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At least 775 records · Page 43Linked to original sources
PubMed1994

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking the hprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and including hprt. We used "bubble" PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream of hprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream of hprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay, hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.

Adult↗
PubMed2005

Draft versus finished sequence data for DNA and protein diagnostic signature development.

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(-3)-10(-5) (approximately 8x coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of approximately 1% (3x to 6x coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures.

Computational Biology↗
PubMed1998

Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility.

Fragile sites are reproducibly expressed and chemically induced decondensations on mitotic chromosomes observed under cytological conditions. They are classified both on the basis of the frequency with which they occur (rare and common) and in terms of the chemical agent used to induce expression in tissue culture cells. Aphidicolin-sensitive common fragile sites appear to be ubiquitous in humans and other mammals and have been considered as candidates of pathological importance. Recently DNA from FRA3B, the most highly expressed constitutive fragile site in the human genome, has been cloned although as yet the cause of the underlying fragility has not been identified. In this study we describe the isolation, using a direct cloning approach, of DNA from a region of the Chinese hamster genome associated with aphidicolin-inducible fragility. Cells of a human-hamster somatic cell hybrid were transfected with a pSV2HPRT vector while exposed to aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon. FISH analysis of stable transfectant clones revealed that the ingoing plasmid DNA had preferentially integrated into fragile site-containing chromosomal bands. Plasmid rescue was used to recover DNA sequences flanking one such integration site in the hamster genome. We demonstrate by FISH analysis of metaphase cells induced with aphidicolin that the rescued DNA is from a region of fragility on Chinese hamster chromosome 2, distal to the DHFR locus. Analysis of the DNA sequences flanking the integration site revealed the overall A+T content of the 3,725 bp region sequenced to be 63.3%, with a highly [A].[T]-rich 156 bp region (86.5%) almost adjacent to the integration site. Computational analyses have identified strong homologies to Saccharomyces cerevisiae autonomous replicating sequences (ARS), polypyrimidine tracts, scaffold attachment site consensus sequences and a 24 bp consensus sequence highly conserved in eukaryotic replication origins, all of which appear to cluster around the [A].[T]-rich sequences. This domain also possesses structural characteristics which are common to both prokaryotic and eukaryotic origins of replications, in particular an unusually straight conformation of low thermal stability flanked either side by highly bent DNA segments. Further isolation and characterisation of DNA sequences from common fragile sites will facilitate studies into the underlying nature of these enigmatic regions of the mammalian genome, leading to a greater understanding of chromatin structure.

Animals↗
PubMed1997

Isolation and genomic analysis of the rat polymeric immunoglobulin receptor gene terminal domain and transcriptional control region.

The polymeric immunoglobulin receptor (pIgR) transports IgA and IgM across secretory epithelial cells and is essential in external immunity maintenance. We report here the structural characterization of the single-copy rat gene distributed over 30 kb of chromosomal DNA and analysis of its transcriptional control region. RNA sequencing and genomic analysis show a 5' terminal region originates at a major (+1) and a minor site producing an unusual 124-bp nontranslated exon I separated from a small 96-bp initiator ATG coding exon II by a 7.5-kb intron. The pIgR 5' region comprises a structured promoter with abundant helix-loop-helix (bHLH) cis elements positioned within an equivalent internal -70, -290, -528, and three centered at -745. The three latter bHLH elements each occur within 30-bp repeats at -690 to -780. Transient expression assays show a 1.3-kb 5' region is sufficient to drive expression in rat primary hepatocyte monolayer cultures, transformed human hepatic (HepG2) cells, and a mammary epithelial tumor cell line MCF-7, but is inactive in the rodent fibroblast 3T3 cell line. A minimal transcriptional promoter domain was deduced from sequentially deleted vectors revealing a +40 to -922 sequence to be sufficient for full activity. Further deletions within this region yield incremental losses in cis activity, indicating that multiple subregions comprise an extended transcriptional control region.

Animals↗
PubMedDate not supplied

Faster algorithms for optimal multiple sequence alignment based on pairwise comparisons.

