Vegetative λ DNA: III. Pulse-labeled Components

The structures of λ DNA labeled during short pulses of [^3H]thymine at various times after infection have been studied by ultracentrifugal analysis. Circular DNA's, both the supercoiled form and an open form, are the main DNA components synthesized in the first half of the latent period. The open circular form is the most rapidly synthesized and may be a precursor to the supercoiled DNA. During the later period of progeny DNA accumulation the open circular form and a heterogeneous, more rapidly sedimenting component(s) are synthesized during such pulses. These are apparently involved in the generation of the linear, viral DNA

A Comparison of the Initial Actions of Spleen Deoxyrihonuclease and Pancreatic Deoxyrihonuclease

The modes of action of several deoxyribonucleases have been studied in varying detail. The manner in which pancreatic deoxyribonuclease degrades deoxyribonucleic acid has been thoroughly analyzed both with regard to the breakdown of secondary structure and to the products formed. The activities of several other deoxyribonucleases have been characterized, especially those from Escherichia coli. A variety of specificities has been observed in which the preference seems to be most directly related to the secondary structure of the substrate and not rigorously to the internucleotide linkage

Promoter Binding by the Adr1 Transcriptional Activator May Be Regulated by Phosphorylation in the DNA-Binding Region

Background: Post-translational modification regulates promoter-binding by Adr1, a Zn-finger transcriptional activator of glucose-regulated genes. Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr1 protein synthesis, and a requirement for the kinase Snf1 for Adr1 DNA-binding. A fusion protein with the Adr1 DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation. Methodology/Principal Findings: Peptide mapping identified serine 98 adjacent to the Zn-fingers as a phosphorylation site. An antibody specific for phosphorylated serine 98 on Adr1 showed that the level of phosphorylated Adr1 relative to the level of total Adr1 decreased with glucose derepression, in a Snf1-dependent manner. Relative phosphorylation decreased in a PHO85 mutant, and this mutant constitutively expressed an Adr1-dependent reporter. Pho85 did not phosphorylate Adr1 in vitro, suggesting that it affects Adr1 indirectly. Mutation of serine 98 to the phosphomimetic amino acid aspartate reduced in vitro DNA-binding of the recombinant Adr1 DNA-binding domain. Mutation to aspartate or alanine affected activation of a reporter by full-length Adr1, and in vivo promoter binding. Conclusions/Significance: Mutation of Adr1 serine 98 affects in vitro and in vivo DNA binding, and phosphorylation of serine 98 in vivo correlates with glucose availability, suggesting that Adr1 promoter-binding is regulated in part by serine 98 phosphorylation

Vegetative λ DNA: III. Pulse-labeled Components

The structures of λ DNA labeled during short pulses of [^3H]thymine at various times after infection have been studied by ultracentrifugal analysis. Circular DNA's, both the supercoiled form and an open form, are the main DNA components synthesized in the first half of the latent period. The open circular form is the most rapidly synthesized and may be a precursor to the supercoiled DNA. During the later period of progeny DNA accumulation the open circular form and a heterogeneous, more rapidly sedimenting component(s) are synthesized during such pulses. These are apparently involved in the generation of the linear, viral DNA

Orbits and phase transitions in the multifractal spectrum

We consider the one dimensional classical Ising model in a symmetric dichotomous random field. The problem is reduced to a random iterated function system for an effective field. The D_q-spectrum of the invariant measure of this effective field exhibits a sharp drop of all D_q with q < 0 at some critical strength of the random field. We introduce the concept of orbits which naturally group the points of the support of the invariant measure. We then show that the pointwise dimension at all points of an orbit has the same value and calculate it for a class of periodic orbits and their so-called offshoots as well as for generic orbits in the non-overlapping case. The sharp drop in the D_q-spectrum is analytically explained by a drastic change of the scaling properties of the measure near the points of a certain periodic orbit at a critical strength of the random field which is explicitly given. A similar drastic change near the points of a special family of periodic orbits explains a second, hitherto unnoticed transition in the D_q-spectrum. As it turns out, a decisive role in this mechanism is played by a specific offshoot. We furthermore give rigorous upper and/or lower bounds on all D_q in a wide parameter range. In most cases the numerically obtained D_q coincide with either the upper or the lower bound. The results in this paper are relevant for the understanding of random iterated function systems in the case of moderate overlap in which periodic orbits with weak singularity can play a decisive role.Comment: The article has been completely rewritten; the title has changed; a section about the typical pointwise dimension as well as several references and remarks about more general systems have been added; to appear in J. Phys. A; 25 pages, 11 figures, LaTeX2

Adr1 and Cat8 Mediate Coactivator Recruitment and Chromatin Remodeling at Glucose-Regulated Genes

Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription.We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1. Through combined expression and recruitment data, along with analysis of chromatin remodeling at two of these genes, ADH2 and FBP1, we clarified how these activators achieve this wide range of co-regulation. We find that Adr1 and Cat8 are not intrinsically different in their abilities to recruit coactivators but rather, promoter context appears to dictate which activator is responsible for recruitment to specific genes. These promoter-specific contributions are also apparent in the chromatin remodeling that accompanies derepression: ADH2 requires both Adr1 and Cat8, whereas, at FBP1, significant remodeling occurs with Cat8 alone. Although over-expression of Adr1 can compensate for loss of Cat8 at many genes in terms of both activation and chromatin remodeling, this over-expression cannot complement all of the cat8Delta phenotypes.Thus, at many of the glucose-repressed genes, Cat8 and Adr1 appear to have interchangeable roles and promoter architecture may dictate the roles of these activators

Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase.

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects

Novel Intra-cellular Forms of Lambda DNA

The DNA complement of bacteriophage lambda is infective if certain conditions of bacteria and "helper" phage are satisfied (Kaiser & Hogness, 1960). However, after infection of sensitive bacteria by lambda phage, most of the injected DNA appears to lose its infectivity, at least as measured under these conditions. Little infective DNA can be recovered from the infected cell until mature phage appear or just prior to this time (Dove & Weigle, personal communication)