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PubMed2003

Diversity of polyketide synthase gene sequences in Aspergillus species.

Fungal polyketide synthases are responsible for the biosynthesis of several mycotoxins and other secondary metabolites. The aim of our work was to investigate the diversity of polyketide synthases in Aspergillus species using two approaches: PCR amplification using oligonucleotide primers, and bioinformatics. Ketosynthase domain probes amplified DNA fragments of about 700 bp in each examined isolate. Sequences of these domains were aligned and analyzed by phylogenetic methods. The ketosynthase domain sequences were highly diverse indicating that they most probably represent polyketide synthases responsible for different functions. A. albertensis and A. niger ketosynthase domain sequences clustered together with sequences of genes required for pigment biosynthesis (wA) in A. nidulans and P. patulum, while the ketosynthase domain sequence of A. muricatus was most closely related to an A. parasiticus wA type domain sequence, and those of the A. ochraceus isolates formed a distinct clade on the tree. These sequences were highly homologous to an A. terreus naphthopyrone synthase gene. An Aspergillus fumigatus genomic database was also searched for ketosynthase domain sequences, which have been included in the phylogenetic analysis. Altogether 14 putative ketosynthase domain sequences were identified. Clustering of the ketosynthase domain sequences correlated well with the type of metabolites produced by the corresponding polyketide synthases. At least 8 clusters with putative ketosynthase domain sequences of unknown function have been identified. Further studies are in progress to clarify the role of some of the identified polyketide synthase genes.

Amino Acid Sequence↗
PubMedDate not supplied

Protein transport via amino-terminal targeting sequences: common themes in diverse systems.

Many proteins that are synthesized in the cytoplasm of cells are ultimately found in non-cytoplasmic locations. The correct targeting and transport of proteins must occur across bacterial cell membranes, the endoplasmic reticulum membrane, and those of mitochondria and chloroplasts. One unifying feature among transported proteins in these systems is the requirement for an amino-terminal targeting signal. Although the primary sequence of targeting signals varies substantially, many patterns involving overall properties are shared. A recent surge in the identification of components of the transport apparatus from many different systems has revealed that these are also closely related. In this review we describe some of the key components of different transport systems and highlight these common features.

Bacteria↗
PubMed1996

Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.

Saccharomyces cerevisiae transcription factor (TF) IIIB, a TATA-binding protein (TBP)-containing multisubunit factor, recruits RNA polymerase (Pol) III for multiple rounds of transcription. TFIIIC is an assembly factor for TFIIIB on TATA-less tRNA gene promoters. To investigate the role of TBP-DNA interactions in tRNA gene transcription, we generated sequence substitutions in the SUP4 tRNATyr gene TFIIIB binding site. Purified transcription proteins were used to analyze the selection of transcription initiation sites and the physical structures of the protein complexes formed on these mutant genes. We show that the association of TFIIIB with tRNA genes proceeds through an initial step of binding-site selection that is codirected by its TBP subunit and by TFIIIC. TFIIIB is assembled in a predominantly metric manner with regard to box A, the start site-proximal binding site of TFIIIC, but TFIIIC opens a window within which wild-type TBP can select the TFIIIB-binding site. Despite its clear preference for AT-rich sequences, TBP can mediate TFIIIB assembly at diverse DNA sequences, including stretches containing only G and C. However, a mutant TBP, m3, which recognizes TATAAA and TGTAAA and is active for Pol III transcription, utilizes other sequences only poorly. We also show that alternative alignments between DNA-bound TFIIIB and TFIIIC are possible, implying a remarkably flexible linkage, and suggest that Tfc4, the TFIIIB-assembling subunit of TFIIIC, could be responsible for such elasticity. The relevance of these findings to alternative initiation of Pol II- and other Pol III-transcribed genes is discussed.

Base Sequence↗
PubMed2002

Antisense treatment of caliciviridae: an emerging disease agent of animals and humans.

The Earth's oceans are the primary reservoir for an emerging family of RNA viruses, the Caliciviridae, which can cause a spectrum of diseases in marine animals, wildlife, farm animals, pets and humans. Certain members of this family have unusually broad host ranges, and some are zoonotic (transmissible from animals to humans). The RNA virus replicative processes lack effective genetic repair mechanisms, and, therefore, virtually every calicivirus replicate is a mutant. Hence, traditional therapeutics dependent on specific nucleic acid sequences or protein epitopes lack the required diversity of sequence or conformational specificity that would be required to reliably detect, prevent or treat infections from these mutant clusters (quasi-species) of RNA viruses, including the Caliciviridae. Antisense technology using phosphorodiamidate morpholino oligomers shows promise in overcoming these current diagnostic and therapeutic problems inherent with newly emerging viral diseases.

Animals↗
PubMed1997

Mutation rate and excess African heterozygosity.

Global studies of within-group genetic variation have revealed a tendency for some traits, but not all, to show higher heterozygosity in sub-Saharan African populations. Although excess African diversity has been interpreted as reflecting a greater "age" of sub-Saharan African populations, more recent research has shown that this excess is more likely a consequence of a larger African long-term effective population size. The observation that certain traits, particularly classic genetic markers and RFLPs, do not show this pattern has been interpreted as ascertainment bias. Here, I examine another possible factor: that excess African heterozygosity is in part a function of mutation rate. Simple equilibrium and nonequilibrium models of absolute excess heterozygosity are examined. The results indicate that there is little excess African heterozygosity for traits with low mutation rates and greater excess heterozygosity for traits with moderate to high aggregate mutation rates. Observed data are consistent with these models. Also, depending on population size and time depth, traits with high levels of mutation might show less excess heterozygosity than those with moderate to high mutation rates. Another measure of diversity, mean sequence divergence, shows an increase in excess diversity for traits with high mutation rates.

Africa South of the Sahara↗
PubMed2006

Analysis of distribution indicates diverse functions of simple sequence repeats in Mycoplasma genomes.

Simple sequence repeats (SSRs) composed of extensive tandem iterations of a single nucleotide or a short oligonucleotide are rare in most bacterial genomes, but they are common among Mycoplasma. Some of these repeats act as contingency loci in association with families of surface antigens. By contraction or expansion during replication, these SSRs increase genetic variance of the population and facilitate avoidance of the immune response of the host. Occurrence and distribution of SSRs are analyzed in complete genomes of 11 Mycoplasma and 3 related Mollicutes in order to gain insights into functional and evolutionary diversity of the SSRs in Mycoplasma. The results revealed an unexpected variety of SSRs with respect to their distribution and composition and suggest that it is unlikely that all SSRs function as contingency loci or recombination hot spots. Various types of SSRs are most abundant in Mycoplasma hyopneumoniae, whereas Mycoplasma penetrans, Mycoplasma mobile, and Mycoplasma synoviae do not contain unusually long SSRs. Mycoplasma hyopneumoniae and Mycoplasma pulmonis feature abundant short adenine and thymine runs periodically spaced at 11 and 12 bp, respectively, which likely affect the supercoiling propensities of the DNA molecule. Physiological roles of long adenine and thymine runs in M. hyopneumoniae appear independent of location upstream or downstream of genes, unlike contingency loci that are typically located in protein-coding regions or upstream regulatory regions. Comparisons among 3 M. hyopneumoniae strains suggest that the adenine and thymine runs are rarely involved in genome rearrangements. The results indicate that the SSRs in the Mycoplasma genomes play diverse roles, including modulating gene expression as contingency loci, facilitating genome rearrangements via recombination, affecting protein structure and possibly protein-protein interactions, and contributing to the organization of the DNA molecule in the cell.

Base Sequence↗
PubMed1997

Diverse function of aromatase and the N-terminal sequence deleted form.

The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

Aromatase↗
PubMed2004

Distribution of genetic variation in the growth hormone 1 gene in Atlantic salmon (Salmo salar) populations from Europe and North America.

The level and hierarchical distribution of genetic variation in complete sequences of the Atlantic salmon (Salmo salar) growth hormone (GH1) gene were investigated in populations from Europe and North America with a view to inferring the major evolutionary forces affecting genetic variation at this locus. Seventeen polymorphic sites were identified in complete sequences from nine populations, with levels of noncoding (intron and untranslated region sequences) nucleotide diversity being similar to those observed in other species. No variation, however, was observed in exonic sequences, indicating that nucleotide diversity in the Atlantic salmon GH1 gene is three and 25 times less than that estimated for human and Drosophila coding sequences, respectively. This suggests that purifying selection is the predominant contemporary force controlling the molecular evolution of GH1 coding sequences. Comparison of haplotype relationships within and between populations indicated that differentiation between populations from Europe and North America was greater than within-continent comparisons. However, several haplotypes observed in the northernmost European populations were more similar to those observed in North American than to any other haplotypes observed in Europe. This is most likely to be a result of historical, rather than contemporary, gene flow. Neutrality test statistics, such as Tajima's D, were significantly positive in the European populations in which North American-like haplotypes were observed. Although a positive Tajima's D is commonly interpreted as the signal of balancing selection, a more likely explanation in this case is that either historical migration or ascertainment bias, rather than within population local adaptation, has given rise to an excess of intermediate frequency alleles.

Amino Acid Sequence↗
PubMed2005

Suppression subtractive hybridization as a basis to assess Mycoplasma agalactiae and Mycoplasma bovis genomic diversity and species-specific sequences.

The phylogenically related Mycoplasma agalactiae and Mycoplasma bovis species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for M. bovis type-strain PG45 or for M. agalactiae type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of M. agalactiae and M. bovis field isolates. Results indicated that approximately 70 % of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one M. bovis isolate, which was first classified as M. agalactiae, reacted with 15 % of the PG2-specific probes, while several M. agalactiae isolates reacted with 15 % of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of Mycoplasma fermentans (ICEF) was found in most M. bovis field isolates and in a few M. agalactiae isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of M. agalactiae- and M. bovis-specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of M. agalactiae and M. bovis.

Animals↗
PubMed2006

Distribution of optineurin sequence variations in an ethnically diverse population of low-tension glaucoma patients from the United States.

PURPOSE: Previous studies have suggested that Optineurin (OPTN) sequence variants contribute to low-tension glaucoma (LTG) in ethnically homogeneous populations. The purpose of this study is to evaluate the prevalence of OPTN sequence variants in an ethnically diverse population of LTG patients from the United States, and to describe the phenotype of patients with OPTN sequence variants preferentially found in LTG patients. METHODS: Genomic DNA purified from 67 LTG patients was screened for DNA sequence variants located in the exons and flanking introns of the OPTN gene using high-performance liquid chromatography analysis and direct genomic DNA sequencing. Eighty-six primary open-angle glaucoma probands and 100 control patients were also analyzed. RESULTS: Nine OPTN DNA sequence variants were identified in this patient population including the 2 previously identified heterozygous nonsynonymous single-nucleotide polymorphisms in exons 4 and 5. Four LTG patients with severe disease and positive family history of glaucoma, were found to have DNA sequence changes not found in primary open-angle glaucoma probands or control individuals including the previously reported E50K variation. CONCLUSIONS: The results of this study support the rare association of OPTN sequence variants with familial forms of LTG. The E50K mutation seems to be associated with a severe form of LTG, and although rare, the identification of this sequence variant in patients at risk may help direct appropriate therapy.

Adult↗
PubMed2003

Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis.

Escherichia coli O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequence-based approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (ompA and espA) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and espA. For ompA, all but five isolates had sequence identical to that of the reference strains. E. coli O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for E. coli 0157:H7 are needed.

DNA, Bacterial↗
PubMed2002

Sequence analysis of an artificial family of RNA-binding peptides.

Diverse peptide sequences recognizing the lambda boxB RNA hairpin were previously isolated from a library encoding the 22-residue lambda N peptide with random amino acids at positions 13-22 using mRNA display. We have statistically analyzed amino acid distributions in 65 unique sequences from rounds 11 and 12 of this selection and evaluated the resulting structural and functional predictions by alanine-scanning mutagenesis and circular dichroism spectrometry. This artificial sequence family has a consensus structure that continues the bent alpha helix of lambda N up to position 17 when bound to lambda boxB. A charge pair (E(14)R(15)) and hydrophobic patch (A(21)L(22) or V(21)L(22)) have important functional roles in this context. Notably, amino acid covariance reveals six specific pairs of random region positions with >95% significant linkage and strong overall helical (i+1, i+3, and i+4) couplings. The covariance analysis suggests that (1) the sequence context of every residue in each insert has been optimized, (2) selected sequences are local optima on a rugged fitness landscape, and (3) it is possible to detect more subtle structural features with artificial protein sequence families than natural homologs. Our results provide a framework for investigating the structures of in vitro selected proteins by functional minimization, reselection, and covariance analysis.

Amino Acid Sequence↗
PubMed2000

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys.

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.

Ammonia↗
PubMed1993

Combinatorial diversity in DR2 haplotypes.