Multiple Sequence Alignment (MSA) is one of the most fundamental problems in computational molecular biology. The running time of the best known scheme for finding an optimal alignment, based on dynamic programming, increases exponentially with the number of input sequences. Hence, many heuristics were suggested for the problem. We consider a version of the MSA problem where the goal is to find an optimal alignment in which matches are restricted to positions in predefined matching segments. We present several techniques for making the dynamic programming algorithm more efficient, while still finding an optimal solution under these restrictions. We prove that it suffices to find an optimal alignment of the predefined sequence segments, rather than single letters, thereby reducing the input size and thus improving the running time. We also identify "shortcuts" that expedite the dynamic programming scheme. Empirical study shows that, taken together, these observations lead to an improved running time over the basic dynamic programming algorithm by 4 to 12 orders of magnitude, while still obtaining an optimal solution. Under the additional assumption that matches between segments are transitive, we further improve the running time for finding the optimal solution by restricting the search space of the dynamic programming algorithm.

Algorithms↗
PubMed2004

MOP-UP: an online tool for finding strain-specific primers or motifs in DNA or protein alignments.

MOP-UP is a web-based application that enables efficient searching of nucleic acid or amino-acid alignments for sequences or motifs that are unique to a subset of the members represented in the alignment. This has applications in the design of assays that aim to detect particular strains or species. Since molecular-based characterisation of microbes is becoming increasingly important, MOP-UP can aid microbiologists in finding the best loci on which to base such assays. The program is accessible at: http://www.hpa.org.uk/srmd/bioinformatics/tools/mop-ups.htm.

Amino Acid Motifs↗
PubMed2003

Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis.

Modern DNA sequencing 'factories' have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a 'sequencing lab-on-a-chip'. Integrated microfluidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.

Base Sequence↗
PubMed2006

PCR-based detection of Angiostrongylus cantonensis in tissue and mucus secretions from molluscan hosts.

Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.

Angiostrongylus cantonensis↗
PubMed1983

Identification of genomic DNA coding for chicken type II procollagen.

A segment of the type II procollagen gene has been isolated by screening a lambda Charon 4A library containing fragments of chicken genomic DNA. The specific clone, LgCOL(II), was selected by hybridization using overlapping inserts from two cDNA clones which are specific for a cartilage procollagen (Vuorio, E., Sandell, L., Kravis, D., Sheffield, V. C., Vuorio, T., Dorfman, A., and Upholt, W. B. (1982) Nucleic Acids Res. 10, 1175-1192). DNA sequence analysis of LgCOL(II) in the COOH-telopeptide region of the protein, shows conclusively that this DNA corresponds to the chicken type II procollagen gene. Hybridization of cDNA probes to restriction fragment gel blots together with DNA sequence analysis have established the orientation and position of the procollagen gene within the lambda Charon 4A vector and indicate that LgCOL(II) contains approximately 6 kilobase pairs of the type II procollagen gene plus additional DNA flanking the 3' end of the gene. DNA sequence analysis shows directly that LgCOL(II) contains DNA sequences identical with those in the cDNA clones. The portion of the gene from amino acid 578 of the triple helical region to the COOH-terminal end of the protein (approximately 700 amino acids) is contained within the clone, corresponding to approximately 50% of the amino acid coding sequence of the gene. This region of the chicken alpha 1 (type II) procollagen gene is encoded within a shorter segment of the chicken genome than is the corresponding region of the alpha 2(type I) procollagen gene.

Amino Acid Sequence↗
PubMed2005

Determining functional specificity from protein sequences.

MOTIVATION: Given a large family of homologous protein sequences, many methods can divide the family into smaller groups that correspond to the different functions carried out by proteins within the family. One important problem, however, has been the absence of a general method for selecting an appropriate level of granularity, or size of the groups. RESULTS: We propose a consistent way of choosing the granularity that is independent of the sequence similarity and sequence clustering method used. We study three large, well-investigated protein families: basic leucine zippers, nuclear receptors and proteins with three consecutive C2H2 zinc fingers. Our method is tested against known functional information, the experimentally determined binding specificities, using a simple scoring method. The significance of the groups is also measured by randomizing the data. Finally, we compare our algorithm against a popular method of grouping proteins, the TRIBE-MCL method. In the end, we determine that dividing the families at the proposed level of granularity creates very significant and useful groups of proteins that correspond to the different DNA-binding motifs. We expect that such groupings will be useful in studying not only DNA binding but also other protein interactions.

