Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis

Bibliography: pages 161-175.A poly(dG).poly(dC)-binding protein (suGF1) had previously been identified in sea urchin (Parenchinus angulosis) embryonic nuclear extracts (J.P. Hapgood, personal communication). suGF1 may be involved in the regulation of early histone gene expression by interaction with a nudeosome which has been shown to be positioned in vitro over the H1-H4 intergenic region of the early histone gene battery of Psammechinus miliaris (189, H.-G. Patterton & J.P. Hapgood, unpublished). In this investigation suGF1 was purified to near homogeneity by DNA-affinity chromatography. The purification procedure involved a cation exchange step, followed by poly(dG).poly(dC)-affinity chromatography, and finally by affinity chromatography utilizing multimerized specific DNA-binding sites of suGF1. The 59,5 kDa purified protein was identified as suGFl by renaturation of sequence-specific DNA-binding activity from a SDSpAGE gel slice, by Southwestern blotting and by DNase I footprinting. Ultraviolet crosslinking of the nuclear extract and purified suGF1 revealed the presence of the same specific bands on SDS-P AGE. Optimal suGF1 DNA-binding was shown to occur at relatively high ionic strength (175 mM). suGF1 DNA-binding was sensitive to EDTA, implying a requirement for a divalent cation for DNA-binding. The suGF1 DNA-binding interaction was investigated by methylation interference, and DNase I and hydroxyl radical footprinting. The footprinting data was analyzed in difference probability plots, from which a model for the suGFl-DNA complex was inferred. In the model suGFl approaches the DNA helix mainly from one side, and interacts with guanine residues in the major groove. Contacts are made to one of the DNA sugar phosphate backbones abutting this major groove. The data is consistent with the DNA in the complex being curved, and/ or exhibiting a sharp bend at the site of suGFl contact in the major groove. suGFl does not seem to bind to DNA as a rotationally symmetrical dimer. The results of this investigation are discussed in terms of the literature. suGFl may be related to the chicken erythrocyte-specific factor BGPl, which has been shown to bind to 16 contiguous guanines in the βᴬ-globin promoter, both in the naked DNA molecule and wrapped around a histone octamer (37, 139)

The effect of negative supercoiling on the formation and positioning of nucleosome cores in vitro

Bibliography: pages 250-279.The effect of the negative supercoiling of DNA on the formation and positioning of nucleosome cores was investigated in a 1915bp plasmid (pHP2) containing a section of the early H1-H4 histone gene spacer of Psammechinus miliaris, previously shown to position the histone octamer (Retief et al., 1987, Biochemistry, 26, 4449-4453). It is shown for the first time, by determinations of the linking difference of reconstituted supercoiled plasmid following topoisomerase I relaxation and the yield and fragment size distribution of micrococcal nuclease digests of supercoiled and linearized plasmid, that nucleosome core reconstitution by urea/salt and salt dialysis proceeds cooperatively on both linearized and supercoiled plasmids. Evidence is further presented which indicates that the nucleosome core.reconstitutes more efficiently on negatively supercoiled plasmids compared to linearized plasmids. The free energy of supercoiling is shown to be sufficient to account for this difference, and may contribut·e to the observed preferential migration of the octamer to negatively supercoiled plasmid compared to linear fragments. This migration is facilitated by high ionic strength, but not by high concentrations of poly[L-glutamate] or 146bp core DNA. It is further shown for the first time, by DNase I digestion and primer extension, that identical translational and rotational positions are adopted by nucleosome cores on linearized plasmids and circular plasmids in the absence and presence of negative superhelical stress. This conservation of the positioning frames is -shown to persist, irrespective of the precision of the core placement, or alterations of the relative angular orientation of the positioning frames of adjacent cores. This finding suggests that the topological and geometric constraints of chromatin loops may be negligible in the determination of nucleosome positioning in vivo. The positioning of the core incorporating a d(A-G)₁₆.d(C-T)₁₆ stretch, shown not to adopt a H-DNA conformation in the reconstituted supercoiled plasmid, is analyzed in terms of known rotational determinants, and possible translational determinants proposed. The biological significance of the determined position of the core is discussed, and lastly, the conclusions related to other studies

Characterization of casein and alpha lactalbumin of African elephant (Loxodonta africana) milk

