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PubMed2006

A survey of mRNA sequences with a non-AUG start codon in RefSeq database.

Alternative initiation in translation is one of the important mechanisms in which multiple proteins are synthesized from a single mRNA. In many cases, translation initiation occurring at a non-AUG codon has been reported by several experimental studies. We have analyzed all mRNA sequences in the RefSeq database and found that coding regions of about 0.1% of the total mRNA sequences begin with a non-AUG codon (nonAUG mRNAs). Major fraction of non-AUG mRNAs is predicted from genomic sequences. More than 100 non-AUG sequences are highly curated and 52 of them are explicitly annotated that they use alternate start codons for translation initiation. Analysis of these sequences reveals that majority of the protein products contain domains that are DNA/RNA-binding, kinases, growth factors, or involved in immune response or cell proliferation. Thus, the proteins translated from non-canonical codons seem to be implicated in regulatory role and/or signaling mechanism. The sequence context of the non-AUG start codons shows that purine at -3 position and/or G at +4 position are strongly conserved and the corresponding genes give rise to alternate transcripts and/or multiple isoforms. We have also developed a database "nonAUG" (http://bioinfo.iitk.ac.in) that contains a collection of all mRNA sequences whose coding regions start with a non-AUG codon. nonAUG database will be continuously updated and is freely available to the scientific community.

Codon, Initiator↗
PubMed1998

Candida dubliniensis: phylogeny and putative virulence factors.

Candida dubliniensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The C. dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral C. dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The C. dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested C. albicans secretory aspartyl proteinase (SAP) genes were detected in C. dubliniensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that C. dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of C. dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.

Animals↗
PubMed1995

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

Base Sequence↗
PubMed2005

Characterization and expression sites of newly identified chicken collectins.

Collectins are members of the family of vertebrate C-type lectins. They have been found almost exclusively in mammals, with the exception of chicken MBL. Because of their important role in innate immunity, we sought to identify other collectins in chicken. Using the amino acid sequences of known collectins, the EST database was searched and related to the chicken genome. Three chicken collectins were found and designated chicken Collectin 1 (cCL-1), chicken Collectin 2 (cCL-2), and chicken Collectin 3 (cCL-3), which resemble the mammalian proteins Collectin Liver 1, Collectin 11 and Collectin Placenta 1, respectively. Additionally, a lectin was found which resembled Surfactant Protein A, but lacked the collagen domain. Therefore, it was named chicken Lung Lectin (cLL). Tissue distribution analysis showed cCL-1, cCL-2 and cCL-3 are expressed in a wide range of tissues throughout the digestive, the reproductive and the lymphatic system. Similar to SP-A, cLL is mainly localized in lung tissue. Phylogenetic analysis indicates that cCL-1, cCL-2 and cCL-3 represent new subgroups within the collectin family. The newly found collectins may have an important function in avian host defence. Elucidation of the role of these pattern-recognition molecules could lead to strategies that thwart infectious diseases in poultry, which could also be beneficial for public health.

Amino Acid Sequence↗
PubMed2004

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori.

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.

Animals↗
PubMed2006

Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data.

Microarrays have enabled the determination of how thousands of genes are expressed to coordinate function within single organisms. Yet applications to natural or engineered communities where different organisms interact to produce complex properties are hampered by theoretical and technological limitations. Here we describe a general method to accurately identify low-abundant targets in systems containing complex mixtures of homologous targets. We combined an analytical predictor of nonspecific probe-target interactions (cross-hybridization) with an optimization algorithm that iteratively deconvolutes true probe-target signal from raw signal affected by spurious contributions (cross-hybridization, noise, background, and unequal specific hybridization response). The method was capable of quantifying, with unprecedented specificity and accuracy, ribosomal RNA (rRNA) sequences in artificial and natural communities. Controlled experiments with spiked rRNA into artificial and natural communities demonstrated the accuracy of identification and quantitative behavior over different concentration ranges. Finally, we illustrated the power of this methodology for accurate detection of low-abundant targets in natural communities. We accurately identified Vibrio taxa in coastal marine samples at their natural concentrations (<0.05% of total bacteria), despite the high potential for cross-hybridization by hundreds of different coexisting rRNAs, suggesting this methodology should be expandable to any microarray platform and system requiring accurate identification of low-abundant targets amid pools of similar sequences.

