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PubMed1984

Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

Amino Acid Sequence↗

PubMed2025

Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).

Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.

Streptococcus pneumoniae↗

PubMed1997

Photoreaction of new psoralen analogs with DNA: sequence and mutation specificity in the Escherichia coli lacZ gene.

New thio- and seleno-analogs of psoralen were synthesized and analyzed for their photoreactivity toward DNA. Using oligonucleotides of defined sequence, we first showed that these derivatives predominantly generated interstrand crosslinks at 5'-TpA sites. We also observed a surprisingly high reactivity of 7H-thiopyrano[3,2-f][1]benzofuran-7-one (PSO[O-S]) with the BamHI and PstI oligomers, giving rise to the formation of crosslinks at 5'-ApT sites and of the thymidine-psoralen-cytosine type. Next, the sequence specificity in the photochemical binding of all the compounds was investigated in two DNA fragments encompassing the lacZ gene of Escherichia coli, using the T4 DNA polymerase sequencing methodology. Resulting maps demonstrated that thio- and seleno-analogs of psoralen preferentially photoreacted with thymine and cytosine residues. The AT-rich sequences proved to be particularly reactive sites as did adjacent thymines, especially at C-surrounding residues. Likewise, photoaddition at cytosines in CA/AC context was observed. It was highly significant that all of the derivatives exhibited similar sequence specificities with only minor differences. However, PSO(O-S) differed from the other heteropsoralens. Photoadducts occurred with a higher frequency at AC and CA dinucleotides, and new sites were detected. A comparison with 8-methoxypsoralen photobinding is also reported. Finally, the mutagenic consequences of photoadducts induced in M13mp19 DNA by PSO(O-S) were determined in a forward system that detects all classes of mutagenic events. The high phototoxicity exhibited by PSO(O-S) could be attributed to crosslinks, and the comparison of the observed mutational specificity with the photoadduct distribution within the same gene showed that mutations were targeted at potential monoadduct sites where photolesions were detected in our footprinting experiments.

DNA Adducts↗

PubMed1990

A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes.

The rat K and T kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the exon next to and upstream from the LMW exon (HMW exon) through alternative splicing and polyadenylation. In contrast, the T gene generates one mRNA by using selectively the LMW exon, although the T gene is extremely homologous to the K gene. In this study, we constructed a series of chimeric kininogen genes by not only exchanging equivalent restriction fragments of the two genes but also replacing nucleotides that differ between the two genes. We then examined the sequences and the mechanisms governing the different expression patterns of the two genes by transfecting the chimeric genes into heterologous COS cells. The results indicated that the different expression patterns of the K and T genes are governed by two separate internal sequences of the HMW and LMW exons. The internal HMW sequence contains a set of five repetitive sequences, and these repetitive sequences are highly complementary to the 5' portion of U1 snRNA. Furthermore, the nucleotide differences in the U1 snRNA-complementary sequences between the K and T genes have marked effects on the relative formation of the HMW and LMW mRNAs; this indicates that the repetitive sequences complementary to U1 snRNA play a crucial role in determining the relative expression of the two mRNAs. Based on these findings, we discuss a novel mechanism for alternative RNA processing, in which splicing efficiency is controlled by the interaction of U1 small nuclear ribonucleoproteins and the U1 snRNA-complementary repetitive sequences of the kininogen pre-mRNA.

Animals↗

PubMed2000

Oxalate decarboxylase from Collybia velutipes. Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato.

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mechanism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5'-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548-23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants expressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotiorum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.

Amino Acid Sequence↗

PubMed1997

The sequence-specific cleavage of RNA by artificial chemical ribonucleases.

