The purpose of this study was to evaluate the test-retest reliability of an instrumented walkway system (the GAITRite mat) for the measurement of temporal and spatial parameters of gait in young and older people. Thirty young subjects (12 males, 18 females) aged between 22 and 40 years (mean 28.5, S.D. 4.8) and 31 older subjects (13 males, 18 females) aged between 76 and 87 years (mean 80.8, S.D. 3.1) walked at a self-selected comfortable walking speed across the pressure-sensor mat three times and repeated the process approximately 2 weeks later. Intra-class correlation coefficients (ICC), coefficients of variation (CV) and 95% limits of agreement were then determined. For both groups of subjects, the reliability of walking speed, cadence and step length was excellent (ICCs between 0.82 and 0.92 and CVs between 1.4 and 3.5%). Base of support and toe in/out angles, although exhibiting high ICCs, were associated with higher CVs (8.3-17.7% in young subjects and 14.3-33.0% in older subjects). It is concluded that the GAITRite mat exhibits excellent reliability for most temporo-spatial gait parameters in both young and older subjects, however, base of support and toe in/out angles need to viewed with some caution, particularly in older people.

Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of PUM to the NRE is thought to target hb(mat) mRNA for repression. Here, we show the RNA-binding domain of PUM to be an evolutionarily conserved, 334-amino acid region at the carboxy-terminus of the approximately 158-kDa PUM protein. This contiguous region of PUM retains the RNA-binding specificity of full-length PUM protein. Proteins with sequences homologous to the PUM RNA-binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the PUM RNA-binding domain in other far-flung species suggests that the domain is an ancient protein motif, and we show that conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila PUM.

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Curaçao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including Clostridium spp., Arcobacter spp., Campylobacter spp., Cytophaga fermentans, Cytophaga columnaris, and Trichodesmium tenue.

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.

S-adenosylmethionine (AdoMet, also known as SAM and SAMe) is the principal biological methyl donor synthesized in all mammalian cells but most abundantly in the liver. Biosynthesis of AdoMet requires the enzyme methionine adenosyltransferase (MAT). In mammals, two genes, MATMATMAT. Patients with chronic liver disease have reduced MAT activity and AdoMet levels. Mice lacking Mat1a have reduced hepatic AdoMet levels and develop oxidative stress, steatohepatitis, and hepatocellular carcinoma (HCC). In these mice, several signaling pathways are abnormal that can contribute to HCC formation. However, injury and HCC also occur if hepatic AdoMet level is excessive chronically. This can result from inactive mutation of the enzyme glycine N-methyltransferase (GNMT). Children with GNMT mutation have elevated liver transaminases, and Gnmt knockout mice develop liver injury, fibrosis, and HCC. Thus a normal hepatic AdoMet level is necessary to maintain liver health and prevent injury and HCC. AdoMet is effective in cholestasis of pregnancy, and its role in other human liver diseases remains to be better defined. In experimental models, it is effective as a chemopreventive agent in HCC and perhaps other forms of cancer as well.

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.

Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT-PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT-PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data.

The adipocyte-derived hormone adiponectin promotes metabolic and cardiovascular health. Circulating adiponectin increases in lean states such as caloric restriction (CR), but the reasons for this paradox remain unclear. Unlike white adipose tissue (WAT), bone marrow adipose tissue (MAT) increases during CR, and both MAT and serum adiponectin increase in many other clinical conditions. Thus, we investigated whether MAT contributes to circulating adiponectin. We find that adiponectin secretion is greater from MAT than WAT. Notably, specific inhibition of MAT formation in mice results in decreased circulating adiponectin during CR despite unaltered adiponectin expression in WAT. Inhibiting MAT formation also alters skeletal muscle adaptation to CR, suggesting that MAT exerts systemic effects. Finally, we reveal that both MAT and serum adiponectin increase during cancer therapy in humans. These observations identify MAT as an endocrine organ that contributes significantly to increased serum adiponectin during CR and perhaps in other adverse states.

FXYD proteins belong to a family of small-membrane proteins. Recent experimental evidence suggests that at least five of the seven members of this family, FXYD1 (phospholemman), FXYD2 (gamma-subunit of Na-K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), and FXYD7, are auxiliary subunits of Na-K-ATPase and regulate Na-K-ATPase activity in a tissue- and isoform-specific way. These results highlight the complexity of the regulation of Na+ and K+ handling by Na-K-ATPase, which is necessary to ensure appropriate tissue functions such as renal Na+ reabsorption, muscle contractility, and neuronal excitability. Moreover, a mutation in FXYD2 has been linked to cases of human hypomagnesemia, indicating that perturbations in the regulation of Na-K-ATPase by FXYD proteins may be critically involved in pathophysiological states. A better understanding of this novel regulatory mechanism of Na-K-ATPase should help in learning more about its role in pathophysiological states. This review summarizes the present knowledge of the role of FXYD proteins in the modulation of Na-K-ATPase as well as of other proteins, their regulation, and their structure-function relationship.

