Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Nucleotide Mapping”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1994

Analysis of respiratory syncytial virus strain variation in successive epidemics in one city.

The variability of respiratory syncytial virus isolates from five successive epidemics in an urban population was determined. A total of 187 isolates of respiratory syncytial virus from the southern part of Birmingham, United Kingdom, were classified into subgroups A and B and were then further assigned to genetic lineages. Allocation of isolates into lineages was achieved by reverse transcription of infected cell RNA and then PCR amplification of selected regions of the genome; PCR products were examined by restriction mapping or nucleotide sequencing of parts of the nucleoprotein gene, the small hydrophobic protein gene, and the attachment protein gene. Previous work has shown that estimations of genetic diversity by analysis of genes coding for proteins likely (attachment protein) and unlikely (nucleoprotein and small hydrophobic protein) to be under immune pressure gave concordant results. Six genetic lineages of subgroup A isolates have been defined by this procedure; these isolates differ by up to 20% in the amino acid sequences of their attachment proteins; likewise, subgroup B isolates can be divided into two categories by restriction mapping of parts of their nucleoprotein and attachment protein genes. The same genetic lineages appeared to be present worldwide during the same period. The analysis of isolates from successive epidemics showed that different lineages predominated in each epidemic and that not all lineages were present in every epidemic. Some lineages appeared to increase in numbers over several years and then decline, possibly indicating a buildup of resistance in the community to a particular genotype.

Capsid↗
PubMed1991

Utility of the polymerase chain reaction (PCR) for prenatal diagnosis of genetic disease.

Gene amplification by the polymerase chain reaction (PCR) has been applied to prenatal diagnosis for alpha and beta thalassemias (1 and 5 cases respectively), Hemoglobin (Hb) Lepore/beta thalassemia (1 case) and cystic fibrosis (14 cases). Chorionic villus samples were obtained in the tenth week of pregnancy and DNA analysed in parallel with conventional gene mapping. Direct diagnosis of the common Mediterranean beta-thalassemia mutations (IVS-1-110 and codon 39), Hb Lepore, and the delta F508 mutation causing cystic fibrosis was achieved by hybridization of amplified material with pairs of allele-specific oligonucleotide (ASO) probes or by restriction enzyme digestion of PCR products. Results were confirmed by DNA mapping. Definitive diagnosis or exclusion of an affected fetus was possible in 17 of 21 cases thus examined. PCR reduces the time required for prenatal diagnosis. DNA contamination is a potential source of error.

Alleles↗
PubMed1984

The nucleotide binding site detected by affinity labeling in the large T proteins of polyoma and SV40 viruses is distinct from their ATPase catalytic site.

Binding of nucleotides to a specific site of the large T proteins of polyoma and SV40 viruses was demonstrated by covalent affinity labeling with periodate-oxidized [alpha-32P]ATP (oxATP) (Clertant, P., and Cuzin, F. (1982) J. Biol. Chem. 257, 6300-6305). This site appears different from the catalytic site of these proteins for ATP hydrolysis: (i) nucleotide binding and ATPase activities exhibited different ionic requirements and kinetic parameters; (ii) different antibodies directed against the polyoma large T protein either completely inhibited ATPase activity and not affinity labeling, or vice versa; (iii) a truncated form of polyoma large T, with its carboxyl-terminal third deleted, does not bind oxATP but exhibits normal ATPase activity; (iv) conversely, a "super T" SV40 protein, resulting from a duplication within the coding region of large T, was efficiently labeled with oxATP, although it lacks detectable ATPase activity. Cyanogen bromide cleavage after affinity labeling mapped the nucleotide binding site of the polyoma and SV40 large T proteins within a carboxyl-terminal amino acid sequence highly homologous between the two polypeptides. A survey of the phenotypes of the known mutations in these multifunctional proteins suggests that their ATPase and nucleotide-binding activities, although distinct, might both be required to ensure crucial steps in the lytic cycle.

Adenosine Triphosphatases↗
PubMed1988

Molecular cloning of human T-cell lymphotrophic virus type I-like proviral genome from the peripheral lymphocyte DNA of a patient with chronic neurologic disorders.