The current research reports partial characterization of the caseins and alpha-lactalbumin (alpha-LA) of the African elephant with proposed unique structure-function properties. Extensive research has been carried out to understand the structure of the casein micelles. Crystallographic structure elucidation of caseins and casein micelles is not possible. Consequently, several models have been developed in an effort to describe the casein micelle, specifically of cow milk. Here we report the characterization of African elephant milk caseins. The kappa-caseins and beta-caseins were investigated, and their relative ratio was found to be approximately 1:8.5, whereas alpha-caseins were not detected. The gene sequence of beta-casein in the NCBI database was revisited, and a different sequence in the N-terminal region is proposed. Amino acid sequence alignment and hydropathy plots showed that the kappa-casein of African elephant milk is similar to that of other mammals, whereas the beta-casein is similar to the human protein, and displayed a section of unique AA composition and additional hydrophilic regions compared with bovine caseins. Elephant milk is destabilized by 62% alcohol, and it is speculated that the beta-casein characteristics may allow maintenance of the colloidal nature of the casein micelle, a role that was previously only associated with K-casein. The oligosaccharide content of milk was reported to be low in dairy animals but high in some other species such as humans and elephants. In the milk of the African elephant, lactose and oligosaccharides both occur at high levels. These levels are typically related to the content of alpha-LA in the mammary gland and thus point to a specialized carbohydrate synthesis, where the whey protein alpha-LA plays a role. We report the characterization of African elephant alpha-LA. Homology modeling of the alpha-LA showed that it is structurally similar to crystal structures of other mammalian species, which in turn may be an indication that its functional properties, such as lactose synthesis, should not be impaired

An LC-MS/MS based survey of contaminants of emerging concern in drinking water in South Africa

Advances in many analytical techniques allow the detection of compounds in water at very low concentrations (ng/L), which has facilitated the identification of many compounds in drinking water that went previously undetected. Some of these compounds are contaminants of emerging concern (CECs), which is broadly defined as any chemical or microorganism that is not currently being routinely monitored but has recently been identified as being present in the environment, and that may pose health or ecological risks. CECs can include pharmaceuticals, personal health care products and pesticides. Some CECs can act as endocrine disruptors, interfering with the normal functioning of the human endocrine system, potentially influencing foetal and child development. Although the level of many of these compounds are orders of magnitude below known acute toxicity levels, the health impact of long term exposure at low levels is mostly unknown. In this study, we present the results of a national survey over four seasons of potential CECs in the drinking water of major South African cities. The contaminants most often detected were the related herbicides atrazine and terbuthylazine, and the anticonvulsant and mood-stabilising drug, carbamazepine. The levels of these CECs were well below maximum levels proposed by the World Health Organization and the US Environmental Protection Agency. However, the range of CECs detected in drinking water, and seasonal and geographic variability in CECs levels, warrant a more frequent screening programme

Nano-electrospray tandem mass spectrometric analysis of the acetylation state of histones H3 and H4 in stationary phase in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in <it>Saccharomyces cerevisiae </it>that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence.</p> <p>Results</p> <p>We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an <it>m/z </it>126 immonium ion, diagnostic of an N^ε-acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and b-ion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones), but could not detect these modifications in histones isolated from stationary phase cells at the sensitivity level of the mass spectrometer. The corresponding un-acetylated peptides were, however, observed. A significantly higher level of acetylation of these residues in exponential phase was confirmed by immuno-blotting.</p> <p>Conclusion</p> <p>H4K16 acetylation was previously shown to disrupt formation of condensed chromatin <it>in vitro</it>. We propose that de-acetylation of H4K16 allowed formation of condensed chromatin in stationary phase, and that acetylation of H3K9, H3K14, H3K36, and H4K12 reflected the active transcriptional state of the yeast genome in exponential phase.</p

Non-random clustering of stress-related genes during evolution of the S. cerevisiae genome

BACKGROUND: Coordinately regulated genes often physically cluster in eukaryotic genomes, for reasons that remain unclear. RESULTS: Here we provide evidence that many S. cerevisiae genes induced by starvation and other stresses reside in non-random clusters, where transcription of these genes is repressed in the absence of stress. Most genes essential for growth or for rapid, post-transcriptional responses to stress in cycling cells map between these gene clusters. Genes that are transcriptionally induced by stresses include a large fraction of rapidly evolving paralogues of duplicated genes that arose during an ancient whole genome duplication event. Many of these rapidly evolving paralogues have acquired new or more specialized functions that are less essential for growth. The slowly evolving paralogues of these genes are less likely to be transcriptionally repressed in the absence of stress, and are frequently essential for growth or for rapid stress responses that may require constitutive expression of these genes in cycling cells. CONCLUSION: Our findings suggest that a fundamental organizing principle during evolution of the S. cerevisiae genome has been clustering of starvation and other stress-induced genes in chromosome regions that are transcriptionally repressed in the absence of stress, from which most genes essential for growth or rapid stress responses have been excluded. Chromatin-mediated repression of many stress-induced genes may have evolved since the whole genome duplication in parallel with functions for proteins encoded by these genes that are incompatible with growth. These functions likely provide fitness effects that escape detection in assays of reproductive capacity routinely employed to assess evolutionary fitness, or to identify genes that confer stress-resistance in cycling cells

