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Results for “second generation sequencing”

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PubMed1993

Linkage analysis by two-dimensional DNA typing.

In two-dimensional (2-D) DNA typing, genomic DNA fragments are separated, first according to size by electrophoresis in a neutral polyacrylamide gel and second according to sequence by denaturing gradient gel electrophoresis, followed by hybridization analysis using micro- and minisatellite core probes. The 2-D DNA typing method generates a large amount of information on polymorphic loci per gel. Here we demonstrate the potential usefulness of 2-D DNA typing in an empirical linkage study on the red factor in cattle, and we show an example of the 2-D DNA typing analysis of a human pedigree. The power efficiency of 2-D DNA typing in general is compared with that of single-locus typing by simulation. The results indicate that, although 2-D DNA typing is very efficient in generating data on polymorphic loci, its power to detect linkage is lower than single-locus typing, because it is not obvious whether a spot represents the presence of one or two alleles. It is possible to compensate for this lower informativeness by increasing the sample size. Genome scanning by 2-D DNA typing has the potential to be more efficient than current genotyping methods in scoring polymorphic loci. Hence, it could become a method of choice in mapping genetic traits in humans and animals.

Alleles↗

PubMed1994

Characterization of partial gene deletions in type III von Willebrand disease with alloantibody inhibitors.

von Willebrand factor gene deletions were characterized in four patients with severe type III von Wilebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an approximately 56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

Base Sequence↗

PubMed1989

Porcine tumor necrosis factor alpha: cloning with the polymerase chain reaction and determination of the nucleotide sequence.

We have cloned the gene for the porcine tumor necrosis factor alpha (TNF-alpha) utilizing the polymerase chain reaction (PCR). Total RNA from stimulated monocytes was used to generate TNF-alpha-specific single-stranded cDNA, with reverse transcriptase and a conserved consensus primer, starting at the TNF-alpha translation stop codon. After adding a second conserved consensus primer to the propeptide region, we amplified the cDNA between the two primers. The isolated fragment was cloned and sequenced. Comparison of the nucleotide sequence indicated an 85% sequence similarity to the human TNF-alpha gene. Eighteen amino acids of the deduced mature peptide sequence of porcine TNF-alpha were different from those in the sequence of man. The technique used in this work allows rapid cloning of specific genes from total RNA, and, additionally, screens for full-length transcripts during the amplification procedure.

Amino Acid Sequence↗

PubMed2004

Prediction of alpha-turns in proteins using PSI-BLAST profiles and secondary structure information.

In this paper a systematic attempt has been made to develop a better method for predicting alpha-turns in proteins. Most of the commonly used approaches in the field of protein structure prediction have been tried in this study, which includes statistical approach "Sequence Coupled Model" and machine learning approaches; i) artificial neural network (ANN); ii) Weka (Waikato Environment for Knowledge Analysis) Classifiers and iii) Parallel Exemplar Based Learning (PEBLS). We have also used multiple sequence alignment obtained from PSIBLAST and secondary structure information predicted by PSIPRED. The training and testing of all methods has been performed on a data set of 193 non-homologous protein X-ray structures using five-fold cross-validation. It has been observed that ANN with multiple sequence alignment and predicted secondary structure information outperforms other methods. Based on our observations we have developed an ANN-based method for predicting alpha-turns in proteins. The main components of the method are two feed-forward back-propagation networks with a single hidden layer. 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 obtained 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. The final network yields an overall prediction accuracy of 78.0% and MCC of 0.16. A web server AlphaPred (http://www.imtech.res.in/raghava/alphapred/) has been developed based on this approach.

Amino Acid Sequence↗

PubMed1983

Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase.

M13 recombinant DNA clones containing a 350-base sequence derived from the EcoRI fragment of two tandemly linked Rous-associated virus 2 (RAV-2) long terminal repeat (LTR) sequences have been used to map reverse transcriptase-associated endonuclease (RT-endonuclease) cleavage sites by primer extension studies. Under appropriate conditions, the alpha beta form of RT-endonuclease (composed of both the alpha and beta subunits) purified from Avian sarcoma virus (Pr-C and B-77 strains) introduces a specific break in the inverted complementary repeat sequence found at the junction of the LTRs. The cleavage sites occur in the same nucleotide sequence in (-) and (+) DNA strands; together they have the potential of generating a 6-base-pair staggered overlap that spans the junction. This supports the notion that the enzyme is involved in viral DNA integration. Other RT-endonuclease sites were analyzed. A second site, which occurs in the lac region of the M13 vector DNA upstream from the unique EcoRI cloning site, bears no apparent sequence homology to the site at the junction of the LTRs. However, it also lies within an inverted complementary repeat and, as is the case for the site in the LTR, the break occurs to the 5' side of the axis of symmetry. Cleavage at this second site is suppressed when the vector contains the RAV-2 LTR insert. Thus, the viral LTR appears to exert a cis effect that can influence a region over 300 base pairs away.

