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PubMed2003

Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin.

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.

Base Sequence↗
PubMed2004

Gene specific siRNA selector.

Small interfering RNA (siRNA) is used in functional genomics applications to decrease the expression of a target gene, which may yield a biological effect that suggests a function for the target gene. The siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest.

Algorithms↗
PubMed1994

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.

Amino Acid Sequence↗
PubMed2026

S100P as a Shared Biomarker in Inflammatory Bowel Disease, Colorectal Cancer, and Pancreatic Adenocarcinoma: An Integrated Transcriptomic Analysis.

Inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD), yet the molecular features shared among these diseases remain incompletely understood. This study aimed to identify common genes and biological pathways associated with IBD, CRC, and PAAD through integrated transcriptomic analysis and experimental validation. Gene expression datasets for IBD, CRC, and PAAD were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. Weighted gene co-expression network analysis and differential expression analysis were performed to identify disease-associated and shared genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (analyses were used to explore enriched biological functions and pathways. Immune cell infiltration was evaluated using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts. Receiver operating characteristic analysis was performed to assess the diagnostic performance of common genes. Single-cell RNA sequencing analysis was conducted to examine the cellular distribution of S100P. In addition, the effects of S100P downregulation were evaluated in lipopolysaccharide (LPS)-stimulated colonic epithelial cells. A total of 162 disease-associated genes and four common genes were identified. Functional enrichment analyses indicated significant enrichment of immune- and inflammation-related pathways, including the interleukin-17 signaling pathway. Immune infiltration analysis revealed similar trends in several immune cell populations across IBD, CRC, and PAAD. Single-cell analysis showed elevated S100P expression in epithelial cells from all three diseases. Downregulation of S100P restored the proliferative capacity of LPS-stimulated colonic epithelial cells and reduced inflammatory cytokine expression. Integrated transcriptomic analysis identified S100P as a biomarker associated with IBD, CRC, and PAAD and highlighted shared immune-related features across these diseases.

Humans↗
PubMed1980

3' Terminal labelling of RNA of RNA with beta-32P-pyrophosphate group and its application to the sequence analysis of 5S RNA from Streptomyces griseus.

Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from gamma-32P-ATP to the 3'-OH of tRNA, generating a strictly terminal label at its 3' end. Using yeast tRNAPhe as model compound, it is demonstrated that the labelled molecule is suitable for rapid gel sequencing by both enzymatic and chemical methods. RNA molecules terminated by pyrimidine nucleoside are poor pyrophosphate acceptors. To label RNAs of this kind, first guanosine 5'-phosphate 3'-(beta-32P)-pyrophosphate (pGpp) is prepared from gamma-32P-ATP and GMP by nucleotide pyrophosphate transferase. pGpp is then ligated to the 3' end of RNA by T4 RNA ligase. The complete nucleotide sequence of 5S RNA from Streptomyces griseus is established by rapid gel sequencing methods performed on 3'-(beta-32P)-pyrophosphate labelled molecule.

Base Sequence↗
PubMed1979

Four T7 RNA polymerase promoters contain an identical 23 bp sequence.

The DNA sequences of four promoters for the bacteriophage-specified T7 RNA polymerase have been determined. All four regions contain an identical 23 bp sequence 5' pTAATACGACTCACTATAGGGAG-A(oh) which includes the 17 bp prior to and the first 6 bp of the transcribed region. They are located at 46.5, 55, 57 and 87% on the standard T7 map, thereby corresponding to the in vitro T7 class III RNA species IIIa, IV, V and IIIb, respectively. The precise RNA initiation sites were determined by 5' terminal RNA sequence analysis of the transcripts synthesized from promoter-containing DNA fragments. The comparative properties of these promoters for the monomeric T7 RNA polymerase and those for the multimeric E. coli RNA polymerase are discussed.

Base Sequence↗
PubMed1990

In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

Animals↗
PubMed1993

Isolation and characterization of cDNA clones encoding jacalin isolectins.

