Deoxyribonuclease probing on sea urchin embryo chromatin

Bibliography: pages 118-143.The role that the sea urchin, Parechinus angulosus, embryo and sperm histone variants play in chromatin structure has been investigated. Chromatin structure has been determined at different levels of resolution in sperm and in developing embryos using micrococcal nuclease, pancreatic deoxyribonuclease (DNase I) and restriction endonucleases. Micrococcal nuclease and restriction endonuclease digestions of sea urchin gastrula chromatin have been analysed and it is shown that it is not possible to isolate large polynucleosomal chromatin complexes which are soluble in low ionic strength buffers. The nucleosomal DNA repeat lengths for sea urchin blastula, gastrula and sperm have been determined using micrococcal nuclease. The repeat length for sperm is significantly larger than blastula and gastrula repeat lengths whereas blastula and gastrula repeat lengths are not significantly different. Nucleosomal core particles have been isolated from early blastula, gastrula and sperm of sea urchins. After DNase I digestion of 51-labelled core particles the rate constants of cutting of the DNA at the susceptible sites on these core particles have been determined. The DNase I digestion kinetics of blastula and gastrula core particles are similar whereas sperm core particles are digested at a slower rate, mainly at the sites which are closest to the ends of the core particle DNA. Also, a site, which is 5 bases on the outside of the core particle and which is partially protected from nuclease attack, has been identified. The implications of these findings in relation to the histone variants in embryos and sperm of sea urchins are discussed

BPST instanton and Spin from inequivalent quantizations

We present a simple alternative to Mackey's account of the (infinite) inequivalent quantizations possible on a coset space G/H. Our reformulation is based on the reduction ${\rm G \rightarrow G/H}$ and employs a generalized form of Dirac's approach to the quantization of constrained systems. When applied to the four-sphere $S^4 \simeq {\rm Spin(5)/Spin(4)}$, the inequivalent quantizations induce relativistic spin and a background BPST instanton; thus they might provide a natural account of both of these physical entities.Comment: 11 pages, plain TeX, PLY-MS-93-04, DIAS-STP-93-21 (This version should now TeX

Many sequence-specific chromatin modifying protein-binding motifs show strong positional preferences for potential regulatory regions in the Saccharomyces cerevisiae genome

Initiation and regulation of gene expression is critically dependent on the binding of transcriptional regulators, which is often temporal and position specific. Many transcriptional regulators recognize and bind specific DNA motifs. The length and degeneracy of these motifs results in their frequent occurrence within the genome, with only a small subset serving as actual binding sites. By occupying potential binding sites, nucleosome placement can specify which sequence motif is available for DNA-binding regulatory factors. Therefore, the specification of nucleosome placement to allow access to transcriptional regulators whenever and wherever required is critical. We show that many DNA-binding motifs in Saccharomyces cerevisiae show a strong positional preference to occur only in potential regulatory regions. Furthermore, using gene ontology enrichment tools, we demonstrate that proteins with binding motifs that show the strongest positional preference also have a tendency to have chromatin-modifying properties and functions. This suggests that some DNA-binding proteins may depend on the distribution of their binding motifs across the genome to assist in the determination of specificity. Since many of these DNA-binding proteins have chromatin remodeling properties, they can alter the local nucleosome structure to a more permissive and/or restrictive state, thereby assisting in determining DNA-binding protein specificity

Identifying related L1 retrotransposons by analyzing 3' transduced sequences

BACKGROUND: A large fraction of the human genome is attributable to L1 retrotransposon sequences. Not only do L1s themselves make up a significant portion of the genome, but L1-encoded proteins are thought to be responsible for the transposition of other repetitive elements and processed pseudogenes. In addition, L1s can mobilize non-L1, 3'-flanking DNA in a process called 3' transduction. Using computational methods, we collected DNA sequences from the human genome for which we have high confidence of their mobilization through L1-mediated 3' transduction. RESULTS: The precursors of L1s with transduced sequence can often be identified, allowing us to reconstruct L1 element families in which a single parent L1 element begot many progeny L1s. Of the L1s exhibiting a sequence structure consistent with 3' transduction (L1 with transduction-derived sequence, L1-TD), the vast majority were located in duplicated regions of the genome and thus did not necessarily represent unique insertion events. Of the remaining L1-TDs, some lack a clear polyadenylation signal, but the alignment between the parent-progeny sequences nevertheless ends in an A-rich tract of DNA. CONCLUSIONS: Sequence data suggest that during the integration into the genome of RNA representing an L1-TD, reverse transcription may be primed internally at A-rich sequences that lie downstream of the L1 3' untranslated region. The occurrence of L1-mediated transduction in the human genome may be less frequent than previously thought, and an accurate estimate is confounded by the frequent occurrence of segmental genomic duplications

Scanning sequences after Gibbs sampling to find multiple occurrences of functional elements

BACKGROUND: Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set. RESULTS: We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM). The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence. CONCLUSION: Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances