We present an update of our method for systematic detection and evaluation potential hellx-turn-helix DNA-binding motifs in protein sequences [Dodd, I. Egan, J. B. (1987) Mol.Biol.194,[557][558][559][560][561][562][563][564].The new is considerably more powerful, detecting approximately 50% likely without Increase In false predictions.This improvement due almost entirely to the use a much larger reference set 91 presumed helix-tumhellx sequences.The scoring matrix derived from this has been...

We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases time and effort needed integration single or multiple fragments. Additionally, facilitates expression genetic elements are impossible propagate typical multicopy plasmids.

In vertebrates, methylation of cytosine at CpG sequences is implicated in stable and heritable patterns gene expression. The classical model for inheritance, which individual sites are independent, provides no explanation the observed non-random methylation. We first investigate exact topology clustering human genome associated to islands. Then, by pooling genomic clusters on basis short distances between CpGs within long outside clusters, we show a strong dependence number density...

Effective repression of cI transcription from PRM by the bacteriophage lambda CI repressor requires binding sites (OL) located 2.4 kb promoter. A tetramer bound to OL1.OL2 interacts with a near (OR1.OR2), looping intervening DNA. We previously proposed that in this octamer:DNA complex, distant OL3 operator and weak OR3 overlapping are juxtaposed so dimer at can cooperate OR3. Here we show is necessary for effective appears interact specifically The OL3-CI-OR3 interaction involves same...

The CI repressor of bacteriophage λ is a model for the role cooperativity in efficient functioning genetic switches. Pairs dimers interact to cooperatively occupy adjacent operator sites at O R and L . These tetramers repress lytic promoters activate transcription cI gene from P RM also able octamerize, forming large DNA loop between , but physiological this unclear. Another puzzle that, although dimer by binding third 3, seems too weak affect production lysogenic state. Here we show that...

Abstract Inheritance of 5-methyl cytosine modification CpG (CG/CG) DNA sequences is needed to maintain early developmental decisions in vertebrates. The standard inheritance model treats CpGs as independent, with methylated maintained by efficient methylation hemimethylated produced after replication, and unmethylated an absence de novo methylation. By stochastic simulations islands over multiple cell cycles systematic sampling reaction parameters, we show that the inconsistent many...

Abstract DNA looping is a ubiquitous and critical feature of gene regulation. Although can be efficiently detected, tools to readily manipulate are limited. Here we develop CRISPR-based reagents for creation programmable loops. Cleavage-defective Cas9 proteins different specificity linked by heterodimerization or translational fusion create bivalent complexes able link two separate regions. After model-directed optimization, the validated using quantitative assay in E. coli . Looping...

CI represses cro ; Cro cI . This double negative feedback loop is the core of classical CI–Cro epigenetic switch bacteriophage λ. Despite status this switch, role in λ development repression P RM promoter for has remained unclear. To address this, we created binding site mutations that strongly impaired with only minimal effects on regulation These had little impact after infection but inhibited transition from lysogeny to lytic pathway. We demonstrate following inactivation by ultraviolet...

The complete cDNA for human tissue-type plasminogen activator (t-PA) was cloned and sequenced.A mutant constructed by using in vitro site-specific mutagenesis to delete the region encoding growth factor domain (amino acids 51-87 inclusive).Normal t-PA species were produced two mammalian expression systems (in HeLa cells mouse C127 cells).The clearance of normal from plasma examined vivo a guinea pig model.Mutant derived or cleared much more slowly than cognate t-PA.The potential role...

Report15 April 2008Open Access Ultrasensitive gene regulation by positive feedback loops in nucleosome modification Kim Sneppen Center for Models of Life, Niels Bohr Institute, Blegdamsvej, Copenhagen, Denmark Search more papers this author Mille A Micheelsen Ian B Dodd Corresponding Author Department Molecular and Biomedical Sciences (Biochemistry), University Adelaide, South Australia, Australia Information Sneppen1, Micheelsen1 2 1Center 2Department *Corresponding author. Science, North...

Cells can often choose among several stably heritable phenotypes. Examples are the expression of genes in eukaryotic cells where long chromosomal regions adopt persistent and silenced or active states, that may be associated with positive feedback dynamic modification nucleosomes. We generalize this mechanism terms bistability valleys an epigenetic landscape. A transfer matrix method was used to rigorously follow system through disruptive process cell division. This combined treatment noisy...

Nucleosomes can be covalently modified by addition of various chemical groups on several their exposed histone amino acids. These modifications are added and removed enzymes (writers) recognized nucleosome-binding proteins (readers). Linking a reader domain writer that recognize create the same modification state should allow nucleosomes in particular to recruit nearby nucleosomes. This positive feedback has potential provide alternative stable heritable states required for epigenetic...

Significance Proteins bound to DNA often interact with proteins elsewhere on the same regulate gene expression. The intervening tethers near each other, making their interaction efficient and specific, but importance of this tethering effect is poorly understood at large separations. We quantitated inside bacterial cells, using two different separations up 10,000 bp, show that strong enough drive interactions over these distances. were ∼10-fold weaker outside implying cellular factors...

Genomic DNA is bound by many proteins that could potentially impede elongation of RNA polymerase (RNAP), but the factors determining magnitude transcriptional roadblocking in vivo are poorly understood. Through systematic experiments and modeling, we analyse how lac repressor (LacI) Escherichia coli cells controlled promoter firing rate, concentration affinity roadblocker protein, transcription-coupled repair protein Mfd, promoter-roadblock spacing. Increased readthrough roadblock at higher...

A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties D1-D4 have defined both global local level. Diffusion studies give an average effective radius 26.2 +/- 0.1 A, approximately 75% larger than that expected for globular this size....

Noise in the expression of a gene produces fluctuations concentration product. These can interfere with optimal function or be exploited to generate beneficial diversity between cells; noise is therefore expected subject evolutionary pressure. Shifts modes high and low rates transcription initiation at promoter appear contribute this both eukaryotes prokaryotes. However, models invoked for eukaryotic such as stable activation scaffolds persistent nucleosome alterations seem unlikely apply...

Phage lambda is among the simplest organisms that make a developmental decision. An infected bacterium goes either into lytic state, where phage particles rapidly replicate and eventually lyse cell, or lysogenic dormant replicates along with cell. Experimental observations by P. Kourilsky are consistent single infection deterministically choosing lysis double resulting in stochastic choice. We argue playing "game" of minimizing chance extinction shift from determinism to stochasticity due...

The basic helix–loop–helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends part on partner protein choices, where dimerization with common Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of interactions, we identified 22 amino acids the...