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genomic DNA

About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.


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Journal ArticleDOI
TL;DR: Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, and the Leishmania bifunctionsal DH FR-TS evolved independently and not through a phage T4-related intermediate.
Abstract: We have determined the nucleotide sequence of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and the rate of evolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.

143 citations

Patent
02 Nov 1999
TL;DR: In this article, a novel method for analyzing genomic DNA and expressed sequences using auxiliary oligonucleotides, preannealed to the single-stranded target nucleic acid to form a partially duplex target molecule, offers several advantages in the analysis of nucleic acids sequences by hybridization to genosensor arrays or DNA chips.
Abstract: The disclosed invention provides a novel method for analyzing genomic DNA and expressed sequences using auxiliary oligonucleotides, preannealed to the single-stranded target nucleic acid to form a partially duplex target molecule, offers several advantages in the analysis of nucleic acid sequences by hybridization to genosensor arrays or “DNA chips”. Also provided is a method for directly analyzing and comparing patterns of gene expression at the level of transcription in different cellular samples.

143 citations

Journal ArticleDOI
TL;DR: Using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method, and genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.
Abstract: The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.

142 citations

Journal ArticleDOI
TL;DR: SFHR-mediated modification of the ΔF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR) and demonstrated that the colony is not yet clonal, but still contains a population of parental, CFBE41o− cells that have not been modified.
Abstract: Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the DeltaF508 mutation, a 3-bp deletion that results in the loss of a phenylalanine at amino acid 508 in the CF transmembrane conductance regulator (CFTR) protein. Direct modification of the DeltaF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR). Transformed human airway epithelial cells (CFBE41o(-)), homozygous for DeltaF508 mutation, were transfected with small fragments (491-bp) of wild-type (WT) CFTR DNA comprising exon 10 and the flanking introns. The DNA fragments were in a liposome-DNA complex at a charge ratio of 6:1 (+:-), respectively). The population of transfected cells was subcloned by limiting dilution at approximately 1 cell/well in 96-well plates. Individual colonies were isolated and analyzed. The DNA from several colonies was characterized by radiolabeled, nonallele-specific and radiolabeled, allele-specific PCR amplification, as well as by genomic DNA fingerprinting. The CFTR-WT allele was detected in five of these colonies by allele-specific PCR amplification thus indicating that the cell lines carried both WT and DeltaF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o(-) cell line. Although, the WT allele was detected by allele-specific PCR, it was not detected initially when the same samples were analyzed by non allele-specific PCR. A sensitivity assay, mixing the genomic DNA of wild-type (16HBE14o(-)) and mutant (CFBE41o(-)) cell lines, indicated that the allele-specific PCR was at least 25-fold more sensitive than non allele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o(-) cells that have not been modified. Based on the mixing analysis, the proportion of corrected cells appears to be between 1 and 10% of the total population. Nonallele-specific reverse transcriptase PCR (RT-PCR) analysis of the CFTR mRNA indicated that two of the colonies expressed both WT and DeltaF508 CFTR mRNA, while one colony appeared to express only the WT mRNA. The mRNA results were confirmed by sequence analysis of 3' end primer extension products from the mRNA of CFTR exon 10 showing that the mRNA containing exon 10. Furthermore, a survey of primer extension products indicated no random insertion of the fragment in an expressed gene. This study demonstrates SFHR-mediated modification of the DeltaF508 allele in DeltaF508 homozygote human airway epithelial cells over multiple generations. The resultant cells express WT-CFTR mRNA and can be subcloned further to isolate isogenic clonal populations of cells.

