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PubMed2005

RNAshapes: an integrated RNA analysis package based on abstract shapes.

We introduce RNAshapes, a new software package that integrates three RNA analysis tools based on the abstract shapes approach: the analysis of shape representatives, the calculation of shape probabilities and the consensus shapes approach. This new package is completely reimplemented in C and outruns the original implementations significantly in runtime and memory requirements. Additionally, we added a number of useful features like suboptimal folding with correct dangling energies, structure graph output, shape matching and a sliding window approach.

Algorithms↗
PubMed1993

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4.

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of the td gene by recombinational marker rescue using plasmid or M13 subclones of the td gene. Phage crosses using deletion mutants with known breakpoints in the 3' end of the td intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5' and 3' ends of the intron sequence, in accordance with the hypothesis that the 5' and 3' domains of the T4 td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mapped td mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within the td gene.

Bacteriophage T4↗
PubMed1981

Yeast ochre suppressor SUQ5-ol is an altered tRNA Ser UCA.

Ochre suppressor tRNA was partially purified from strains of Saccharomyces cerevisiae containing the serine-inserting class III suppressor SUQ5-ol. RNA sequence analysis of this tRNA indicated that the suppressor is derived from a UCA-decoding tRNA Ser by a G leads to U substitution in the middle position of the anticodon. The suppressor further differs from the wild-type UCA-decoding tRNA Ser in that the mutant anticodon lacks the modified uridine found in the wobble position of the wild-type tRNA and contains instead another modification in or near the anticodon.

Anticodon↗
PubMed2004

Evidence of avian pneumovirus spread beyond Minnesota among wild and domestic birds in central North America.

To detect avian pneumovirus (APV) in central North America, nasal turbinates or choanal deft tissues from domestic turkeys and wild birds were examined for the presence of APV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas serum samples from domestic turkeys were analyzed for APV antibodies by enzyme-linked immunosorbent assay (ELISA). In 2002, the seroprevalence of disease in domestic turkeys in Minnesota remained high (42.3% of the flocks). In addition, there is evidence the disease has spread to turkey flocks in North Dakota (8.2%), South Dakota (7%), Iowa (10%), and Wisconsin (8.6%) as detected by RT-PCR and/or ELISA. House sparrows and ring-billed gulls sampled in Minnesota and snow geese from Saskatchewan, Canada, were found to harbor APV RNA. Sequence analysis of wild bird APV strains showed high amino acid sequence identity among wild bird isolates (<97%) and between wild bird and turkey viral isolates (93.2%-99.3%). This study demonstrated that APV infections were present in domestic turkey flocks and wild birds outside the state of Minnesota; however, the role of wild birds in spreading APV to domestic turkeys remains unclear.

Amino Acid Sequence↗
PubMed2025

The impact of Iso-mukaadial acetate on Plasmodium falciparum transcriptional gene regulation.

Malaria remains prevalent globally despite various intervention strategies aimed at preventing its transmission. With the decreasing effectiveness of antimalarial drugs, medicinal plant extracts have been proposed as alternatives. Iso-mukaadial acetate extracted from Warburgia salutaris has shown anti-plasmodial activity, but the mechanism of inhibition is unknown. In this study, RNA sequencing analysis of P. falciparum NF54 strain treated with IMA was conducted to determine the possible targets of IMA. The expression profiles of P. falciparum genes regulated by IMA and chloroquine (antimalarial control) during the intraerythrocytic stage were analyzed with gene ontology tools, including PlasmoDB, ShinyGO and g: Profiler. IMA and chloroquine upregulated genes linked to parasite biological processes and cell adhesion molecular binding functions, including PfEMP1, RIFIN, and STEVOR. Chloroquine specifically downregulated DNA replication processes involving DNA replication licensing factors MCM3 and DNA helicase, while IMA downregulated peptidyl-proline modification and glycolytic pathways. KEGG analysis suggested glycolysis-gluconeogenesis and pentose phosphate pathway enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PD)-6-phosphogluconolactonase) as theoretical IMA targets, whose suppression could hypothetically reduce ATP and NADPH production, weakening parasite energy supply and antioxidant defenses. The inhibition of DNA replication components (MCM complex, DNA topoisomerases) by IMA, and the downregulation of DNA replication/repair proteins by chloroquine, may both impair genome integrity, contributing to the observed anti-plasmodial effects. IMA treatment was assumed to be associated with impairment of parasite energy metabolism, redox balance and DNA replication machinery. These effects differ from chloroquine, which primarily targeted DNA replication and repair processes, yet both drugs upregulated adhesion-associated gene families. Changes in the expression of metabolic and replication genes induced by IMA suggest the compounds potential as an anti-plasmodial candidate, warranting further biochemical validation of its mechanism of effect.

Plasmodium falciparum↗
PubMed1988

Relative frequencies of rotavirus serotypes 1, 2, 3, and 4 in Venezuelan infants with gastroenteritis.

We have used a recently developed monoclonal antibody enzyme-linked immunosorbent assay (K. Taniguchi, T. Urasawa, Y. Morita, H. B. Greenberg, and S. Urasawa, J. Infect. Dis. 155:1159-1166, 1987) for serotyping rotaviruses recovered from 134 Venezuelan infants over a period of 15 months. One hundred and nine of the specimens were typed with the following distribution: serotype 1, 48%; serotype 2, 16%; serotype 3, 22%; and serotype 4, 14%. Three specimens reacted with two different monoclonal antibodies. In addition, 6 specimens (5%) containing enough outer capsid antigen could not be typed; partial RNA sequence analysis of the glycoprotein gene from three of these six strains failed to reveal sequence differences with prototype strains that could be serotyped with the monoclonal antibodies. Variations in the recovery rates of the different serotypes were observed. Serotypes 2, 3, and 4 predominated at the beginning of the study, and serotype 1 predominated at the end of the study. Diarrheal illness appeared to be more prolonged in infants shedding rotavirus serotypes 1 and 3 than in those shedding serotypes 2 and 4.

Base Sequence↗
PubMed1999

[The feature of influenza virus activity and genetic characterization of HA1 gene of influenza A H3N2 viruses isolated from 1994-1997 in Shenzhen].

OBJECTIVE: To understand the feature of influenza virus activity and genetic characterization of HA1 gene of influenza A H3N2 viruses isolated in recently years in Shenzhen. METHODS: The specimens were inoculated into amniotic and allantoic cavities of the embryonated chicken eggs for viral isolation. The isolates were typed with HI test. Virion RNA extracted from allantoic fluid by Rneasy RNA extraction kit was used for RT-PCR amplification. Afterward, the product of PCR was purified with purification kit. RNA sequence analysis was performed by Sanger dideoxy-mediated nucleotide chain termination method. RESULTS: Influenza A H3N2, H1N1 and B viruses were cocirculating in men in Shenzhen in recent years. However, the extent of their activities was not the same in a single year. The influenza B virus was the predominant strain in 1994 and 1997, but the predominant strain was H3N2 virus in 1996. The genetic analysis showed that the substitution of amino acids occured year by year and was mainly located at antigenic determinants or receptor binding site on HA1 domain of H3N2 viruses. CONCLUSION: Influenza A H3N2, H1N1 and B viruses were still co-circulating in men in Shenzhen in recent years, the predominant strain of influenza virus was different in different years. An increase of influenza A H3N2 virus activity in 1996 was due to the substitution of amino acids sequences on viral HA1 domain.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of a truncated dopamine D1 receptor from goldfish retina: stimulation of cyclic AMP production and calcium mobilization.

Receptors for dopamine are present on horizontal cells of fish retina that are linked to the activation of adenylate cyclase. In the present study, the goldfish (Carassius auratus) gene that encodes these receptors, referred to as gfD1, was isolated and analyzed. A single open reading frame within the gfD1 gene encodes a protein of 363 amino acids that is highly homologous with dopamine D1 receptors from rats and humans. Interestingly, the carboxyl terminus of gfD1 lacks 80 amino acids that are present in the mammalian receptor sequences. RNA analysis using the polymerase chain reaction demonstrated that the gene is expressed in the goldfish retina and is intronless within the coding region. The fact that gfD1 encodes a dopamine D1 receptor was demonstrated through pharmacological analysis of transfected cells. Both the gfD1 receptor and the human D1 receptor expressed in mammalian cells had high affinity for SCH-23390 and other D1-specific ligands. In addition, the gfD1 receptor and the human D1 receptor were able to stimulate the accumulation of cAMP in response to SKF-38393 or dopamine. Interestingly, stimulation of both the gfD1 and human receptors with dopamine also resulted in an increase in intracellular Ca2+. Finally, long term pretreatment of transfected cells with dopamine resulted in the desensitization and down-regulation of both the goldfish and human receptors.

Amino Acid Sequence↗
PubMed1993

Characterization of a human corneal metalloproteinase inhibitor (TIMP-1).

The gradual corneal thinning seen in keratoconus may be due to altered degradation of the corneal extracellular matrix. Studies have shown that human keratocytes produce matrix metalloproteinase-2 (MMP-2) and two proteins (28 kDa and 21 kDa) that are capable of inhibiting the activity of MMP-2. In the present study, the 28 kDa inhibitor from keratoconus keratocyte cultures has been characterized as it may be important to the elevated MMP-2 activity seen in these cultures. Biochemical analyses indicated that this keratoconus corneal inhibitor was similar to TIMP-1 from other sources. Oligonucleotides to the reported sequence of human tumor cell TIMP-1 were used for reverse-transcriptase PCR to generate a 700 bp clone of the 28 kDa inhibitor from keratoconus keratocyte cytoplasmic RNA. Sequence analysis verified that the clone was nearly identical to the reported human TIMP-1 with a single base substitution that did not affect the predicted amino acid sequence. In addition, protein translated from the clone corresponded to the expected size. This data suggests that the elevated levels of gelatinolytic activity in these keratoconus keratocyte cultures is not due to a primary alteration of the TIMP-1 molecule. Protein expression studies of the TIMP-1 clone are currently underway.

Amino Acid Sequence↗
PubMed2005

Identification and characterization of vascular calcification-associated factor, a novel gene upregulated during vascular calcification in vitro and in vivo.

OBJECTIVE: Vascular calcification, with its increasing clinical sequelae, presents an important and unresolved dilemma in cardiac and vascular practice. We aimed to identify molecules involved in this process to develop strategies for treatment or prevention. METHODS AND RESULTS: Using subtractive hybridization, a novel cDNA, designated vascular calcification-associated factor (VCAF), has been isolated from a bovine retinal pericyte cDNA library generated during the differentiation and mineralization of these cells in vitro. RNA ligase-mediated rapid amplification of cDNA ends was used to compile the 740-bp bovine cDNA sequence. Database searching reveals that VCAF has novel nucleotide/amino acid sequences. RNA analysis confirms that VCAF is upregulated in mineralized pericytes and is present in human calcified arteries but not noncalcified arteries. Protein analysis using a VCAF antibody confirms the presence of an 18-kDa protein in calcified nodules but not in confluent pericytes. Adenoviral antisense VCAF gene delivery reduces VCAF protein levels and accelerates pericyte differentiation compared with controls. CONCLUSIONS: We demonstrate the isolation of a novel gene, VCAF, which is upregulated during vascular calcification in vitro and in vivo. Antisense VCAF gene delivery accelerates pericyte differentiation, implicating a role for VCAF in this clinically significant pathological process.

Adenoviridae↗
PubMed1996

Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists.

High-density arrays of oligonucleotide probes are proving to be powerful new tools for large-scale DNA and RNA sequence analysis. A method for constructing these arrays, using light-directed DNA synthesis with photo-activatable monomers, can currently achieve densities on the order of 10(6) sequences/cm2. One of the challenges facing this technology is to further increase the volume, complexity, and density of sequence information encoded in these arrays. Here we demonstrate a new approach for synthesizing DNA probe arrays that combines standard solid-phase oligonucleotide synthesis with polymeric photoresist films serving as the photoimageable component. This opens the way to exploiting high-resolution imaging materials and processes from the microelectronics industry for the fabrication of DNA probe arrays with substantially higher densities than are currently available.

Base Sequence↗
PubMed2005

Tree decomposition based fast search of RNA structures including pseudoknots in genomes.

Searching genomes for RNA secondary structure with computational methods has become an important approach to the annotation of non-coding RNAs. However, due to the lack of efficient algorithms for accurate RNA structure-sequence alignment, computer programs capable of fast and effectively searching genomes for RNA secondary structures have not been available. In this paper, a novel RNA structure profiling model is introduced based on the notion of a conformational graph to specify the consensus structure of an RNA family. Tree decomposition yields a small tree width t for such conformation graphs (e.g., t = 2 for stem loops and only a slight increase for pseudo-knots). Within this modelling framework, the optimal alignment of a sequence to the structure model corresponds to finding a maximum valued isomorphic subgraph and consequently can be accomplished through dynamic programming on the tree decomposition of the conformational graph in time O(k(t)N(2)), where k is a small parameter; and N is the size of the projiled RNA structure. Experiments show that the application of the alignment algorithm to search in genomes yields the same search accuracy as methods based on a Covariance model with a significant reduction in computation time. In particular; very accurate searches of tmRNAs in bacteria genomes and of telomerase RNAs in yeast genomes can be accomplished in days, as opposed to months required by other methods. The tree decomposition based searching tool is free upon request and can be downloaded at our site h t t p ://w.uga.edu/RNA-informatics/software/index.php.

Algorithms↗
PubMed1989

The mouse carbonic anhydrase I gene contains two tissue-specific promoters.

We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.

Animals↗
PubMed1989

The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme.

Rice seed ADP-glucose pyrophosphorylase cDNA clones were isolated by screening a lambda expression library prepared from rice endosperm poly(A+) RNA with a heterologous antibody raised against the spinach leaf enzyme and subsequently by nucleic acid hybridization. One cDNA plasmid, possessing about 1650 nucleotides, was shown by both DNA and RNA sequence analysis to contain the complete ADP-glucose pyrophosphorylase coding sequence of 483 amino acids. The primary sequence displayed a putative leader peptide presumably required for transport of this nuclear encoded protein into the amyloplasts, a differentiated starch containing plastid. The leader peptide, however, showed little sequence homology with transit peptides displayed by other known nuclear encoded proteins localized in the chloroplasts. A comparison of the primary sequence of the putative mature subunit to the Escherichia coli pyrophosphorylase showed two regions displaying significant homology. These two conserved regions contain residues shown previously to be essential for the allosteric regulation and catalytic activity of the E. coli enzyme. Differences in the primary sequences of the plant and bacterial enzyme may reflect the distinct nature of the allosteric effectors that control these enzymes.

Amino Acid Sequence↗
PubMed1995

An aspartic acid at amino acid 108 is required to rescue infectious virus after transfection of a poliovirus cDNA containing a CGDD but not SGDD amino acid motif in 3Dpol.

The poliovirus RNA-dependent RNA polymerase (3Dpol) contains a region of homology centered around the amino acid motif YGDD (amino acids 326 to 329), which has been postulated to be involved in the catalytic activity of the enzyme. Previous studies from this laboratory have used oligonucleotide site-directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S. A. Jablonski and C. D. Morrow, J. Virol. 67:373-381, 1993). The viruses recovered with 3Dpol genes with a methionine mutation also contained a second mutation at amino acid 108 resulting in a glutamic acid-to-aspartic acid change (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase. On the basis of these results, we suggested that the amino acid at position 108 might interact with the YGDD region of the poliovirus polymerase. To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutation at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in which the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326). The mutant 3Dpol polymerases were expressed in Escherichia coli, and in vitro enzyme activity was analyzed. Enzymes containing the 3D-D-108 mutation with the wild-type amino acid (3D-Y-326) demonstrated in vitro enzyme activity similar to that of the wild-type enzyme containing 3D-E-108. In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type. The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation. Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without the second mutation at amino acid 108 were performed. Consistent with previous findings, we found that transfection of poliovirus cDNAs containing the 3D-C-326 or 3D-S-326 mutation in 3Dpol did not result in the production of virus. Surprisingly, transfection of the poliovirus cDNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-108/S-326 mutation, resulted in the production of virus. The virus obtained from transfection of polio-virus cDNAs containing 3D-D-108/C-326 mutation replicated with kinetics similar to that of the wild-type virus. RNA sequence analysis of the region of the 3Dpol containing the 3D-C-326 mutation revealed that the codon for cysteine (UGC) reverted to the codon for tyrosine (UAC). The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3Dpol gene and further strengthen the idea that interaction between amino acid 108 and the YGDD region of 3Dpol is required for viral replication.

