23 Results
for author "Bruce L. Miller"
- Table 1A. nidulans strains used in this study
Figure 1Duplication of the matA gene in the haploid genome of A. nidulans prevents ascospore formation. Differentiation of cleistothecia and ascospores are compared in the wild type (WT) and a transformant with a matA duplication (T). (Top panels) Mature, dark-pigmented cleistothecia and mature white conidiophores. (Middle panels) (Left) Contents of the individual broken cleistothecium with mature ascospores (arrow). (Right) Undeveloped ascogenous tissue with orange debris (arrow). (Bottom panels) (Left) Mature ascospores (arrow) produced by wild-type strain. (Right) Debris (arrow) with absence of ascospores. Magnification bars are shown.
Figure 2Nuclei carrying duplications of matA do not undergo karyogamy and meiosis. Development of the ascogenous tissue and distribution of nuclei were analyzed at 4 days PI of sexual development. Nuclei are visualized with DAPI. Different stages of ascus development are shown in the wild-type (WT) strain. Prezygotic cell (arrowhead), zygote (thin arrow), and ascus with ascospores (thick arrow) are indicated. Strains carrying a matA gene duplication differentiate ascogenous tissue with normal nuclear distribution up to the prezygotic stage (arrowhead). Nuclei do not undergo karyogamy and meiosis; therefore, neither zygotes nor asci are recognized. Magnification bar: 10 µm.
Figure 3(A and B) Duplication of matA triggers gene silencing and has a downstream effect on the gprA target gene. Developmental expression of the matA transcript (A) and downstream target gprA (B) over the time course of 2, 4, and 6 days PI of sexual development.
Figure 4(A and B) Ectopically introduced matA transgene induces gene silencing at both resident and ectopic matA. (A) Schematic representation of the removal of the ectopic matA transgene using 5′-FOA selection. (B) Expression analysis of the matA gene in a strain having duplication (before FOA) and a strain that was recovered upon excision of the ectopic matA (after 5′-FOA). Transcript levels were analyzed in undifferentiated hyphae (H) and in reproductive tissue 6 days PI of sexual development (R6).- Table 2Fertility in strains carrying an extra copy of matA
Figure 5(A and B) Transgene-derived matA transcript is involved in gene silencing. Schematic summary of the complementation studies is presented. (A) Resident matA wild-type allele or deletion mutant alleles matA(0) or matAΔ, respectively, were complemented in separate experiments by a complete matA transgene that was introduced ectopically. (B) matA deletions introduced ectopically into a wild-type matA [chromosome (chr) III] background. Genetic distance is marked (−1001 to +3056 bp). The solid bar indicates the matA-coding region. The shaded flanking regions represent 5′ and 3′ UTRs. Chromosomal position in the genome is indicated (chr III, chr I). Deleted regions of matA sequence are indicated by dashed lines. RNA status and silencing effect associated with each complementation experiment are shown: present (+), absent (−), silencing present (YES), no silencing (NO), does not apply (N/A), and 70–80% reduced fertility (R).
Figure 6MatA protein is not involved in HDGS phenomenon. Graphic representation of the wild-type matA gene at the resident locus and the ectopically integrated matAfs transgene carrying a frameshift mutation and therefore deficient in native MatA protein. Analyses of matA transcript level in the undifferentiated hyphae (H) and in the reproductive tissue 4 days PI of sexual development (R4).
Figure 7Model of the nucleus-restricted, RNA-mediated premeiotic silencing in A. nidulans. Schematic representation of the prezygotic cell containing two haploid nuclei from a cross between two parental strains is shown. One nucleus carries duplication of the matA gene (solid bars); the other nucleus carries a single copy of matA. Gene silencing is mediated by a transgene-derived matA transcript and appears to be restricted to the nucleus of origin. The silencing effect does not spread between nuclei in the common cytoplasm of the syncytium or dikaryon. Therefore, a parental nucleus with a single wild-type matA gene retains active mat function that is fully able to complement the silenced matA function of the other parental nucleus, resulting in a wild-type sexual phenotype. Framed box with dashed line indicates a potential cytoplasmic compartment that might contribute to nucleus-restricted MatIS in the prezygotic cell. Refer to Discussion for details.