The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling.

Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.

Use of DNA photoaffinity labeling to study nucleosome remodeling by SWI/SNF.

The ability of large protein assemblies to reorganize chromatin in an ATP-dependent manner is an important process for regulation of gene expression and potentially for DNA replication and recombination. The manner in which these proteins remodel chromatin to make the DNA accessible to DNA binding proteins such as transcription factors is not well understood. Site-directed DNA-protein cross-linking has been used to understand the architecture of the SWI/SNF-nucleosomal complex and the mechanistic details of how the nucleosome is remodeled. A detailed protocol for such a study is presented along with examples of the extent of this approach.

Spatial organization of the core region of yeast TFIIIB-DNA complexes.

The interaction of yeast TFIIIB with the region upstream of the SUP4 tRNATyr gene was extensively probed by use of photoreactive phosphodiesters, deoxyuridines, and deoxycytidines that are site specifically incorporated into DNA. The TATA binding protein (TBP) was found to be in close proximity to the minor groove of a TATA-like DNA sequence that starts 30 nucleotides upstream of the start site of transcription. TBP was cross-linked to the phosphate backbone of DNA from bp -30 to -20 in the nontranscribed strand and from bp -28 to -24 in the transcribed strand (+1 denotes the start site of transcription). Most of the major groove of DNA in this region was shown not to be in close proximity to TBP, thus resembling the binding of TBP to the TATA box, with one notable exception. TBP was shown to interact with the major groove of DNA primarily at bp -23 and to a lesser degree at bp -25 in the transcribed strand. The stable interaction of TBP with the major groove at bp -23 was shown to require the B" subunit of TFIIIB. The S4 helix and flanking region of TBP were shown to be proximal to the major groove of DNA by peptide mapping of the region of TBP cross-linked at bp -23. Thus, TBP in the TFIIIB-SUP4 gene promoter region is bound in the same direction as TBP bound to the TATA box with respect to the transcription start site. The B" and TFIIB-related factor (BRF) subunits of TFIIIB are positioned on opposite sides of the TBP-DNA core of the TFIIIB complex, as indicated by correlation of cross-linking data to the crystal structure of the TBP-TATA box complex. Evidence is given for BRF binding near the C-terminal stirrup of TBP, similar to that of TFIIB near the TBP-TATA box complex. The protein clamp formed around the TBP-DNA complex by BRF and B" would help explain the long half-life of the TFIIIB-DNA complex and its resistance to polyanions and high salt. The path of DNA traversing the surface of TBP at the 3' end of the TATA-like element in the SUP4 tRNA gene is not the same as that of TBP bound to a TATA box element, as shown by the cross-linking of TBP at bp -23.

Long-term clinical and hemodynamic evaluation of the Ionescu-Schiley pericardial xenograft and the Braunwald-Cutter and Björk-Shiley prostheses in the mitral position.

Single mitral valve replacement was undertaken in 220 patients between March, 1971, and October, 1977. Bjork-Shiley prostheses (BS) were inserted in 42 patients, Braunwald-Cutter prostheses (BC) in 52, and Ionescu-Shiley pericardial xenografts (PX) in 126. No attempt was made at randomization. The actuarial survival rate was 81.9 +/- 12.8 percent at 7 years for patients with BS prostheses, 41.7 +/- 22.9 percent at 6 years for patients with BC prostheses, and 89.0 +/- 9.3 percent for patients with PX valves 7 years following valve replacement. Of the late deaths in patients with BC prostheses, 62.5 percent were valve related. The incidence of thromboembolism was 4.7, 1.8, and 1.5 episodes per 100 patient-years in the BS, BC, and PX groups, respectively. Long-term anticoagulation was used only in patients with BS and BC prostheses. Late postoperative hemodynamic studies were performed in six patients each with BS and BC prostheses and in 29 patients with PX valves. The mean diastolic gradients at rest were 6.2, 8.3, and 6.4 mm. Hg in the respective groups. The corresponding figures for calculated valve area were 1.8, 1.6, and 2.0 sq. cm. There was no statistically significant difference between the data recorded from the three groups of patients except for the survival rate of the BC prosthesis and the PX valve. The BS prosthesis and the PX valve have similar durability and hemodynamic performance for an almost identical duration of follow-up. Because of the improved quality of life and reduced morbidity without anticoagulants, we are using the PX valve exclusively for heart valve replacement.

Natamycin in genital candidosis in men.

In a trial of natamycin, an antifungal antibiotic in a vanishing cream base, assessment was possible in 66 men with genital or anal candidosis. The overall cure rate was 82 percent, In 43 patients with culturally proven candidosis it was 98 percent. but in 23 patients treated solely on clinical impression it was only 52 percent. Symptoms were rapidly relieved in those who responded and there were no side-effects. In our hands, natamycin 2 per cent cream has proved to be a valuable preparation in the treatment of candidal balanitis.