Avian Sarcoma Viruses↗

PubMed1993

Eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae. Cloning, characterization, and expression of the gene encoding the 45,346-Da protein.

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes hydrolysis of GTP bound to a 40 S ribosomal initiation complex with the subsequent joining of a 60 S ribosomal subunit to form an 80 S initiation complex. The yeast gene that encodes eIF-5, designated TIF5, has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. TIF5 is a single-copy gene that maps on yeast chromosome XVI and is essential for cell viability. The gene contains an intron-free open reading frame that encodes a protein of calculated M(r) 45,346 in close agreement with the apparent molecular weight of eIF-5 isolated from yeast cells. Sequence analysis of the gene reveals several interesting features. First, the presence of two in-frame translational start sites located 51 base pairs apart suggests the possibility that two proteins, differing by an amino-terminal extension of 17 amino acids, could be generated from the TIF5 gene via differential translational starts. This would explain the presence, in yeast cell lysates, of two forms of eIF-5 differing in molecular weight by about 2,000. Second, the predicted amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.

Amino Acid Sequence↗

PubMed2000

Cloning and sequencing of partial genes of hepatitis C virus genome in patients with acute hepatitis C.

OBJECTIVE: To explore the etiological role of HCV in patients with acute hepatitis. METHODS: The prevalence of HCV infection in 89 patients with acute hepatitis was investigated by analysis of HCV RNA and HCV second generation antibody. HCV RNAs extracted from the sera of 5 patients with NANB acute hepatitis, which were positive for HCV RNA, were converted to cDNA by reverse transcription with random primer and genotyping by PCR with type-specific primers. The partial genes of HCV genome were amplified. The PCR products were expressed in E. coli with p-GEM-T vector, and their nucleotide sequences were determined by dideoxynucleotide chain-termination method. RESULTS: The incidence of hepatitis virus infection was 47. 2% in HAV, 28.1% in HBV and 15.7% in HCV, respectively. The incidence of HAV and HBV coinfection was 14.6% and the rate of non-A, non-B and non-C hepatitis was 9% in all patients. The genotype of HCV-RNA positive patients was 85.8% in HCV-II, 7.1% in HCV-III and 7. 1% in combining HCV-II/III, respectively. The partial sequence of HCV genome in 5 patients with non-A and non-B acute hepatitis was amplified and the fragment was 424 bp in accordance with original design. The homology of the sequences was 98.1%-99.5% in nucleotide acid and 97.6%-99.2% in amino acid among five isolates. The average homology was 91.9% or 94.3%-95.6% for nucleotide sequences between HCV-I or HCV-II and the 5 isolates, and 92.3%-95.8% for amino acid sequences between the 5 isolates and HCV-I or HCV-II, respectively. CONCLUSION: HCV infection is one of the main hepatitis viruses in patients with acute hepatitis, in which the HCV-II genotype is dominant and should be paid attention to it.

Acute Disease↗

PubMed1991

Mapping using unique sequences.

Theoretical predictions are given for the progress expected, when mapping DNA by identifying clones containing specific unique sequences. Progress is measured in three ways; however, all results depend on (dimensionless counterparts of) the number of clones and the number of unique sequences used. Furthermore, the effects of clone length dispersion are included in the theoretical predictions. Both the clones in the library and the unique sequences are assumed to be generated randomly, with uniform probability of originating at any base in the region to be mapped. The first measure of progress is the expected length fraction of the region to be mapped covered by at least one clone, when clones containing at least one unique sequence are included in the map. The second measure of progress is the expected length fraction of the region to be mapped in "covered intervals", an interval being the region between adjacent unique sequences. Alternative definitions for clones covering an interval are analyzed. The third measure of progress is the expected number of clone islands generated; an island covers successive intervals. Finally, using these measures of progress, we compare the efficiency of this new mapping strategy with conventional clone mapping strategies.

Chromosome Mapping↗

PubMed1997

Multiple transcripts of the murine immunoglobulin epsilon membrane locus are generated by alternative splicing and differential usage of two polyadenylation sites.

