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PubMed2005

Tiling microarray analysis of rice chromosome 10 to identify the transcriptome and relate its expression to chromosomal architecture.

BACKGROUND: Sequencing and annotation of the genome of rice (Oryza sativa) have generated gene models in numbers that top all other fully sequenced species, with many lacking recognizable sequence homology to known genes. Experimental evaluation of these gene models and identification of new models will facilitate rice genome annotation and the application of this knowledge to other more complex cereal genomes. RESULTS: We report here an analysis of the chromosome 10 transcriptome of the two major rice subspecies, japonica and indica, using oligonucleotide tiling microarrays. This analysis detected expression of approximately three-quarters of the gene models without previous experimental evidence in both subspecies. Cloning and sequence analysis of the previously unsupported models suggests that the predicted gene structure of nearly half of those models needs improvement. Coupled with comparative gene model mapping, the tiling microarray analysis identified 549 new models for the japonica chromosome, representing an 18% increase in the annotated protein-coding capacity. Furthermore, an asymmetric distribution of genome elements along the chromosome was found that coincides with the cytological definition of the heterochromatin and euchromatin domains. The heterochromatin domain appears to associate with distinct chromosome level transcriptional activities under normal and stress conditions. CONCLUSION: These results demonstrated the utility of genome tiling microarray in evaluating annotated rice gene models and in identifying novel transcriptional units. The tiling microarray sanalysis further revealed a chromosome-wide transcription pattern that suggests a role for transposable element-enriched heterochromatin in shaping global transcription in response to environmental changes in rice.

Chromosomes, Plant↗
PubMed2006

Spliceosomal small nuclear RNA genes in 11 insect genomes.

The removal of introns from the primary transcripts of protein-coding genes is accomplished by the spliceosome, a large macromolecular complex of which small nuclear RNAs (snRNAs) are crucial components. Following the recent sequencing of the honeybee (Apis mellifera) genome, we used various computational methods, ranging from sequence similarity search to RNA secondary structure prediction, to search for putative snRNA genes (including their promoters) and to examine their pattern of conservation among 11 available insect genomes (A. mellifera, Tribolium castaneum, Bombyx mori, Anopheles gambiae, Aedes aegypti, and six Drosophila species). We identified candidates for all nine spliceosomal snRNA genes in all the analyzed genomes. All the species contain a similar number of snRNA genes, with the exception of A. aegypti, whose genome contains more U1, U2, and U5 genes, and A. mellifera, whose genome contains fewer U2 and U5 genes. We found that snRNA genes are generally more closely related to homologs within the same genus than to those in other genera. Promoter regions for all spliceosomal snRNA genes within each insect species share similar sequence motifs that are likely to correspond to the PSEA (proximal sequence element A), the binding site for snRNA activating protein complex, but these promoter elements vary in sequence among the five insect families surveyed here. In contrast to the other insect species investigated, Dipteran genomes are characterized by a rapid evolution (or loss) of components of the U12 spliceosome and a striking loss of U12-type introns.

Animals↗
PubMed2004

Principles of 3' splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis.

We investigated the self-splicing properties of two introns from the bacterium Bacillus anthracis. One intron (B.a.I1) splices poorly in vitro despite having typical structural motifs, while the second (B.a.I2) splices well while having apparently degenerated features. The spliced exons of B.a.I2 were sequenced, and splicing was found to occur at a 3' site shifted one nucleotide from the expected position, thus restoring missing gamma-gamma' and IBS3-EBS3 pairings, but leaving the two conserved exonic ORFs out of frame. Because of the unexpected splice site, the principles for 3' intron definition were examined, which showed that the 3' splice site is flexible but contingent on gamma-gamma' and IBS3-EBS3 pairings, and can be as far away as four nucleotides from the wild-type site. Surprisingly, alternative splicing occurs at position +4 for wild-type B.a.I2 intron, both in vitro and in vivo, and the alternative event fuses the two conserved exon ORFs, presumably leading to translation of the downstream ORF. The finding suggests that the structural irregularities of B.a.I2 may be an adaptation to facilitate gene expression in vivo.

Alternative Splicing↗
PubMed2004

Detection of a novel sense-antisense RNA-hybrid structure by RACE experiments on endogenous troponin I antisense RNA.

Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this "hybrid RNA." Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and antisense troponin I RNA in vivo, which we discuss in the article.

Animals↗
PubMed2000

Prevalence of Ehrlichia chaffeensis (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from the Georgia coast and Barrier Islands.

