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PubMed1992

Two mRNAs are transcribed from the human gene for choline acetyltransferase.

The product of the choline acetyltransferase (ChAT) gene is the enzyme that synthesizes the neurotransmitter acetylcholine. A 14.4-kb portion of the human ChAT gene contains 7 exons, which are estimated to comprise approximately one-third of the human protein coding sequence by comparison with porcine ChAT mRNA. Two of the exons were used to identify polyadenylated human ChAT gene transcripts on Northern blots. An exon with 84% identity to the region of porcine ChAT mRNA that codes for the amino terminus of the corresponding protein detected 6,000- and 2,300-nucleotide mRNAs in RNA isolated from human CHP134 neuroblastoma cells. Only the 2,300-nucleotide mRNA was detected by a second probe containing an exon with 96% identity to porcine ChAT mRNA in the domain that encodes amino acids 204-263 of the predicted porcine ChAT protein. Further evidence that two species of human mRNA are produced from the human ChAT gene was obtained from nuclease protection assays using an antisense RNA probe prepared from a human ChAT cDNA clone. Total RNA isolated from either CHP134 cells or adult human nucleus basalis protected 525- and 400-nucleotide fragments of this probe, confirming the presence of two species of RNA that differ by the inclusion of an internal exon. cDNA clones of each of these transcripts have been isolated. Their sequences suggest that the 2,300-nucleotide mRNA encodes enzymatically active human ChAT, while translation of the 6,000-nucleotide mRNA would be terminated prematurely by a shift in the reading frame. These results indicate that a complex pattern of transcription produces two mRNAs with different coding potentials from the human ChAT gene.

Blotting, Northern↗
PubMed2006

IWoCS: analyzing ribosomal intergenic transcribed spacers configuration and taxonomic relationships.

MOTIVATION: Lately the use of 16S-23S Intergenic Transcribed Spacer (ITS) sequences for bacterial typing purposes has increased. The presence of conserved regions like tRNA genes or boxes together with hypervariable regions allows performing intraspecific discrimination of very close bacterial strains. On the other hand this mosaic of variability makes the ITS a sequence difficult to analyze and compare. RESULTS: A software to study ITSs by a Word Count based System (IWoCS) is proposed. A large dataset of ITS was created (comprising 7355 sequences). A database indicating all the occurrences of possible n-mers (tags), describing each ITS sequence, was created (with n going from 5 to 13) including 32 061 819 entries. The database allows to analyze ITS sequences submitted by users using a web-based interface. The abundance in the database of each n-mer is given in a one-base sliding frame. A dominance plot reflects how common the tags are within different taxonomic levels. The obtained profile identifies highly repeated tags as evolutionarily conserved regions (like tRNA or boxes) or low frequency tags as regions specifically associated to taxonomic groups. The study of the dominance and abundance profiles combined with the taxonomy reports provides a novel tool for the use of the ITS in bacteria typing and identification. AVAILABILITY: The database is freely accessible at http://egg.umh.es/iwocs/.

Algorithms↗
PubMed2006

Experimental approaches to identify non-coding RNAs.

Cellular RNAs that do not function as messenger RNAs (mRNAs), transfer RNAs (tRNAs) or ribosomal RNAs (rRNAs) comprise a diverse class of molecules that are commonly referred to as non-protein-coding RNAs (ncRNAs). These molecules have been known for quite a while, but their importance was not fully appreciated until recent genome-wide searches discovered thousands of these molecules and their genes in a variety of model organisms. Some of these screens were based on biocomputational prediction of ncRNA candidates within entire genomes of model organisms. Alternatively, direct biochemical isolation of expressed ncRNAs from cells, tissues or entire organisms has been shown to be a powerful approach to identify ncRNAs both at the level of individual molecules and at a global scale. In this review, we will survey several such wet-lab strategies, i.e. direct sequencing of ncRNAs, shotgun cloning of small-sized ncRNAs (cDNA libraries), microarray analysis and genomic SELEX to identify novel ncRNAs, and discuss the advantages and limits of these approaches.

Gene Expression Profiling↗
PubMed1995

Oligonucleotide probes to the 16S ribosomal RNA: implications of sequence homology and secondary structure with particular reference to the oral species Prevotella intermedia and Prevotella nigrescens.

