Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (+/- standard errors) of 5.3 (+/- 4.8) and 4.8 (+/- 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R(2) = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4 degrees C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.

Four different types of adaptation to sulfide among cyanobacteria are described based on the differential toxicity to sulfide of photosystems I and II and the capacity for the induction of anoxygenic photosynthesis. Most cyanobacteria are highly sensitive to sulfide toxicity, and brief exposures to low concentrations cause complete and irreversible cessation of CO(2) photoassimilation. Resistance of photosystem II to sulfide toxicity, allowing for oxygenic photosynthesis under sulfide, is found in cyanobacteria exposed to low H(2)S concentrations in various hot springs. When H(2)S levels exceed 200 muM another type of adaptation involving partial induction of anoxygenic photosynthesis, operating in concert with partially inhibited oxygenic photosynthesis, is found in cyanobacterial strains isolated from both hot springs and hypersaline cyanobacterial mats. The fourth type of adaptation to sulfide is found at H(2)S concentrations higher than 1 mM and involves a complete replacement of oxygenic photosynthesis by an effective sulfide-dependent, photosystem II-independent anoxygenic photosynthesis. The ecophysiology of the various sulfide-adapted cyanobacteria may point to their uniqueness within the division of cyanobacteria.

This review covers briefly the major conditions, genetic and non-genetic, sometimes leading to abnormally elevated methionine, with emphasis on recent developments. A major aim is to assist in the differential diagnosis of hypermethioninemia. The genetic conditions are: (1) Homocystinuria due to cystathionine β-synthase (CBS) deficiency. At least 150 different mutations in the CBS gene have been identified since this deficiency was established in 1964. Hypermethioninemia is due chiefly to remethylation of the accumulated homocysteine. (2) Deficient activity of methionine adenosyltransferases I and III (MAT I/III), the isoenzymes the catalytic subunit of which are encoded by MATMAT. Additional possible genetic causes of hypermethioninemia accompanied by elevations of plasma AdoMet include mitochondrial disorders (the specificity and frequency of which remain to be elucidated). Non-genetic conditions include: (a) Liver disease, which may cause hypermethioninemia, mild, or severe. (b) Low-birth-weight and/or prematurity which may cause transient hypermethioninemia. (c) Ingestion of relatively large amounts of methionine which, even in full-term, normal-birth-weight babies may cause hypermethioninemia.

The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser->>Asn-117 and Ser->>Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/'India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).

Conductive electrodes and electric circuits that can remain active and electrically stable under large mechanical deformations are highly desirable for applications such as flexible displays, field-effect transistors, energy-related devices, smart clothing and actuators. However, high conductivity and stretchability seem to be mutually exclusive parameters. The most promising solution to this problem has been to use one-dimensional nanostructures such as carbon nanotubes and metal nanowires coated on a stretchable fabric, metal stripes with a wavy geometry, composite elastomers embedding conductive fillers and interpenetrating networks of a liquid metal and rubber. At present, the conductivity values at large strains remain too low to satisfy requirements for practical applications. Moreover, the ability to make arbitrary patterns over large areas is also desirable. Here, we introduce a conductive composite mat of silver nanoparticles and rubber fibres that allows the formation of highly stretchable circuits through a fabrication process that is compatible with any substrate and scalable for large-area applications. A silver nanoparticle precursor is absorbed in electrospun poly (styrene-block-butadiene-block-styrene) (SBS) rubber fibres and then converted into silver nanoparticles directly in the fibre mat. Percolation of the silver nanoparticles inside the fibres leads to a high bulk conductivity, which is preserved at large deformations (σ ≈ 2,200 S cm(-1) at 100% strain for a 150-µm-thick mat). We design electric circuits directly on the electrospun fibre mat by nozzle printing, inkjet printing and spray printing of the precursor solution and fabricate a highly stretchable antenna, a strain sensor and a highly stretchable light-emitting diode as examples of applications.

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.

Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival. This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae. Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression. We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus. In a sir-3 mutant, in which both loci are active this preference is not observed.

Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electron-translucent middle plate from an inner plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous corona of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and Colcemid-treated vertebrate somatic (PtK1) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.

A genomic clone (lambda ScG7) from Saccharomyces cerevisiae encoded a 650-nucleotide poly(A)-containing [poly(A)+] RNA that was about 50 times more abundant in MATa cells that had been exposed to the peptide pheromone alpha-factor than in untreated cells. This RNA was transcribed from a cluster of repetitive sequences: both intact and truncated delta and sigma elements adjacent to a tRNATrp gene. Strand-specific probes indicated that this RNA initiated within an intact sigma element and contained sigma sequences at its 5' end. MATa cells produced two other prominent poly(A)+ RNAs (500 and 5,300 bases) in response to alpha-factor that were homologous to the same strand of sigma but transcribed from other locations in the genome. Induction of the sigma-related transcripts was rapid, was not blocked by inhibition of protein synthesis, required a functional receptor (STE2 gene product), and hence appeared to be a primary response to pheromone. Pulse-labeling confirmed that accumulation of sigma RNA following alpha-factor administration was accounted for by an increase in its rate of transcription. The sigma RNAs also were induced in MAT alpha cells that had been treated with a-factor, but were not present at significant levels in MATa/MAT alpha diploids. In MATa cells transformed with a plasmid in which the lambda ScG7 sigma element was inserted just upstream of a gene coding for the intracellular form of invertase (SUC2) lacking its own promoter, a new poly(A)+ RNA (2.2 kilobases) appeared in response to alpha-factor that hybridized to both sigma and SUC2 probes, and intracellular invertase activity was elevated about 10-fold within 30 min. Primer extension showed that transcription from the hybrid gene initiated exclusively within the sigma sequence (117 nucleotides from the 3' end of the element).

When cyanobacteria originated and diversified, and what their ancient traits were, remain critical unresolved problems. Here, we used a phylogenomic approach to construct a well-resolved 'core' cyanobacterial tree. The branching positions of four lineages (Thermosynechococcus elongatus, Synechococcus elongatus, Synechococcus PCC 7335 and Acaryochloris marina) were problematic, probably due to long branch attraction artifacts. A consensus genomic tree was used to study trait evolution using ancestral state reconstruction (ASR). The early cyanobacteria were probably unicellular, freshwater, had small cell diameters, and lacked the traits to form thick microbial mats. Relaxed molecular clock analyses suggested that early cyanobacterial lineages were restricted to freshwater ecosystems until at least 2.4 Ga, before diversifying into coastal brackish and marine environments. The resultant increases in niche space and nutrient availability, and consequent sedimentation of organic carbon into the deep oceans, would have generated large pulses of oxygen into the biosphere, possibly explaining why oxygen rose so rapidly. Rapid atmospheric oxidation could have destroyed the methane-driven greenhouse with simultaneous drawdown in pCO(2), precipitating 'Snowball Earth' conditions. The traits associated with the formation of thick, laminated microbial mats (large cell diameters, filamentous growth, sheaths, motility and nitrogen fixation) were not seen until after diversification of the LPP, SPM and PNT clades, after 2.32 Ga. The appearance of these traits overlaps with a global carbon isotopic excursion between 2.2 and 2.1 Ga. Thus, a massive re-ordering of biogeochemical cycles caused by the appearance of complex laminated microbial communities in marine environments may have caused this excursion. Finally, we show that ASR may provide an explanation for why cyanobacterial microfossils have not been observed until after 2.0 Ga, and make suggestions for how future paleobiological searches for early cyanobacteria might proceed. In summary, key evolutionary events in the microbial world may have triggered some of the key geologic upheavals on the Paleoproterozoic Earth.

Cyanobacterial-bacterial consortial associations are taxonomically complex, metabolically interactive, self-sustaining prokaryotic communities representing pioneer and often the only biota inhabiting extreme aquatic and terrestrial environments. Laminated mats and aggregates exemplify such communities. The fossil record indicates that these associations represent the earliest extant inhabitants and modifiers (i.e. anoxic to oxic conditions) of the Earth's biosphere. Present-day consortia flourish in physically and chemically stressed environments, including nutrient-deplete, hypersaline, calcified, desiccated and high-irradiance ecosystems ranging from the tropics to polar regions. Consortial members exhibit extensive metabolic diversification, but have remained structurally simple. Structural simplicity, while advantageous in countering environmental extremes, presents a 'packaging problem' with regard to compartmentalizing potentially cross-inhibitory aerobic versus anaerobic growth processes. To circumvent these metabolic constraints, phototrophic cyanobacteria and microheterotrophs orient along microscale chemical (i.e. O2, pH, Eh) gradients to meet and optimize the biogeochemical processes (C, N, S cycling) essential for survival, growth and the maintenance of genetic diversity, needed to sustain life. Microscale ecophysiological, analytical, molecular (immunological and nucleic acid) techniques have helped to develop a mechanistic basis for understanding consortial growth and survival under extreme environmental conditions on Earth. Consortia are ideal model systems for developing a process-based understanding of the structural and functional requirements for life in extreme environments representative of the Earth's earliest biosphere and possibly other planets.

We have shown previously that voltage-gated Na(+) channels (VGSCs) are up-regulated in human metastatic disease (prostate, breast and small-cell lung cancers), and that VGSC activity potentiates metastatic cell behaviours. However, the mechanism(s) regulating functional VGSC expression in cancer cells remains unknown. We investigated the possibility of activity-dependent (auto)regulation of VGSC functional expression in the strongly metastatic Mat-LyLu model of rat prostate cancer. Pretreatment with tetrodotoxin (TTX) for 24-72 h subsequently suppressed peak VGSC current density without affecting voltage dependence. The hypothesis was tested that the VGSC auto-regulation occurred via VGSC-mediated Na(+) influx and subsequent activation of protein kinase A (PKA). Indeed, TTX pretreatment reduced the level of phosphorylated PKA, and the PKA inhibitor KT5720 decreased, whilst the adenylate cyclase activator forskolin and the Na(+) ionophore monensin both increased the peak VGSC current density. TTX reduced the mRNA level of Nav1.7, predominant in these cells, and VGSC protein expression at the plasma membrane, although the total VGSC protein level remained unchanged. TTX pretreatment eliminated the VGSC-dependent component of the cells' migration in Transwell assays. We concluded that the VGSC activity in Mat-LyLu rat prostate cancer cells was up-regulated in steady-state via a positive feedback mechanism involving PKA, and this enhanced the cells' migratory potential.

The interactions between colorless sulfur bacteria and the chemical microgradients at the oxygen-sulfide interface were studied in Beggiatoa mats from marine sediments and in Thiovulum veils developing above the sediments. The gradients of O(2), H(2)S, and pH were measured by microelectrodes at depth increments of 50 mum. An unstirred boundary layer in the water surrounding the mats and veils prevented microturbulent or convective mixing of O(2) and H(2)S. The two substrates reached the bacteria only by molecular diffusion through the boundary layer. The bacteria lived as microaerophiles or anaerobes even under stirred, oxic water. Oxygen and sulfide zones overlapped by 50 mum in the bacterial layers. Both compounds had concentrations in the range of 0 to 10 mumol liter and residence times of 0.1 to 0.6 s in the overlapping zone. The sulfide oxidation was purely biological. Diffusion calculations showed that formation of mats on solid substrates or of veils in the water represented optimal strategies for the bacteria to achieve a stable microenvironment, a high substrate supply, and an efficient competition with chemical sulfide oxidation. The continuous gliding movement of Beggiatoa cells in mats or the flickering motion of Thiovulum cells in veils were important for the availability of both O(2) and H(2)S for the individual bacteria.