Oligonucleotide Array Sequence Analysis↗
PubMed1996

An IVS1-116 (A-->G) acceptor splice site mutation in the alpha 2 globin gene causing alpha + thalassaemia in two Dutch families.

We report the characterization of an alpha +(-)thalassaemia determinant due to a transition A-->G of the acceptor splice consensus site sequence (IVS1-116) of the first intron of the alpha 2-globin gene. The mutation, found in two apparently unrelated Dutch Caucasian families, was detected by DGGE analysis followed by direct sequencing. Haplotype analysis suggests a common origin of the mutation in both families. The disruption of the acceptor splice site consensus sequence interferes with the correct splicing and leads to the retention of the first intron in the abnormally spliced mRNA. The alpha +(-)thalassaemia phenotype observed in the carriers is caused by the absence of functional mRNA which cannot be replaced by the abnormally spliced mRNA. The low amounts of abnormal mRNA found in reticulocytes is, most probably, due to the post-transcriptional instability which follows the presence of a termination codon in the retained intronic sequence. This situation is often associated with a decreased mRNA stability as observed for several nonsense mutations of the beta-globin gene.

Blotting, Southern↗
PubMed2006

The tmRDB and SRPDB resources.

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Royal Veterinary and Agricultural University, Denmark (http://tmrdb.kvl.dk/). The signal recognition particle database (SRPDB) at http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html is mirrored at http://srpdb.kvl.dk/ and the University of Goteborg (http://bio.lundberg.gu.se/dbs/SRPDB/SRPDB.html). The databases assist in investigations of the tmRNP (a ribonucleoprotein complex which liberates stalled bacterial ribosomes) and the SRP (a particle which recognizes signal sequences and directs secretory proteins to cell membranes). The curated tmRNA and SRP RNA alignments consider base pairs supported by comparative sequence analysis. Also shown are alignments of the tmRNA-associated proteins SmpB, ribosomal protein S1, alanyl-tRNA synthetase and Elongation Factor Tu, as well as the SRP proteins SRP9, SRP14, SRP19, SRP21, SRP54 (Ffh), SRP68, SRP72, cpSRP43, Flhf, SRP receptor (alpha) and SRP receptor (beta). All alignments can be easily examined using a new exploratory browser. The databases provide links to high-resolution structures and serve as depositories for structures obtained by molecular modeling.

Amino Acid Sequence↗
PubMed1998

A test of Archonta monophyly and the phylogenetic utility of the mitochondrial gene 12S rRNA.

The relationships within the superorder Archonta, which contains the orders Dermoptera (flying lemurs), Scandentia (tree shrews), Chiroptera (bats), and Primates, were examined through the analysis of five newly derived and complete mitochondrial 12S rRNA sequences. The new data is combined with 83 additional known mammalian sequences to provide a full phylogenetic sampling. Phylogenetic hypotheses are generated using PAUP 3.1.1 (Swofford [1993] Illinois Natural History Survey, Champaign, IL) through analyses of all characters equally weighted, transversions only, and the effect of alignment gaps on phylogeny. The Parsimony Jackknifer (Farris et al. [1996] Cladistics 12:99-124) was used to assess the level of ambiguity present in the sequence data, and therefore the strength of the tree topologies. The conclusions of Springer and Douzery (1996, J. Mol. Evol. 43:357-373) which states that 12S rRNA is reliable to a time depth of 100 mya is unsupported by these analyses. The usefulness of 12S rRNA to aid in solving Archonta relationships and others of similar time depth is found to be suspect.

Animals↗
PubMed1996

Cloning and sequencing of a developmentally regulated avian mRNA containing the LEA motif found in plant seed proteins.

