178 research outputs found

    Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block

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    Queuosine (Q), a hypermodified nucleoside, occurs at the wobble position of transfer RNAs (tRNAs) with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells, changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut microflora. However, the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study, we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C. elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C. elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA

    Metal-ion-dependent oxidative DNA cleavage by transition metal complexes of a new water-soluble salen derivative

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    A new water-soluble, salen [salen = bis(salicylidene) ethylenediamine]-based ligand, 3 was developed. Two of the metal complexes of this ligand, i.e., 3a, [Mn(III)] and 3b, [Ni(II)], in the presence of cooxidant magnesium monoperoxyphthalate (MMPP) cleaved plasmid DNA pTZ19R efficiently and rapidly at a concentration ≈ 1 μM. In contrast, under comparable conditions, other metal complexes 3c, [Cu(II)] or 3d, [Cr(III)] could not induce any significant DNA nicking. The findings with Ni(II) complexes suggest that the DNA cleavage processes can be modulated by the disposition of charges around the ligand

    Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

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    Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1′β2, mβ1–β5, mβ1–β6 and mβ4–β5) by transplanting β1, β1′β2, β1–β5, β1–β6 and β4–β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1′β2ESWR SSB was generated by mutating the MtuSSB specific ‘PRIY’ sequence in the β2 strand of mβ1′β2 SSB to EcoSSB specific ‘ESWR’ sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1–β6, MtuSSB, mβ1′β2 and mβ1–β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1–β5 SSB was poor. In contrast, mβ1′β2ESWR SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function

    Eugene L. Opie, 1910

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    Eugene L. Opie, M.D. Inflammation Lecture delivered February 10th, 1910https://digitalcommons.rockefeller.edu/harvey-lectures/1014/thumbnail.jp

    Left ventricle hydatid cyst of heart removed under cardiopulmonary bypass: anesthesia management

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    Hydatid cyst uncommon in heart, echinococcosis is endemine in our country. Hydatid cyst in a heart may causes disturbances in conducting system, Pericarditis congestive cardiac failure. The surgery and anesthetic management become very challenging in this case. We are describing the successful management of such a case of left ventricle hydatid cyst remove under cardio pulmonary bypass in a middle age female

    Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose

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    Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel+) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the Cel+ derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N,N'-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single-amino-acid substitutions. Introduction of chbR from two Cel+ mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel+ phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-β-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-β-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator

    A Single Mammalian Mitochondrial Translation Initiation Factor Functionally Replaces Two Bacterial Factors

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    The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2mt) complements E. coli containing a deletion of the IF2 gene (E. coli ΔinfB). We find that IF1 is no longer essential in an IF2mt-supported E. coli ΔinfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2mt substitutes for the function of IF1. Deletion of this insertion from IF2mt supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2mt) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2

    Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

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    Uracil DNA glycosylase (Ung) initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by ∼7- and ∼170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed ∼5-, ∼3.0- and ∼2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair
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