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Example sentences for: cacet
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Here we have shown that the RNA triphosphatases Pct1 and CaCet1 are essential for viability of S. pombe and C. albicans, respectively The conclusion that CaCet1 is essential is based on a finding that none of the 54 independent isolates in the single-transformation test were homozygous for cacet1Δ; our interpretation is consistent with criteria established by Mitchell and colleagues for inference of essentiality using this genetic approach.
The yeast triphosphatase has a novel tertiary structure in which the active site is situated within a topologically closed hydrophilic tunnel composed of 8 antiparallel β strands, which are conserved in CaCet1 and Pct1 [ 2 ] . Mutational analysis of Cet1 has identified 15 individual side chains within the tunnel that are important for Cet1 function in vitro and in vivo [ 7 8 9 ] . Each of the 8 strands contributes at least one functional group to the active site.
The CaCET1 gene was disrupted by insertion of a UAU1 cassette [ 16 ] . We first constructed plasmid pKS-5'3'CaCET1, in which a 665-bp PCR fragment derived from the 5' end of the CaCET1 gene (from nucleotides -50 to +615 of the open reading frame, with the A residue of the ATG translation start codon defined as position +1) was cloned between the Kpn I and Xba I sites of pBluescript KS +and a 720-bp fragment extending from position +1518 of the 1560-nt CaCET1 coding sequence into the 3' flanking genomic region was inserted between the Sac I and Sac II sites of pBluescript KS +The 3.8-kbp UAU1 gene was excised from pBME101 with Xba I and Sac II and inserted between the Xba I and Sac II sites of pKS-5'3'CaCET1 to yield pCaCET1::UAU1.
This analysis identified 16/25 of the Arg +transformants as CaCET1/cacet1::UAU1 heterozygotes.
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