We report the cloning of a bromodeoxyuridine (BrdU)-sensitive transcript of 918 bp from an immortalized quail heart cell line containing an open reading frame (ORF) of 215 amino acids (aa) (approximately 23 kDa). Analysis of the secondary structure predicts two amphipathic alpha-helices with oppositely oriented amphipathic surfaces at the C-terminus of the protein. Each of the helices contains an LEA (late embryogenesis abundant) consensus sequence (A/TAEKAK/RETKD) which has been previously described only in a group of plant seed-specific proteins. Temporal and spatial distribution patterns of the transcript during chick embryo development were examined by whole-mount in situ hybridization and Northern blot analysis. At H&H (Hamburger and Hamilton, 1951) stages 11-14, the message was expressed strongly in blood islands in the area opaca. At day 5, strong signals were found in the liver primordia, mesonephrons, and nephric duct. Frozen sections of whole mount-stained embryonic liver demonstrated that the message was restricted to developing blood cells. The expression pattern of this transcript suggests that its protein product may be involved in hematopoiesis during avian development.

Amino Acid Sequence↗
PubMed2003

Purification of Mycobacterium leprae RNA for gene expression analysis from leprosy biopsy specimens.

Gene expression analysis in Mycobacterium leprae, an obligate intracellular pathogen and the etiologic agent of leprosy, has been hampered by the lack of an efficient method to purify RNA from leprosy lesions. Therefore to date, transcripts for only a few genes have been identified. We report the use of a single-tube homogenization/RNA extraction method that produces enough RNA to study the expression of 30 genes from a single skin biopsy specimen of a multibacillary leprosy patient and demonstrate that RNA can be purified after fixation of biopsies in 70% ethanol for up to a year. This represents a major advancement in the ability to study M. leprae gene expression directly from biopsy material and should help to define genes that are associated with intracellular survival of this human pathogen.

Biopsy↗
PubMed2006

Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure.

A greenhouse experiment was performed to analyze a potential effect of genetically modified potatoes expressing antibacterial compounds (attacin/cecropin, T4 lysozyme) and their nearly isogenic, nontransformed parental wild types on rhizosphere bacterial communities. To compare plant transformation-related variations with commonly accepted impacts caused by altered environmental conditions, potatoes were cultivated under different environmental conditions, for example using contrasting soil types. Further, plants were challenged with the blackleg pathogen Erwinia carotovora ssp. atroseptica. Rhizosphere soil samples were obtained at the stem elongation and early flowering stages. The activities of various extracellular rhizosphere enzymes involved in the C-, P- and N-nutrient cycles were determined as the rates of fluorescence of enzymatically hydrolyzed substrates containing the highly fluorescent compounds 4-methylumbelliferone or 7-amino-4-methyl coumarin. The structural diversity of the bacterial communities was assessed by 16S rRNA-based terminal restriction fragment length polymorphism analysis, and 16S rRNA gene clone libraries were established for the flowering conventional and T4 lysozyme-expressing Desirée lines grown on the chernozem soil, each line treated with and without E. carotovora ssp. atroseptica. Both genetic transformation events induced a differentiation in the activity rates and structures of associated bacterial communities. In general, T4 lysozyme had a stronger effect than attacin/cecropin. In comparison with the other factors, the impact of the genetic modification was only transient and minor, or comparable to the dominant variations caused by soil type, plant genotype, vegetation stage and pathogen exposure.

Anti-Bacterial Agents↗
PubMed1997

Normal FHIT transcripts in renal cell cancer- and lung cancer-derived cell lines, including a cell line with a homozygous deletion in the FRA3B region.

The recently identified FHIT gene encompasses the FRA3B region and the breakpoint of a constitutive t(3;8) occurring in a family with hereditary renal cell cancer. Occurrence of aberrant transcripts in different types of tumours has led to the suggestion that FHIT might play a critical role in the development of various types of cancer. We have analyzed the gene and its transcripts in lung cancers and renal cell cancer-derived cell lines. A lung adenocarcinoma cell line, GLC-A2, appeared to have a homozygous deletion in intron 5 of FHIT. RT-PCR analysis revealed a normal-sized PCR product in all of the cell lines: Including GLC-A2. A number of them had an additional aberrant product. Analysis of a great number of control cell lines and tissues showed that the majority of these also had aberrant PCR products in addition to a normal-sized PCR product. Different specimens of the same cell type showed variable additional RT-PCR products. Normal-sized PCR products had a sequence identical to the FHIT sequence. PCR products longer than normal had insertions of different sizes at different positions. With three exceptions, PCR products shorter than normal represented FHIT sequences missing one or more entire exons. Thus, the presence of aberrant transcripts is not cancer-specific. Conceivably, sequence responsible for the instability of the FRA3B region are being transcribed into FHIT pre-mRNA and may cause the abnormal splicing and processing of the transcripts.