Human T-cell lymphotropic virus type 1 (HTLV-I), the etiologic agent of human T-cell leukemia, has recently been shown to be associated with neurologic disorders such as tropical spastic paraparesis, HTLV-associated myelopathy, and possibly with multiple sclerosis. In this communication, we have examined one specific case of neurologic disorder that can be classified as multiple sclerosis or tropical spastic paraparesis. The patient suffering from chronic neurologic disorder was found to contain antibodies to HTLV-I envelope and gag proteins in his serum and cerebrospinal fluid. Lymphocytes from peripheral blood and cerebrospinal fluid of the patient were shown to express viral RNA sequences by in situ hybridization. Southern blot analysis of the patient lymphocyte DNA revealed the presence of HTLV-I-related sequences. Blot-hybridization analysis of the RNA from fresh peripheral lymphocytes stimulated with interleukin 2 revealed the presence of abundant amounts of genomic viral RNA with little or no subgenomic RNA. We have cloned the proviral genome from the DNA of the peripheral lymphocytes and determined its restriction map. This analysis shows that this proviral genome is very similar if not identical to that of the prototype HTLV-I genome.

Adult↗
PubMed1997

Stimulation of gene induction and cell growth by the Ras effector Rlf.

Rlf is a ubiquitously expressed distinct relative of RalGDS that interacts with active Ras in vitro. We now demonstrate that Rlf, when co-expressed with Ras mutants, associates in vivo with RasV12 and the effector-domain mutant RasV12G37, but not with RasV12E38 or RasV12C40. Rlf exhibits guanine nucleotide exchange activity towards the small GTPase Ral and, importantly, Rlf-induced Ral activation is stimulated by active Ras. In addition, RasV12 and RasV12G37 synergize with Rlf in the transcriptional activation of the c-fos promoter. Rlf, when targeted to the plasma membrane using the Ras farnesyl attachment site (Rlf-CAAX), is constitutively active, inducing both Ral activation and c-fos promoter activity. Rlf-CAAX-induced gene expression is insensitive to dominant negative Ras and the MEK inhibitor PD98059, and involves activation of the serum response element. Furthermore, expression of Rlf-CAAX is sufficient to induce proliferation of NIH 3T3 cells under low-serum conditions. These data demonstrate that Rlf is an effector of Ras which functions as an exchange factor for Ral. Rlf mediates a distinct Ras-induced signalling pathway to gene induction. Finally, a constitutively active form of Rlf can stimulate transcriptional activation and cell growth.

3T3 Cells↗
PubMed2000

Haplotype dimorphism in a SNP collection from Drosophila melanogaster.

A moderate resolution single nucleotide polymorphism (SNP) map of the genome of Drosophila melanogaster that is designed for use in quantitative genetic mapping is described. Seventeen approximately 500 nucleotide gene sequences spaced at 10 to 20 centimorgan intervals were combined with 49 shorter sequence tag sites (STSs) at 5 to 10 centimorgan intervals to generate a map that should not leave any gaps greater than one half of a chromosome arm when any two wild type lines are compared. Of 20 markers with sufficient polymorphism to construct haplotype cladograms, 13 showed evidence for two divergent classes of haplotype. The possible mechanisms for and implications of the unexpected finding that two thirds of all short gene sequences in D. melanogaster may be dimorphic are discussed, including the suggestion that admixture between two separate lineages may have been a major event in the history of the species.

Animals↗
PubMed1989

Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector.

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.

Animals↗
PubMed1989

Characterization of a rat myosin alkali light chain gene expressed in ventricular and slow twitch skeletal muscles.

Mammalian cardiac muscle contains two myosin alkali light chains: 1) the atrial light chain (MLC1A), and 2) the ventricular light chain (MLC1V) predominantly expressed either in the atrium or in the ventricle. In this report we describe the isolation and characterization of the complete gene for rat MLC1V. The rat MLC1V gene is approximately 6.5 kb long and the mRNA coding sequences are organized in 7 different exons. Comparison of this gene sequence with other known MLC1 gene sequences revealed that the exon-intron organization is highly conserved within the MLC1 gene family. The derived protein sequence of rat MLC1V showed a higher sequence homology with human ventricular (96%) MLC1V than with rat fast skeletal MLC1f (74%), suggesting functional similarities between different MLC1V proteins. S1 nuclease mapping and primer extension analysis demonstrated that this gene is expressed only in ventricular and slow twitch skeletal muscle tissues and is transcribed from the same promoter and transcription initiation site.