Algorithms↗
PubMed1999

TT virus sequence heterogeneity in vivo: evidence for co-infection with multiple genetic types.

TT virus (TTV) is a newly described DNA virus of humans that exhibits an unusually high degree of genetic heterogeneity. We have performed extensive analysis of the TTV populations present in samples, taken over a period of 2 to 6 years, from three individuals with persistent TTV infection. TTV DNA titres estimated for sequential samples were found to be quite stable over the entire study period in two patients, but fluctuated considerably in the third. DNA sequence analysis revealed different genetic diversity among TTV populations from samples from the three patients. In one case, absolute sequence homogeneity was observed among samples over a 3 year period. In a second, a limited amount of heterogeneity was found, including one sequence exhibiting G-->A hypermutation. TTV DNA sequences from the third patient exhibited quite remarkable genetic heterogeneity: evidence was found of seven distinct infecting viruses, representing four of the six TTV genotypes that have been described. In addition, minor variants of three of these seven sequences were observed. The heterogeneity of the viral population in this individual declined steadily over a 6 year period. This patient infected with a genetically diverse TTV population had the highest viral DNA titre.

Amino Acid Sequence↗
PubMed1995

A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping or DNA sequencing. V. Analysis of case/control sampling designs: Alzheimer's disease and the apoprotein E locus.

Present-day associations between haplotypes at a candidate locus and phenotypes exist when phenotypically important mutations occurred at some point during the evolution of the current array of genetic variation. A cladistic statistical design can be defined that focuses power by using the evolutionary history of the candidate DNA region. This paper shows how cladistic methodology is used for the analysis of case/control data, a common sampling design in genetic/disease association studies. A worked example is presented of the associations for sporadic early and late-onset forms of Alzheimer's disease with the 19q13.2 chromosomal region that includes the loci for apoproteins E, CI, and CII. This analysis confirms earlier reports of a strong association of the ApoE epsilon 4 allele with Alzheimer's disease but indicates that it is premature to consider this association causal, particularly for early onset cases. Associations were also found with the epsilon 2 allele, as previously reported, and with the 1 allele at the ApoCI locus. However, this analysis indicates that it is inappropriate both statistically and medically to use single markers as risk predictors when haplotype data are available, even when the mutation leading to the marker is identified as having a strong phenotypic association.

Age of Onset↗
PubMed1996

Identification and isolation of the FEEBLY gene from tomato by transposon tagging.

The Ac/Ds transposon system from maize was used for insertional mutagenesis in tomato. Marker genes were employed for the selection of plants carrying a total of 471 unique Ds elements. Three mutants were obtained with Ds insertions closely linked to recessive mutations: feebly (fb), yellow jim (yj) and dopey (dp). The fb seedlings produced high anthocyanin levels, developed into small fragile plants, and were insensitive to the herbicide phosphinothricin. The yj plants had yellow leaves as a result of reduced levels of chlorophyll. The dp mutants completely or partially lacked inflorescences. The fb and yj loci were genetically linked to the Ds donor site on chromosome 3. Reactivation of the Ds element in the fb mutants by crosses with an Ac-containing line resulted in restoration of the wild-type phenotypes. Plant DNA fragments flanking both sides of the Ds element in the fb mutant were isolated by the inverse polymerase chain reaction. Molecular analysis showed that phenotypic reversions of fb were correlated with excisions of Ds. DNA sequence analysis of Fb reversion alleles showed the characteristic Ds footprints. Northern and cDNA sequence analysis indicated that transcription of the FEEBLY (FB) gene was impeded by the insertion of Ds in an intron. Comparison of the predicted amino acid sequence of the FB protein with other database sequences indicated that FB is a novel gene.

Aminobutyrates↗
PubMed2002

[The detection of the frequent mutations of iduronate-2-sulphatase gene in mucopolysaccharidosis type II patients in Chinese].