Amino Acid Sequence↗
PubMed2005

Improvement in the accuracy of multiple sequence alignment program MAFFT.

In 2002, we developed and released a rapid multiple sequence alignment program MAFFT that was designed to handle a huge (up to approximately 5,000 sequences) and long data (approximately 2,000 aa or approximately 5,000 nt) in a reasonable time on a standard desktop PC. As for the accuracy, however, the previous versions (v.4 and lower) of MAFFT were outperformed by ProbCons and TCoffee v.2, both of which were released in 2004, in several benchmark tests. Here we report a recent extension of MAFFT that aims to improve the accuracy with as little cost of calculation time as possible. The extended version of MAFFT (v.5) has new iterative refinement options, G-INS-i and L-INS-i (collectively denoted as [GL]-INS-i in this report). These options use a new objective function combining the weighted sum-of-pairs (WSP) score and a score similar to COFFEE derived from all pairwise alignments. We discuss the improvement in accuracy brought by this extension, mainly using two benchmark tests released very recently, BAliBASE v.3 (for protein alignments) and BRAliBASE (for RNA alignments). According to BAliBASE v.3, the overall average accuracy of L-INS-i was higher than those of other methods successively released in 2004, although the difference among the most accurate methods (ProbCons, TCoffee v.2 and new options of MAFFT) was small. The advantage in accuracy of [GL]-INS-i became greater for the alignments consisting of approximately 50-100 sequences. By utilizing this feature of MAFFT, we also examined another possible approach to improve the accuracy by incorporating homolog information collected from database. The [GL]-INS-i options are applicable to aligning up to approximately 200 sequences, although not applicable to thousands of sequences because of time and space complexities.

Amino Acid Sequence↗
PubMed1985

Two initiation sites for foot-and-mouth disease virus polyprotein in vivo.

Typically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P16, are translated from the 5' end of the coding region of the genome of foot-and-mouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (A10 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported.

Aphthovirus↗
PubMed2001

The API2/MALT1 fusion product may lead to germinal center B cell lymphomas by suppression of apoptosis.

Low-grade B cell lymphomas of mucosa-associated lymphoid tissue (MALT) represent a distinct clinicopathological entity that arises in a wide variety of extranodal sites. Genetically, MALT lymphomas are characterized by the t(11;18)(q21;q21). The genes involved in this translocation have been identified to be API2 on chromosome 11, which encodes an apoptotic inhibitor, and MALT1, a novel gene on chromosome 18. We identified the t(11;18)(q21;q21) by Southern blot analysis and reverse transcriptase PCR in 42% of a panel of extranodal MALT lymphomas. We also identified the breakpoints within the API2 and MALT1 genes in 7 patients, which revealed a consistent breakpoint after the third baculoviral inhibitor of apoptosis repeat domain within API2, and variable breakpoints in MALT1. We determined the API2/MALT1 fusion transcript in 2 cases by Northern blot analysis and also showed that MALT1 mRNA is constitutively expressed in a variety of human tissues. To understand the functional consequence of the translocation, we determined the pattern of expression of API2 and MALT1 through B lineage differentiation. API2 was expressed only in cell lines which correspond to mature B cells, whereas MALT1 mRNA was detectable in pre-B cells, mature B cells and plasma cells. These results suggest that fusion of MALT1 to API2 mediated by the t(11;18)(q21;q21) may result in an increased inhibition of germinal center B cell apoptosis and subsequent development of MALT lymphomas.

Apoptosis↗