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Results for “second generation sequencing”

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At least 487 records · Page 27Linked to original sources

PubMed1996

Hepatitis C virus genotype in anti-HCV-positive hemodialysed patients.

We investigated the influence of hepatitis C virus (HCV) genotypes on the clinical course of HCV infection in a haemodialysis population. In June 1991, a 4 year prospective follow-up programme was implemented in 184 consecutive haemodialysis patients. Alanine aminotransferase (ALT) and gamma glutamine transferase (GGT) were performed every 2 months. When HCV antibody (Ab) (by second-generation ELISA) was positive, it was confirmed by RIBA 2 and HCV RNA amplification by PCR. The pattern of nucleotide sequence variability in the 5' non-coding region was categorized according to Simmonds' genotype classification. Risk factors including blood transfusions were evaluated. The levels of hepatic enzymes in HCV Ab-positive patients were retrospectively studied over a mean period of 11.8 years. ALT and GGT levels were assigned a score for every year of infection (0 = normal, 1 = fluctuating 2 = high levels). Fifty-two patients were HCV Ab reactive (30.4%), eight were RIBA undetermined and 44 were RIBA positive; 40 of these were HCV RNA positive (91%). Twelve patients were HCV RNA negative, suggesting that they had recovered from the infection. Four genotypes were identified: 1b [26 patients (65%)], 1a (one patient), 2 [12 patients (30%)] and 3 (one patient). The genotype distribution was not different from that found in patients with chronic hepatitis C and normal renal function of the same geographical area. Genotype 1b accounted for 75% of the cases before 1985 and an equal prevalence of the two major genotypes was observed after 1985. Patients infected with HCV subtype 1 had normal mean ALT levels, but higher levels in the follow-up period (28 +/- 15.6 IU/l) and higher ALT and GGT personal scores in the retrospective study. Genotype 1 patients had higher mean ALT levels after 6 months. HCV RNA-negative patients had lower ALT levels after 24 months. RIBA pattern could differentiate the patients. Patients with genotype 1 received a higher number of transfusions, while only 50% of HCV RNA-negative patients had been transfused. Our data suggest a worse course of HCV infection in haemodialysis patients infected with HCV subtype 1, but the severity of HCV infection can only be assessed by histology. Transaminases are only loosely correlated with severity.

Antibodies, Viral↗

PubMed1982

A structural approach to the single-parent family.

Although the single-parent family is becoming a predominant family structure, there is a surprising scarcity of articles discussing its typical problems or detailing specific therapeutic approaches. This paper attempts, first, to list some core problems encountered by all single parents and, second, to present a sequence of therapeutic approaches geared toward dealing with these problems. Structural theory, with its emphasis on subsystems, generational boundaries, and organizational patterns, provides a framework for viewing the built-in vulnerabilities of the single-parent family unit. Minuchin's (3) emphasis on generational boundaries and the problem of enmeshment underlies the theoretical and therapeutic approach presented here. I have drawn heavily on Haley's (2) idea that the therapist must be an authority, a problem-solver, and a teacher. Finally, the emphasis on creating new organizational patterns is most thoroughly explored in Aponte's (1) discussions of "underorganized families" (many of whom are single-parent families).

Adolescent↗

PubMed2005

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency.

BACKGROUND: Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations. METHODS: Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 microg/ml but remained sensitive to rifabutin (MIC<0.8 microg/ml). This mutant was subjected to a second round of selection with rifabutin. RESULTS: All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain. CONCLUSION: First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.

Bacterial Proteins↗

PubMed1998

Molecular characterization of the operon comprising the spoIV gene of Bacillus megaterium DSM319 and generation of a deletion mutant.

According to sequence analysis, the spoIV-locus of Bacillus megaterium DSM 319 is 1,185 bp long; it is the second gene of a sporulation operon, which altogether contains three open reading frames. The ORF preceding spoIV encodes a putative polypeptide with 94 amino acids; the 3rd ORF of the operon has 972 bp corresponding to 324 amino acids. The operon is flanked on both sides by palindromic sequences, probably representing Rho-independent terminators. A primer extension analysis revealed that mRNA synthesis starts immediately downstream of a promoter, which is similar to the consensus sequence of Bacillus subtilis sigma(E) dependent promoters. Both the -35 and the -10 region are within the terminator region of the preceding operon. Gene knockout experiments and reporter gene assays with a newly developed system based on the heterologous Paenibacillus macerans glucanase gene (bgl) confirmed sigma(E)-dependent transcription. Two open reading frames of a further upstream operon were also identified. Northern analysis revealed that transcription of these ORFs comes about in late sporulation phases. The genetic organization of the spoIV comprising operon and adjacent loci clearly resembles that of the B. subtilis yqfa-phoH gene cluster. Thus our findings are of general significance for endospore-forming bacteria.