Based on work spanning 50 years, several groups have recently achieved the specific cleavage of RNA by attaching RNA-cleaving chemical moieties to antisense oligonucleotides. Such artificial chemical ribonucleases have potential as a possible next generation of antisense compounds and also as probes for structural and functional investigations of RNA. Different chemical moieties, such as polyamines, imidazoles, and metal complexes, have been used as the catalytic part of the artificial nucleases. To be of practical use as therapeutics, however, the conjugates must fulfil a number of strict requirements, such as ease of preparation, chemical stability, selectivity, nontoxicity, and, for metal complexes, inertness to loss of cation from the ligand. In addition, high cleavage efficiency is essential to overcome short lifetimes of cellular mRNA targets, and the reaction should not depend on additional cofactors. Based on these criteria, we believe that metal complexes, in particular macrocyclic lanthanide complexes, have the best chance of success for said purpose.

Base Sequence↗

PubMed1993

Recombinant-DNA-derived insulin analogues as potentially useful therapeutic agents.

Whilst insulin is a spectacularly successful drug for the management of diabetes mellitus, it remains difficult to mimic the physiological pattern of insulin secretion, even using the various quick-acting and insoluble formulations that are available. The introduction of recombinant-DNA technology to the manufacture of therapeutic insulin has made the rational design and production of insulin analogues with altered pharmacokinetic and pharmacological properties possible. Such analogues include 'monomeric' insulins, which do not form the insulin-zinc hexamer in solution and are absorbed more rapidly from the injection site, and long-acting insulins, which are absorbed very slowly at physiological pH. Many of these analogues are being tested clinically, and it is possible that the next generation of insulin therapy will be various combinations of rationally designed insulin analogues produced by industrial biotechnology.

Amino Acid Sequence↗

PubMed1998

NMR analysis of a potent decapeptide agonist of human C5a anaphylatoxin.

A NMR investigation in H2O, TFE and DMSO of a conformationally constrained, potent decapeptide agonist of human C5a, YSFKDMPLaR (C5a65-74, Y65, F67, P71, D-Ala73) showed that its N-terminal region (YSFKD) exhibited an extended backbone conformation in H2O and a more twisted conformation in both TFE/H2O (30:70, v/v; referred to as TFE) and DMSO. The C-terminal region (MPLaR) of the peptide adopted compact, turn-like structures. In H2O, the C-terminal region adopted a type II beta-turn or a distorted type V/II beta-turn involving residues PLaR. In the distorted type V/II beta-turn, Leu72 exhibited a conformation typical of a type V beta-turn, whereas D-Ala73 exhibited a conformation typical of a type II beta-turn. The distorted type V/II beta-turn overlapped with an inverse gamma-turn involving residues MPL. In DMSO, the C-terminal region had the analogous inverse gamma-turn and the V/II beta-turn found in H2O. In many of the DMSO structures, two inverse gamma-turns in the MPL and PLa positions formed a double-inverse gamma-turn. None of the turns observed in H2O were present in TFE. However, in TFE, the PLa residues formed an inverse gamma-turn. Overall, the turn-like structural motifs in the C-terminal region of the peptide in both H2O and DMSO (but not in TFE) agreed with the biologically important conformations obtained earlier by the structure-function analysis of a panel of C5a agonist peptides. These motifs may represent key structural elements important for C5a agonist activity and may be used to design the next generation of C5a agonist and antagonist analogues.

Amino Acid Sequence↗

PubMed1993

Characterization of pertussis toxin analogs containing mutations in B-oligomer subunits.

The S2, S3, and S4 subunit genes of pertussis toxin (PT) from Bordetella pertussis were subjected to site-directed mutagenesis, and the resultant PT analogs were assayed for altered biological properties. PT analogs S2(T91,R92,N93) delta and S2(Y102A,Y103A) exhibited reduced binding to fetuin. Several PT analogs with mutations in the S2, S3, or S4 subunit showed reduced in vitro toxicity, as measured in the Chinese hamster ovary (CHO) cell clustering assay. In particular, PT analogs S3(Y82A) and S3(I91,Y92,K93) delta retained 10% or less residual toxicity. These mutants also exhibited significantly lower mitogenic and hemagglutinating activities and reduced in vivo activities, as measured by the histamine sensitization and leukocytosis assays. The S4(K54A,K57A) PT analog had significantly reduced CHO cell clustering activity, though other biological activities remained unaffected. PT analogs S1(E129G)/S3(Y82A) and S1(E129G)/S3(I91,Y92,K93) delta displayed a cumulative effect of the S1 and S3 mutations for both in vitro and in vivo toxic activities. These PT analogs, as well as S1(R9K,E129G)/S3(K82A) and S1(R9K,E129G)/S3(I91,Y92,K93) delta, still expressed an epitope which elicits a neutralizing antitoxin antibody and were protective in the mouse intracerebral challenge test. Recombinant pertussis vaccines based on PT analogs with detoxifying mutations in multiple subunits may thus represent the next generation of improved whooping cough vaccines.