Acid Anhydride Hydrolases↗
PubMed2006

Anticodons, frameshifts, and hidden periodicities in tRNA sequences.

Fourier analysis of the short-range periodicities for the complete set of sequences coding for tRNA genes in genome of Bacillus subtilis proves that periodicities with periods p = 2, 3, 4, and 6 sites are the inherent properties of tRNAs. The related periodicities should be understood in a broad statistical sense and their identifying needs the elaborate statistical methods. To improve the statistics, the analysis of significant periodicities was performed for the binary R-Y, S-W, and K-M sequences. Generally, such short-range periodicities are produced via biased positioning of particular nucleotides rather than via the tandem multiplication and subsequent modifications of repeats, though the latter mechanism may also be realized. Quasi-coherently piercing long segments of tRNA, the short-range periodicities create the effective long-range structural coupling between the acceptor stem and the anticodon loop and may participate in the mechanisms of molecular recognition. The periodicities with p = 2 and 4 provide the natural ground for the translation with spontaneous or programmed frameshifting and are present in tRNAs decoding the most frameshift-prone codons. The observation of short-range periodicities suggests that the mechanisms of amino-acylation of tRNAs and codon-anticodon pairing are not independent. Their study may also provide the important information related to the origin and evolution of the genetic code.

Anticodon↗
PubMed2006

Detection and genetic characterization of norovirus strains circulating among infants and children with acute gastroenteritis in Japan during 2004-2005.

A total of 752 fecal specimens collected during the period of July 2004 to June 2005 from infants and children with acute gastroenteritis from four different regions (Maizuru, Tokyo, Sapporo, and Osaka) of Japan were tested for the presence of norovirus by RT-PCR. It was found that 139 (18.5%) fecal specimens were positive for norovirus. Norovirus infection was detected almost all year round with the highest prevalence in January. Norovirus GII was the most predominant genogroup (98.6%; 137 of 139). The genotypes detected in this study were GI/1, GII/1, GII/3, GII/4, and GII/6. Of these, NoV GII/4 (known as the Lordsdale virus cluster) was re-emerging and became the leading genotype (77.7%). Meanwhile, the incidence of NoV GII/3 (known as the Arg320 virus cluster) has dropped rapidly, accounting for only 15.8%. Another interesting feature of the study was the identification of Picton03/AU-like recombinant NoV for the first time in Japan. Based on the genetic analysis, it was interesting to note that NoV GII/4 in 2004-2005 made a distinct cluster in comparison to other NoV GII/4 circulating in 2002-2003 and 2003-2004. Of note, "new recombinant variant designated GIIb" within NoV GII/3, which was first detected in Saga City, Japan in 2003-2004 in only one case, had increased, spreading widely in Japan and representing 45.5% (10 of 22). Further epidemiological studies should be conducted to determine whether this new recombinant variant strain will be dominant in Japan in the coming year.

Acute Disease↗
PubMed2003

Sequence context dependence of tandem guanine:adenine mismatch conformations in RNA: a continuum solvent analysis.