Four jacalin cDNA clones (pSKcJA1, pSKcJA3, pSKcJA15, and pSKcJA17) have been obtained from an Artocarpus integrifolia (jackfruit) seed cDNA library. These clones share over 94% sequence homology, and their deduced polypeptide sequences confirm the existence of multiple jacalin isolectins in jackfruit seeds. The deduced amino acid sequences show that jacalin appears to be initially synthesized as a prepropeptide with the following structure: N-signal (21 residues)-->propeptide (39 residues)-->beta-peptide (20 residues)-->linker region (4 residues)-->alpha-peptide (133 residues). These observations are supported by Western blot analysis of jackfruit seed extract and by immunoprecipitation of in vitro translated products of both pSKcJA3 transcript and jackfruit seed poly(A)+ RNA. Sequence analysis of the 39-residue propeptide reveals that it has the potential to facilitate proper folding of jacalin protein. The unusual primary structure of jacalin prepropeptide suggests a quite interesting processing of this lectin precursor into mature alpha- and beta-subunits.

Amino Acid Sequence↗
PubMed2000

[Study on the origin of influenza A(H1N2) virus HA and NA genes].

OBJECTIVE: To determine the origins of the HA and NA genes of new subtype (H1N2) of influenza A virus. METHODS: Virus was amplified in embryonated chicken eggs, then virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, the products of PCR were purified. Afterward, RNA sequence analysis was performed by the dideoxynucleotide chain termination method, using synthetic oligodideoxynucleotide primers. Finally, phylogenetic analysis of the sequencing data was per-formed with MegAlign (Version 1.03) and Editseq (Version 3.69) software. RESULTS: The homology of amino acid sequences of protein molecules on HAl domains when compared, with A/PR/8/34(H1N1) and A/Guangdong/6/91(H1 N1) viruses was 98.2% and 99.4%, respectively. The homology of amino acid sequences of NA protein molecules between the novel reassortant and H1N1 virus was only 83.4 %, but the homology of those between H1N2 strain and H3N2 virus circulating in men in 1995 was as high as 99.1 %. CONCLUSIONS: The new reassortant (H1N2) virus HA gene was derived from A/PR/8/34(H1N1) like virus, but its NA gene was derived from H3N2 virus circulating in men in 1995.

Adult↗
PubMed2005

Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development.

We have characterized a cDNA encoding a Xenopus laevis apyrase (XAPY) that is expressed during embryogenesis. XAPY is highly homologous to two recently described mammalian apyrases, human SCAN-1 and rat Ca2+-NDPase, and to a lesser extent the salivary apyrase of the blood-feeding arthropod Cimex lectularis. RT-PCR analysis shows that Xapy is expressed at all the developmental stages tested, from oocytes through to tadpoles. Xapy transcripts are widely distributed in the embryo, but from late neurulae through to late tailbud stages they are highly enriched in the cement gland, an adhesive organ in the epidermis of the head. When expressed in HEK 293 cells, XAPY is largely retained in the endoplasmic reticulum, although some is also secreted. XAPY conditioned media hydrolyses UDP and UTP, confirming that it is a functional apyrase.

Amino Acid Sequence↗
PubMed2000

[Studies on the basis of molecular biology of the phase change of influenza A(H1N1) viruses].

OBJECTIVE: To reveal the basis of molecular biology of the phase change of influenza A (H1N1) viruses. METHODS: Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, the products of PCR were purified. Afterward, RNA sequence analysis was performed by the dideoxynucleotide chain termination method, using synthetic oligodeoxynucleotide primers. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software. RESULTS: No special amino acid on HA1 protein molecule determining the influenza A(H1N1) virus with O or D phase feature was found. However, there are differences of amino acid sequences at -2, -7, 130 and 139 positions between H1N1 viruses isolated in or before end after 1995. It is very interesting that since 1995, two different genetic lineages of influenza A (H1N1) virus HA gene have been cocirculating in men in China. One lineage is no any deletion of amino acid on HA1 protein molecule. The other one has a deletion at 130 position. Their amino acid sequences were compared with those of H1N1 viruses isolated before 1995. CONCLUSIONS: There is no special amino acid on HA1 domain protein molecule determining the influenza A (H1N1) virus with O or D phase feature was found. Since 1995, two distinguished genetic lineages of influenza A H1N1 virus HA gene have been cocirculating in men in China.

Amino Acid Sequence↗
PubMed2006

Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences.

We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design.

Base Sequence↗
PubMed2004

Evaluation of several lightweight stochastic context-free grammars for RNA secondary structure prediction.