We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life>> 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.

Most microbes have complex life cycles with multiple modes of reproduction that differ in their effects on DNA sequence variation. Population genomic analyses can therefore be used to estimate the relative frequencies of these different modes in nature. The life cycle of the wild yeast Saccharomyces paradoxus is complex, including clonal reproduction, outcrossing, and two different modes of inbreeding. To quantify these different aspects we analyzed DNA sequence variation in the third chromosome among 20 isolates from two populations. Measures of mutational and recombinational diversity were used to make two independent estimates of the population size. In an obligately sexual population these values should be approximately equal. Instead there is a discrepancy of about three orders of magnitude between our two estimates of population size, indicating that S. paradoxus goes through a sexual cycle approximately once in every 1,000 asexual generations. Chromosome III also contains the mating type locus (MAT), which is the most outbred part in the entire genome, and by comparing recombinational diversity as a function of distance from MAT we estimate the frequency of matings to be approximately 94% from within the same tetrad, 5% with a clonemate after switching the mating type, and 1% outcrossed. Our study illustrates the utility of population genomic data in quantifying life cycles.

The yeast Doa10 ubiquitin (Ub) ligase resides in the endoplasmic reticulum (ER)/nuclear envelope (NE), where it functions in ER-associated degradation (ERAD). Doa10 substrates include non-ER proteins such as the transcription factor Mat alpha2. Here, we expand the range of Doa10 substrates to include a defective kinetochore component, a mutant NE membrane protein, and a substrate-regulated human ER enzyme. For all these substrates, Doa10 requires two Ub-conjugating enzymes, Ubc6 and Ubc7, as well as the Ubc7 cofactor Cue1. Based on a novel genomic screen of a comprehensive gene deletion library and other data, these four proteins appear to be the only nonessential and nonredundant factors generally required for Doa10-mediated ubiquitination. Notably, the Cdc48 ATPase facilitates degradation of membrane-embedded Doa10 substrates, but is not required for any tested soluble Doa10 substrates. This distinction is maintained even when comparing membrane and soluble proteins bearing the same degradation signal. Thus, while Doa10 ubiquitinates both membrane and soluble proteins, the mechanisms of subsequent proteasome targeting differ.

In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.

Only five bacterial phyla with members capable of chlorophyll (Chl)-based phototrophy are presently known. Metagenomic data from the phototrophic microbial mats of alkaline siliceous hot springs in Yellowstone National Park revealed the existence of a distinctive bacteriochlorophyll (BChl)-synthesizing, phototrophic bacterium. A highly enriched culture of this bacterium grew photoheterotrophically, synthesized BChls a and c under oxic conditions, and had chlorosomes and type 1 reaction centers. "Candidatus Chloracidobacterium thermophilum" is a BChl-producing member of the poorly characterized phylum Acidobacteria.

This paper reviews the occurrence and properties of cyanobacterial toxins, with reference to the recognition and management of the human health risks which they may present. Mass populations of toxin-producing cyanobacteria in natural and controlled waterbodies include blooms and scums of planktonic species, and mats and biofilms of benthic species. Toxic cyanobacterial populations have been reported in freshwaters in over 45 countries, and in numerous brackish, coastal, and marine environments. The principal toxigenic genera are listed. Known sources of the families of cyanobacterial toxins (hepato-, neuro-, and cytotoxins, irritants, and gastrointestinal toxins) are briefly discussed. Key procedures in the risk management of cyanobacterial toxins and cells are reviewed, including derivations (where sufficient data are available) of tolerable daily intakes (TDIs) and guideline values (GVs) with reference to the toxins in drinking water, and guideline levels for toxigenic cyanobacteria in bathing waters. Uncertainties and some gaps in knowledge are also discussed, including the importance of exposure media (animal and plant foods), in addition to potable and recreational waters. Finally, we present an outline of steps to develop and implement risk management strategies for cyanobacterial cells and toxins in waterbodies, with recent applications and the integration of Hazard Assessment Critical Control Point (HACCP) principles.

Cells of the yeast Saccharomyces cerevisiae are delayed in the G2 phase of the cell cycle following chromosomal DNA damage. This arrest is RAD9-dependent and suggests a signaling mechanism(s) between chromosomal lesions and cell cycling. We examined the global nature of growth inhibition caused by an HO endonuclease-induced double-strand break (DSB) at a 45-bp YZ sequence (from MAT YZ) in a non-yeast region of a dispensable single-copy plasmid. The presence of an unrepaired DSB results in cellular death even though the plasmid is dispensable. Loss of cell viability is partially dependent on the RAD9 gene product. Following induction of the DSB, 40% of RAD+ and 49% of rad9 delta cells [including both unbudded (G1) and budded (S plus G2) cells] did not progress further in the cell cycle. The remaining RAD+ cells progressed to form microcolonies (< 30 cells) containing aberrantly shaped inviable cells. For the rad9 delta mutant, the majority of the remaining cells produced viable colonies accounting for the greater survival of the rad9 delta strain. Based on the profound effects of a single nonchromosomal DNA lesion, this system provides a convenient means for studying the signaling effects of a DNA lesion, as well as for designing strategies for modulating cell proliferation.