Sequence analysis has identified multiple alleles at two loci that encode for the DR2 specificity. The loci, DRB1 and DRB5, are in linkage disequilibrium which can extend to alleles of the DQ loci. Serologic, cellular, and sequence-specific oligonucleotide probe (SSOP) typing techniques have been used to identify the DR2 haplotypes. In this report, we have characterized by SSOP typing and cDNA/DNA sequence analyses the combinatorial diversity of DR2 haplotypes. Cells were selected on the basis of unique serologic reactivity, unique associations of alleles of DR and DQ loci, and/or presence in populations which have not been extensively characterized for HLA diversity. An asymmetric polymerase chain reaction (PCR) amplification was applied to rapidly screen unique cells and to characterize DNA sequence in conjunction with more conventional cDNA sequence analysis. The sequence data confirm the lack of a DRB5 locus in the DR2"LUM" specificity, the unexpected association of DRB1*1602 and DRB5*010 alleles in a nonCaucasoid population, and the association of the allele DRB1*1503 with DRB5*0101 in black African, African American and native American individuals. The DRB1*1503 and DRB5*0101 alleles were identified in an unusual haplotype, DR2,DQ2. The combinatorial diversity of the DR2 haplotypes is extended by these studies in nonCaucasoid populations.

Alleles↗
PubMed1998

Diploid Musa acuminata genetic diversity assayed with sequence-tagged microsatellite sites.

The sequence-tagged microsatellite site (STMS) discrimination potential was explored using nine microsatellite primer pairs. STMS polymorphism was assayed by nonradioactive urea-polyacrylamide gel electrophoresis. Genetic relationships were examined among 59 genotypes of wild or cultivated accessions of diploid Musa acuminata. The organization of the subspecies was confirmed and some clone relationships were clarified.

DNA, Plant↗
PubMed1995

Unexpected diversity in U6 snRNA sequences from trypanosomatids.

We have isolated and sequenced the genes for the trans-spliceosomal U6 small nuclear RNAs (snRNAs) from the trypanosomatid species Leishmania mexicana (Lm) and Phytomonas sp. (Ps). Compared with the Trypanosoma brucei (Tb) U6 snRNA, the Lm U6 snRNA contains only a single additional G-C bp in the 5' terminal stem-loop. In contrast, the Ps U6 snRNA sequence contains a G-->C change at the last nucleotide of the highly conserved and functionally important ACAGAG hexanucleotide and three additional changes in conserved positions. Our results indicate that trans-spliceosomal U6 snRNAs from trypanosomatid species do not always conform to the consensus sequence of cis-spliceosomal U6 snRNAs.

Animals↗
PubMed2004

Diversity of 16S rDNA sequences of Rhizobium spp. implications for species determinations.

Comparative analysis of 70 16S rDNA sequences representing 20 Rhizobium species (including pathogenic Agrobacterium spp.) was conducted using Maximum Likelihood to establish relationships of species using multiple sequences. There is no significant internal division of the Rhizobium clade to suggest that it represents more than one genus. Plant pathogenic (Agrobacterium) species are distributed within the genus. The analysis supported the synonymy of some species (Rhizobium gallicum and Rhizobium mongolense) and the need for comparative investigations of the tumorigenic and nodulating properties of Rhizobium tropici and Rhizobium rhizogenes. Misidentification of some sequences may conceal one or more putative novel species. Some sequences appear to be misidentified because of faulty sequencing or incomplete or inadequate analysis.

DNA, Bacterial↗
PubMed2002

Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns.

Adenosine deaminase that acts on RNA -2 (ADAR2) is a member of a family of vertebrate genes that encode adenosine (A)-to-inosine (I) RNA deaminases, enzymes that deaminate specific A residues in specific pre-mRNAs to produce I. Known substrates of ADAR2 include sites within the coding regions of pre-mRNAs of the ionotropic glutamate receptors, GluR2-6, and the serotonin receptor, 5HT2C. Mammalian ADAR2 expression is itself regulated by A-to-I editing and by several alternative splicing events. Because the biological consequences of ADAR2 function are significant, we have undertaken a phylogenetic comparison of these features. Here we report a comparison of cDNA sequences, genomic organization, editing site sequences and patterns of alternative splicing of ADAR2 genes from human, mouse, chicken, pufferfish and zebrafish. Coding sequences and intron/exon organization are highly conserved. All ADAR2 genes show evidence of transcript editing with required sequences and predicted secondary structures very highly conserved. Patterns and levels of editing and alternative splicing vary among organisms, and include novel N-terminal exons and splicing events.

Adenosine Deaminase↗