The advent of oxygenic photosynthesis on Earth may have increased global biological productivity by a factor of 100-1,000 (ref. 1), profoundly affecting both geochemical and biological evolution. Much of this new productivity probably occurred in microbial mats, which incorporate a range of photosynthetic and anaerobic microorganisms in extremely close physical proximity. The potential contribution of these systems to global biogeochemical change would have depended on the nature of the interactions among these mat microorganisms. Here we report that in modern, cyanobacteria-dominated mats from hypersaline environments in Guerrero Negro, Mexico, photosynthetic microorganisms generate H2 and CO-gases that provide a basis for direct chemical interactions with neighbouring chemotrophic and heterotrophic microbes. We also observe an unexpected flux of CH4, which is probably related to H2-based alteration of the redox potential within the mats. These fluxes would have been most important during the nearly 2-billion-year period during which photosynthetic mats contributed substantially to biological productivity-and hence, to biogeochemistry-on Earth. In particular, the large fluxes of H2 that we observe could, with subsequent escape to space, represent a potentially important mechanism for oxidation of the primitive oceans and atmosphere.

Saccharomyces cerevisiae haploid cells, alpha and a, mate after being appropriately stimulated by the pheromone secreted by the opposite cell type (a-factor and alpha-factor, respectively). The binding of a pheromone to its receptor is a signal that initiates a series of intracellular changes that lead to the specific physiological alterations required for mating. To identify components of the signal transduction pathway, we sought pseudorevertants that restored mating competence to receptor mutants (MAT alpha ste3::LEU2). The suppressor srm1-1 was isolated as a recessive mutation that conferred temperature-sensitive growth to all strains and mating ability to MAT alpha ste3::LEU2 strains at the nonpermissive temperature. In addition, when srm1-1 mutants were shifted to the nonpermissive temperature, they exhibited two phenotypes characteristic of pheromone response, induction of FUS1 transcription and accumulation of cells in the G1 phase of the cell cycle. The srm1-1 mutation also suppressed a deletion of the alpha-factor-receptor gene in a cells. Together, these phenotypes suggest that the wild-type SRM1 product is a component of the pheromone response pathway. Deletion of STE4 or STE5, which are required in both haploid cell types for mating and response to pheromone, was not suppressed by srm1-1, suggesting that the SRM1 product may function before the STE4 and STE5 products. SRM1 is an essential gene and is expressed in both haploid cell types as well as in the product of their mating, a/alpha diploids. Homozygous srm1-1 a/alpha diploids were temperature sensitive although they did not arrest in G1. Thus, the SRM1 product may also have a role in the vegetative life cycle of cells.

Protein methylation pathways comprise methionine adenosyltransferase (MAT), which produces S-adenosylmethionine (SAM) and SAM-dependent substrate-specific methyltransferases. However, the function of MAT in the nucleus is largely unknown. MafK represses or activates expression of heme oxygenase-1 (HO-1) gene, depending on its heterodimer partners. Proteomics analysis of MafK revealed its interaction with MATIIα, a MAT isozyme. MATIIα was localized in nuclei and found to form a dense network with chromatin-related proteins including Swi/Snf and NuRD complexes. MATIIα was recruited to Maf recognition element (MARE) at HO-1 gene. When MATIIα was knocked down in murine hepatoma cell line, expression of HO-1 was derepressed at both basal and induced levels. The catalytic activity of MATIIα, as well as its interacting factors such as MATIIβ, BAF53a, CHD4, and PARP1, was required for HO-1 repression. MATII serves as a transcriptional corepressor of MafK by interacting with chromatin regulators and supplying SAM for methyltransferases.

Homothallic switching in S. cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR. The MATa and MAT alpha alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR. Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region. Coconversion events occur on both sides of the double-strand cleavage by the HO gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate.

Stably maintaining specific states of gene expression during cell division is crucial for cellular differentiation. In fission yeast, such patterns result from directed gene rearrangements and chromosomally inherited epigenetic gene control mechanisms that control mating cell type. Recent advances have shown that a specific DNA strand at the matmatids are produced. Therefore, cellular differentiation is a natural consequence of the fact that DNA strands are complementary and nonequivalent. Another epigenetic control that "silences" library copies of mat-information is due to heterochromatin organization. This is a clear case where Mendel's gene is composed of DNA plus the associated epigenetic moiety. Following up on initial genetic studies with more recent molecular investigations, this system has become one of the prominent models to understand mechanisms of gene regulation, genome integrity, and cellular differentiation. By applying lessons learned from these studies, such epigenetic gene control mechanisms, which must be installed in somatic cells, might explain mechanisms of cellular differentiation and development in higher eukaryotes.