Human monocytic ehrlichiosis is an emerging zoonosis caused by infection with Ehrlichia chaffeensis Anderson, Dawson, Jones & Wilson, which is transmitted to mammals by ixodid ticks. Prevalence of infected ticks and distribution of infection foci indicate relative risk of human exposure to ehrlichiosis and may be influenced by factors such as geographic isolation, human disturbance, and the availability of suitable mammalian reservoir hosts. To test, this, individual and pooled lone star ticks, Amblyomma americanum (L.), were collected from three populations from the Georgia coast (1 mainland site and two barrier islands) and screened for E. chaffeensis. A species-specific, nested polymerase chain reaction (PCR) assay was used to amplify a 572 bp fragment of the E. chaffeensis 16S rRNA gene from infected ticks. PCR product specificity was confirmed by nucleotide sequencing. Our results showed the prevalence of infected ticks to be 0.0% (n = 151), 0.9% (n = 111), and 9.3% (n = 129) for Sapelo Island, St. Catherine's Island, and Fort McAllister, respectively.

Animals↗
PubMed2002

Guiding revision of regulatory models with expression data.

BioLingua is a computational system designed to support biologists' efforts to construct models, make predictions, and interpret data. In this paper, we focus on the specific task of revising an initial model of gene regulation based on expression levels from gene microarrays. We describe BioLingua's formalism for representing process models, its method for predicting qualitative correlations from such models, and its use of data to constrain search through the space of revised models. We also report experimental results on revising a model of photosynthetic regulation in Cyanobacteria to better fit expression data for both wild and mutant strains, along with model mutilation studies designed to test our method's robustness. In closing, we discuss related work on representing, discovering, and revising biological models, after which we propose some directions for future research.

Computational Biology↗
PubMed2001

[Sequences of 5S-rRNA gene spacer region and comparison of alkaloid content in Fritillaria thunbergii from different habitats].

OBJECTIVE: To study the genetic basis of the formation of indigenous Chinese medicine materials. METHODS: The 5S-rRNA gene spacer regions in F. thunbergii from different habitats were amplified with AS and AS-1 as primers, and then sequenced. Total alkaloid contents were assayed by acid dye colorimetry, and 2 main alkaloid contents were assayed by pre-column derivatization and gas chromatographic method. RESULT: The sequenues of 5S-rRNA gene spacer regions in F. thunbergii from different habitats were same, and the length of them was 588 bp. They had same content total alkaloid. The results of gas chromatography showed that they had same kinds of monomer alkaloids, but the contents of different monomer alkaloids were different. CONCLUSION: The difference of alkaloid content in F. thunbergii from various habitats isn't resulted from base sequence variation, but from microenvironment.

Alkaloids↗
PubMed2005

[The application of bioinformatics in the research of alternative splicing].

Alternative splicing, a fundamental and important regulatory mechanism in eukaryotes, allows one pre-mRNA to be processed into many different mature forms within a cell, each of which can have distinct functions. As alternative splicing is associated with human diseases, the study of alternative splicing becomes quite important. Bioinformatics is a new subject for the study of alternative splicing, especially for its regulatory mechanism, prediction and origin. Of course, bioinformatics must be combined with experimental research so as to clarify these aspects of alternative splicing. This paper reviewed the recent research progress in this field in the hope to gain a deeper understanding of eukaryotic gene expression regulation.

Alternative Splicing↗
PubMed2004

Pair stochastic tree adjoining grammars for aligning and predicting pseudoknot RNA structures.

MOTIVATION: Since the whole genome sequences for many species are currently available, computational predictions of RNA secondary structures and computational identifications of those non-coding RNA regions by comparative genomics become important, and require more advanced alignment methods. Recently, an approach of structural alignments for RNA sequences has been introduced to solve these problems. By structural alignments, we mean a pairwise alignment to align an unfolded RNA sequence into a folded RNA sequence of known secondary structure. Pair HMMs on tree structures (PHMMTSs) proposed by Sakakibara are efficient automata-theoretic models for structural alignments of RNA secondary structures, but are incapable of handling pseudoknots. On the other hand, tree adjoining grammars (TAGs) is a subclass of context-sensitive grammar, which is suitable for modeling pseudoknots. Our goal is to extend PHMMTSs by incorporating TAGs to be able to handle pseudoknots. RESULTS: We propose the pair stochastic tree adjoining grammars (PSTAGs) for modeling RNA secondary structures including pseudoknots and show the strong experimental evidences that modeling pseudoknot structures significantly improves the prediction accuracies of RNA secondary structures. First, we extend the notion of PHMMTSs defined on alignments of 'trees' to PSTAGs defined on alignments of "TAG (derivation) trees", which represent a top-down parsing process of TAGs and are functionally equivalent to derived trees of TAGs. Second, we modify PSTAGs so that it takes as input a pair of a linear sequence and a TAG tree representing a pseudoknot structure of RNA to produce a structural alignment. Then, we develop a polynomial-time algorithm for obtaining an optimal structural alignment by PSTAGs, based on dynamic programming parser. We have done several computational experiments for predicting pseudoknots by PSTAGs, and our computational experiments suggests that prediction of RNA pseudoknot structures by our method are more efficient and biologically plausible than by other conventional methods. The binary code for PSTAG method is freely available from our website at http://www.dna.bio.keio.ac.jp/pstag/.

Algorithms↗
PubMed1993

Apolipoprotein B mRNA editing: a key controlling element targeting fats to proper tissue.