Calculating the statistical significance of physical clusters of co-regulated genes in the genome: The role of chromatin in domain-wide gene regulation

Physical clusters of co‐regulated, but apparently functionally unrelated, genes are present in many genomes. Despite the important implication that the genomic environment contributes appreciably to the regulation of gene expression, no simple statistical method has been described to identify physical clusters of co‐regulated genes. Here we report the development of a model that allows the direct calculation of the significance of such clusters. We have implemented the derived statistical relation in a software program, Pyxis, and have analyzed a selection of Saccharomyces cerevisiae gene expression microarray data sets. We have identified many gene clusters where constituent genes exhibited a regulatory dependence on proteins previously implicated in chromatin structure. Specifically, we found that Tup1p‐dependent gene domains were enriched close to telomeres, which suggested a new role for Tup1p in telomere silencing. In addition, we identified Sir2p‐, Sir3p‐ and Sir4p‐dependent clusters, which suggested the presence of Sir‐mediated heterochromatin in previously unidentified regions of the yeast genome. We also showed the presence of Sir4p‐dependent gene clusters bordering the HMRaheterothallic locus, which suggested leaky termination of the heterochromatin by the boundary elements. These results demonstrate the utility of Pyxis in identifying possible higher order genomic features that may contribute to gene regulation in extended domains

Enrichment of a fraction toxic to guinea-pigs from Pachystigma pygmaeum (Schltr.) Robyns

Pachystigma pygmaeum is one of several species of rubiaceous plants which cause delayed heart failure among ruminants after their ingestion at relatively high doses. Using guinea-pigs for toxicity determinations, we were able to separate and enrich a toxic fraction from a fermentation extract of the plant material by countercurrent distribution. It contained virtually no potassium salts, passed through a 500 dalton selective membrane, exhibited lability under acid conditions and was toxic at 1 g/kg per os, with a delayed response of 3-4 days.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Department of Agriculture

Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei

Background: The compaction of DNA in chromatin in eukaryotes allowed the expansion of genome size and coincided with significant evolutionary diversification. However, chromatin generally represses DNA function, and mechanisms coevolved to regulate chromatin structure and its impact on DNA. This included the selection of specific nucleosome positions to modulate accessibility to the DNA molecule. Trypanosoma brucei, a member of the Excavates supergroup, falls in an ancient evolutionary branch of eukaryotes and provides valuable insight into the organization of chromatin in early genomes. Results: We have mapped nucleosome positions in T. brucei and identified important differences compared to other eukaryotes: The RNA polymerase II initiation regions in T. brucei do not exhibit pronounced nucleosome depletion, and show little evidence for defined −1 and +1 nucleosomes. In contrast, a well-positioned nucleosome is present directly on the splice acceptor sites within the polycistronic transcription units. The RNA polyadenylation sites were depleted of nucleosomes, with a single well-positioned nucleosome present immediately downstream of the predicted sites. The regions flanking the silent variant surface glycoprotein (VSG) gene cassettes showed extensive arrays of well-positioned nucleosomes, which may repress cryptic transcription initiation. The silent VSG genes themselves exhibited a less regular nucleosomal pattern in both bloodstream and procyclic form trypanosomes. The DNA replication origins, when present within silent VSG gene cassettes, displayed a defined nucleosomal organization compared with replication origins in other chromosomal core regions. Conclusions: Our results indicate that some organizational features of chromatin are evolutionarily ancient, and may already have been present in the last eukaryotic common ancestor

Bioinformatics education—perspectives and challenges out of Africa

The discipline of bioinformatics has developed rapidly since the complete sequencing of the first genomes in the 1990s.The development of many high-throughput techniques during the last decades has ensured that bioinformatics has grown into a discipline that overlaps with, and is required for, the modern practice of virtually every field in the life sciences. This has placed a scientific premium on the availability of skilled bioinformaticians, a qualification that is extremely scarce on the African continent. The reasons for this are numerous, although the absence of a skilled bioinformatician at academic institutions to initiate a training process and build sustained capacity seems to be a common African shortcoming.This dearth of bioinformatics expertise has had a knock-on effect on the establishment of many modern high-throughput projects at African institutes, including the comprehensive and systematic analysis of genomes from African populations, which are among the most genetically diverse anywhere on the planet. Recent funding initiatives from the National Institutes of Health and theWellcomeTrust are aimed at ameliorating this shortcoming. In this paper, we discuss the problems that have limited the establishment of the bioinformatics field in Africa, as well as propose specific actions that will help with the education and training of bioinformaticians on the continent. This is an absolute requirement in anticipation of a boom in high-throughput approaches to human health issues unique to data from African populations