genus, have been included in the study. The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis. Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1, gyrB, rpoD, nosZ, catA, and nahH) confirmed the monophyletic origin of the genomovars within the Pseudomonas branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species.

Alleles↗
PubMed1993

Prediction of common folding structures of homologous RNAs.

We have developed an algorithm and a computer program for simultaneously folding homologous RNA sequences. Given an alignment of M homologous sequences of length N, the program performs phylogenetic comparative analysis and predicts a common secondary structure conserved in the sequences. When the structure is not uniquely determined, it infers multiple structures which appear most plausible. This method is superior to energy minimization methods in the sense that it is not sensitive to point mutation of a sequence. It is also superior to usual phylogenetic comparative methods in that it does not require manual scrutiny for covariation or secondary structures. The most plausible 1-5 structures are produced in O(MN2 + N3) time and O(N2) space, which are the same requirements as those of widely used dynamic programs based on energy minimization for folding a single sequence. This is the first algorithm probably practical both in terms of time and space for finding secondary structures of homologous RNA sequences. The algorithm has been implemented in C on a Sun SparcStation, and has been verified by testing on tRNAs, 5S rRNAs, 16S rRNAs, TAR RNAs of human immunodeficiency virus type 1 (HIV-1), and RRE RNAs of HIV-1. We have also applied the program to cis-acting packaging sequences of HIV-1, for which no generally accepted structures yet exist, and propose potentially stable structures. Simulation of the program with random sequences with the same base composition and the same degree of similarity as the above sequences shows that structures common to homologous sequences are very unlikely to occur by chance in random sequences.

Algorithms↗
PubMed2005

Computational analysis of protein tyrosine phosphatases: practical guide to bioinformatics and data resources.

The exponential growth of sequence data has become a challenge to database curators and end-users alike and biologists seeking to utilize the data effectively are faced with numerous analysis methods. Here, with practical examples from our bioinformatics analysis of the protein tyrosine phosphatases (PTPs), we show how computational analysis can be exploited to fuel hypothesis-driven experimental research through the exploration of online databases. We cover the following elements: (i) similarity searches and strategies to collect a non-redundant database of tyrosine-specific PTP domains; (ii) utilization of this database to classify human, fly, and worm PTPs (based on alignments and phylogenetic analysis); (iii) three-dimensional structural analysis to identify conserved regions (structure-function) and non-conserved selectivity-determining regions (substrate specificity); and (iv) genomic analysis, including mapping of exon structure, identification of pseudogenes, and exploration of disease databases. We discuss the importance of manual curation, illustrating examples in which pseudogenes give rise to predicted proteins in GenBank and note that domain servers, such as PFAM and SMART, erroneously include dual-specificity and lipid phosphatases in their collection of tyrosine-specific PTPs. To capitalize on our annotated set of 402 PTP domains (from 47 species and five phyla), we identify sequence conservation across taxonomic categories and explore structure-function relationships among tandem domain receptor-like PTPs. We define three Src homology 2 domain-containing PTP genes in stingray, zebrafish, and fugu and speculate on their evolutionary relationship with human pseudogenes. Our annotated sequences, along with a web service for phylogenetic classification of PTP domains, are available online (http://ptp.cshl.edu and http://science.novonordisk.com/ptp).

Amino Acid Sequence↗
PubMed1995

Genetic diversity and population structure of Trypanosoma brucei: clonality versus sexuality.

Genomic fingerprinting by arbitrarily primed PCR was used to analyze the genetic variability among 59 Trypanosoma brucei stocks representing the three T. brucei subspecies isolated from various hosts and different countries in Africa. 14 oligonucleotide primers revealed 355 polymorphic binary characters which were used for phenetic and phylogenetic analysis and to perform recombination tests exploring the linkage disequilibrium in the sample. There was good concordance between arbitrarily primed PCR polymorphisms and isoenzyme data previously collected for many of the same strains [1]. However, the arbitrarily primed PCR typing was more discerning than multilocus enzyme electrophoresis typing. Phenetic and phylogenetic analysis using arbitrarily primed PCR markers did not confirm T. brucei brucei and T. brucei rhodesiense as separate subspecies, but T. brucei gambiense group I was monophyletic, confirming this group as suitable for the subspecies status. With this exception, there were no clear lineages among the sample, other than clustering of East African stocks and clustering of West African stocks. Some features of the phylogenetic analysis suggested that the population structure was not strictly clonal though recombination tests showed linkage disequilibrium, even in the absence of repeated genotypes. While genotypes appear stable enough for tracking in applied studies, sexuality will impact at the evolutionary time scale, and may be more frequent under some ecological conditions. The arbitrarily primed PCR approach should be an effective and simple approach to follow epidemics and to quantify the role of sexuality in T. brucei populations.

Africa↗
PubMed1997

Evolutionary shifts in three major structural features of the mitochondrial genome among iguanian lizards.

A phylogenetic tree for major lineages of iguanian lizards is estimated from 1,488 aligned base positions (858 informative) of newly reported mitochondrial DNA sequences representing coding regions for eight tRNAs, ND2, and portions of ND1 and COI. Two well-supported groups are defined, the Acrodonta and the Iguanidae (sensu lato). This phylogenetic hypothesis is used to investigate evolutionary shifts in mitochondrial gene order, origin for light-strand replication, and secondary structure of tRNACys. These three characters shift together on the branch leading to acrodont lizards. Plate tectonics and the fossil record indicate that these characters changed in the Jurassic. We propose that changes to the secondary structure of tRNACys may destroy function of the origin for light-strand replication which, in turn, may facilitate shifts in gene order.

Animals↗
PubMed1992

Cross-linking of U1 snRNA using nitrogen mustard. Evidence for higher order structure.

