Characterization of the SarA virulence gene regulator of Staphylococcus aureus

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum-sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA-binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of alpha-helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high-affinity binding sites composed of two half-sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (KD) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half-sites and may involve other sites located downstream of the promoters.     

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.     

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.     

Site-directed mutagenesis of the ''zinc finger'' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.     

Four distinct nuclear proteins recognize in vitro the proximal promoter of the bean seed storage protein β-phaseolin gene conferring spatial and temporal control

A proximal promoter (-422/-13) of the bean seed storage protein beta-phaseolin gene contains cis-regulatory elements conferring spatial and temporal gene regulation. To correlate trans-acting elements with these cis-elements, we performed gel mobility shift and exonuclease III protection assays using bean seed nuclear proteins, and identified target sequences of four DNA-binding proteins associated with this promoter. Three CANNTG motifs, CACGTG (-248/-243), CACCTG (-163/-158), and CATATG (-100/-95), were determined as target sequences of the same DNA-binding protein designated CAN. Competition assays using oligonucleotides containing the wild-type or mutated CANNTG motif indicated that the CANNTG motif appears to be a preferred target sequence for CAN binding. Competition assays also demonstrated that DNA-binding protein AG-1 binds to AAAAAG(A/G)CAA (-356/-347, -191/-182), CA-1 binds to two CA-rich sequences (-201/-192, -175/-160), and that a TATA-box binding protein binds to either TATATAA (-43/-37) or TATAAA (-32/-27) or both. Based on these and other results, it is proposed that CACGTG motif (-248/-243) is a major cis-acting regulatory element conferring spatial and temporal control of the beta-phaseolin gene.     

The Optimedin gene is a downstream target of Pax6

The Optimedin gene, also known as Olfactomedin 3, encodes an olfactomedin domain-containing protein. There are two major splice variants of the Optimedin mRNA, Optimedin A and Optimedin B, transcribed from different promoters. The expression pattern of the Optimedin A variant in the eye and brain overlaps with that for Pax6, which encodes a protein containing the paired and homeobox DNA-binding domains. The Pax6 gene plays a critical role for the development of eyes, central nervous system, and endocrine glands. The proximal promoter of the Optimedin A variant contains a putative Pax6 binding site in position -86/-70. Pax6 binds this site through the paired domain in vitro as judged by electrophoretic mobility shift assay. Mutations in this site eliminate Pax6 binding as well as stimulation of the Optimedin promoter activity by Pax6 in transfection experiments. Pax6 occupies the binding site in the proximal promoter in vivo as demonstrated by the chromatin immunoprecipitation assay. Altogether these results identify the Optimedin gene as a downstream target regulated by Pax6. Although the function of optimedin is still not clear, it is suggested to be involved in cell-cell adhesion and cell attachment to the extracellular matrix. Pax6 regulation of Optimedin in the eye and brain may directly affect multiple developmental processes, including cell migration and axon growth.