Journal Article↗

PubMed1997

First- and second-generation antisense oligonucleotide inhibitors targeted against human c-raf kinase.

Following extensive screening of more than 50 antisense-designed phosphorothioate oligodeoxynucleotides targeted to human c-raf mRNA, one oligodeoxynucleotide (ISIS 5132/CGP 69846A) was identified as being the most potent inhibitor of c-raf gene expression both in vitro and in vivo. ISIS 5132 is a highly sequence-specific and target-specific inhibitor of c-raf mRNA and protein levels. c-raf inhibition results in dramatic alteration of downstream signalling events within the MAP kinase signalling pathway. Moreover, this oligodeoxynucleotide displays potent antitumour activity against a broad spectrum of tumour types in mouse models and has progressed to Phase I clinical trails. In an effort to identify potential back-up compounds to ISIS 5132, a variety of second-generation 2' sugar modifications have been evaluated for activity against c-raf in cell culture. We have identified a number of second-generation oligonucleotides with improved biophysical characteristics that result in enhanced activity against c-raf in cell culture. Activity enhancement was most pronounced for 2'-O-methoxyethyl-modified oligonucleotides and this modification also resulted in significantly improved antitumour activity in vivo.

Animals↗

PubMed1989

Structural requirements for membrane assembly of proteins spanning the membrane several times.

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.

Cell Membrane↗

PubMed1989

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome.

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.

Amino Acid Sequence↗

PubMed1996

Control of remembered reaching sequences in monkey. II. Storage and preparation before movement in motor and premotor cortex.

Single-neuron responses in motor and premotor cortex were recorded during a movement-sequence delay task. On each trial the monkey viewed a randomly selected sequence of target lights arrayed in two-dimensional space, remembered the sequence during a delay period, and then generated a coordinated sequence of movements to the remembered targets. Of 307 neurons studied, 25% were tuned specifically for either the first or the second target, but not both. In particular, for neurons tuned during both target presentations, tuned activity related to a particular first target direction were maintained during the presentation of a second target in a different direction. During the delay period, 32% of the neurons were tuned for upcoming movement in a single direction. These delay period responses often reflected activity patterns that first developed during target presentations and may therefore act to maintain target period information during the delay. Neurons with tuned activity during both the delay and movement periods exhibited two patterns: the first exhibited tuned responses during the delay that were correlated with the tuning of first-movement responses, while the second pattern showed delay-period tuning that was better correlated with tuned responses during second movements. This indicates that, before movement, distinct neural populations are correlated with specific movements in a sequence. About half the neurons studied were not directionally tuned during the initiation, target, or delay periods, but did show systematic changes in activity during task performance. Some (34%) were exclusively tuned during movement and appear to be involved in the direct control of movement. Others (17%) showed changes in firing rate from period to period within a trial but showed no directional preference for a particular direction of movement. Population analyses of tuned activity during the target and delay periods indicated that accurate directional information about both first and second movements was available in the neuronal ensemble well before reaching began. These results extend the idea that both motor and premotor cortex play a role in reaching behavior other than the direct control of muscles. While some early neural responses resembled muscle activation patterns involved in maintaining fixed postures before movement, others probably relate to the sensory-to-motor transformations, information storage in short-term memory, and movement preparation required to generate accurate reaching to remembered locations in space.

Animals↗

PubMed1994

Mapping of 386 kb of genomic DNA in the human dystrophin-encoding gene (DYS) using an ordered phage lambda sublibrary of a YAC clone containing the DYS region.

