Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y⋅) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to ≈90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 μmol⋅min−1⋅mg−1 and of (His)6-Y2 [(His)6-Y2-K387N] from yeast is 0.037 μmol⋅min−1⋅mg−1 (0.125 μmol⋅min−1⋅mg−1). Y2, Y3, and Y4 isolated from E. coli have no measurable activity. Efforts to generate Y⋅ in Y2 or Y4 using Fe2+, O2, and reductant have been unsuccessful. However, preliminary studies show that incubation of Y4 and Fe2+ with inactive E. coli Y2 followed by addition of O2 generates Y2 with a specific activity of 0.069 μmol⋅min−1⋅mg−1 and a Y⋅. A similar experiment with (His)6-Y2-K387N, Y4, O2, and Fe2+ results in an increase in its specific activity to 0.30 μmol⋅min−1⋅mg−1. Studies with antibodies to Y4 and Y2 reveal that they can form a complex in vivo. Y4 appears to play an important role in diiron-Y⋅ assembly of Y2.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

The second STE12 homologue of Cryptococcus neoformans is MATa-specific and plays an important role in virulence

Cryptococcus neoformans STE12α, a homologue of Saccharomyces cerevisiae STE12, exists only in MATα strains. We identified another STE12 homologue, STE12a, which is MATa specific. As in the case with Δste12α, the mating efficiency for Δste12a was reduced significantly. The Δste12a strains surprisingly still mated with Δste12α strains. In MATα strains, STE12a functionally complemented STE12α for mating efficacy, haploid fruiting, and regulation of capsule size in the mouse brain. Furthermore, when STE12a was replaced with two copies of STE12α, the resulting MATa strain produced hyphae on filament agar. STE12a regulates mRNA levels of several genes that are important for virulence including CNLAC1 and CAP genes. STE12a also modulates enzyme activities of phospholipase and superoxide dismutase. Importantly, deletion of STE12a markedly reduced the virulence in mice, as is the case with STE12α. Brain smears of mice infected with the Δste12a strain showed yeast cells with a considerable reduction in capsule size compared with those infected with STE12a strains. When the disrupted locus of ste12a was replaced with a wild-type STE12a gene, both in vivo and in vitro mutant phenotypes were reversed. These results suggest that STE12a and STE12α have similar functions, and that the mating type of the cells influences the alleles to exert their biological effects. C. neoformans, thus, is the first fungal species that contains a mating-type-specific STE12 homologue in each mating type. Our results demonstrate that mating-type-specific genes are not only important for saprobic reproduction but also play an important role for survival of the organism in host tissue.

Plays and poetry : manuscript, [17--]

Plays and poems, chiefly satires, in support of American independence and patriotic causes.

ISDS and TTIP – A miracle cure for a systemic challenge? EPC Policy Brief, 14 July 2014

The European Commission decided to carry out a public consultation, which closed on 13 July, on the possible inclusion of investor-to-state dispute settlement (ISDS) provisions in the Transatlantic Trade and Investment Partnership (TTIP). This decision came in a context of polarised public debate around this procedural mechanism which enables investors to bring a case against a country that hosts their investments. Even though the main question is whether or not to include ISDS in TTIP, the Commission eyes the public consultation as a way to correct the inconsistencies of the mechanism by modernising it. However, the inclusion of ISDS in TTIP will not be a miracle cure because ISDS is a complex multilayered systemic challenge which requires a multilateral solution. In this Policy Brief, Romain Pardo explores which challenges are posed by ISDS and the extent at which TTIP’s contribution can tackle these challenges.

Dates and performances of Thomas D'Urfey's plays.

"Intended to confirm, to supplement, and occasionally to correct the information contained in Genest's account of the English stage and in Allardyce Nicoll's histories of the restoration and eighteenth-century drama."

How to teach kitchen garden; or, Object lessons in household work, including songs, plays, exercises, and games illustrating household occupations.

Mode of access: Internet.

Recital pieces the whole world plays : containing forty-seven compositions, especially adapted for solo playing, by the most celebrated modern and contemporary composers /

Contains works by Moussorgsky, Arensky, Brahms, Debussy, Dvorak, Gliére, Grieg and others.

The Plays, histories, and novels of the ingenious Mrs. Aphra Behn : with life and memoirs /

Each volume has also, as special t.-p., a reproduction of the original t.p.; v. 1-4: Plays written by the late ingenious Mrs. Behn. In four volumes, 3rd ed., London, 1724; v. 5-6: All the histories and novels written by the late ingenious Mrs. Behn, intire in two volumes. Pub. by Mr. Charles Gildon.

Archaelogic and historic fragments. Containing, inter alia, a facsimile of a rare ms. page dated 1638, having reference to two of Shakespeare's most famous plays, with notes thereon.

On a ms. list of plays of the year 1638. -- Sir Anthony Browne, standard bearer to king Henry VIII, and his descendants. -- On the source and nomenclature of the river Thames. -- On Sir Philip Sidney and his father, Sir Henry Sidney, in relation to Ludlow castle. -- Episodes in the career of Humphrey duke of Gloucester, and his first duchess, and their connection with the abbey of St. Albans. -- On the involuntary visit of Philip of Austria and Juana of Spain to Weymouth in 1506, and its consequences. -- Recuiver, the Regulbium of the Romans. -- Appendix.

Books of the time of the restoration, being a collection of plays, poems and prose works produced between the years 1660 and 1700, by the contemporaries of John Dryden /

The entries supplement and illustrate the works of the authors in "The literature of the restoration" issued May 1918. cf. Prefatory note.

Key to ghostism. : Science and art unlock its mysteries /

Mode of access: Internet.

Pawns and Cophetua : four poetic plays /

The storm.--The god of quiet.--X=0.--Cophetua.

Chief contemporary dramatists; twenty plays from the recent drama of England, Ireland, America, Germany, France, Belgium, Norway, Sweden, and Russia,

--Appendix; Authors and plays. Notes on the production of plays. A reading list in contemporary dramatists. A working book list in contemporary drama. Index to charaters.

Children's piano pieces the whole world plays : a collection of more than one hundred and fifty tuneful piano pieces for juvenile players, including standard teaching and recreation pieces, and in addition a considerable number of new compositions /

Mode of access: Internet.