The human C epsilon gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed epsilon mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary epsilon chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in the present study by RACE-PCR analysis. The first poly(A) site is located 743 nt downstream from the beginning of the second membrane exon (M2) and contains the same non-consensus AGTAAA signal sequence as the single poly(A) site of the human membrane transcripts. The second poly(A) site is located almost 500nt further downstream and is characterized by an AAGAAA hexamer. This poly(A) site contains a (G+T) rich element downstream to the site of cleavage and polyadenylation and is preferentially utilized by the membrane epsilon transcripts. Additional diversity of epsilon transcripts is generated by alternative splicing between the last constant region exon (CH4) and the two membrane exons (M1 and M2). The alternatively spliced transcripts include two variants that skip the first membrane exon and encode epsilon heavy chains that lack the transmembrane domain. The third variant is generated by splicing to an internal site in M2 and codes for a membrane isoform that is 10 amino acids shorter in the cytoplasmic domain than the classical membrane IgE. Although little amino-acid sequence homology exists between the murine epsilon chain isoforms and their human counterparts, the pattern of splicing is rather conserved between the two species.

Alternative Splicing↗

PubMedDate not supplied

Generation of DM-20 splice site in myelin proteolipid protein gene: a hypothesis based on analysis of the amphibian protein.

The DM-20 isoform of proteolipid protein (PLP) of central nervous system myelin is undetectable in amphibia, but readily detectable in reptiles, birds and mammals. To explain the phylogenetic origin of DM-20, it has been proposed that a donor-acceptor RNA splice site was generated by the introduction of a single base change in the codon encoding amino acid 116 of PLP. We tested this hypothesis by isolating and sequencing peptides from bullfrog PLP. One of the peptides corresponded to mammalian residues 110-122, which contain the N-terminal boundary region for the domain excluded from DM-20 in higher vertebrates. We found that the Thr115-Val116 sequence is conserved between frog and mammal. Therefore, we propose that a single base change in the third position of the codon for Thr115 would account for the appearance of the new donor-acceptor splice site. Three amino acids elsewhere in this thirteen-residue peptide were found to differ between bullfrog and mammal, which could account for the previously reported weak recognition of amphibian PLP by an antiserum specific for peptide 109-128. A second peptide from bullfrog PLP had a sequence identical to that of residues 45-52 in mammalian PLP. Our findings explain how a specific change in the myelin PLP gene could generate a new form of the PLP protein.

Amino Acid Sequence↗

PubMed1989

Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae.

Alleles↗

PubMed1986

Localization of action of the IS50-encoded transposase protein.

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp+), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp- IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules.

Alleles↗

PubMed2007

Soil fungal communities underneath willow canopies on a primary successional glacier forefront: rDNA sequence results can be affected by primer selection and chimeric data.

Soil fungal communities underneath willow canopies that had established on the forefront of a receding glacier were analyzed by cloning the polymerase chain reaction (PCR)-amplified partial small subunit (18S) of the ribosomal (rRNA) genes. Congruence between two sets of fungus-specific primers targeting the same gene region was analyzed by comparisons of inferred neighbor-joining topologies. The importance of chimeric sequences was evaluated by Chimera Check (Ribosomal Database Project) and by data reanalyses after omission of potentially chimeric regions at the 5'- and 3'-ends of the cloned amplicons. Diverse communities of fungi representing Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota were detected. Ectomycorrhizal fungi comprised a major component in the early plant communities in primary successional ecosystems, as both primer sets frequently detected basidiomycetes (Russulaceae and Thelephoraceae) forming mycorrhizal symbioses. Various ascomycetes (Ophiostomatales, Pezizales, and Sordariales) of uncertain function dominated the clone libraries amplified from the willow canopy soil with one set of primers, whereas the clone libraries of the amplicons generated with the second primer set were dominated by basidiomycetes. Accordingly, primer bias is an important factor in fungal community analyses using DNA extracted from environmental samples. A large proportion (>30%) of the cloned sequences were concluded to be chimeric based on their changing positions in inferred phylogenies after omission of possibly chimeric data. Many chimeric sequences were positioned basal to existing classes of fungi, suggesting that PCR artifacts may cause frequent discovery of new, higher level taxa (order, class) in direct PCR analyses. Longer extension times during the PCR amplification and a smaller number of PCR cycles are necessary precautions to allow collection of reliable environmental sequence data.

Artifacts↗

PubMed1996

Methodology citations and the quality of randomized controlled trials in obstetrics and gynecology.

OBJECTIVES: Randomized controlled trials offer the best chance for valid treatment comparisons, yet most trials are of poor quality. This may reflect a lack of awareness of the requirements for conducting and reporting this type of research. If so, then citation of methodology references might indicate knowledge of how to conduct these studies and vice versa. Our study tests the hypothesis that the methodologic quality of published trials is related to citation of methodology references. STUDY DESIGN: We performed a hand search of the American Journal of Obstetrics and Gynecology, the British Journal of Obstetrics and Gynaecology, the Journal of Obstetrics and Gynaecology, and Obstetrics and Gynecology to identify all randomized controlled trials published in 1990 and 1991 (N = 206). We reviewed the reference lists of all reports of randomized controlled trials and evaluated the adequacy of randomization methods by accepted criteria. RESULTS: Most reports (81.6%) cited no methodology text or article. Although lack of any methodology reference was not significantly related to failure to report an adequate random method of sequence generation, this was highly related (p < 0.001) to failure to report adequate allocation concealment. Scanning the reference list of reports took a mean of 16 seconds and identified most poorly done trials. CONCLUSIONS: Investigators who conduct randomized controlled trials should be thoroughly familiar with this type of research or should get expert help. Poorly done trials are wasteful and often misleading.