OBJECTIVE: To investigate and establish the gene diagnosis methods for the frequent mutations of iduronate-2-sulphatase(IDS) gene in mucopolysaccharidosis type II patients. METHODS: polymerase chain- reaction-single strand conformation polymorphism PCR-SSCP) analysis was applied to detect the mutations of exons 3, 8 and 9 which were hot spots in the iduronate-2-sulfatase gene; DNA sequencing was applied to analyze the mutations which had been detected by PCR-SSCP; PCR-restriction fragment length polymorphism (PCR-RFLP) was applied to detect the results of DNA sequencing. RESULTS: Obvious and abnormal bands in exon 9 of the IDS gene were found by applying PCR-SSCP; the mutation(C1672T) of exon 9 was found in the patient through DNA sequencing, which led to amino acid replacement(R468W); the PCR-restriction enzyme digestion showed that only one band(554 bp) appeared in the patient, but there were two bands (257 bp and 297 bp) in his parents, and it verified the results of sequencing analysis. CONCLUSION: PCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion are effective methods for MPS II diagnosis. Combined applications of these methods can verify and complement each other and improve the accuracy of diagnosis.

Amino Acid Substitution↗
PubMed2004

Identification of transthyretin variants by sequential proteomic and genomic analysis.

BACKGROUND: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. METHODS: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 microL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13,761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. RESULTS: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. CONCLUSIONS: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Val94Ala.

Amyloidosis, Familial↗
PubMed2002

Expression of human VEGF(121) cDNA in mouse bone marrow stromal cells.

OBJECTIVE: To construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF (121)) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease. METHODS: hVEGF(121) cDNA was obtained from the plasmid pCDI/VEGF(121) and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retro virus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF(121) cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF(121) in culture medium were performed. RESULTS: Recombinant pLXSN/VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF(121) gene was integrated into MSC genomic DNA after transfection, and the VEGF(121) protein was expressed. Proliferation assays showed VEGF(121) in culture medium was a biologically active protein and had a mitogenic effect on HUVEC. CONCLUSIONS: Recombinant retroviral vector carrying hVEGF(121) cDNA was successfully constructed. VEGF (121) protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.

Animals↗
PubMed2001

Mutations of the p53 gene in malignant rhabdoid tumors of soft tissue and the kidney: immunohistochemical and DNA direct sequencing analysis.

Malignant rhabdoid tumor (MRT) is characterized by the presence of intracytoplasmic eosinophilic inclusions composed of whorls of intermediate filaments. This tumor was originally described as an entity of the abortive type of Wilms' tumor in childhood. Recently, it has been proved that these rhabdoid cells can be observed in various types of malignant tumors, including soft tissue sarcoma or carcinoma. To investigate the oncogenesis of this tumor, we examined the p53 gene alteration by means of immunohistochemical analysis and DNA direct sequencing in three cases of malignant rhabdoid tumor (MRT) of the soft tissue and three cases of MRT of the kidney. All the cases of MRT of the soft tissue and two of the cases of MRT of the kidney showed immunopositivity for p53 protein. Among them, one of the cases of MRT of the soft tissue and two of the cases of MRT of the kidney showed missense mutations of the p53 gene. These results strongly suggest that p53 gene alterations may have an important role to play in the aggressive biological behavior and poor prognosis of this tumor.

Adult↗
PubMed2003

Detection of identical Helicobacter DNA in the stomach and in the non-cirrhotic liver of patients with hepatocellular carcinoma.

OBJECTIVES: Studies have implied the presence of Helicobacter species in the human liver. The possible relationship between the presence of Helicobacter species in the non-cirrhotic liver of patients with hepatocellular carcinoma (HCC) and the detection of Helicobacter species in their stomach was explored. PATIENTS AND METHODS: A 16S rDNA-based polymerase chain reaction (PCR) followed by DNA sequence analysis of the obtained PCR fragments was performed on 51 surgically obtained non-cirrhotic liver specimens and 14 gastric samples. RESULTS: Analysis indicated a significant difference in the presence of Helicobacter-species-specific DNA in the liver of patients with HCC compared with controls. Sequence analysis of these PCR products obtained from HCC patients indicated that they were related most closely to the 16S rDNA sequence of Helicobacter pylori but that they always differed at the same two positions. This same aberrant Helicobacter-species-specific 16S rDNA could be isolated in gastric samples of patients with HCC. CONCLUSION: These data suggest that gastric colonization with a specific subset of Helicobacter strains is associated with the induction of HCC, either directly via colonization of the liver or indirectly, e.g. via secretion of specific toxins by Helicobacter residing in the stomach.

Carcinoma, Hepatocellular↗