In response to DNA damage, eukaryotic cells activate checkpoint pathways that arrest cell cycle progression and induce the expression of genes required for DNA repair. In budding yeast, the homothallic switching (HO) endonuclease creates a site-specific double-strand break at the mating type (MAT) locus. Continuous HO expression results in the phosphorylation of Rad53, which is dependent on products of the ataxia telangiectasia mutated-related MEC1 gene and other checkpoint genes, including DDC1, RAD9, and RAD24. Chromatin immunoprecipitation experiments revealed that the Ddc1 protein associates with a region near the MAT locus after HO expression. Ddc1 association required Rad24 but not Mec1 or Rad9. Mec1 also associated with a region near the cleavage site after HO expression, but this association is independent of Ddc1, Rad9, and Rad24. Thus, Mec1 and Ddc1 are recruited independently to sites of DNA damage, suggesting the existence of two separate mechanisms involved in recognition of DNA damage.

We show by electrophoresis mobility shift and by DNAase I footprinting assays that the alpha 1 product of the yeast alpha mating-type locus binds to homologous sequences within the control regions of the three known alpha-specific genes. Binding requires both alpha 1 and a second yeast protein(s) (called PRTF) that is present in all three cell types (a, alpha, and a/alpha); neither protein binds alone. Binding and competition experiments using synthetic oligonucleotides indicate that PRTF binds to only part of the homology found at alpha-specific genes and imply that alpha 1 binds to the remainder. Our results suggest that alpha 1 renders gene expression alpha-specific by creating a binding site for PRTF. Similar experiments lead to the idea that PRTF also plays a role in transcription of a-specific genes. Perhaps a-specificity is achieved through the occlusion of the PRTF binding site by alpha 2, the negative regulator encoded by the alpha mating-type locus.

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MAT alpha 2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein. We now demonstrate that alpha 2 is conjugated to ubiquitin in vivo. More than one lysine residue of alpha 2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain. Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha 2 degradation is dependent on its ubiquitination.

Myxococcus xanthus develops species-specific multicellular fruiting bodies. Starting from a uniform mat of cells, some cells enter into nascent fruiting body aggregates, whereas other cells remain outside. The cells within the fruiting body differentiate from rods into spherical, heat-resistant spores, whereas the cells outside the aggregates, called peripheral cells, remain rod-shaped. Early developmentally regulated genes are expressed in peripheral cells as well as by cells in the fruiting bodies. By contrast, late developmental genes are only expressed by cells within the nascent fruiting bodies. The data show that peripheral cells begin to develop, but are unable to express genes that are switched on later than about 6 h after the start of development. All of the genes whose expression is limited to the fruiting body are dependent on C-signaling either directly or indirectly, whereas the genes that are equally expressed in peripheral rods and in fruiting body cells are not. One of the C-signal-dependent and spatially patterned operons is called dev, and the dev operon has been implicated in the process of sporulation. It is proposed that expression of certain genes, including those of the dev operon, is limited to the nascent fruiting body because fruiting body cells engage in a high level of C-signaling. Peripheral cells do less C-signaling than fruiting body cells, because they have a different spatial arrangement and are at lower density. As a consequence, peripheral cells fail to express the late genes necessary for spore differentiation.

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, alpha and a. The alpha and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The alpha and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.

BACKGROUND

Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.

METHODS

Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.

RESULTS

Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4 degrees C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.

CONCLUSIONS

This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.

The two yeast mating-type alleles MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters. Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed, except in sir- strains. Since SIR represses not only transcription from a silent copy but also its ability to act as a recipient in a mating-type interconversion, we have investigated whether it might act by regulating the entire chromatin structure of a silent locus. We have therefore compared the profile of DNAase I and micrococcal nuclease cleavage at HML alpha with MAT alpha and HMRa with MATa in sir- and SIR+ strains. We find that SIR is necessary for the maintenance of a different chromatin structure at HM loci from their active counterparts at MAT. One particularly striking change that SIR induces provides a simple explanation for one of its biological properties: control of directionality of switching. SIR causes the disappearance of a DNAase I-hypersensitive site at Y-Z boundary (found at MAT or HM sir-) that is coincident with a double-strand cleavage possibly created by HO in the initiation of a mating-type switch.