Amino Acid Sequence↗

PubMed2006

EpiJen: a server for multistep T cell epitope prediction.

BACKGROUND: The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP) to the endoplasmic reticulum (ER), where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. RESULTS: To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM), EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. CONCLUSION: EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

Computer Simulation↗

PubMed2001

Sequence variation and disease in the wake of the draft human genome.

The sequencing phase of the human genome project will soon be over. In its wake, repertoires of sequence polymorphisms among the human population are being sampled and a battery of functional genomics projects, from gene and protein expression studies to whole proteome interaction experiments, are generating vast quantities of data. Now that the data, or the means to generate data, are available it is the application of this information in enhancing our understanding of biology that represents the next formidable challenge. Two prominent issues should be considered. First, existing data must be analysed using the best methods available. The prediction of enzymatic activity for bestrophin, whose gene is mutated in Best macular dystrophy, is described in this review. This is an example of the experimentally testable hypotheses that can result from such detailed and exhaustive analyses. Secondly, the torrents of data from high-throughput studies will need to be made more accessible to all using web-based resources that integrate and digest complementary data types. The internet sites that showcase the human genome sequence are blazing a new trail. Ultimately, the success of genome sequencing and functional genomics will be measured not by the quantity and accuracy of raw data generated, but how rapidly they can be harnessed to span the divide between genotype and phenotype.

Alleles↗

PubMed2026

Programmable enzymes for targeted gene insertion.

Genome editing technologies have advanced from nuclease-based reagents that generate programmed DNA double-strand breaks, which can cause deleterious effects, to next-generation reagents that perform controlled DNA modification through double-strand break-independent mechanisms, such as base editing and prime editing. Although these approaches enable precise small-scale sequence changes, methods for programmable insertion of large DNA cargos have been limited. The ability to write entire genes or large regions into the genome could transform the treatment of genetically heterogeneous disorders, for which numerous pathogenic variants underlie a common disease and mutation-specific editing strategies are impractical. Recent advances in computational genome mining have accelerated the discovery of naturally occurring enzymes with novel biochemical and functional properties, including recombinases and transposases capable of large-scale modifications. Moreover, directed evolution, rational engineering and expanded homologue discovery are enabling the repurposing and optimization of these systems for genome engineering. Here we review recent technology development efforts that harness diverse enzymes for kilobase-scale genome engineering, with a particular focus on CRISPR-associated transposase systems.

Journal Article↗

PubMed1990

Ventricular tachycardia and fibrillation detection by a sequential hypothesis testing algorithm.

An algorithm for detecting ventricular fibrillation (VF) and ventricular tachycardia (VT) by the method of sequential hypothesis testing is presented. The algorithm first generates a binary sequence by comparing the signal to a threshold. The probability distribution of the time intervals of the binary sequence is obtained, and Wald's sequential hypothesis testing procedure is next employed to discriminate the arrhythmias. Sequential hypothesis testing of 85 cases resulted in identification of 1) 97.64% VF and 97.65% VT episodes after 5 s, and 2) 100% identification of both VF and VT after 7 s. The desired false positive and false negative error probabilities can be preprogrammed into the algorithm. An important feature of the sequential method is that extra time for detection can be traded off for improved accuracy, and vice versa.