Apolipoprotein B (apo B) circulates in two distinct forms referred to as apo B100 and apo B48. Apo B48 is colinear with the amino-terminal half of apo B100 and arises as a result of a post-transcriptional modification, termed apo B mRNA editing. This process changes a single cytidine nucleotide in apo B100 mRNA thereby altering a CAA codon, encoding glutamine in apo B100, to a UAA codon, which specifies an in-frame stop codon in apo B48. The functional consequences of apo B mRNA editing include the divergent catabolism of plasma lipoproteins expressing either apo B100 or B48, and also the ability to generate the hybrid lipoprotein, Lp(a). These differences arise because the requisite regions of apo B for interaction either with the low-density lipoprotein receptor or with apolipoprotein (a) are contained within the carboxyl terminus of apo B100. Apo B mRNA editing is regulated by species, tissue and cell-specific factors, one of which has been recently cloned. The further characterization of apo B mRNA editing, the first example of a mammalian gene regulated by post-transcriptional nucleotide alteration, will be important for an understanding of lipoprotein assembly.

APOBEC-1 Deaminase↗
PubMed1999

Identification of clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene. Experience from a clinical laboratory.

Twenty-one mycobacterial type strains and 334 clinical isolates of mycobacteria were identified by standardized sequence analysis using part of the gene encoding 16S rRNA. Apart from two clinical isolates, the resulting sequences corresponded to previously published sequences. The results of the molecular determinations of the type strains completely overlapped the identities obtained using conventional techniques (cultural characteristics, biochemical tests, commercial DNA probes, and gas chromatographic lipid profiles). Of 323 isolates conventionally identified as slow-growing mycobacteria, 318 (98.5%) were identified to the same species or group level by 16S rDNA sequence analysis, while 6 of the 11 strains of rapid growers obtained a corresponding identity with the two approaches. The sequencing protocol combined with a few cultural characteristics (i.e. growth rate, pigmentation and susceptibility testing) offers a rapid, reliable and usually definite identification of mycobacterial isolates.

Amino Acid Sequence↗
PubMed1991

Eukaryotic origins: string analysis of 5S ribosomal RNA sequences from some relevant organisms.

Using the PHYLOGEN tree-forming programs, we evaluate the published 5S rRNA sequences in certain of the files in the Berlin DataBank in an attempt to identify the connection between archaebacteria and the eukaryotic protists. These programs are based on methods of string analysis developed by Sankoff and others. Their discriminatory power is derived from their continuous realignment of sequences through repeated assessment of insertions and deletions as well as substitutions. The programs demonstrate that even these small molecules (ca. 120 bases) retain substantial records of evolutionary events that occurred over a billion years ago. The eukaryotes seem to have been derived from ancestors near the common origins of the halobacterial and Methanococcales groups. Identifying what might have been a primordial eukaryote is more difficult because several of the species considered as early derivatives from the common root are isolated species with large genetic differences from each other and from all other extant forms that have been sequenced. The ameboid, flagellated, and ciliated protists seem to have emerged nearly simultaneously from an ancient cluster, but the sarcodinid protozoa have preference as the group of most ancient origin. The euglenozoa and the ciliates are of later derivation. Our ability to tease plausible trees from such small molecules suggests that the mode of analysis rather than the size of the molecule is often a major limitation in the reconstruction of acceptable ancient phylogenics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2026

Integrated single-cell RNA sequencing and mendelian randomization analysis identifies causal immune-related driver genes in the heart failure inflammatory microenvironment.

BACKGROUND: Heart failure (HF) is a major global cause of cardiovascular death and disability. Chronic inflammation and immune dysregulation are critical in its development. The cardiac immune microenvironment, especially macrophages, drives HF progression, yet its molecular mechanisms and prognostic impact are not fully clear. This study aimed to identify causal immune-related driver genes in the HF inflammatory microenvironment. METHODS: We combined single-cell RNA sequencing (scRNA-seq) and Mendelian randomization (MR) to study how the inflammatory immune microenvironment affects HF risk. Using two public scRNA-seq datasets, we identified differentially expressed genes (DEGs) in HF heart tissues and selected 489 candidate genes. Causal relationships between these genes and HF were tested using expression quantitative trait loci (eQTL) data and HF genome-wide association study (GWAS) summary statistics. RESULTS: MR analysis showed that 65 genes were causally linked to HF risk. These genes were enriched in pathways related to cardiomyopathy, leukocyte migration, natural killer (NK) cell cytotoxicity, neutrophil extracellular traps, and NF-κB signaling. HF hearts displayed increased levels of macrophages, T cells, B cells, lymphoid cells, and mast cells, while neutrophils were reduced. CONCLUSIONS: Our integrated analysis reveals the central role of the cardiac inflammatory immune microenvironment in HF and identifies 65 key genes causally associated with HF susceptibility. These genes influence specific immune pathways and cell infiltration, shaping HF progression, and provide a basis for developing new biomarkers and immune-targeted therapies.

Heart failure (HF)↗