An integrated restriction map for HindIII and EcoRI has been constructed for 386 kb of the human dystrophin-encoding gene by partial digest mapping of 35 overlapping lambda EMBL3cosW phage clones derived from a yeast artificial chromosome containing this region. Map construction was simplified in two ways. Firstly, the sequence arrangement of lambda EMBL3cosW is such that only map data from cloned inserts are generated using partial digests of lambda phage DNA asymmetrically labelled at the left cos end with a complementary 32P-labelled oligodeoxyribonucleotide. Secondly, the degree of partial digestion was standardised for each restriction enzyme by using ultraviolet light-induced formation of thymine dimers in the recognition sequence to partially block the cleavage reaction. The map provides the basis for work on the analysis of chromosomal rearrangements in this region which give rise to Duchenne muscular dystrophy, and for studies of chromosome structure and function.

Animals↗

PubMed1998

HLA class II polymorphism in the Ticuna of Brazil: evolutionary implications of the DRB1*0807 allele.

The alleles at the HLA class II loci HLA-DRB1, DQA1, DQB1 and DPB1 were determined for 49 individuals of the Ticuna, a Native South American population living in Brazil, using PCR/SSO probe hybridization and DNA sequencing. A newly described DRB1*08 variant, DRB1*0807, which has previously been reported only in native Colombians and contemporary Brazilians of African and Caucasian descent, was identified in the Ticuna at a high frequency (f=0.225). Because *0807 has been observed only in South American populations, we propose that it was generated from a parental *0802 allele recently, after the isolation of various Native South American populations, and infer that the DRB1*0807 allele was generated by a C to T change at codon 57 (Asp-->Val, GAT-->GTT) from the ancestral *0802. This inference is supported by the sequence of a complex VNTR in the second intron of the DRB1 gene. The DPB1 alleles *0401, *0402 and *1401 constituted 76% of the observed Ticuna DPB1 alleles (f=0.166, 0.427 and 0.166 respectively). In addition, the DPB1 allele *3501, which has been observed in a few other Native South American groups, was observed at a frequency of 0.053 and may have been generated from the putative ancestral allele *1401 allele in South America. The DRB1 and DPB1 allele frequencies for the Ticuna deviate from expected Hardy-Weinberg equilibrium proportions, while DQA1 and DQB1 allele frequencies do not. When this deviation, which involves an observed excess of DRB1*0807 heterozygotes, is considered with the high frequency of the DRB1*0807 and DPB1*1401 alleles, we infer that native South American populations may have been under selection pressure for increased allele diversity.

Africa↗

PubMed1986

Tissue contrast enhancement: image reconstruction algorithm and selection of TI in inversion recovery MRI.

It is clearly demonstrated that the proper application of the inversion recovery imaging pulse sequence is dependent on the method of image reconstruction and the selection of TI for optimum tissue contrast. There are two methods of 2DFT image reconstruction of IR sequence time-domain raw data. The first is a modulus-image reconstruction algorithm (contrast-obliterating option), and the second is a phase-correction routine for reconstructing "phase-sensitive" true IR-images. The second option generates proper "in-phase" images, retains proper scale of contrast, but can invert the algebraic sign of image-values under certain conditions. A series of "phase-sensitive" and "modulus" reconstructed brain images, obtained with conventional and optimized new IR pulse sequences, are shown to demonstrate these effects. They illustrate the considerable advantages gained, in practical clinical situations, if one generates "phase-sensitive" true IR-images from IR-sequence raw data at optimum TI for tissue contrast enhancement.

Algorithms↗

PubMed1992

Characterization of an endogenous RNA transcript with homology to the antisense strand of the human c-myc gene.

In addition to being regulated by a complex array of cis- and trans-acting factors, c-myc protooncogene expression may be modulated by antisense RNA transcripts. Our previous studies have determined that depletion of intracellular polyamines by alpha-difluoromethylornithine results in a marked decrease in the transcription of the human c-myc gene. Because of reports that antisense transcription occurs in the 5' and 3' regions of this gene, we used a genomic clone of the human c-myc gene to ascertain whether polyamine depletion might induce an antisense RNA transcript. These studies demonstrate that polyamine depletion of the human colon cancer cell line COLO 320 results in induction of an endogenous RNA transcript with high homology to the antisense strand of the second intervening sequence (PvuII-RsaI) of the c-myc gene. Furthermore, during such depletion, steady state levels of this transcript vary inversely to the sense direction c-myc RNA. RNase protection studies suggest that the antisense transcript may arise from a different gene locus than the c-myc gene. To further identify the origins of this RNA, a cDNA library was generated from size-selected RNA and screened with c-myc sequences. A 438-base pair cDNA was isolated with approximately 85% homology, to a 285-base region in the second intron of the c-myc gene. Computer homology analysis further reveals that a 120-base region within this cDNA also has approximately 85% homology to the antisense strands of a number of genes, including the growth-related genes, N-myc, p53, and thymidine kinase. These studies provide the initial characterization of an endogenous antisense RNA transcript which could influence cell growth by modulating the expression of c-myc and other genes.