142 citations

01 Jan 1986
TL;DR: For example, Marchionni et al. as mentioned in this paper have shown that the triosephosphate isomerase (TIM) gene in maize roots is interrupted by eight introns and that the introns were in place before the plant-animal divergence.
Abstract: Mark Marchionni and Walter Gilbert Department of Cellular and Developmental Biology Harvard University Biological Laboratories 16 Divinity Avenue Cambridge, Massachusetts 02138 Summary We have cloned and characterized a cDNA and genomic DNA for the triosephosphate isomerase ex- pressed in maize roots. The gene is interrupted by eight introns. If we compare this gene with that for the protein in chicken, which has six intmns, we see that five of the introns are at identical places, one has shifted by three codons, and two are totally new. This great matching leads us to conclude that the introns were in place before the plant-animal divergence, and that the parental gene had at least eight introns, two of which were lost in the line that leads to animals. Introduction Genes of higher eukaryotes are discontinuous: regions of noncoding DNA (introns) separate the coding sequence into discrete segments (exons). Gilbert (1978) proposed that introns are vestigial DNA sequence, remnants of a recombination process that accelerated molecular evolu- tion by assembling new linkage groups from the exons, creating novel gene products. That hypothesis of exon shuffling predicts that positions of introns should portray the evolutionary history of a gene; the exons themselves would encode distinct functional elements (Gilbert, 1978, 1979), stably folding peptides (Blake, 1978) or compact modules (Go, 1981, 1983). Evidence in support of these ideas derives from molecular studies on genes and pro- teins as diverse as immunoglobulins (reviewed by Tone- gawa, 1983, and Honjo, 1983) collagen (Yamada et al., 1980) serum albumin-a-fetoprotein (Sargent et al., 1981; Eiferman et al., 1981) ovomucoid (Stein et al., 1980), glo- bins (Go, 1981) intermediate filaments (Marchuk et al., 1984; Balcarek and Cowan, 1985), lysozyme (Jung et al., 1980) and crystallins (Moormann et al,, 1983). Moreover, recent studies (Siidhof et al., 1985a, 1985b) comparing the gene structure of low density lipoprotein receptor with those of epidermal growth factor and blood coagulation factors 9 and 10 provide a dramatic example of exon shuf- fling in recent evolutionary history. Although there are a few exceptions (Kaine et al., 1983; Chu et al., 1984; Nellen et al., 1981; Miller, 1984), the con- spicuous absence of introns in bacteria and yeast poses questions as to their age and origins. Were introns present as the first genes formed? Or have they more recently taken up residence in genomic DNA by invading continu- ous gene sequences? Doolittle (1978) suggested that the genomes of primitive cells contained introns but that rap- idly dividing organisms lost them by streamlining in re- sponse to the selection pressure of rapid reproduction. The highly conserved, ubiquitous glycolytic enzymes most likely evolved completely before the archaebacteria-pro- karyotic-eukaryotic division and thus represent extremely ancient genes. In vertebrates, introns punctuate nonran- domly the sequences encoding chicken pyruvate kinase (Lonberg and Gilbert, 1985), chicken glyceraldehyde phosphate dehydrogenase (Stone et al., 1985) chicken triosephosphate isomerase (Straus and Gilbert, 1985b), as well human phosphoglycerate kinase (Michelson et al., 1985), suggesting that the introns were not inserted into preexisting genes. To push our view of one these glycolytic enzyme genes back in time, we have character- ized a gene encoding triosephosphate isomerase (EC 5.3.1.1, TIM) in maize in order to compare its structure to the chicken gene. A remarkable conservation of intron po- sitions between these distant relatives indicates that the introns were in place before the plant-animal divergence, more than one billion years ago. Results Cloning and Structure of Maize TIM cDNA During glycolysis, triosephosphate isomerase (TIM) cata- lyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. In addition to a cytosolic TIM, plant cells active in photosynthesis express a plastid enzyme encoded in the nucleus (Pichersky and Gottlieb, 1982). That isozyme functions in the dark reactions that fix carbon dioxide into sugars. As is true for other pho- tosynthetic proteins, the chloroplast-specific TIM is light inducible (e.g., Nelson et al., 1984; Berry-Lowe et al., 1982). Hence, mRNA from roots grown in the dark should be enriched in the cytosolic form, which ought to cog- nate to the chicken enzyme. Because TIM is less than 60% diverged across the most distant species (Pichersky et al., 1984; Straus and Gilbert, 1985b), cross-hybridiza- tion from chicken to maize root cDNA was a plausible ap- proach to cloning the maize gene. Using a chicken probe (Straus et al., 1985a), we screened 80,000 phage cDNA recombinants that repre- sented mRNA extracted from maize root seedlings grown in the dark for 7 days. We chose hybridization and wash- ing conditions of medium stringency (see Experimental Procedures) and detected 18 potential TIM clones. After partially mapping all of the inserts, we focused on two large (>800 bp) overlapping clones, subcloned each of these into the EcoRl site of pUC8 (Vieira and Messing, 1982), and sequenced (Maxam Gilbert, 1980) both strands of the DNA. Figure 1 displays a partial restriction map and the com- plete nucleotide sequence of a full-length maize root TIM cDNA clone, designated pMRT1. Within the 123 bases of 5’ untranslated sequence is a 37 base pyrimidine tract, which is flanked by two overlapping, imperfect, direct repeats of 20 bases each. A similar alternating pyrimidine

142 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023258
2022431
2021232
2020261
2019273
2018339