Eight oligonucleotides based upon regions of the small subunit 16S ribosomal RNA gene sequences were analysed against a background of their position within the molecule and their two-dimensional structure to rationalise their use in recognising Prevotella intermedia and Prevotella nigrescens. The 41 clinical isolates from both oral and respiratory sites and two reference strains were subjected to DNA-DNA hybridisation and multilocus enzyme electrophoresis to confirm their identity. Alignment of oligonucleotide probes designated I Bi-2 to I Bi-6 (for P. intermedia) and 2Bi-2 (for P. nigrescens) with the 16S rRNA suggested that these probes lacked specificity or were constructed from hypervariable regions. A 52-mer oligonucleotide (designated Bi) reliably detected both species. Because of the high degree of concordance between the 16S rRNAs of both species, it was necessary to vary the stringency of hybridisation conditions for detection of both species. Thus probe I Bi-I recognised P. intermedia while I Bi-I detected both P. intermedia and P. nigrescens at low stringency. However, under conditions of high stringency only P. nigrescens was recognised by probe 2Bi-I. These probes were highly specific and did not hybridise with DNA from the closely related P. corporis, nor other periodontal pathogens such as Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola and several pigmented species such as Prevotella melaninogenica, P. denticola, P. loescheii, Porphyromonas asaccharolytica, Py. endodontalis, Py. gingivalis, Py. levii, and Py. macacae.

Bacterial Typing Techniques↗
PubMed2005

Lost in translation: translational interference from a recurrent mutation in exon 1 of MECP2.

BACKGROUND: Rett syndrome (RTT) is an X linked neuro-developmental disorder affecting mostly girls. Mutations in the coding region of MECP2 are found in 80% of classic RTT patients. Until recently, the region encoding MECP2 was believed to comprise exons 2, 3, and 4 with the ATG start site located at the end of exon 2 (MeCP2_e2). METHODS: Recent reports of another mRNA transcript transcribed from exon 1 (MeCP2_e1) prompted us to screen exon 1 among RNA samples from 20 females with classic or atypical RTT. RESULTS: A previously reported 11 base pair deletion in exon 1 was detected in one subject with a milder phenotype. Although RNA expression for both protein isoforms was detected from the mutant allele, evaluation of MeCP2 protein in uncultured patient lymphocytes by immunocytochemistry revealed that MeCP2 protein production was restricted to only 74-76% of lymphocytes. X chromosome inactivation studies of genomic DNA revealed similar XCI ratios at the HUMARA locus (73:27 with HpaII and 74:26 with McrBC). We have demonstrated that translation but not transcription of the MeCP2_e2 isoform is ablated by the 11 nucleotide deletion, 103 nucleotides upstream of the e2 translation start site. CONCLUSIONS: These findings reveal that nucleotides within the deleted sequence in the 5'-UTR of the MeCP2_e2 transcript, while not required for transcription, are essential for translation.

Adolescent↗
PubMed2004

[Identification of bacteria through 16S rRNA sequencing: principles, methods and applications in clinical microbiology].

Phylogenetic relationships among prokaryotes can be inferred from comparisons of their 16S rRNA (or 16S rDNA) sequences. This has had an enormous repercussion on bacterial taxonomy, leading to the currently applied system of classification, and allowing a rapid and precise identification of bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques turns out impossible, difficult, or requires a lot of time. Amplification of the gene to be sequenced uses preferably DNA extracted from a bacterial pure culture, but can be achieved also directly from a clinical sample. The latter has led to the discovery of new pathogens. Bearing in mind its potential, as the technical resources improve and the prize becomes more competitive, the identification based on 16S rDNA sequencing will certainly find a wider application in the clinical microbiology laboratory.

DNA Primers↗
PubMed2001

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells.

Using differential display polymerase chain reaction, a gene was identified in CD34(+)-enriched populations that had with low or absent expression in CD34(-) populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34(+) cells than in CD34(-) cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34(+)38(-) phenotype and was down-regulated with the proliferation and differentiation of CD34(+) cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Antigens, CD34↗
PubMed2005

HotKnots: heuristic prediction of RNA secondary structures including pseudoknots.