The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4',6'-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of alpha-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic gamma- and delta-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.

The yeast S. cerevisiae switches a and alpha cell types by a transposition mechanism that replaces genetic information residing at the mating-type locus (MAT) with information copied from either of the two donor loci, HML and HMR. The donor HML and HMR loci contain the same genetic information as the MATa and MAT alpha alleles, yet they do not switch. Additionally, Strathern et al. (1982) have described an in vivo double-strand DNA break found at subgenomic levels (approximately 2% of MAT DNA) within the MAT locus but not within HML and HMR. We have examined the role of this double-strand DNA break in the switching process. Cell lineage studies show that strains containing deletions of the donor HML and HMR loci produce lethal progeny in the exact pattern described for MAT switching in standard strains. Our interpretation is that the double-strand MAT break in the deletion strains cannot be repaired because of the lack of the donor loci, resulting in cell death. We suggest that the double-strand DNA break is an initiating event for switching and that this event is lethal in the absence of the donor loci. MAT mutants isolated as survivors from this "pedigree of death" define a site required for switching where the double-strand break occurs. We have also examined marl mutant strains in which the donor loci are expressed and observed to switch (Klar et al. 1981a). The double-strand DNA cut appears at the HM loci in these strains. Thus, there is a strong correlation between the presence or absence of the double-strand break at each cassette and its ability or inability to switch as observed at the single cell level.

MCM1 is an essential yeast DNA-binding protein that affects both minichromosome maintenance, in a manner suggesting that it has DNA replication initiation function, and gene expression. It activates alpha-specific genes together with MAT alpha 1, and represses a-specific genes together with MAT alpha 2. Alone, MCM1 can activate transcription. To determine whether different domains of the protein mediate these diverse functions, we constructed and analyzed several mcm1 mutants. The gene expression and minichromosome maintenance phenotypes of these mutants suggest that the role of MCM1 in DNA replication initiation may not involve transcriptional activation. However, both transcription and replication activities require only the 80-amino-acid fragment of MCM1 homologous to the DNA-binding domain of the serum response factor (SRF). This small fragment is also sufficient for cell viability and repression of a-specific genes. A polyacidic amino acid stretch immediately adjacent to the SRF homologous domain of MCM1 was found to be important for activation of alpha-specific genes in alpha cells. Mutants lacking the acidic stretch confer higher expression from an alpha-specific UAS in a cells in addition to lower expression in alpha cells, suggesting that negative regulation at this site occurs in a cells, in addition to the well-documented positive regulation in alpha cells.

Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.

Owing to its superior tribological and mechanical properties with corrosion resistance, biocompatibility, and hemocompatibility, diamond-like carbon (DLC) has emerged as a promising material for biomedical applications. DLC films with various atomic bond structures and compositions are finding places in orthopedic, cardiovascular, and dental applications. Cells grew on to DLC coating without any cytotoxity and inflammation. DLC coatings in orthopedic applications reduced wear, corrosion, and debris formation. DLC coating also reduced thrombogenicity by minimizing the platelet adhesion and activation. However, some contradictory results (Airoldi et al., Am J Cardiol 2004;93:474-477, Taeger et al., Mat-wiss u Werkstofftech 2003;34:1094-1100) were also reported that no significant improvement was observed in the performance of DLC-coated stainless stent or DLC-coated femoral head. This controversy should be discussed based on the detailed information of the coating such as atomic bond structure, composition, and/or electronic structure. In addition, instability of the DLC coating caused by its high level of residual stress and poor adhesion in aqueous environment should be carefully considered. Further in vitro and in vivo studies are thus required to confirm its use for medical devices.