In order to fully understand how vision is used to guide locomotion it is necessary to know what people look at as they move through the environment. This study provides information, hitherto lacking, regarding gaze behaviour associated with both maintaining and changing the direction of locomotion: activities that are essential for efficient navigation through our cluttered environment. Participants' spatiotemporal gaze patterns were recorded whilst they performed a task requiring that they either maintained a straight walking trajectory or changed their direction of walking by 30 degrees or 60 degrees, left or right, at the midpoint of a 9-m path. Participants were either visually cued to turn when they stepped on a trigger mat placed one step before the mid-point of the walkway (cued trials) or given verbal instruction about the required route prior to the start of each trial (advance knowledge trials). Our clear finding was that for the large majority of the time participants' gaze was aligned with environmental features lying in their current plane of progression both prior to and following the onset of the transition stride during which the direction change was implemented. This gaze behaviour was observed both during cued trials (78% of total fixation time prior to the transition stride onset and 89% following the transition stride onset) and advance knowledge trials (67% prior to transition stride onset, 92% following transition stride onset). When not aligned with the plane of progression, gaze was normally fixated on environmental features related to either known or potential future routes. Prior to changing the direction of walking, individuals invariably made saccadic eye movements in order to align gaze with the end-point of the required travel path. This gaze realignment was invariably accompanied by head reorientation, which was initiated, on average, at the same time as the saccade. On average, participants fixated gaze on their goal (represented by the cue light at the travel path end-point) until after head realignment with the new path was achieved. Additionally, the head was consistently aligned with participants' current walking direction prior to and following the transition stride even on the minority of occasions when they were looking elsewhere. These findings challenge the ecological validity of existing theories of how visual information is used to determine heading direction and are consistent with the proposal that aligning the head with the desired travel direction through coordinated eye and head movements provides the CNS with an allocentric frame of reference that is used to control the movement of the body in space.

Biofilms, accumulations of microorganisms at interfaces, have been described for every aqueous system supporting life. The structure of these microbial communities ranges from monolayers of scattered single cells to thick, mucous structures of macroscopic dimensions (microbial mats; algal-microbial associations; trickling filter biofilms). During recent years the structure of biofilms from many different environments has been documented and evaluated by use of a broad variety of microscopic, physico-chemical and molecular biological techniques, revealing a generally complex 3D structure. Parallel to these investigations more and more complex mathematical models and simulations were developed to explain the development, structures, and interactions of biofilms. The forces determining the spatial structure of biofilms, including microcolonies, extracellular polymeric substances (EPS), and channels, are still the subject of controversy. To achieve conclusive explanations for the structures observed in biofilms the cooperation of both fields of investigation, modelling and experimental research, is necessary. The expanding field of molecular techniques not only allows more and more detailed documentation of the spatial distribution of species, but also of functional activities of single cells in their biofilm environment. These new methods will certainly reveal new insights in the mechanisms involved in the developmental processes involved in the formation and behavior of biofilms.

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.

Genome sequences of two Synechococcus ecotypes inhabiting the Octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C); transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at >50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (ldh, pdhB, ald, and ackA) decreased in the evening, whereas others (pflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.

Molecular oxygen (O2) is a potent inhibitor of key microbial processes, including photosynthesis, N2 fixation, denitrification, sulfate reduction, methanogenesis, iron, and metal reduction reactions. Prokaryote survival and proliferation in aquatic environments is often controlled by the ability to tolerate exposure to oxic conditions. Many prokaryotes do not have subcellular organelles for isolating O2-producing from O2-consuming processes and have developed consortial associations with other prokaryotes and eukaryotes that alleviate metabolic constraints of high O2. Nutrient transformations often rely on appropriate cellular and microenvironmental, or microzonal, redox conditions. The spatial and temporal requirements for microenvironmental overlap among microbial groups involved in nutrient transformations necessitates close proximity and diffusional exchange with other biogeochemically distinct, yet complementary, microbial groups. Microbial consortia exist at different levels of community and metabolic complexity, as shown for detrital, microbial mat, biofilm, and planktonic microalgal-bacterial assemblages. To assess the macroscale impacts of consortial interactions, studies should focus on the range of relevant temporal (minutes to hours) and spatial (microns to centimeters) scales controlling microbial production, nutrient exchange, and cycling. In this review, we discuss the utility and application of techniques suitable for determining microscale consortial activity, production, community composition, and interactions in the context of larger scale aquatic ecosystem structure and function.

