The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex.

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

The complete nucleotide sequence of the Bacillus licheniformis NM105 S-layer-encoding gene.

A protein present on the cell surface of Bacillus licheniformis (Bl) NM105 was identified as an S-layer (OlpA in this paper), a protein present on many bacterial cell surfaces. Purification, SDS-PAGE and isoelectrofocusing showed one 94-kDa, slightly acidic (pI 6.5) protein band (defined as OlpA). The pure protein OlpA, has a tetragonal symmetry of its morphological subunits. Following Edman degradation, three 17-mer oligodeoxyribonucleotide (oligo) probes corresponding to the N-terminal sequence of Olpa were synthesized and used for gene cloning. The nucleotide (nt) sequence of the cloned gene (olpA) showed an ORF and encoded an 874 amino acid (aa) protein. In the promoter region of olpA, there appear to be -10 and -35 sigmaA-binding sites, as well as -10 and -35 regions specific for sigmaH. The existence of these two potential promoters suggests that OlpA would be produced during both the vegetative and sporulating stages of growth. The ribosome-binding site (RBS) sequence perfectly matched its consensus sequence, suggesting a high efficiency of translation of olpA. A typical 29-aa leader peptide, characteristic of secretory proteins in Bacilli, is present in the OlpA pre-protein sequence. In olpA, there are two stem-loop structures in tandem, downstream from the stop codon. These stem-loops are probably involved in prolonged olpA expression, by extending the half life of the mRNA.

Phenotypic differentiation of "smart" versus "naive" bacteriophages of Bacillus subtilis.

The temperate bacteriophages of Bacillus subtilis differ dramatically in their response to the induction of the SOS system during the development of competence and following DNA damage. While all temperate bacteriophages are induced following DNA damage, the "naive" bacteriophages (i.e., phi105 and SPO2) are also induced during the development of competence. On the other hand, "smart" bacteriophages (i.e., phi3T and SPbeta) are not induced during the development of competence, and furthermore, once competence has developed, these prophages can no longer be induced by DNA damage.

Cloning of the crystalline cell wall protein gene of Bacillus licheniformis NM 105.

A protein with a tetragonal pattern, defined as RS protein, was found on the wall surface of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C. The protein was present on the wall surface of the exponential-growth-phase cells, but at the stationary growth phase it was overproduced and hypersecreted. This protein was precipitated to homogeneity from the culture fluid by 80% ammonium sulfate saturation and chilled acetone. The molecular mass of the protein was 98 kilodaltons, and it had a single subunit in a sodium dodecyl sulfate gel. Specific anti-RS antibody was generated in rabbits and used to immunolabel the RS protein on the cells at different growth phases. In early-exponential-growth-phase cells, the outside surface of the wall, the cytoplasm, and the inside surface of the cytoplasmic membrane were labeled. In stationary-growth-phase cells, the cytoplasm was poorly labeled, but the labeling on the outside surface of the wall was high. AB. licheniformis NM 105 gene library was made by using the lambda phage EMBL3. The RS protein expression from this gene library was detected by a modified autoradiographic procedure. One of the amplified RS protein-positive plaques (4213-1) containing recombinant DNA was chosen, and the restriction map of this DNA was prepared. The RS protein expressed in Escherichia coli NM 539 infected with 4213-1 recombinant phage had a lower molecular mass than the purified authentic RS protein. The 4.5-kilobase-pair (kbp) SalI-EcoRI fragment of the recombinant DNA was cloned in the shuttle plasmid pMK4 to construct pMK462, which was expressed in B. subtilis MI112 and produced the RS protein identical in molecular mass to the purified authentic RS protein. The RS protein expression was also demonstrated in cryosections of transformed E. coli and B. subtilis cells by immunoelectron microscopy. The 1.2-kbp SalI-HindIII and 1.8-kbp HindIII-HindIII recombinant DNA restriction enzyme fragments, respectively, from the right of the restriction map produced anti-RS antibody cross-reacting proteins. The expression of the 1.2-kbp SalI-HindIII DNA fragment cloned in pUC8 could be induced with isopropyl-beta-D-thiogalactopyranoside. The 1.8-kbp DNA restriction fragment hybridized with both the chromosomal DNA of strain NM 105 and the recombinant phage 4213-1 DNA. The RS gene expression was finally demonstrated in transformed E. coli 539 cells by in situ hybridization of frozen thin sections with the 1.8-kbp HindIII biotin-dATP probe and immunolabeling these with anti-biotin immunoglobulin G and protein A-gold.

Transient superior vena cava syndrome due to propylthiouracil therapy in intrathoracic goiter.

Intrathoracic goiter is a rare cause of superior vena cava syndrome. We present the findings in a patient in whom the syndrome was precipitated by therapy with propylthiouracil and remitted on withdrawal of the medication. The superior vena cava syndrome did not recur on medical management, suggesting that surgery is not always indicated in this setting.