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C Sasho

Publications and source records attributed to C Sasho.

5 recordsLinked to original sources
PubMed2001

[Colony and phage-plaque direct sequencings by dye-terminator methods].

Direct sequencing using lambda phage DNA and E. coli colonies with plasmid DNA is a very powerful technique. Almost all of the reported direct sequencing methods involve either radioactive sequencing or fluorescent dye-primer sequencing. We present a direct colony sequencing strategy that uses a dye terminator (BigDye terminator kit) together with dye primer sequencing. We found that single-colony sequencing with the terminator yielded about 500 base pairs of sequence information. Signal strength was not improved when the number of cycles increased to 40. The colony used for the sequencing was estimated to contain about 5.6 x 10(7) cells. In addition, although a single plaque consisted of 2 x 10(6) cells, the pfu was not high enough to read with single-cycle sequencing, and only about 300 base pairs of sequence information were obtained from a single plaque using two cycle-sequencing reactions (re-cycle sequencing). The optimal amounts of the template were 500 ng of purified lambda DNA and 1 x 10(7) pfu of the lambda phage suspension, but with BigDye terminator it was possible to detect as little as 50 ng of purified lambda DNA and 2 x 10(6) pfu for lambda phage suspensions. Thus, colony direct sequencing and plaque direct sequencing are estimated to be very useful for rapid and high-throughout screening of genomic and cDNA libraries.

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PubMed2000

Genomic structure and promoter analysis of the human alpha1, 6-fucosyltransferase gene (FUT8).

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1,6-fucosyltransferase (alpha1,6FucT) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the asparagine-linked GlcNAc residue of complex N-glycans via alpha1,6-linkage. We have cloned the genomic DNA which encodes the human alpha1,6FucT gene ( FUT8 ) and analyzed its structure. It was found that the gene consists of at least nine exons spanning more than a 50 kbp genomic region, and the coding sequence is divided into eight exons. The translation initiation codon was located at exon 2, and thus exon 1 encodes only 5'-untranslated sequences. Transcription initiation site of FUT8 was determined by 5'-rapid amplification of the cDNA end and a primer-extension analysis using the total RNA isolated from SK-OV-3 cells, which have a high level of alpha1,6FucT activity. We then characterized the FUT8 promoter region by a reporter gene assay. The luciferase reporter assay indicated that the 5'-flanking region of exon 1, which covered about 1 kbp, conferred the promoter activity in SK-OV-3 cells. This region contains potential binding sites for some transcription factors, such as bHLH, cMyb, GATA-1, as well as a TATA-box, but not a CCAAT motif. 5'-Untranslated sequences found in ESTs and the cDNA for the FUT8 suggest the presence of an additional exon(s) at the upstream of the first exon identified in this study, and therefore, the transcription of the gene would be regulated by multiple promoters.

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