Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown.

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.

Purification of an active monomeric recombinant HIV-1 trans-activator.

A method for the purification of a truncated, biologically active human immunodeficiency virus type 1 (HIV-1) trans-activator (rTAT) from recombinant Escherichia coli is reported here. The purification steps utilized include mild extraction (French press), concentration by ammonium sulfate precipitation, chromatography in 8 M urea on an S-Sepharose fast-protein liquid chromatography column, and finally, resolution by C-4 reverse-phase high-performance liquid chromatography. After the final step, the rTAT is dried and stored under salt-free conditions. Amino acid compositional analysis and N-terminal sequence analysis confirm that the purified protein is rTAT. Unlike other methods reported for purification of recombinant HIV-1 trans-activator, our protocol uses urea instead of guanidine HCl. The rTAT is fully soluble in buffered solutions at concentrations exceeding 10 mg/ml, migrates as a single 14 kDa species on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE gels with a pI of 9.3 +/- 0.3. Additionally, the rTAT migrates as a monomer on size-exclusion chromatography columns under native conditions. Finally, purified rTAT exhibits trans-activator activity when introduced into appropriate reporter cells. Since rTAT is monomeric when tested by gel filtration, and yet exhibits biological activity, we conclude that the method of purification we have utilized is distinct from all other methods reported to date.

The binding of a novel bisheteroarylpiperazine mediates inhibition of human immunodeficiency virus type 1 reverse transcriptase.

The bisheteroarylpiperazines (BHAPs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and specifically block HIV-1 replication (Romero, D. L., Busso, M., Tan, C.-K., Reusser, F., Palmer, J. R., Poppe, S. M., Aristoff, P. A., Downey, K. M., So, A. G., Resnick, L., and Tarpley, W. G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8806-8810). Here we show that the radiolabeled BHAP [3H]U-88204 binds specifically to HIV-1 RT with high affinity (KD of 50 nM) and a stoichiometry of 1 mol of U-88204 per 1 mol of p66/p51 RT heterodimer. Binding of [3H]U-88204 to RT is unaffected by the presence of saturating poly(rC).oligo (dG)12-18 template-primer. Direct measurement of competition between [3H]U-88204 and other RT inhibitors for binding to RT reveals mutually exclusive competition between [3H]U-88204 and the non-nucleoside RT inhibitor BI-RG-587 (Kopp, E. B., Miglietta, J. J., Shrutkowski, A. G., Shih, C.-K., Grob, P. M. and Skoog, M.T. (1991) Nucleic Acids Res. 19, 3035-3039), indicating that both share the same binding site. Phosphonoformate in concentrations up to 50 microM shows no competition with [3H]U-88204 for binding to RT either alone or in the presence of template-primer. Dideoxynucleotide RT inhibitors affect the binding of [3H]U-88204 to RT when complementary template-primer is present. [3H]U-88204 and the dideoxynucleotide ddGTP can bind RT simultaneously, but the presence of one ligand decreases the affinity of RT for the second. Inasmuch as ddGTP approximates the nucleotide substrate of RT, the direct demonstration of an RT-dideoxynucleotide-[3H]U-88204 complex validates the use of indirect kinetic methods to assess the strength of BHAP interaction with RT and suggests that RT inhibition by U-88204 is achieved via effects on nucleotide substrate binding.

Isolation of transformation-deficient Streptococcus pneumoniae mutants defective in control of competence, using insertion-duplication mutagenesis with the erythromycin resistance determinant of pAM beta 1.

Several transformation-deficient mutants of Streptococcus pneumoniae were isolated after insertion-duplication mutagenesis. Mutagenesis was accomplished by transformation of competent cells with chimeric DNA formed by the ligation of TaqI fragments of pneumococcal DNA to the erythromycin resistance determinant of the streptococcal plasmid pAM beta 1. The two mutants described were characterized as defective in the control of competence induction, possibly due to a block in the production of the intercellular competence-inducing protein.