FIG. 4.

Determination of empA transcriptional start by RT-PCR and 5′ RACE analysis. (A) Eight different empA sense-strand primers (lanes 1 to 8, primers empAUSP1, empADSP1, empAUSP2, empADSP2, empAP2-1, empAP2-2, empAP2-3, and empAP2-4, respectively) were used with an empA antisense strand primer (empA-R) to determine the approximate transcriptional start for empA. RNA from LB20- and NSSM-grown M93Sm (top panel) or NB10 (bottom panel) cells was prepared and used in the RT-PCRs. The RT-PCR products were visualized on a 1% agarose TAE gel containing ethidium bromide. Molecular weight standards (indicated in kilobase pairs) are in lanes S. (B) One percent agarose TAE gel showing 5′ RACE products (even-numbered lanes) from M93Sm and NB10 in NSSM. As a negative control, the tailing enzyme, terminal deoxynucleotidyl transferase, was left out of the reaction (odd-numbered lanes). A 1-kbp DNA ladder was used as the size standard (lane S). (C) DNA sequence alignments of dC-tailed and (D) dA-tailed 5′ RACE products showing the empA transcriptional start site (underlined) for NB10 in LB20 and NSSM and M93Sm under NSSM growth conditions.