We present HotKnots, a new heuristic algorithm for the prediction of RNA secondary structures including pseudoknots. Based on the simple idea of iteratively forming stable stems, our algorithm explores many alternative secondary structures, using a free energy minimization algorithm for pseudoknot free secondary structures to identify promising candidate stems. In an empirical evaluation of the algorithm with 43 sequences taken from the Pseudobase database and from the literature on pseudoknotted structures, we found that overall, in terms of the sensitivity and specificity of predictions, HotKnots outperforms the well-known Pseudoknots algorithm of Rivas and Eddy and the NUPACK algorithm of Dirks and Pierce, both based on dynamic programming approaches for limited classes of pseudoknotted structures. It also outperforms the heuristic Iterated Loop Matching algorithm of Ruan and colleagues, and in many cases gives better results than the genetic algorithm from the STAR package of van Batenburg and colleagues and the recent pknotsRG-mfe algorithm of Reeder and Giegerich. The HotKnots algorithm has been implemented in C/C++ and is available from http://www.cs.ubc.ca/labs/beta/Software/HotKnots.

Algorithms↗
PubMed1994

The 16S rRNA sequence and genome sizing of tributyltin resistant marine bacterium, strain M-1.

The 16S rRNA of the tributyltin resistant marine bacterium, strain M-1, was partly sequenced to confirm the taxonomic status. The results indicated that this bacterium should be classified under the genus Alteromonas, instead of a previous report in which this strain was identified as a Vibrio. The genome size of this strain was also measured by pulsed field gel electrophoresis (PFGE) using a contoureclamped homogeneous electric field. The strain was found to contain a genome size of 2,240 kilo base pairs, whereas Alteromonas nigrifaciens and Shewanella putrefaciens had 2,040 and 2,383 kilo base pairs, respectively. This is the first report of the genome sizing of the genus Alteromonas by PFGE.

Base Sequence↗
PubMed1989

Laboratory characterization of a swine influenza virus isolated from a fatal case of human influenza.

A swine influenza virus-like type A (H1N1) virus, designated A/Wisconsin/3523/88, was isolated in September 1988 from a Wisconsin woman who had died with primary viral pneumonia. Antigenic analyses with hemagglutinin-specific monoclonal antibodies and postinfection ferret serum indicated that the hemagglutinin of A/Wisconsin/3523/88 was antigenically closely related to viruses currently circulating in swine. Genetic analysis of the A/Wisconsin/3523/88 virus by RNA fingerprinting and partial RNA sequence analysis of seven of the eight segments indicated that the genome of the human isolate was similar to that of enzootic swine viruses. These laboratory data supported the epidemiologic findings that this human infection occurred by transmission of an enzootic swine influenza virus and that the virus showed no major genetic changes potentially related to increased pathogenesis.

Adult↗
PubMed1996

Compilation and analysis of viroid and viroid-like RNA sequences.

We have created a catalogue comprising all viroid and viroid-like RNA sequences which to our knowledge have been either published or were available from on-line sequence libraries as of October 1, 1995. In the development of this catalogue nomenclature ambiguities were removed, the likely ancestral sequence of most species was determined and the most stable secondary structures of these sequences were predicted using the MulFold package. Only viroids of PSTVd-type possessed a rod-like secondary structure, while most other viroids adopted branched secondary structures. Several viroids have predicted secondary structures that include either a Y or cruciform structure reminiscent of the tRNA-like end of virus genomes at an extremity. However, it remains unknown whether or not these predicted structures are adopted in solution, and if they serve a particular function in vivo. Additional information such as the position of the self-catalytic domains are included in the catalogue. An analysis of the data compilated in the catalogue is included. The catalogue will be available on the world wide web (http://www.callistro.si.usherb.ca/jpperra), on computer disk and in printed form. It should provide an excellent reference point for further studies.

Base Sequence↗
PubMed2006

QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences.