A functional copy of the alpha mating type gene of Saccharomyces cerevisiae has been cloned by transformation in yeast. Using the Southern Blotting procedure it has been shown that three distinct genetic loci implicated in mating type interconversion (HML, HMR and MAT) contain sequences homologous to the clone fragment. The restriction fragment associated with each locus exhibits a characteristic size which can be correlated with the mating type allele present at that locus. The characteristic size difference between the a and alpha genetic elements made it possible to demonstrate that the homothallic interconversion of mating types in this yeast occurs by DNA rearrangement as proposed in the 'cassette hypothesis'.

Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate-reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME-1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82-90% of a composite ANME-1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME-1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron-accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]-hydrogenases are lacking in the dataset, genes for an [FeFe]-hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c-type cytochromes were identified, which have not yet been correlated with methanogenesis-related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME-1 to the bacterial sulfate-reducing partner.

Lithification in microbial ecosystems occurs when precipitation of minerals outweighs dissolution. Although the formation of various minerals can result from microbial metabolism, carbonate precipitation is possibly the most important process that impacts global carbon cycling. Recent investigations have produced models for stromatolite formation in open marine environments and lithification in shallow hypersaline lakes, which could be highly relevant for interpreting the rock record and searching for extraterrestrial life. Two factors that are controlled by microbial processes and physicochemical characteristics determine precipitation: exopolymeric substances and the saturation index, the latter being determined by the pH, {Ca(2+)} and {CO(3)(2-)}. Here, we evaluate community metabolism in microbial mats and hypothesize why these organosedimentary biofilms sometimes lithify and sometimes do not.

BACKGROUND

Besides significantly reducing malaria vector densities, prolonged usage of bed nets has been linked to decline of Anopheles gambiae s.s. relative to Anopheles arabiensis, changes in host feeding preference of malaria vectors, and behavioural shifts to exophagy (outdoor biting) for the two important malaria vectors in Africa, An. gambiae s.l. and Anopheles funestus. In southern coastal Kenya, bed net use was negligible in 1997-1998 when Anopheles funestus and An. gambiae s.s. were the primary malaria vectors, with An. arabiensis and Anopheles merus playing a secondary role. Since 2001, bed net use has increased progressively and reached high levels by 2009-2010 with corresponding decline in malaria transmission.

METHODS

To evaluate the impact of the substantial increase in household bed net use within this area on vector density, vector composition, and human-vector contact, indoor and outdoor resting mosquitoes were collected in the same region during 2009-2010 using pyrethrum spray catches and clay pots for indoor and outdoor collections respectively. Information on bed net use per sleeping spaces and factors influencing mosquito density were determined in the same houses using Poisson regression analysis. Species distribution was determined, and number of mosquitoes per house, human-biting rates (HBR), and entomological inoculation rate (EIR) were compared to those reported for the same area during 1997-1998, when bed net coverage had been minimal.

RESULTS

Compared to 1997-1998, a significant decline in the relative proportion of An. gambiae s.s. among collected mosquitoes was noted, coupled with a proportionate increase of An. arabiensis. Following>> 5 years of 60-86% coverage with bed nets, the density, human biting rate and EIR of indoor resting mosquitoes were reduced by more than 92% for An. funestus and by 75% for An. gambiae s.l. In addition, the host feeding choice of both vectors shifted more toward non-human vertebrates. Besides bed net use, malaria vector abundance was also influenced by type of house construction and according to whether one sleeps on a bed or a mat (both of these are associated with household wealth). Mosquito density was positively associated with presence of domestic animals.