Bibliographies as Topic↗

PubMed2003

The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by nonomuraea species.

The glycopeptide A40926 is the precursor of dalbavancin, a second-generation glycopeptide currently under clinical development. The dbv gene cluster, devoted to A40926 biosynthesis, was isolated and characterized from the actinomycete Nonomuraea species ATCC39727. From sequence analysis, 37 open reading frames (ORFs) participate in A40926 biosynthesis, regulation, resistance, and export. Of these, 27 ORFs find a match in at least one of the previously characterized glycopeptide gene clusters, while 10 ORFs are, so far, unique to the dbv cluster. Putative genes could be identified responsible for some of the tailoring steps (attachment of glucosamine, sugar oxidation, and mannosylation) expected during A40926 biosynthesis. After constructing a Nonomuraea mutant by deleting dbv ORFs 8 to 10, the novel compound dechloromannosyl-A40926 aglycone was isolated.

Actinomycetales↗

PubMed2003

Total synthesis of (+)-spongistatin 1. An effective second-generation construction of an advanced EF Wittig salt, fragment union, and final elaboration.

A stereocontrolled, total synthesis of (+)-spongistatin 1 (1) has been achieved. Union of a second-generation EF Wittig salt (+)-3 with the advanced ABCD aldehyde (-)-4, followed by regioselective macrolactonization and global deprotection afforded (+)-spongistatin 1 (1). The longest linear sequence, 29 steps, proceeded in 0.5% overall yield.

Aldehydes↗

PubMed2000

Opposing roles of Elk-1 and its brain-specific isoform, short Elk-1, in nerve growth factor-induced PC12 differentiation.

The ternary complex factor Elk-1, a major nuclear target of extracellular signal-regulated kinases, is a strong transactivator of serum-responsive element (SRE) driven gene expression. We report here that mature brain neurons and nerve growth factor (NGF)-differentiated PC12 cells also express a second, smaller isoform of Elk-1, short Elk-1 (sElk-1). sElk-1 arises from an internal translation start site in the Elk-1 sequence, which generates a protein lacking the first 54 amino acids of the DNA-binding domain. This deletion severely compromises the ability of sElk-1 to form complexes with serum response factor on the SRE in vitro and to activate SRE reporter genes in the presence of activated Ras. Instead, sElk, but not a mutant that cannot be phosphorylated, inhibits transactivation driven by Elk-1. More pertinent to the neuronal-specific expression of sElk-1, we show it plays an opposite role to Elk-1 in potentiating NGF-driven PC12 neuronal differentiation. Overexpression of sElk-1 but not Elk-1 increases neurite extension, an effect critically linked to its phosphorylation. Interestingly, in the presence of sElk-1, Elk-1 loses its strictly nuclear localization to resemble the nuclear/cytoplasm pattern observed in the mature brain. This is blocked by mutating a normally cryptic nuclear export signal in Elk-1. These data provide new insights into molecular events underlying neuronal differentiation of PC12 cells mediated by the NGF-ERK signaling cascade.

Active Transport, Cell Nucleus↗

PubMed2002

Sequential muscle activity and its functional role in the upper extremity and trunk during overarm throwing.

The proximal-to-distal segmental sequence has been identified in many sports activities, including baseball pitching and ball kicking. However, proximal-to-distal sequential muscle activity has not been identified. The aims of this study were to establish whether sequential muscle activity does occur and, if it does, to determine its functional role. We recorded surface electromyograms (EMGs) for 17 muscles from the upper extremity and abdomen during overarm throwing and detected the onset and peak times as indices of muscle activity. The following electromyographic properties were commonly identified in the participants. First, sequential muscle activity was observed from the scapular protractors to the shoulder horizontal flexors and from the shoulder horizontal flexors to the elbow extensor, but not from the elbow extensor to the wrist flexor or forearm pronator. Secondly, the external oblique contralateral to the throwing arm became activated before the ipsilateral external oblique. This sequence is considered to be very effective for the generation of high force and energy in the trunk. Thirdly, the ipsilateral external oblique began its activity almost at foot strike. Finally, the main activity of the rectus abdominis appeared just before the point of release.

Abdominal Muscles↗