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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: High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.
Abstract: High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.

143 citations

Book ChapterDOI
TL;DR: The purpose of this protocol is the isolation of bulk cellular DNA from bacteria and preparation of genomic DNA from Saccharomyces cerevisiae.
Abstract: The purpose of this protocol is the isolation of bulk cellular DNA from bacteria (alternatively see Preparation of genomic DNA from Saccharomyces cerevisiae or Isolation of Genomic DNA from Mammalian Cells protocols).

143 citations

Journal ArticleDOI
TL;DR: It is demonstrated that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae and postulate that the APOBec3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.
Abstract: Human cells harbor a variety of factors that function to block the proliferation of foreign nucleic acid. The APOBEC3G enzyme inhibits the replication of retroviruses by deaminating nascent retroviral cDNA cytosines to uracils, lesions that can result in lethal levels of hypermutation. Here, we demonstrate that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae. APOBEC3G expression caused a 20-fold increase in frequency of mutation to canavanine-resistance, which was further elevated in a uracil DNA glycosylase-deficient background. All APOBEC3G-induced base substitution mutations mapped to the nuclear CAN1 gene and were exclusively C/G → T/A transition mutations within a 5′-CC consensus. The APOBEC3G preferred sites were found on both strands of the DNA duplex, but were otherwise located in hotspots nearly identical to those found previously in retroviral cDNA. This unique genetic system further enabled us to show that expression of APOBEC3G or its homolog APOBEC3F was able to inhibit the mobility of the retrotransposon Ty1 by a mechanism that involves the deamination of cDNA cytosines. Thus, these data expand the range of likely APOBEC3 targets to include nuclear DNA and endogenous retroelements, which have pathological and physiological implications, respectively. We postulate that the APOBEC3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.

143 citations

Journal ArticleDOI
TL;DR: Surprisingly, among class A beta-lactamases, PER-1 was most closely related to the recently reported CFXA from Bacteroides vulgatus, with which it shared 40% amino acid identity.
Abstract: We have determined the nucleotide sequence (EMBL accession number, Z 21957) of the cloned chromosomal PER-1 extended-spectrum beta-lactamase gene from a Pseudomonas aeruginosa RNL-1 clinical isolate, blaPER-1 corresponds to a 924-bp open reading frame which encodes a polypeptide of 308 amino acids. This open reading frame is preceded by a -10 and a -35 region consistent with a putative P. aeruginosa promoter. Primer extension analysis of the PER-1 mRNA start revealed that this promoter was active in P. aeruginosa but not in Escherichia coli, in which PER-1 expression was driven by vector promoter sequences. N-terminal sequencing identified the PER-1 26-amino-acid leader peptide and enabled us to calculate the molecular mass (30.8 kDa) of the PER-1 mature form. Analysis of the percent GC content of blaPER-1 and of its 5' upstream sequences, as well as the codon usage for blaPER-1, indicated that blaPER-1 may have been inserted into P. aeruginosa genomic DNA from a nonpseudomonad bacterium. The PER-1 gene showed very low homology with other beta-lactamase genes at the DNA level. By using computer methods, assessment of the extent of identity between PER-1 and 10 beta-lactamase amino acid sequences indicated that PER-1 is a class A beta-lactamase. PER-1 shares around 27% amino acid identity with the sequenced extended-spectrum beta-lactamases of the TEM-SHV series and MEN-1 from Enterobacteriaceae species. The use of parsimony methods showed that PER-1 is not more closely related to gram-negative than to gram-positive bacterial class A beta-lactamases. Surprisingly, among class A beta-lactamases, PER-1 was most closely related to the recently reported CFXA from Bacteroides vulgatus, with which it shared 40% amino acid identity. This work indicates that non-Enterobacteriaceae species such as P. aeruginosa may possess class A extended-spectrum beta-lactamase genes possibly resulting from intergeneric DNA transfer.

143 citations

Journal ArticleDOI
TL;DR: The results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect.
Abstract: Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 105 to 106 copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 μg of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 105 to 106 copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 105 to 106 copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.

143 citations


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