We were unable to map more than three or four alpha-tubulin loci because only four loci were contained in restriction fragments polymorphic in the two parents in HindlII and EcoRI
5-1 [micro]g) was digested by restriction enzymes EcoRI
and MseI for 1 h at 37[degrees]C.
The pre-amplification DNA was diluted 5-fold with ddH2O and selective amplifications were carried out by using different EcoRI
and Tru9' primer combination (Alpha DNA).
The presence and size of the inserted DNA was confirmed by gel electrophoresis of plasmids digested with EcoRI
for 2 h at 37[degrees]C.
Eight Msel and EcoRI
selective primers with sixty-four primer combinations were tested.
Ten micrograms of high-molecular-weight DNA was digested with 200 units of EcoRI
, HindIII, and BamHI (Promega, Madison, Wis) in the presence of 4 mmol/L spermidine (Sigma) and 100 mg/mL bovine serum albumin (Sigma) for 3 to 4 hours, precipitated in ethanol, and reconstituted in TE buffer (10 mmol/L Tris-HCl, pH 7.
380 was generated; two restriction sites, EcoRI
and NheI, were found to flank the promoter sequence.
Seven clones with inserts of the expected size were identified after EcoRI
and HindIII digestion.
Plasmid Miniprep Kit (Omega, Norcross, GA, USA) and identified by EcoRI
restriction digestion (Promega).
Both strains were made self-bioluminescent by transformation, using standard methods previously described (9), with the broad-host vector pBBR1MCS-2 (17) carrying the luxCDABE cassette from Photorhabdus luminescens as an EcoRI
primer Mse I primer Total No selective selective number of nucleotides nucleotides bands 1.
Each amplification product was digested by EcoRI
, and restriction profiles were observed by gel electrophoresis.