Analysis of a Human Transfer RNA Gene Cluster and Characterization of the Transcription Unit and Two Processed Pseudogenes of Chimpanzee Triosephosphate Isomerase Page: 9
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was sequenced using the universal primer to confirm the
overlap between the 2.1-kb BamHI and 1.6-kb Smal/BamHI
fragments. The M13mpl9 subclone containing the 1.9-kb
insert was sequenced using the primer 5'-CCAGGCTGAAGTGCAGTGG-
3' (around residue 1909 in Fig. 2) to confirm the overlap
between the 1.6-kb Smal/BamHI and 1.5-kb Smal fragments.
After the completion of the sequence of the 1.5-kb Smal
fragment, it was necessary to extend the sequence, since
only about 90 bp of the flanking region upstream from the
valine tRNAcAc gene was contained in the 1.5-kb Smal
fragment. To accomplish this, the 5.6-kb AccI fragment
(Fig. 1A) was filled-in with the Klenow fragment of E. coli
DNA polymerase I (Maniatis et al., 1982). One aliquot of
the blunt-ended AccI fragment was digested with Kpnl, and
the resulting fragments cloned into Kpnl/Smal-digested
M13mpl9RF for transformation into E. coli JM101. The single-
strand phage DNAs were size selected for the 2.0-kb
Kpnl/AccI insert. The primer 5'-GCCGGGATAGCTCTAGGC-3'
(around nucleotide 473 in Fig. 2) was used to extend the
sequence of one strand. To sequence the complementary
strand, the other aliquot of the blunt-ended AccI fragment
was cloned into the Smal site of pUC18 for double-stranded
sequencing (Chen and Seeburg, 1985; Zhang et al., 1988).
The amplified and HPLC-purified plasmid DNA was passed
through an Elutip-d column (Schleicher and Schuell) prior to
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Craig, Leonard C. (Leonard Callaway). Analysis of a Human Transfer RNA Gene Cluster and Characterization of the Transcription Unit and Two Processed Pseudogenes of Chimpanzee Triosephosphate Isomerase, dissertation, August 1990; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc331579/m1/15/: accessed July 18, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .