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PubMed2026

Amplification-Free Nanopore Sequencing for Herpesvirus DNA Detection in Intraocular Fluids.

PURPOSE: To evaluate the feasibility of amplification-free nanopore sequencing for detecting herpesvirus DNA in intraocular fluid using multiplex polymerase chain reaction (mPCR)-characterized herpesvirus-positive and herpesvirus-negative samples. DESIGN: Retrospective, single-center, cross-sectional study. PARTICIPANTS: This study included 42 patients with uveitis whose intraocular fluid samples were examined by mPCR, including 20 mPCR-positive samples (all positive for herpesviruses) and 22 mPCR-negative samples. METHODS INTERVENTION OR TESTING: DNA extracted from intraocular fluid samples underwent ligation-based library preparation without whole-genome amplification and was sequenced on the MinION platform with Flongle flow cells for untargeted analysis. Nanopore sequencing results were compared with mPCR findings, and associations between nanopore-derived virus-specific read counts and corresponding herpesvirus DNA copy numbers measured by mPCR were assessed. MAIN OUTCOME MEASURES: Primary outcome measure was concordance between nanopore sequencing and mPCR in herpesvirus species identification. Secondary outcome measures included nanopore sequencing detection rates stratified according to mPCR-measured herpesvirus DNA copy numbers and correlations between nanopore sequencing-derived virus-specific read counts and mPCR-measured herpesvirus DNA copy numbers. RESULTS: Among 20 mPCR-positive intraocular fluid samples, nanopore sequencing identified viral DNA from the same herpesvirus species detected by mPCR in 15 (75.0%), indicating species-level concordance. None of the 22 mPCR-negative samples contained virus-specific reads. Among the 22 herpesvirus targets identified in the 20 mPCR-positive samples, herpesvirus DNA copy numbers measured by mPCR were significantly higher in nanopore-positive than in nanopore-negative targets (P = 0.015). Nanopore detection rates increased with increasing herpesvirus DNA copy numbers measured by mPCR: 3 of 6 targets (50.0%) with <105 copies/mL, 2 of 4 (50.0%) with 105-106 copies/mL, and 12 of 12 (100%) with >106 copies/mL (P = 0.021). Nanopore sequencing-derived virus-specific read counts correlated positively with herpesvirus DNA copy numbers measured by mPCR (r = 0.76, P = 0.0004). CONCLUSIONS: Amplification-free nanopore sequencing demonstrated the feasibility of detecting herpesvirus DNA in intraocular fluid samples, with detection performance dependent on herpesvirus DNA load. This simplified workflow may provide complementary information regarding viral DNA burden in minute ocular samples. FINANCIAL DISCLOSURES: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Herpesvirus↗

PubMed2024

Protocol to decode the role of transcriptionally active microbes in SARS-CoV-2-positive patients using an RNA-seq-based approach.

The elucidation of the role of microorganisms in human infections has been hindered by difficulties using conventional culture-based techniques. Here, we present a protocol for the investigation of transcriptionally active microbes (TAMs) using an RNA sequencing (RNA-seq)-based approach. We describe the steps for RNA isolation, viral genome sequencing, RNA-seq library preparation, and metatranscriptomic and transcriptomic analysis. This protocol permits a comprehensive evaluation of TAMs' contributions to the differential severity of infectious diseases, with a particular focus on diseases such as COVID-19. For complete details on the use and execution of this protocol, please refer to Devi et al.1.

Humans↗

PubMed1997

Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.

Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.

Amino Acid Sequence↗

PubMed1983

Cloning of interleukin 2 mRNAs from human tonsils.

Human interleukin 2 (IL-2) mRNAs were cloned from a cDNA library prepared from mitogen-stimulated tonsillar mononuclear cells. One of these clones was sequenced and, using this cDNA as a hybridization probe, Southern blot analysis of the human placental DNA was performed. Our results indicate that there is only one IL-2 gene in the human genome.

Cloning, Molecular↗

PubMed1988

Cloning, structure and expression of a cDNA encoding the human androgen receptor.

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.

Amino Acid Sequence↗

PubMed1988

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum as deduced from the cDNA sequence.

S-Adenosyl-L-homocysteine hydrolase has been cloned from a lambda gt11 cDNA library prepared from Dictyostelium discoideum that had been starved for 3 hours. The sequence of the cloned cDNA was determined and the deduced amino acid sequence was compared to the amino acid sequence of rat AdoHcy hydrolase. When the sequences from the two species were aligned, 74% of the amino acids were in identical positions. If conservative changes were taken into account the homology was 84%. Because differences have been reported in the binding characteristics of NAD+ to the D. discoideum and rat AdoHcy hydrolases, changes in the amino acids of the putative NAD+-binding site were of particular interest. Six changes were observed in this region but the changes appeared to be in regions that are not critical to the three dimensional folding of the NAD+-binding site.

Adenosylhomocysteinase↗

PubMed1995

Lens epithelial cell mRNA. I. Cloning and sequencing of a messenger RNA with a basic motif/leucine-rich domain specifically expressed in rat lens epithelial cells.

