F. Galibert et al.

摘要: Usually-10 pmol of DNA were restricted with a given endonuclease. The fragments were dephosphorylated and labelled with ['y-32P] ATP and polynu-cleotide kinase as described (Hrisse et al., 1980). To separate the two labelled ends, fragments were denatured at 920C in 30% dimethyl sulfoxide and fractionated in a polyacrylamide gel (Maxam and Gilbert, 1980). 5'-labelled single-stranded fragments were recovered from the gel and subjected to chemical degradation with reagents specific for G, AG, CT, C and AC. The enzymatic method of Sanger et al.(1977) was also used. To this end the PstI fragment was digested with HaeIII, or FnuDII or partially digested with Bal3 1. Products were fractionated on polyacrylamide or agarose gels and subcloned in the SmaI site of vector Ml3mp9. After transformation of JM10I E. coli cells, white plaques were used to make templates which were sequenced using the M13 sequencing kit of Amersham. As a result of this, both strands were independently analyzed several times. All restriction sites used as starting points were analyzed as interval sites within overlapping fragments. Figure 1 summarizes the sequencing strategy and indicates the sites of restriction used for these analyses.