Having identified the genes which regulate conidiation, the time of their expression could be studied. Total RNA was isolated from cultures and analyzed by Northern blots using brlA, abaA and wetA probes. The experiment detected transcripts of brlA at 10 hours, of abaA at 15 hours and of wetA at 25 hours. The brlA mutation blocked the accumulation of all three RNAs. The abaA and wetA mutations reduced the accumulations of the brlA and abaA RNAs and blocked accumulation of the wetA RNA. The brlA and abaA mutations affected their own expression and the expression of one another. The wetA transcript was absent in wetA temperature-sensitive mutants grown at a restrictive temperature, implying that the wetA gene is autoregulatory (Mirabito et al., 1989). Subsequently, mutations in six genes were isolated that affect conidiophore development and result in cotton-like colonies with a "fluffy" morphology. These were designated flbA, flbB, flbC, flbD, flbE and fluG and the expression of brlA was reduced in these mutants. The non-regulatory developmentally activated genes were divided into early, middle and late depending on the timing of their expression. The three genes brlA, abaA and wetA define a linear dependent pathway (Figure 7.4) in which the activation of brlA is sufficient to initiate a cascade of events that involve other genes (Adams, 1995). By examining patterns of RNA accumulation in mutant strains, the developmentally activated genes were divided into four categories (Timberlake and Marshall, 1988; Mirabito et al., 1989). Class A genes are activated by either brlA or abaA or both, independent of wetA (Figure 7.5). wetA activates Class B genes, independent of brlA and abaA. The brlA, abaA and wetA together activate Class C and Class D genes. The accumulation of wetA mRNA requires wetA+ activity, suggesting that wetA is autogenously regulated (Marshall and Timberlake, 1991).
The primary structure of the brlA gene was determined and the inferred polypeptide sequence resembled coordination sites that are typical of "zinc finger" DNA binding motifs, indicating that the brlA protein is a sequence specific DNA-binding protein—a transcription factor—that activates the expression of a conidiation-specific gene cluster.
PROBE 0 brlA abaA wetA
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