By methods of subtraction-hybridization of lambda ZAP cDNA libraries, prepared from 4-week-old rat lens epithelial cells (capsule) and lens fiber cells (decapsulated lens), we have isolated a specific cDNA clone whose target mRNA is about 600 b long. Northern blot hybridization analysis data showed that the target mRNA was preferentially expressed in the lens epithelial cells; it was not found in the retina or in non-ocular tissues. The complete sequence of the mRNA was obtained both by the 5' and 3' rapid amplification of cDNA ends (5'-RACE, 3'-RACE), and by sequencing of a clone containing the full-length cDNA insert. It contains 599 b. with an open reading frame at nucleotide (nt) 30 (ORF30). The base sequence appears to represent a complete sequence of the target mRNA, with a poly(A) signal at nt 563-569, and a poly(A) tail at nt 582 (GenBank accession #U15149). In situ hybridization showed that the target mRNA was localized in the anterior epithelial cells, and in the elongating cells in the bow region. The encoded protein of 57 amino acids (aa) contains a basic and an acidic domain. It is rich in leucine, which is located at the basic region of the ORF at every fourth aa, and at the acidic region at eight aa intervals. Therefore, there is a periodicity in the appearance of leucine residues in the deduced primary sequence, as has been seen in DNA binding proteins. Search of GenBank, EMBL and Swiss-Prot did not yield significant homology to any of the entered sequences in those databanks. Our data demonstrate that we have isolated a cDNA clone containing novel sequences specifically expressed in lens epithelial cells.

Animals↗

PubMed1995

Coordinated activation of corneal wound response genes in vivo as observed by in situ hybridization.

We used subtractive screening of a cDNA library prepared from corneoscleral rims after cauterizing rat corneas. We identified 76 clones whose corresponding mRNA increased during the wound healing process in an in vivo model of injury which damages the corneal epithelium, stroma, and endothelium. Of these clones, 31 sequences encode known proteins. Another 45 clones are novel sequences based on comparison with the GenBank/EMBL databases. Changes in the level of expression of the novel genes, and a selected number of the known genes, were examined by in situ hybridization 22 and 72 hr after corneal injury. The majority produced a 'wound pattern' of expression such that the mRNAs were highly induced in all cell types adjacent to the wound site at 22 hr post injury. This signal decreased in intensity with distance from the wound site. In a subset of corneoslceral rims examined by in situ hybridization, the mRNAs for these genes were also highly induced in the limbal epithelium, where the progenitor corneal epithelial stem cells reside. By 72 hr, when acute tissue damage had been repaired, the induced mRNA was only faintly present in the thickened epithelium. Our results provide a useful framework for further studies defining the pathophysiological roles of the known and novel proteins encoded by the isolated cDNA clones.

Animals↗

PubMed2001

A naturally occurring non-coding fusion transcript derived from scorpion venom gland: implication for the regulation of scorpion toxin gene expression.

Scorpion venom glands synthesize and secrete a great number of low molecular mass toxic peptides for prey and defense. Many cDNAs and genomic genes encoding these toxins have been isolated and sequenced. However, their expression regulation mechanism is not yet known at present. During screening of a cDNA library prepared from venom glands of the scorpion Buthus martensii Karsch, we isolated a natural fusion cDNA composed of the 5'-untranslated region (UTR) and upstream coding sequence of a long-chain toxin transcript and the downstream coding sequence and 3'-UTR of a short-chain toxin transcript. The junction site is just the overlapping region of 11 nucleotides (GGCAAGGAAAT) between the two wild transcripts, and thus leads to the formation of an early stop codon, which will cause premature translation. Based on the above observations, combined with the genomic data, we proposed a characteristic regulation mechanism of scorpion toxin genes, in which trans-splicing and nonsense mediated mRNA decay are involved.

3' Untranslated Regions↗

PubMed2002

Apisimin, a new serine-valine-rich peptide from honeybee (Apis mellifera L.) royal jelly: purification and molecular characterization.

A peptide named apisimin was found in honeybee (Apis mellifera L.) royal jelly (RJ). N-terminal sequencing showed that this peptide corresponded to the sequence of a cDNA clone isolated from an expression cDNA library prepared from heads of nurse honeybees. No homology was found between the protein sequence of apisimin with a molecular mass of 5540.4 Da and sequences deposited in the Swiss-Prot database. The 54 amino acids of apisimin do not include Cys, Met, Pro, Arg, His, Tyr, and Trp residues. The peptide shows a well-defined secondary structure as observed by CD spectroscopy, and has the tendency to form oligomers. Isoelectrofocusing showed apisimin to be an acidic peptide.

Amino Acid Sequence↗

PubMed1997

A new transglutaminase-like from the ascidian Ciona intestinalis.

