In an approach to clone and characterize centromeric DNA sequences of Candida albicans by chromatin immunoprecipitation, we have used antibodies directed against an evolutionarily conserved histone H3-like protein, CaCse4p (CENP-A homolog). Sequence analysis of clones obtained by this procedure reveals that only eight relatively small regions (Ϸ3 kb each) of the Can. albicans genome are selectively enriched. These CaCse4-bound sequences are located within 4-to 18-kb regions lacking ORFs and occur once in each of the eight chromosomes of Can. albicans. Binding of another evolutionarily conserved kinetochore protein, CaMif2p (CENP-C homolog), colocalizes with CaCse4p. Deletion of the CaCse4p-binding region of chromosome 7 results in a high rate of loss of the altered chromosome, confirming that CaCse4p, a centromeric histone in the CENP-A family, indeed identifies the functional centromeric DNA of Can. albicans. The CaCse4p-rich regions not only lack conserved DNA motifs of point (<400 bp) centromeres and repeated elements of regional (>40 kb) centromeres, but also each chromosome of Can. albicans contains a different and unique CaCse4p-rich centromeric DNA sequence, a centromeric property previously unobserved in other organisms.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome H behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes. (8,9). In contrast, however, the centromere regions of the fission yeast Schizosaccharomyces pombe resemble those of higher eucaryotes in containing several classes of repeated DNA sequences which encompass many kilobases (kb) of DNA on each of the three S. pombe chromosomes (6,7,10,24). Centromeric repeats designated K, L, and B are heterochromatic-like and untranscribed (10). The centromere regions of the three S. pombe chromosomes, including all the centromere-specific DNA sequence repeats, reside on three large genomic SalI restriction fragments of 65, 100, and 150 kb, derived from chromosomes I, II, and III, respectively (10).Recently Gutz et al. (12). DNA transformations of S. pombe were performed as described before (16). Stable Ura+ transformants obtained by site-directed integration of pSp500 (Fig. 1) into SBPD400 were identified as follows. A total of 240 Ura+ transformant colonies were picked onto nonselective medium and subsequently replica-plated for seven rounds of nonselective growth before they were replica-plated to medium selecting for the Ura+ phenotype. Those clones (14 total) that were still Ura+ after several rounds of nonselective growth and that contained sequences hybridizing to pBR322 were characterized further.

The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system. Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat. The S. pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes. Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy. Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences. Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S. pombe.

The oncoprotein E7 of human papilloma viruses (HPV) is involved in the pathogenesis and maintenance of human cervical cancers. The most prevalent HPV types found in cervix carcinomas are HPV16, 18 and 45. The structure of the E7 dimer from HPV45 (PDB 2F8B) was determined by nuclear magnetic resonance spectroscopy. Each monomer comprises an unfolded N-terminus and a well-structured C-terminal domain with a b1b2a1b3a2 topology representing a unique zinc-binding fold found only for E7. Dimerization occurs through the a1/a1 0 helices and intermolecular b-sheet formation but excludes the zinc-binding sites. E7 is reported to interact with a number of cellular proteins (e.g. pRb, p21 CIP1 ). Binding of a peptide derived from the C-terminus of p21CIP1 to the Cterminal domain of E7 was characterized by monitoring chemical shift perturbations of the amide groups of E7. This provides direct evidence that a shallow groove situated between a1 and b1 of the E7 C-terminal domain is interacting with the C-terminus of p21 CIP1. Intriguingly, this binding site overlaps with the low-affinity binding site on E7 for the C-domain of pRb.

We have developed a dual-chamber microfluidic microbial fuel cell (MFC) system that allows on-chip bacterial culture and conversion of bacterial metabolism into electricity. The micro-MFC contains a vertically stacked 1.5 microL anode chamber and 4 microL cathode chamber, and represents the smallest MFC device to our knowledge. Microfluidic deliveries of growth medium and catholyte were achieved in separate flow channels without cross-channel mass exchange. After inoculation of electrogenic Shewanella oneidensis strain MR-1, current generation was observed on an external load for up to two weeks. Current production was repeatable with replenishment of organic substrates. A maximum current density of 1300 A/m(3) and power density of 15 W/m(3) were achieved. Electron microscopic studies confirmed large-scale, uniform biofilm growth on the gold anode, and suggested that the enhanced cell/anode interaction in the small volume may accelerate start-up. Our result demonstrates a versatile platform for studying the fundamental issues in MFCs on the micro-scale, and suggests the possibility of powering nanodevices using on-chip bioenergy.