CONCLUSIONS

These entomological indices indicate a much reduced human biting rate and a diminishing role of An. gambiae s.s. in malaria transmission following high bed net coverage. While increasing bed net coverage beyond the current levels may not significantly reduce the transmission potential of An. arabiensis, it is anticipated that increasing or at least sustaining high bed net coverage will result in a diminished role for An. funestus in malaria transmission.

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F(430) was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.

In yeast, the mRNA encoded by the MAT alpha 1 gene is unstable (t1/2 = 5 min) and the mRNAs encoded by the ACT1 gene (t1/2 = 30 min) and the PGK1 gene (t1/2 = 45 min) are stable. To understand the RNA structural features that dictate mRNA decay rates in yeast, we have constructed PGK1/MAT alpha 1 and ACT1/MAT alpha 1 gene fusions and analyzed the decay rates of the resultant chimeric transcripts. Fusion of a MAT alpha 1 segment containing 73% of the coding region and the 3' untranslated region to either of the stable genes is sufficient to cause rapid decay of the chimeric mRNAs (t1/2 = 6-7.5 min). Sequences required for this rapid decay are not found in the MAT alpha 1 3' untranslated region but are located within a 42-nucleotide segment of the coding region that has a high content (8 out of 14) of rare codons. Introduction of a translational stop codon upstream of this region stabilizes the hybrid mRNAs, indicating that the rapid decay promoted by these sequences is dependent on ribosomal translocation.

Sexual reproduction occurs in two fundamentally different ways: by outcrossing, in which two distinct partners contribute nuclei, or by self-fertilization (selfing), in which both nuclei are derived from the same individual. Selfing is common in flowering plants, fungi, and some animal taxa. We investigated the genetic basis of selfing in the homothallic fungus Aspergillus nidulans. We demonstrate that alpha and high-mobility group domain mating-type (MAT) genes, found in outcrossing species, are both present in the genome of A. nidulans and that their expression is required for normal sexual development and ascospore production. Balanced overexpression of MAT genes suppressed vegetative growth and stimulated sexual differentiation under conditions unfavorable for sex. Sexual reproduction was correlated with significantly increased expression of MAT genes and key genes of a pheromone-response MAP-kinase signaling pathway involved in heterothallic outcrossing. Mutation of a component MAP-kinase mpkB gene resulted in sterility. These results indicate that selfing in A. nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e., self-fertilization does not bypass requirements for outcrossing sex but instead requires activation of these pathways within a single individual. However, unlike heterothallic species, aspects of pheromone signaling appeared to be independent of MAT control.

We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A. Percival-Smith and J. Segall, Mol. Cell. Biol. 4:142-150, 1984). This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium. Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease. In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated. The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation. The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation. A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability. However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation. The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain. This phenotype is presumably a reflection of a defect in spore wall maturation. This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression pattern, contributes to spore development.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(L-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.

To investigate the extent of genetic stratification in structured microbial communities, we compared the metagenomes of 10 successive layers of a phylogenetically complex hypersaline mat from Guerrero Negro, Mexico. We found pronounced millimeter-scale genetic gradients that were consistent with the physicochemical profile of the mat. Despite these gradients, all layers displayed near-identical and acid-shifted isoelectric point profiles due to a molecular convergence of amino-acid usage, indicating that hypersalinity enforces an overriding selective pressure on the mat community.

S-Adenosylmethionine (SAMe), the principal biological methyl donor, is synthesized from methionine and ATP in a reaction catalyzed by methionine adenosyltransferase (MAT). In mammals, two genes (MATMATMAT catalytic subunits, while a third gene MATMATMATMATMATMATMAT activity and SAMe biosynthesis. Consequences of hepatic SAMe deficiency as illustrated by the Mat1a knock-out mouse model include increased susceptibility to steatosis and oxidative liver injury, spontaneous development of steatohepatitis and HCC. Predisposition to HCC can be partly explained by the effect of SAMe on growth. Thus, SAMe inhibits the mitogenic effect of growth factors such as hepatocyte growth factor and, following partial hepatectomy, a fall in SAMe level is required for the liver to regenerate. During liver regeneration, the fall in hepatic SAMe is transient. If the fall were to persist, it would favor a proliferative phenotype and, ultimately, development of HCC. Not only does SAMe control liver growth, it also regulates apoptosis. Interestingly, SAMe is anti-apoptotic in normal hepatocytes but pro-apoptotic in liver cancer cells. In liver cancer cells but not in normal human hepatocytes, SAMe can selectively induce Bcl-x(S), an alternatively spliced isoform of Bcl-x(L) that promotes apoptosis. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80 degrees C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90 degrees C, and a Chloroflexus mat is formed at 65 to 70 degrees C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter(-1)) below 70 degrees C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter(-1)) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.