A cDNA clone encoding a transglutaminase (TGase) was isolated from a cDNA library prepared from the larval stage of Ciona intestinalis. The cDNA sequence has an open reading frame encoding a protein of 696 amino acids and is about 36% identical to 11 other TGase sequences. In addition, the critical residues thought to form the catalytic center are conserved. The Ciona TGase (CiTGase) has an extension of 39 amino acids in the NH2-terminal region similar to that reported for keratinocyte TGases. A phylogenetic analysis among other types of TGases demonstrated that CiTGase represents a new type of the enzyme.

Amino Acid Sequence↗

PubMed1997

cDNA cloning of Clavanins: antimicrobial peptides of tunicate hemocytes.

Clavanins are a family of alpha-helical antimicrobial peptides found in hemocytes of the tunicate, Styela clava. We examined a cDNA library prepared from pharyngeal tissues of S. clava and sequenced 24 clones that encoded prepropeptides of Clavanins A, C, D or E. These sequences indicated that Clavanins are synthesized as 9.2 kDa prepropeptides which contain a 19-residue signal peptide, followed in turn by a highly polar 'pro' region (LEERKSEEEK) with five glutamic acid residues, the 23 residues of the mature Clavanin peptide, the glycine residue needed for its amidation and a 27-residue polar C-terminal extension that is removed in later processing. Although the signal sequence and anionic propiece of Clavanin precursors share features with corresponding regions in precursors of the certain frog peptides, including ranalexin, gaegurins, dermaseptins and deltorphins, their unique multipartite structure suggests that they are not actually homologues of these amphibian peptides.

Amino Acid Sequence↗

PubMed1998

A novel lipopolysaccharide inducible C-C chemokine receptor related gene in murine macrophages.

To identify genes induced in activated macrophages, we screened a cDNA library prepared from the lipopolysaccharide (LPS)-treated cell line, RAW264, using the suppression subtractive hybridization technique. One of the clones isolated was dramatically induced by LPS in macrophages. The predicted protein sequence of this gene contains the domain unique to seven transmembrane receptors, and shows similarity with mouse C-C chemokine receptor 5 (CCR5). Therefore, we designated it LPS inducible C-C chemokine receptor related gene (L-CCR). Northern blot analysis revealed that L-CCR was specifically expressed in differentiated macrophages after LPS stimulation. These results show that L-CCR is a novel C-C chemokine receptor related gene induced by LPS in macrophages and may play an important role in inflammatory responses.

Amino Acid Sequence↗

PubMed1998

Isolation and sequence of cDNA encoding the motilin precursor from monkey intestine. Demonstration of the motilin precursor in the monkey brain.

The motilin precursor cDNA has been isolated and sequenced from a cDNA library prepared from monkey small intestine. The sequence indicates a 345 bp open reading frame, a 63 bp 5' untranslated region and a 154 bp 3' untranslated region. The sequence encodes a 115 amino acid motilin precursor composed of a 25 amino acid signal peptide, the 22 amino acid motilin peptide and a 68 amino acid motilin associated peptide (MAP). Compared with the human motilin precursor cDNA, there are two amino acid substitutions in the signal peptide, one in motilin and four in the MAP. The presence of the motilin precursor in hypothalamus, hippocampus and cerebellum was demonstrated by RT-PCR.

Amino Acid Sequence↗

PubMed1999

Identification of novel 70-kDa heat shock protein-encoding cDNAs from Schistosoma japonicum.

A diverse range of organisms respond to a variety of chemical, physiological and temperature-associated stresses by a rapid and transient increase in the synthesis of heat shock proteins. We immunoscreened a Uni-ZAP XR cDNA library, prepared from mRNA isolated from the Philippine strain of the Asian bloodfluke, Schistosoma japonicum, using hyperimmune rabbit sera raised against soluble adult S. japonicum proteins. Six 70-kDa heat shock protein-encoding cDNA clones were identified which, upon further analysis, were separated into two distinct protein groups within the 70-kDa heat shock protein family, the 70-kDa heat shock proteins and the immunoglobulin heavy chain-binding proteins/glucose-related proteins (Grp78). A representative from both groups was fully sequenced and compared with homologous sequences available in the GenBank/EMBL database as the first stage in determining the role of their expression products in the regulation of S. japonicum development, in the induction of immunity, and whether they act as molecular chaperones capable of modulating the correct folding or repair of proteins within this species of schistosome.

Amino Acid Sequence↗

PubMed2000

Solid-phase extraction for combinatorial libraries.

Solid-phase extraction (SPE) has during the last three years emerged as a convenient method for the purification of compound libraries prepared by solution synthesis. The widespread use of SPE in combinatorial chemistry can be explained by straightforward SPE method development facilitated by the availability of numerous commercial SPE resins. High-speed automated SPE is readily accomplished by taking advantage of commercial laboratory robot systems. The present review summarizes and discusses advancements made in the use of different SPE resins and molecule tagging techniques for optimization of ion-exchange, reversed-phase, normal-phase and fluorous-phase SPE in combinatorial chemistry.

Chromatography, Liquid↗

PubMed1990

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA.

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.

Base Sequence↗

PubMed2001

cDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6.

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5'-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, confirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.

Amino Acid Sequence↗