Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The plasmid pAZ1, which determines trimethoprim and sulfonamide resistance, was characterized by restriction endonuclease mapping. The restriction map was identical to that of the incQ plasmid RSF1010 over a 5.1-kbp region. The type III dihydrofolate reductase gene was cloned, and the DNA sequence was determined. The predicted protein had 162 amino acid residues, and it was more closely related to the gram-negative bacterial chromosomal dihydrofolate reductases than to other plasmid or vertebrate dihydrofolate reductases. Sequence identity was 51% with the Escherichia coli enzyme and 44% with the Neisseria gonorrhoeae enzyme.
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PMID:Characterization of plasmid pAZ1 and the type III dihydrofolate reductase gene. 284 Jun 79

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.
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PMID:The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism. 330 67

Transposon Tn4132, encoding the type Ib trimethoprim-resistant, dihydrofolate reductase, was transposed from the clinical plasmid pUK163 to a small recombinant plasmid pZMR12. Restriction endonuclease and partial sequence analysis of Tn4132 revealed a close relationship to Tn7. The nucleotide sequence of the dhfrIb trimethoprim-resistance gene was determined and the gene and its product were found to share significant homology with the dhfrIa, V, VI and VII plasmid-encoded dihydrofolate reductases. Extensive sequence homology (88%) was observed with the type V dihydrofolate reductase at both the nucleotide and amino acid level. Oligonucleotide probes, distinguishing between the dhfrIb and dhfrV genes, were designed. The discriminatory capabilities of these probes in future epidemiological studies will permit a more accurate determination of the dissemination of these two closely related trimethoprim-resistance genes than has previously been possible.
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PMID:Nucleotide sequence and genetic analysis of the type Ib trimethoprim-resistant, Tn4132-encoded dihydrofolate reductase. 770 67

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR-Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod-2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off-target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9-mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.
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PMID:First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites. 2593 77