Marrow adipose tissue (MAT) is functionally distinct from both white and brown adipose tissue and can contribute to systemic and skeletal metabolism. MAT formation is a spatially and temporally defined developmental event, suggesting that MAT is an organ that serves important functions and, like other organs, can undergo pathologic change. The well-documented inverse relationship between MAT and bone mineral density has been interpreted to mean that MAT removal is a possible therapeutic target for osteoporosis. However, the bone and metabolic phenotypes of patients with lipodystrophy argues that retention of MAT may actually be beneficial in some circumstances. Furthermore, MAT may exist in two forms, regulated and constitutive, with divergent responses to hematopoietic and nutritional demands. In this review, we discuss the role of MAT in lipodystrophy, bone loss, and metabolism, and highlight our current understanding of this unique adipose tissue depot.

Recent re-evaluations of the geological record of the earliest life on Earth have led to the suggestion that some of the oldest putative microfossils and carbonaceous matter were formed through abiotic hydrothermal processes. Similarly, many early Archaean (more than 3,400-Myr-old) cherts have been reinterpreted as hydrothermal deposits rather than products of normal marine sedimentary processes. Here we present the results of a field, petrographic and geochemical study testing these hypotheses for the 3,416-Myr-old Buck Reef Chert, South Africa. From sedimentary structures and distributions of sand and mud, we infer that deposition occurred in normal open shallow to deep marine environments. The siderite enrichment that we observe in deep-water sediments is consistent with a stratified early ocean. We show that most carbonaceous matter was formed by photosynthetic mats within the euphotic zone and distributed as detrital matter by waves and currents to surrounding environments. We find no evidence that hydrothermal processes had any direct role in the deposition of either the carbonaceous matter or the enclosing sediments. Instead, we conclude that photosynthetic organisms had evolved and were living in a stratified ocean supersaturated in dissolved silica 3,416 Myr ago.

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5' to 3' exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.

The yeast alpha 2 protein, the product of the MAT alpha 2 gene, is a regulator of yeast cell type; it turns off transcription of the a-specific genes by binding to an operator located upstream of each gene. In this paper we describe the domain structure, subunit organization, and some unusual features of the way this protein contacts its operator. We show that the protein is folded into two domains. The carboxy-terminal domain binds specifically to the operator; the amino-terminal domain contains dimerization contacts. The alpha 2 dimer differs from those of the phage repressors in that it is flexible and therefore is able to bind tightly to differently spaced operator half-sites. In the natural operator, the centers of the operator half-sites are two and one-half turns of DNA apart, exposing them on opposite sides of the DNA helix. We show that the design of alpha 2 allows a dimer to reach across its operator such that it occupies the two half-sites but leaves the middle of the operator available to other proteins.

The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.

Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products a1 and alpha 2, which determine the a/alpha cell type. These signals lead to increased expression of the IME1 (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IME1 expression from the GAL1 promoter. The deduced IME1 product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression of PGAL1-IME1 in vegetative a/alpha cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPO11, SPO13, and HOP1); the transcripts accumulated 3- to 10-fold more after starvation. Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGAL1-IME1 strains were starved, and the intact IME1 gene was not activated by PGAL1-IME1 expression. In a or alpha cells, which lack alpha 2 or a1, expression of PGAL1-IME1 led to the same pattern of IME2 and SPO13 expression as in a/alpha cells, as measured with ime2::lacZ and spo13::lacZ fusions. Thus, in wild-type strains, the increased expression of IME1 in starved a/alpha cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression. PGAL1-IME1 expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly. We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.