Algorithms↗

PubMed2003

Trans allele methylation and paramutation-like effects in mice.

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele. At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele. Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants.

Alleles↗

PubMed1999

Synthetic peptides identify the minimal substrate requirements of tubulin polyglutamylase in side chain elongation.

The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free alpha-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per alphabeta-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the isopeptide bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.

Amino Acid Sequence↗

PubMed1996

Establishment and characterization of transgenic mice expressing human platelet glycoprotein Ib alpha.

The platelet glycoprotein (GP) Ib/IX/V is a hetero-oligomeric receptor complex for von Willebrand factor (vWF) and mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of alpha and beta chain of GP Ib, GP IX, AND and GP V. To establish transgenic mice carrying human GP Ib alpha, we injected into mouse zygotes a 6 kb DNA fragment containing human GP Ib alpha gene that included entire coding sequence and putative promoter region. One hundred and thirteen offsprings were screened, and only one was found to express human GP Ib alpha protein and has passed the human GP Ib alpha gene as well as the expression of the gene to next generation. The expression of human GP Ib alpha in transgenic mice was limited to platelets and megakaryocytes. Glycocalicin, a proteolytic fragment of human GP Ib alpha found in normal human plasma, was not detected in transgenic mouse plasma. Human vWF in the presence of ristocetin supported agglutination of transgenic mouse platelets, but not of control mouse platelets.

Animals↗

PubMed2000

Biology of lumbar spine fusion and use of bone graft substitutes: present, future, and next generation.

Posterolateral lumbar spine arthrodesis is a commonly performed procedure, yet the biology of healing is poorly understood. Nonunion, or failure to achieve a solid bony fusion, occurs in up to 40% of patients. We first developed and validated a rabbit model to characterize the healing process by measuring macroscopic parameters, microscopic parameters, and gene expression. We found that presently available osteoconductive and weakly osteoinductive materials were insufficient to replace autografts, but could in some cases serve as bone graft extenders. In contrast, two osteoinductive growth factors currently in development could replace autograft in non-human primates and in humans, but may be limited by the high dose required, carrier variability, and high cost. We identified, cloned, and sequenced a novel complementary DNA (cDNA) encoding for an intracellular protein LMP-1, which is expressed during the first few hours of osteoblast differentiation. LMP-1 expression is able to induce many BMPs, their receptors, and other bone growth factors. Local implantation of bone marrow cells transfected with LMP-1 cDNA induced spine fusion in 100% of sites tested; no bone formed at the control sites without LMP-1. This strategy of local gene therapy may provide a basis for the next generation of bone graft substitutes.

Adaptor Proteins, Signal Transducing↗

PubMed1998

X-linked recessive bulbospinal neuronopathy: clinical and molecular studies in a Taiwanese family.

We describe clinical, biochemical, and molecular studies on a Taiwanese family with X-linked recessive bulbospinal neuronopathy. There were three probands and five female carriers among the 23 members examined. The clinical manifestations included progressive muscle weakness, diffuse fasciculation, postural tremor, muscle cramps, dysarthria, dysphagia, diabetes, and gynecomastia. Phenotypic expression varied among the affected subjects. Two carriers also had postural tremor and perioral fasciculation. Endocrine tests were normal except for a mild increase in serum testosterone and/or growth hormone in one patient and one carrier. Type IV hyperlipoproteinemia was observed in two patients, one carrier, and one healthy offspring. Molecular genetic studies confirmed elongation of the CAG triplet repeat in exon 1 of the gene for the androgen receptor. Sequence analysis showed that there were 42 to 43 CAG repeats in the three probands and 42 to 45 in the five carriers. The mutant allele had a tendency to increase by one or two repeats from one generation to the next. The length of CAG repeats at which the mutant allele became unstable was shorter in our family than in previous reports. The normal allele was also unstable and had a tendency to shrink by one of five repeats during transmission. These findings suggest that the number of CAG triplet repeats is variable in both the mutant and normal alleles.

Adolescent↗