Guanine:adenine (G:A) mismatches and in particular tandem G:A (tG:A) mismatches are frequently observed in biological RNA molecules and can serve as sites for tertiary interaction, metal binding and protein recognition. Depending on the surrounding sequence tG:A mismatches can adopt different basepairing topologies. In the sequence context (5'-) GGAC (tandem G:A in bold) a face-to-face (imino or Watson-Crick-like) pairing is preferred whereas in the CGAG context, G and A adopt a sheared arrangement. Systematic conformational searches with a generalized Born continuum model and molecular dynamics simulations including explicit water molecules and ions have been used to generate face-to-face and sheared tG:A mismatches in both CGAG and GGAC sequence contexts. Conformations from both approaches were evaluated using the same force field and a Poisson-Boltzmann continuum solvent model. Although the substate analysis predicted the sheared arrangement to be energetically preferred in both sequence contexts, a significantly greater preference of the sheared form was found for the CGAG context. In agreement with the experimental observation, the analysis of molecular dynamics trajectories indicated a preference of the sheared form in the case of the CGAG-context and a favorization of the face-to-face form in the case of the GGAC context. The computational studies allowed to identify energetic contributions that stabilize or destabilize the face-to-face and sheared tandem mismatch topologies. The calculated nonpolar solvation and Lennard-Jones packing interaction were found to stabilize the sheared topology independent of the sequence context. Electrostatic contributions are predicted to make the most significant contribution to the sequence context dependence on the structural preference of tG:A mismatches.

Adenine↗
PubMed2000

The molecular basis for the aberrant production of plasminogen activator inhibitor type 2 in THP-1 monocytes.

Plasminogen activator inhibitor type 2 (PAI-2) is a urokinase inhibitor that is expressed primarily in monocytes. THP-1 monocytes, however. contain a unique defect in the production of PAI-2 in that the PAI-2 transcript is truncated and the expressed protein inactive (1). Here we describe the basis of this mutation in THP-1 cells. Southern blot analysis of THP-1-derived genomic DNA indicated that there were no obvious deletions in the structure of the PAI-2 gene. However, assessment of the THP-1-derived PAI-2 transcript by RT-PCR indicated that only exons seven and eight of the normal PAI-2 mRNA could be detected. Cloning of the 5' region of the PAI-2 mRNA by 5-'RACE indicated that the PAI-2 cDNA derived from THP-1 cells is approximately 1329 bp long and contains 180 bp of sequence derived from intron 5. followed by sequences corresponding to exons seven and eight of the normal PAI-2 mRNA. The presence of the intron five fragment in endogenous THP-1 derived PAI-2 mRNA was confirmed by Northern blotting. The absence of any wild-type PAI-2 mRNA in these cells suggests that one copy of the PAI-2 allele has been deleted. The remaining allele producing the truncated mRNA appears to have undergone a translocation event and contains a mutation that has disrupted the splicing of the PAI-2 primary transcript.

Cloning, Molecular↗
PubMed2006

Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells.

Antiretroviral therapy can reduce human immunodeficiency virus type 1 (HIV-1) viremia to below the detection limit of ultrasensitive clinical assays (50 copies of HIV-1 RNA/ml). However, latent HIV-1 persists in resting CD4+ T cells, and low residual levels of free virus are found in the plasma. Limited characterization of this residual viremia has been done because of the low number of virions per sample. Using intensive sampling, we analyzed residual viremia and compared these viruses to latent proviruses in resting CD4+ T cells in peripheral blood. For each patient, we found some viruses in the plasma that were identical to viruses in resting CD4+ T cells by pol gene sequencing. However, in a majority of patients, the most common viruses in the plasma were rarely found in resting CD4+ T cells even when the resting cell compartment was analyzed with assays that detect replication-competent viruses. Despite the large diversity of pol sequences in resting CD4+ T cells, the residual viremia was dominated by a homogeneous population of viruses with identical pol sequences. In the most extensively studied case, a predominant plasma sequence was also found in analysis of the env gene, and linkage by long-distance reverse transcriptase PCR established that these predominant plasma sequences represented a single predominant plasma virus clone. The predominant plasma clones were released for months to years without evident sequence change. Thus, in some patients on antiretroviral therapy, the major mechanism for residual viremia involves prolonged production of a small number of viral clones without evident evolution, possibly by cells other than circulating CD4+ T cells. The sequences have been deposited in GenBank. The accession numbers are DQ 391282 to DQ 391351 (for env) and DQ 391352 to DQ 392955 (for RT).

Antiretroviral Therapy, Highly Active↗