Amino Acid Sequence↗
PubMed1989

The 85-kd surface antigen gene of Trypanosoma cruzi is telomeric and a member of a multigene family.

In previous studies we identified a 500 bp DNA fragment from the genome of Trypanosoma cruzi which encoded epitopes present in an 85 kd trypomastigote-specific surface antigen. A unique feature of this DNA insert was the presence of a 27 bp tandem repeat unit within the putative coding region. The findings presented here show that the gene which encodes this particular surface protein is a member of a multigene family, and that the 27 bp repeat unit defines a subset of this family. Only four separate members of the family contain sequences homologous to the 27 bp repeat unit. Of these, three have been cloned and shown by direct nucleotide sequence analysis not to contain the original 500 bp fragment. By restriction enzyme analysis, the 500 bp fragment is inferred to be present in a 5.4 kb EcoRI genomic DNA fragment that is refractory to isolation by standard cloning procedures. Preferential sensitivity of this fragment to digestion with Bal31 nuclease indicates that it is likely to be telomeric, thus explaining the inability to obtain it in several different recombinant DNA libraries. In order to determine which of the four members were transcribed, 26 cDNA recombinants having sequence homology with the 27 bp repeat were examined. Restriction enzyme maps and nucleotide sequence analysis of these cDNAs indicate that transcription occurs almost exclusively from the telomeric member of the family.

Animals↗
PubMed1987

DNA changes during blastic transformation in chronic granulocytic leukemia.

Immunoglobulin and T cell receptor gene mapping were undertaken in 10 patients during both chronic and blastic phases of chronic granulocytic leukemia. Twenty percent were shown to undergo lymphoblastic transformation. DNA changes did not predict blastic transformation or those who would respond favourably to treatment with vincristine and prednisone.

Adolescent↗
PubMed1975

Role of DNA sequences in genetic recombination in the iso-1-cytochrome c gene of yeast. I. Discrepancies between physical distances and genetic distances determined by five mapping procedures.

Recombination rates have been examined in two-point crosses of various defined cyc1 mutants using five mapping methods. Nucleotide sequences of mutant codons were identified in previous studies from alterations in functional iso-1-cytochromes c produced by intragenic revertants. Heteroallelic diploids were analyzed for rates of mitotic recombination that occurred spontaneously and that were induced with x-rays, ultraviolet light and the near-ultraviolet light emitted by sunlamps, as well as rates of meiotic recombination that occur after sporulation. Frequencies of both mitotic and meiotic recombination do not necessarily correspond with physical distances separating altered nucleotides. The most extreme discrepancy involved two adjacent intervals of thirteen basepairs which differed approximately thirty-fold in their spontaneous and X-ray-induced recombination rates. Marked disproportions between genetic and physical distances appear to be due to the interaction of the two nucleotide sequences in the heteroallelic combination and not to the sequences of the mutant codons alone. Recombination values that were obtained by all five methods could not be used to establish to correct order of mutant sitesmrelationships of the recombination rates for the various pairwise crosses are different after mitosis from those after meiosis, suggesting that these two recombinational processes are to some extent different in their dependence on particular nucleotide configurations. On the other hand, the relationships of the rates induced by UV-, sunlamp- and X-irradiation were identical or very similar. In addition to the intrinsic properties of the alleles affecting frequencies of mitotic and meiotic recombination rates, two- to threefold variations in recombination rates could be attributed to genetic backgrounds.

Base Sequence↗
PubMed1987

5-Fluorouracil augmentation of dihydrofolate reductase RNA containing contiguous exon and intron sequences in KB7B cells.