Base Sequence↗

PubMed1991

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola.

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.

Amino Acid Sequence↗

PubMed1997

Better 1D predictions by experts with machines.

Accuracy of predicting protein secondary structure and solvent accessibility has been improved significantly by using evolutionary information contained in multiple sequence alignments. For the second Asilomar meeting, predictions were made automatically for all targets using the publicly available prediction service PredictProtein. Additionally, a semiautomatic procedure for generating more informative alignments was used in combination with the PHD prediction methods. Results confirmed the estimates for prediction accuracy. Furthermore, the more informative alignments yielded better predictions. The fairly accurate predictions of 1D structure were successfully used by various groups for the Asilomar meeting as first step toward predicting higher dimensions of protein structure.

Expert Systems↗

PubMed1997

On the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides.

BACKGROUND: Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (Im) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G.C and A.T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G.C base pairs. RESULTS: Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G.C and A.T base pairs in the central position. We observed that a G.C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A.T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im. CONCLUSIONS: An Im/Im pairing is energetically disfavored for the recognition of both A.T and G.C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.

Base Composition↗

PubMed2006

The second half of the fourth period of tropomyosin is a key region for Ca(2+)-dependent regulation of striated muscle thin filaments.

Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.

Actin Cytoskeleton↗

PubMed2003

Prediction of beta-turns in proteins from multiple alignment using neural network.

A neural network-based method has been developed for the prediction of beta-turns in proteins by using multiple sequence alignment. Two feed-forward back-propagation networks with a single hidden layer are used where the first-sequence structure network is trained with the multiple sequence alignment in the form of PSI-BLAST-generated position-specific scoring matrices. The initial predictions from the first network and PSIPRED-predicted secondary structure are used as input to the second structure-structure network to refine the predictions obtained from the first net. A significant improvement in prediction accuracy has been achieved by using evolutionary information contained in the multiple sequence alignment. The final network yields an overall prediction accuracy of 75.5% when tested by sevenfold cross-validation on a set of 426 nonhomologous protein chains. The corresponding Q(pred), Q(obs), and Matthews correlation coefficient values are 49.8%, 72.3%, and 0.43, respectively, and are the best among all the previously published beta-turn prediction methods. The Web server BetaTPred2 (http://www.imtech.res.in/raghava/betatpred2/) has been developed based on this approach.

Algorithms↗

PubMed1995

Detection of related positive-strand RNA virus genomes by reverse transcription/polymerase chain reaction using degenerate primers for common replicase sequences.

A set of degenerate sense and antisense primers were designed on the basis of short segments with identical amino acids in the predicted ORF 1b replicase proteins of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus, strain Lelystad virus (PRRSV-LV), which are members of a new group of positive-strand RNA viruses. Reverse transcription/polymerase chain reaction amplification using this set of degenerate primers yielded products of the expected size from the genomes of all three viruses. It also yielded a product of appropriate size from the genome of another strain of PRRSV (VR2332), the ORF 1b sequence of which is unknown, but the 3' end of the genome of which differs from that of the PRRSV-LV genome by about 50%. No products were generated from the genome of simian hemorrhagic fever virus (SHFV), another member of this virus group. However, an appropriate product was generated with a second set of degenerate primers which was designed from the same ORF 1b segments of LDV, EAV and PRRSV-LV as the first set but on the basis of human codon preferences. Sequence analysis showed that the amplified SHFV ORF 1b segment exhibited about 50% nucleotide identity with the corresponding segments of ORF 1b of LDV, EAV and PRRSV. The results show that these and other degenerate primer sets might be useful for the search of related viruses in other mammalian species.

Amino Acid Sequence↗