24 Results
for author "Bruce L. Miller"
- Table 1A. nidulans strains used in this study
- Table 2Primers used in this study
Figure 1Genetic context of the matA gene on chromosome III. Black arrows indicate arrangement and orientation of genes (coding regions) flanking the matA locus. The matA transcript and protein structure are shown. Solid triangle indicates the reference transcriptional start site 1 (TSS1), the start of the transcriptional unit. Open triangle indicates the end of the matA transcript.
Figure 2Schematic representation of the deletion and complementation of matA. (A) Wild type matA allele; solid bar represents coding region (ORF), lightly shaded bars represent matA 5′- and 3′-UTRs, and open bars represent upstream 5′ and downstream 3′ genomic sequences flanking the transcriptional unit. Horizontal solid line represents vector sequence of pWP3. Physical distance is marked (−1001 to +3056 bp). (B) Null matA(0) allele; the matA transcriptional unit was deleted and replaced with the Aspergillus fumigatus argB (AfargB) marker. (C) Complementation via ectopic integration; matA complementing transgene was integrated ectopically at the pyrG89 locus on chromosome I. (D) Complementation via 3′ integration at the matA(0) locus; matA transgene carried on pWP3 was integrated at matA(0) locus via homology between 3′ flanking sequences. The corresponding phenotype is shown: C, cleistothecia; A, ascospores; (+), presence; (−), absence.
Figure 3Sexual development in wild type (wt), matA(0), and complemented A. nidulans strains. Representative strains (A–D) were induced to undergo sexual development as described in Materials and Methods. Abundance of cleistothecia and internal reproductive tissue were examined under specific levels of magnification as indicated. (A, C, and D, top row) Morphology and abundance of mature cleistothecia on rich medium agar plates in wild-type and complemented strains (arrow in A indicates a single cleistothecium). (B, top row) Cluster of hülle cells in the matA(0) strain. (A, C, and D, middle row) Individual mature cleistothecia in wild-type and complemented strains. (B, middle row) Mass of hülle cells in the matA(0) strain. (A, C, and D, bottom row) Efficiency of ascospore production in wild-type and complemented strains, respectively. (B, bottom row) Sexual development in the matA(0) strain is aborted at the stage of unfertilized protocleistothecia, shown as brown spherical structures (thick black arrow) with individual hülle cells (thin black arrow).
Figure 4Comparative transcriptional expression of matA at resident and ectopic loci. Phenotype associated with a specific abundance of matA transcript in the reproductive tissue is indicated. (A) Relative quantitation (RQ) of matA expression from the endogenous matA locus on chromosome (chr) III and from the ectopically integrated matA transgene on chr I. The transgene carries 1000 bp of 5′ flanking upstream sequences. H, undifferentiated hyphae; (R6) reproductive tissue, 6 days postinduction. (B) Relative quantitation of matA expression over a developmental time course at 2, 4, and 6 days postinduction of sexual development. Expression profiles of the endogenous matA, ectopically integrated matA transgene and the matA transgene integrated at resident or ectopic loci are shown.
Figure 5Deletion analysis of the 5′ flanking region of matA. The solid bar indicates the matA coding region, lightly shaded bars represent 5′- and 3′-UTRs. The silencer element is indicated as a small shaded box. Chromosomal positions in the genome are indicated (chr III and chr I). Positions of 5′ deletion end points and internal deletions of upstream flanking sequences are indicated by − and Δ, respectively. Transgene deletions were integrated ectopically at the pyrG locus on chr I. Complementation was observed for all transgene constructs. Transcript presence (+) or absence (−) is indicated. Phenotypes associated with each deletion are indicated. Horizontal brackets indicate positions of mRNA cap sites determined for hyphal and developmental RNAs using RACE (refer to Figure 7).
Figure 6Comparative expression of matA transgene deletions. (A) Developmental expression of matA in transgene strains lacking either 830 bp of upstream sequence or the internal 170 bp proximal to the start of transcription. The level of matA transcript was analyzed in the undifferentiated hyphae, H, and in the reproductive tissue, D, at 4 days postinduction of sexual development. The dashed line box indicates expression of matA at the resident locus in the wild-type strain. The solid line box indicates expression of matA transgenes at the ectopic locus. (B) Expression in additional 5′ deletion strains used to determine position of upstream repressor and promoter elements. Specific 5′ deletion end points or internal deletions are indicated by − or Δ, respectively.
Figure 7Identification of both Inr and novel promoter elements that regulate matA transcription. Mapping of mRNA cap sites identified three zones of transcriptional initiation in hyphae and developmental tissue from wild-type and deletion strains (A–C). Each zone is indicated by brackets. An asterisk marks a fourth zone near position −170 observed in hyphal tissue from derepressed strains matA (−248) and matA (−170). The position of the majority of cap sites within a zone is indicated by a thick arrow. Thin arrows mark two minor sites of mRNA start sites. The reference +1 represents the 5′ end of the largest cDNA previously identified from a sexual development library. Potential Inrs are indicated in blue; novel promoter elements are underlined. The translational start site (TSL) and beginning of the ORF are in red text. A black dot below the text marks the −170 position for reference.