Quantitative S1 nuclease mapping studies were performed with uniformly labeled RNA probes, containing contiguous dihydrofolate reductase exon and intron sequences, and total RNA isolated from KB7B cells exposed to 5-fluorouracil for 5 days. Dihydrofolate reductase RNA containing both exon 1 and intron I, or exon 5 and a portion of intron V, increased up to 5-fold in cells grown in the presence of 2.0 to 3.0 microM 5-fluorouracil. Dihydrofolate reductase RNA containing exon 1 or exon 5, but lacking intron I or intron V, respectively, increased 2-fold in cells grown in the presence of 0.65 to 3.0 microM 5-fluorouracil. Primer extension analysis and S1 mapping studies revealed two major transcriptional start sites at positions -72 and -69 and minor start sites upstream from position -183, for dihydrofolate reductase RNA isolated from methotrexate-resistant KB7B cells. The results of these studies demonstrate that 5-fluorouracil alters the metabolism of dihydrofolate reductase precursor mRNA and/or processing intermediates.

Endonucleases↗
PubMed1990

Alternative splicing generates two forms of mRNA coding for human heparin-binding growth factor 1.

Human class 1 heparin-binding growth factor (HBGF-1), also known as acidic fibroblast growth factor, is a mitogen for a variety of mesoderm- and neutroectoderm-derived cells in vitro as well as an angiogenic factor in vivo. Several oncogenes and growth factors have been shown to be homologous to HBGF-1. Four cDNA clones coding for HBGF-1 have been isolated from a human brain stem cDNA library. Nucleotide sequence analysis revealed that alternative splicing generated at least two different forms of HBGF-1 mRNA. Because the difference occurs in the 5'-untranslated regions, these transcripts may result from the usage of alternative promoters. One of the cDNA clones contains the polyadenylation signal, AATAAA, and a poly(A) tail, representing the 3'-end of an HBGF-1 mRNA. RNAase protection assays suggested this cDNA clone corresponds to a minor transcript, and the majority of the HBGF-1 mRNA terminates at 3.1 kbp downstream from the translation termination codon. The biological significance of this unusually long 3'-untranslated sequence is not known. To study the HBGF-1 gene structure, we have isolated 50 kbp of contiguous genomic DNA coding for the HBGF-1 protein. Both restriction enzyme mapping and nucleotide sequencing established that the distance between the first and second protein-coding exons is 13.6 kbp while that between the second and third is 5.3 kbp. By using the HBGF-1 cDNA as a probe, we showed that human fetal heart expresses high levels of HBGF-1 mRNA. Thus, HBGF-1 may be involved in mediating processes such as embryonic development and vascular growth in the heart.

Amino Acid Sequence↗
PubMed1986

[Mutations predetermined by the primary structure of DNA].

This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions. It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair. These hairpin structures can be eliminated by nucleases or during DNA replication. Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure. Model experiments were carried out with the pBR322 plasmid. A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment. The plasmid was used for transformation of Escherichia coli. Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions. The end points of the deletions coincide with the palindrome. To model homologous recombination, a plasmid with D-loop was constructed. A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex. When E. coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose. The evolutionary role of mutations predetermined by primary DNA structure is discussed.

Base Sequence↗
PubMed1988

Dispersed localization of D segments in the human immunoglobulin heavy-chain locus.

We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.

Amino Acid Sequence↗
PubMed1989

Structure of the promoter region and tissue specificity of the human glycophorin C gene.

Glycophorin C (GPC) is an integral membrane protein of human erythrocytes which plays an important role in regulating the deformability and mechanical stability of red cells. Recently, the structural gene for this glycoprotein has been cloned (Colin, Y., Le Van Kim, C., Tsapis, A., Clerget, M., d'Auriol, L., London, J., Galibert, F., and Cartron, J. P. (1989) J. Biol. Chem. 264, 3773-3780), and we have now determined the sequence of the 1050 base pairs of DNA preceding the transcription initiation site mapped in erythroid cells. This region contains different potential regulatory cis-acting elements found in a variety of eukaryotic promoters (TATA box, CAAT box, Sp1-binding site) as well as sequences present in the promoter and enhancer regions of genes specific for the erythroid lineage (CACCC box and NF-E1-binding site). Northern blot analysis and immunological studies indicate that the GPC gene is expressed in a large number of cells and tissues. However, the level of transcription as well as the glycosylation of the mature GPC differ in erythroid and nonerythroid cells. Primer extension analysis and mapping of the 5' end GPC mRNA by the polymerase chain reaction indicate that different transcription sites are utilized for the expression of the GPC gene in erythroid and nonerythroid tissues and cell lines.

