Figure 3.

Validation of the classification of induced transcripts in the transition library. A – Analysis by northern blot was carried out with RNA from mycelium during transition to yeast colleted at 22 h, 48 h and 6 days after the temperature shift. Total RNA was fractionated on a 1.2% formaldehyde agarose gel and hybridized to the cDNA inserts Aspartyl proteinase (asp) and Sugar transporter protein (stl). Ribosomal RNAs are shown as the loading control. The sizes of the transcripts are as follows: asp 1.7 kb; stl 2.65 kb. B – Validation of some novel genes of P. brasiliensis. Semi-quantitative RT-PCR of RNAs obtained from mycelium in transition to yeast. Semi-quantitative RT-PCR analysis was carried out with specific primers, as described. Gray bars indicate the transcript level for the L34 ribosomal protein and black bars refers to the described new transcript. Numbers associated with the bars indicate fold differences relative to the data for the reference mycelium, which were established by densitometry analysis. Using varied number of cycle numbers, the exponential phase of each primer was determined and used to allow semi-quantitative analysis of the respective reactions. The same amount of cDNA was used for all PCRs. The RNAs used for RT-PCR were obtained from samples of: mycelium (M) and mycelium in transition to yeast after 22 h of the temperature shift (T). Genes and sizes of the respective amplified fragments are as follows in bp: dead: 408; hlp: 274; uvsB: 318; cts3: 268; gma12: 152; mnn2: 363; gdpase: 126; samB: 114; dphs: 284; pss: 281; glcaseI: 359; glnl: 368.