Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.     

Susceptibility of Campylobacter jejuni and Yersinia enterocolitica to UV radiation

Two enteric pathogens, Campylobacter jejuni and Yersinia enterocolitica serogroup O:3, together with Escherichia coli, were investigated for susceptibility to UV radiation at 254 nm. The UV dose required for a 3-log reduction (99.9% inactivation) of C. jejuni, Y. enterocolitica, and E. coli was 1.8, 2.7, and 5.0 mWs/cm2, respectively. Using E. coli as the basis for comparison, it appears that C. jejuni and Y. enterocolitica serogroup O:3 are more sensitive to UV than many of the pathogens associated with waterborne disease outbreaks and can be easily inactivated in most commercially available UV reactors. No association was found between the sensitivity of Y. enterocolitica to UV and the presence of a 40- to 50-megadalton virulence plasmid.     

Efficiency in cloning and sequencing using the single-stranded bacteriophage M13

A number of approaches to sequence DNA by the chain termination method are based on cloning into M13 phage vectors and the use of a universal primer. In this paper we investigate some of the factors which influence the speed and efficiency of these approaches. A modification of the template preparation, sequencing reaction, and gel system was used to obtain more reliable and clearer ladder gels. Redundancy and deficiency of shotgun DNA sequencing were reduced by a mapping technique and the use of synthetic primers. The mapping and cloning technique was used to organize the data entry so that the computer time necessary to reconstruct the sequence out of overlaps was reduced.     

A monoclonal antibody stains myogenic cells in regenerating newt muscle

Monoclonal antibodies have been used to study minced muscle regeneration in the adult newt, Notophthalmus viridescens. The contralateral limb was amputated and the immunostaining patterns in the regenerating blastema were compared with the minced tissue in sectioned material. Staining with a myofibre-specific antibody, called 12/101 (Kintner & Brockes, 1984), showed that myofibre degeneration was complete by 8-10 days after mincing, with myogenesis commencing 2 days later. Another monoclonal antibody, called 22/18, previously shown to label a subset of cells in the regeneration blastema of the newt (Kintner & Brockes, 1984, 1985), was found also to recognize a population of cells in regenerating minced muscle. At 6 days after mincing, the number of 22/18-positive (22/18+) cells was low but by days 12-16, during the period of myogenesis, their number had increased to become a major population within the minced tissue. A small number of the 22/18+ cells could be double labelled with 12/101 at this time. Prior to this, there was a phase in which 12/101 staining had disappeared from the mince. Cells immunoreactive with both antibodies after this phase confirm that at least some of the 22/18+ cells are myogenic. The number of 22/18+ cells decreased as muscle repair and maturation progressed. These results show that 22/18 is not specifically associated with blastemal cells but is a more general marker for regenerating systems in the newt. They further suggest an alternative interpretation of the double-labelled cells used by Kintner & Brockes (1984) as evidence for myofibre dedifferentiation in limb regeneration. Instead, we propose that such cells represent new myogenesis occurring by tissue repair of locally damaged muscle fibres.     

Utilization of conical equilibrium dialysis cells to shorten equilibration time

Equilibrium dialysis is commonly used to characterize the binding properties of macromolecules; however, it is not always preferred because of the lengthy time required to reach equilibrium. Consequently, other methods have been developed. This paper compares the disadvantages of various methods for obtaining binding data and shows that utilization of a conically shaped chamber can accelerate the process of equilibrium dialysis. For cylindrical and conical cells of different dimensions a linear relationship was found for the time of equilibration versus the ratio of volume to surface area. Minimizing the volume to surface area ratio by using a conical chamber shortens the time of equilibration by more than half and facilitates sampling of the chambers' contents.     

Evidence that physiologic levels of circulating estrogens and neonatal sex-imprinting modify postpubertal hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme a reductase activity

Ontact, but sham-operated female rats had 2- to 3-fold higher levels of hepatic 3-hydroxy-3-methylglutary CoA reductase activity than their male couterparts (15–21.5 vs. 6.7–8.7 nmol mevalonate/mg protein per h). The activity of the hepatic enzyme declined to about the same relative degree (40–60%) in male and female rats that were gonadectomized after puberty (53 days of age) and killed 5 weeks later. Implantion of silastic capsules containing 17β-estradiol increased the level of hepatic 3-hydroxy-3-methylglutaryl CoA reductase to levels found in sham-operated controls. In rats that were gonadectomized in infacny (12 h old) and killed 7–8 weeks later, the level of enzyme activity was not altered in females, but it was increased from 60–240% in males. Consequently, following neonatal gonadectomy, male-female differences in enzyme activity were no longer apparent. Implantation of islastic capsules containing estradiol in neonatally gonadectomized rats resulted in a doubling of enzyme activity in both males and females. Ovariectomy reduced plasma estrogen levels, but implantation of estradiol in gonadectomized males and behavioral characteristics. We found in confirmation of an earlier study [20], that in comparison to females, the higher body weight of males and presumably their increased food intake, was also dependent on sex imprinting that occured prior to birth. This observation takes on particular significance in view of the recent report that the amount and quality of food eaten during infancy exerted a long lasting effect on the post-pubertal regulation of 3-hydroxy-3-methyl-glutaryl CoA reductase activity [21,22] and bile acid synthesis [23]. Thus, while a direct effect of neonatal sex imprinting on the regulation of 3-hydroxy-3-methyglutaryl CoA reductase activity is still possible, more indirect mechanisms [24] should also be considered.     

BICAR: A New Algorithm for Multiresolution Spatiotemporal Data Fusion

We introduce a method for spatiotemporal data fusion and demonstrate its performance on three constructed data sets: one entirely simulated, one with temporal speech signals and simulated spatial images, and another with recorded music time series and astronomical images defining the spatial patterns. Each case study is constructed to present specific challenges to test the method and demonstrate its capabilities. Our algorithm, BICAR (Bidirectional Independent Component Averaged Representation), is based on independent component analysis (ICA) and extracts pairs of temporal and spatial sources from two data matrices with arbitrarily different spatiotemporal resolution. We pair the temporal and spatial sources using a physical transfer function that connects the dynamics of the two. BICAR produces a hierarchy of sources ranked according to reproducibility; we show that sources which are more reproducible are more similar to true (known) sources. BICAR is robust to added noise, even in a “worst case” scenario where all physical sources are equally noisy. BICAR is also relatively robust to misspecification of the transfer function. BICAR holds promise as a useful data-driven assimilation method in neuroscience, earth science, astronomy, and other signal processing domains.     

In vivo restriction. Sequence and structure of endonuclease II-dependent cleavage sites in bacteriophage T4 DNA.

Endonuclease II of bacteriophage T4 is required for in vivo restriction of cytosine-containing DNA from its host, Escherichia coli, (as well as from phage mutants lacking cytosine modification), normally the first step in the reutilization of host DNA nucleotides for synthesis of phage DNA in infected cells. The phage cytosine-DNA is fragmented incompletely to yield genetically defined fragments. This restriction is different from that of type I, II, or III restriction enzymes. We have located seven major endonuclease II-dependent restriction sites in the T4 genome, of which three were analyzed in detail; in addition, abundant sites were cleaved in less than or equal to 5% of all molecules. Sites I, II, and III shared the sequence 5'-CCGNNTTGGC-3' and were cleaved in about 25% (I and III) and 65% (II) of all molecules, predominantly staggered around the first or second of the central unspecified base pairs to yield fragments with one 5' base. The less frequently cleaved sites I and III deviated from site II in predicted helical structure when viewed from the consensus strand, and in sequence when viewed from the opposite strand. Thus, interaction with a particular helical structure as well as recognition of the bases in DNA appears important for efficient cleavage.     

A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50–70MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasm id-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. cob strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The 8FP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.