The quadruplex structures formed by guanine-rich nucleic acid sequences have received significant attention recently because of growing evidence for their role in important biological processes and as therapeutic targets. G-quadruplex DNA has been suggested to regulate DNA replication and may control cellular proliferation. Sequences capable of forming G-quadruplexes in the RNA have been shown to play significant roles in regulation of polyadenylation and splicing events in mammalian transcripts. Whether quadruplex structure directly plays a role in regulating RNA processing requires investigation. Computational approaches to study G-quadruplexes allow detailed analysis of mammalian genomes. There are no known easily accessible user-friendly tools that can compute G-quadruplexes in the nucleotide sequences. We have developed a web-based server, QGRS Mapper, that predicts quadruplex forming G-rich sequences (QGRS) in nucleotide sequences. It is a user-friendly application that provides many options for defining and studying G-quadruplexes. It performs analysis of the user provided genomic sequences, e.g. promoter and telomeric regions, as well as RNA sequences. It is also useful for predicting G-quadruplex structures in oligonucleotides. The program provides options to search and retrieve desired gene/nucleotide sequence entries from NCBI databases for mapping G-quadruplexes in the context of RNA processing sites. This feature is very useful for investigating the functional relevance of G-quadruplex structure, in particular its role in regulating the gene expression by alternative processing. In addition to providing data on composition and locations of QGRS relative to the processing sites in the pre-mRNA sequence, QGRS Mapper features interactive graphic representation of the data. The user can also use the graphics module to visualize QGRS distribution patterns among all the alternative RNA products of a gene simultaneously on a single screen. QGRS Mapper can be accessed at http://bioinformatics.ramapo.edu/QGRS/.

Computer Graphics↗
PubMed1996

Template strand gap bypass is a general property of prokaryotic RNA polymerases: implications for elongation mechanisms.

It has previously been shown that T7 RNA polymerase is capable of bypassing gaps on the template strand ranging in size from 1 to 24 nucleotides. This as well as other observations suggested a role for the nontemplate strand during elongation. To establish the generality of this gap bypassing event, we have extended these studies to SP6 and Escherichia coli RNA polymerases. SP6 RNA polymerase bypasses template gaps from 1 to 19 nucleotides in size with various degrees of efficiency and produces runoff transcripts of decreasing length corresponding to increasing gap size. RNA sequence analysis of the resulting runoff transcripts revealed that SP6 RNA polymerase faithfully transcribed both parts of the template strand flanking the gapped region. Similar experiments were carried out with E. coli RNA polymerase (a multiple subunit enzyme) and indicate that it is also capable of gap bypass albeit with reduced efficiency compared to T7 and SP6 RNA polymerases. It appears that the ability to bypass gaps present on the DNA template strand is a general property of prokaryotic RNA polymerases. These results have implications with respect to the mechanism of elongation and the role of the nontemplate strand in transcription.

Base Sequence↗
PubMed2002

[Results of the hepatitis A epidemic in Blekinge. Transmission links surveyed with the help of sequence analysis of viral RNA].

In 1994-1995 there was an epidemic of hepatitis A (HAV) among drug abusers in the county of Blekinge, Sweden. There were also a number of other cases in which the epidemiological links were unclear. In order to uncover possible links between unclear cases retrospective sequencing was carried out of the VP1 region of HAV-RNA from patient sera. It was found that the epidemic among drug abusers spread beyond their closest contacts, revealing links that would otherwise not have been suspected. The extent of the epidemic also demonstrated the need to vaccinate injecting drug users against hepatitis A.

Adolescent↗
PubMed1995

Identification of a new noradrenaline induced gene in the rat heart by differential mRNA display.

OBJECTIVE: Noradrenaline treatment of animals results in postnatal hypertrophy of the heart. This process requires many qualitative and quantitative changes in gene expression; however, the identities of the key regulatory genes which modulate the process are not known. The aim of this study was to investigate whether a recently developed technique, differential display, could provide a new route to the identification and characterisation of these critical genes. METHODS: The technique of differential display was modified for use on cardiac RNA samples and the expression of clones identified by this approach was characterised by northern analysis. RESULTS: Differential display was successfully adapted to the study of noradrenaline induced cardiac gene expression. A previously unsuspected gene was identified, the expression of which appears to be strongly modified during the onset of this process. CONCLUSIONS: Differential display offers the potential to identify and clone many of the genes critically important in regulation of growth of the mammalian heart.

Animals↗
PubMed2006

Quadfinder: server for identification and analysis of quadruplex-forming motifs in nucleotide sequences.

G-quadruplex secondary structures, which play a structural role in repetitive DNA such as telomeres, may also play a functional role at other genomic locations as targetable regulatory elements which control gene expression. The recent interest in application of quadruplexes in biological systems prompted us to develop a tool for the identification and analysis of quadruplex-forming nucleotide sequences especially in the RNA. Here we present Quadfinder, an online server for prediction and bioinformatics of uni-molecular quadruplex-forming nucleotide sequences. The server is designed to be user-friendly and needs minimal intervention by the user, while providing flexibility of defining the variants of the motif. The server is freely available at URL http://miracle.igib.res.in/quadfinder/.

DNA↗