Secondary structures for all five spliceosomal small nuclear (sn) RNAs (U1, U2, U4, U5, and U6 snRNAs) have been derived from phylogenetic, biochemical, and genetic data, but tertiary structure information has been more difficult to obtain. Here we have used the general cross-linking reagent nitrogen mustard (bis-(2-chloroethyl)methylamine) to explore the tertiary conformation of naked U1 snRNA. We detected two intramolecularly cross-linked U1 species (X-U1#1 and X-U1#2) after cross-linking of deproteinized HeLa nuclear extract. We determined the cross-linked sites and found that X-U1#1 is cross-linked between the C82-A85 and U129, while X-U1#2 is cross-linked between U105-G108 and A118. We then tested the ability of these two cross-linked species to bind small nuclear ribonucleo-protein (snRNP) proteins in vitro (in HeLa nuclear extract or S100) and in vivo (in Xenopus oocytes). Both X-U1#1 and X-U1#2 were found to reconstitute efficiently in vitro and in vivo, as judged by immunoprecipitation with antibodies specific for Sm and U1-specific proteins. Our data suggest that (i) the Sm-binding site lies on the surface of the native U1 snRNP, since the cross-link in X-U1#1 involves the Sm-binding site but does not block snRNP assembly, and (ii) U1 snRNA may adopt the correct tertiary conformation even in the absence of U1 snRNP proteins.

Base Sequence↗
PubMed1995

Translational control of maturation-protein synthesis in phage MS2: a role for the kinetics of RNA folding?

The gene for the maturation (A) protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt. Secondary structure of the leader was deduced by phylogenetic comparison and by probing with enzymes and chemicals. The RNA folds into a cloverleaf, i.e., three stem-loop structures enclosed by a long-distance interaction (LDI). This LDI is essential for translational control. Its 3'moiety contains the Shine-Dalgarno region of the A-protein gene, whereas its complement is located 80 nt upstream, i.e., about 30 nt from the 5'-terminus of the RNA chain. Mutational analysis shows that this base pairing represses expression of the A-protein gene. We present a model in which translational starts can only take place on nonequilibrated RNA, in which base pairing between the complementary regions has not yet taken place. We suggest that this pairing is kinetically delayed by the intervening sequence, which contains the three hairpins of the cloverleaf. The model is mainly based on the observation that reducing the length of the intervening sequence reduces expression, whereas increasing the length has the opposite effect. In addition, further stabilization of the LDI by a stronger base pair does not lead to a decrease in A-protein synthesis. Such a decrease is predicted to occur if translation would be controlled by the equilibrium structure of the leader RNA. These and other observations fit a kinetic model of translational control by RNA folding.

Base Sequence↗
PubMed2002

[Association between NS5A gene sequence and response to interferon therapy in chronic hepatitis C patients in Shanghai].

BACKGROUND: To elucidate relationship between amino acid sequence of non-structural protein 5A (NS5A) and outcome of HCV (1 b) patients after interferon (IFNa) therapy. METHODS: Sera of 24 patients were collected before, during and after IFNa therapy. Pretreatment RNA levels and the sequences of HCV NS5A interferon sensitivity determining region (ISDR) were determined. NS5A full-length sequences of 5 HCV isolates from 3 patients with different response types were also analyzed. Phylogenetic tree analysis and protein secondary structure prediction were undertaken. RESULTS: Pretreatment RNA levels of sustained response group were significantly lower than that of non-response group and relapse group (4.50X104 copies/ml versus 1.82X107 copies/ml, P < 0.01).ISDR sequences of NS5A from pretreatment sera were compared with HCV-J strain (prototype). Thirteen of 24 isolates were wild type,11 of 24 were intermediate type and none of them was mutant type. 3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. Phylogenetic tree based on NS5A full-length sequences classified 5 isolates with 3 different response types into 3 groups. Non-response isolates belonged to the same group as HCV-J. Secondary structure prediction of 5 isolates revealed significant differences existing in 2 255- 2 289. This region was partly overlapped with PKR-binding domain. CONCLUSIONS: Low HCV RNA levels in serum are associated with favorable outcome of IFNa therapy. ISDR sequence alone could not predict outcome of IFN treatment. Combination of determination of HCV RNA levels in serum with sequence analysis of PKR-binding domain may be helpful in predicting the efficacy of IFN therapy.

Amino Acid Sequence↗
PubMed1992

Duplication of the structural gene for glucosephosphate isomerase and phosphogluconate dehydrogenase in Scabiosa columbaria and their phylogenetic implications in the dipsacaceae.

Zymograms of glucosephosphate isomerase (GPI) and phosphogluconate dehydrogenase (PGD) revealed three isozymes for each enzyme in the plant species Scabiosa columbaria. Intergenic heterodimers are formed between the polypeptides coded by Gpi-1 and Gpi-2 and between those coded by Pgd-1 and Pgd-2, indicating that a GPI and a PGD locus have been duplicated in the past. The ancestral genes assort independently with their duplicated gene, suggesting that the duplications have originated from a process of translocation. Linkage was found only between Gpi-1 and Pgd-2 and between Gpi-2 and Pgd-1, suggesting that the duplicated loci were located on the same translocated chromosomal segment. Both duplications are present in all other examined species of Scabiosa and in Cephalaria and Knautia, two other genera of the Dipsacaceae. The genera Succisa and Dipsacus, also belonging to the Dipsacaceae, do not show Gpi-1 activity, making Gpi-2 and Pgd-1 the most likely ancestral genes. In Succisa, the isozymes of Gpi-1 and Gpi-2 either overlap or Gpi-1 has been silenced. The combined results suggest that a chromosomal segment containing Gpi-2 and Pgd-1 has been translocated before the divergence of Scabiosa, Cephalaria, Knautia, and Succisa.

Genes, Plant↗