For A. niger
and previously characterized gene products, given names are also included. This phylogenetic tree was built using the neighbor joining algorithm with 32 000 Entospletinib bootstrap replicates. Based on sequence identities, the S. cerevisiae Tps1 protein was selected by the software as outgroup. Optional settings or use of other algorithms gave identical, or very similar, results. Two-hybrid assay to reveal putative protein-protein interactions In order to determine whether the homologous proteins physically interact, as has been reported in S. cerevisiae, we performed a bacterial-based two-hybrid assay screening for interactions between all six A. niger proteins. For each protein, the full-length open reading frame was cloned into an expression vector and co-transformed into E. coli cells. All 36 possible combinations of A. niger proteins were screened, together with two clones containing different subunits of the leucine zipper GCN4 serving as a positive control and four combinations of one A. niger
protein and one bacterial protein serving as negative controls. Results with no interactions were repeated at least once in an additional independent two-hybrid assay. Where interactions were detected, the assay was repeated in at least two independent assays. Results indicated that TpsB interacts click here with TpsA, TpsB and TppA, and that all Tps units Adriamycin molecular weight interact with themselves (Table 4). All putative interactions involving either TppB or TppC did not score any signals above the negative controls (data not shown). why Table 4 Protein-protein interactions assayed by Bacterial adenylate cyclase two-hybrid system Protein TpsA TpsB TpsC TppA TpsA 418 (210–863)* 1746 (1582–1799) 113 (77–135) 71 (43–89) TpsB 1593 (1467–1832) 1776 (1658–1988) 441 (341–560) 581 (322–714) TpsC 172 (101–244) 688 (315–980) 1214 (861–1551) 80 (67–102) TppA 429 (167–656) 691 (462–987) 156 (133–198) 83 (58–98) *Estimated values are in units/mg dry weight
bacteria. Values in parentheses are the highest and lowest scores for each based on three to four independent assays. The positive control zip-zip (T18 and T25 fragments of the leucine zipper of GCN4) was scored to 3429 (2938–4270). Negative controls and remaining protein interactions scored at maximum 220 (zip-tpsA) but usually less than 50. Values in bold are considered true protein-protein interactions. Gene expression during conidial outgrowth Gene expressions were quantified during different stages of A. niger development. Preliminary results showed that due to the extractability of different structures, two RNA extraction protocols (see Methods) were required: The first included high force to break the tough cell walls of conidia and early germination structures; and, the second was more efficient for fragile structures. Notably, the second protocol was not vigorous enough to extract any RNA from spores (data not shown).