BACKGROUND: RNA secondary structure prediction methods based on probabilistic modeling can be developed using stochastic context-free grammars (SCFGs). Such methods can readily combine different sources of information that can be expressed probabilistically, such as an evolutionary model of comparative RNA sequence analysis and a biophysical model of structure plausibility. However, the number of free parameters in an integrated model for consensus RNA structure prediction can become untenable if the underlying SCFG design is too complex. Thus a key question is, what small, simple SCFG designs perform best for RNA secondary structure prediction? RESULTS: Nine different small SCFGs were implemented to explore the tradeoffs between model complexity and prediction accuracy. Each model was tested for single sequence structure prediction accuracy on a benchmark set of RNA secondary structures. CONCLUSIONS: Four SCFG designs had prediction accuracies near the performance of current energy minimization programs. One of these designs, introduced by Knudsen and Hein in their PFOLD algorithm, has only 21 free parameters and is significantly simpler than the others.

Computational Biology↗
PubMed1984

Locations and nucleotide sequences of three major class III promoters for bacteriophage T3 RNA polymerase on T3 DNA.

The DNA sequences of three major class III T3 RNA polymerase promoters located at 45.0, 55.0, and 64.8% on the standard T3 genetic map have been determined. The precise RNA initiation sites were also determined by 5'-terminal RNA sequence analysis of the transcripts synthesized from the promoter-containing DNA fragments. Alignment of these three class III promoters and a previously determined T3 RNA polymerase promoter at 1.05% on T3 genetic map, with start points of transcription (+1) in register, indicates a high degree of sequence conservation among the four T3 RNA polymerase promoters. The sequences are identical between positions -12 and +4 and are uniformly A-T between -12 and -17. The conserved portion of the (-) strand sequence is 5' (sequence in text) Upstream from -17 and downstream from +4 the sequences diverge. Comparison of this sequence with a prototype 23-base pair promoter sequence for T7 RNA polymerase shows overall homology between positions -17 and +4 with conserved divergence at residues -2 and between -10 and -12. Furthermore, careful examination of the nucleotide sequences around 45.0 and 64.8 T3 map units shows that the putative RNA sequences arising from these regions by overlapping transcription from upstream promoters can be arranged into stable stemloop structures thought to be required for RNase III cleavage. This pattern is similar to those reported for the corresponding T7 RNA polymerase promoters on T7 DNA (Dunn. J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535).

Base Sequence↗
PubMed2000

The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma. Cloning and identification of a transcribed trna-like element that controls its expression.

U4 small nuclear RNA is essential for trans-splicing. Here we report the cloning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, it carries an Sm-like site, and has the potential for extensive intermolecular base pairing with the U6 RNA. Sequence analysis of the U4 locus indicates the presence of a tRNA-like element 86 base pairs upstream of the gene that is divergently transcribed to yield a stable small tRNA-like RNA. Two additional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this element. By stable expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene, but not the upstream tRNA genes, is essential for U4 expression and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl groups on U4 and U6 small nuclear RNAs suggests the presence of modifications in canonical positions. However, the number of modified nucleotides is fewer than in mammalian homologues. The U4 genomic organization including both tRNA-like and tRNA genes may represent a relic whereby trypanosomatids "hired" tRNA genes to provide extragenic promoter elements. The close proximity of tRNA genes to the tRNA-like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.

Animals↗
PubMed1999

Rupestris stem pitting associated virus-1 consists of a family of sequence variants.

Rupestris stem pitting (RSP) seems to be one of the most widespread virus diseases of grapevines. A virus, designated as rupestris stem pitting associated virus-1 (RSPaV-1), is consistently associated with, and likely to be the causative agent of RSP. Sequence analyses of cDNA clones derived from several RSP-affected grapevines suggested that a family of sequence variants of RSPaV-1 was associated with RSP. The genome structure of the sequence variants is identical to that of RSPaV-1 in that they had five open reading frames (ORF) and sequence identities ranging from 75 to 93% in nucleotide sequence and from 80 to 99% in amino acid sequence. ORF5 (coat protein) and the carboxyl-terminal portion of ORF1 (replicase) appeared to be the most conserved regions. The coat proteins of the sequence variants exhibited highly similar antigenic indices, suggesting serological relatedness among them. The cDNA clones obtained through reverse transcription-polymerase chain reaction from RSP-infected grapevines were heterogeneous in nt sequence with identities of 77-99% relative to RSPaV-1. Furthermore, a number of sequence variants were identified in several grapevines infected with RSP. Baselines for defining RSPaV-1 and possible mechanisms accounting for infection of grapevines with multiple sequence variants of RSPaV-1 are proposed. Findings from this study should have practical applications toward understanding the etiology of RSP and developing reliable assays to rapidly detect the disease.

Amino Acid Sequence↗
PubMed2023

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

RNA↗