Base Sequence↗
PubMed1988

Identity of Karelian fever and Ockelbo viruses determined by serum dilution-plaque reduction neutralization tests and oligonucleotide mapping.

The causative agents of Ockelbo disease in Sweden, Pogosta disease in Finland, and Karelian fever in the USSR have been attributed to alphaviruses (family Togaviridae) related to Sindbis virus. We compared prototypes Sindbis, Ockelbo, and Karelian fever viruses by neutralization tests. We also analyzed oligonucleotide fingerprint maps of prototypes Ockelbo and Karelian fever viruses and a strain of Sindbis virus from Czechoslovakia. The results indicate that Ockelbo and Karelian fever viruses are essentially identical and suggest that Ockelbo disease, Pogosta disease, and Karelian fever are synonyms for the same disease.

Alphavirus↗
PubMed2001

Antisense targeting of c-fos transcripts inhibits serum- and TGF-beta 1-stimulated PAI-1 gene expression and directed motility in renal epithelial cells.

Plasminogen activator inhibitor type-1 (PAI-1), the major regulator of pericellular plasmin generation, and the c-FOS transcription factor are expressed by migrating cells in response to monolayer wounding. Induced c-fos and PAI-1 transcripts were evident within 30 min and 2 h, respectively, of scrape injury to confluent, growth-arrested, cultures of NRK epithelial cells. Since c-FOS/AP-1 DNA-binding activity modulates both basal and inducible modes of PAI-1 gene control, and AP-1 motif binding factors were present in quiescent as well as stimulated NRK cells, a model of directionally regulated cell movement (migration into scrape-denuded "wounds") was used to assess the consequences of c-fos transcript targeting on PAI-1 expression and cell motility. This in vitro model of epithelial injury closely approximated in vivo wound repair with regard to the spatial and temporal emergence of cohorts of cells involved in migration, proliferation, and PAI-1 expression. Stable cell lines (NRKsof) were generated by transfection of parental NRK cells with a c-fos antisense expression vector. Serum-inducible c-fos transcripts and PAI-1 protein levels were significantly attenuated in NRKsof transfectants relative to parental controls or cells transfected with a neo(R) vector without the sof insert. NRKsof cells had a markedly impaired ability to repair scrape-generated monolayer wounds under basal, serum-stimulated, or TGF-beta 1-supplemented culture conditions. Since injury closure and PAI-1 induction were attenuated in c-fos antisense cells, it was important to clarify the role of specific AP-1 sites in serum-mediated PAI-1 transcription. PAI-1 "promoter"-driven CAT reporter expression was assessed within the real time of serum-stimulated PAI-1 induction. A segment of the PAI-1 promoter corresponding to nucleotides -533 to -764 upstream of the transcription start site functioned as a prominent serum-responsive region (SSR). The 9-fold increase in CAT mRNA levels attained with the -533 to -764 bp PAI-1 SRR ligated to a minimal PAI-1 promoter (i.e., 162 bp of 5' flanking sequence containing the basal transcription complex) closely approximated the serum-induced transcriptional activity of a fully responsive (1,230 bp) PAI-1 promoter construct as well as the endogenous PAI-1 gene. AP-1-like, CTF/NF-1-like, and AP-2 sites were identified in the SRR. The SRR AP-1 motif was homologous to the sequence TGACACA that mapped between nucleotides -740 and -703 in the human PAI-1 gene, a region essential for growth factor-inducible PAI-1 transcription. While the functionality of this AP-1 site in wound-regulated PAI-1 synthesis remains to be determined, antisense c-fos transcripts effectively attenuated PAI-1 induction and basal as well as growth factor-stimulated cell locomotion, indicating that expression of both the PAI-1 and c-fos genes is necessary for wound-initiated NRK cell migration.

Animals↗