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The bis-guanidinium toxins are a collection of natural products that display nanomolar potency against select isoforms of eukaryotic voltage-gated Na+ ion channels. We describe a synthetic strategy that enables access to four of these poisons, namely 11-saxitoxinethanoic acid, C13-acetoxy saxitoxin, decarbamoyl saxitoxin, and saxitoxin. Highlights of this work include an unusual Mislow-Evans rearrangement and a late-stage Stille ketene acetal coupling. The IC50 value of 11-saxitoxinethanoic acid was measured against rat NaV 1.4, and found to be 17.0 nm, similar to those of the sulfated toxins gonyautoxin II and III.

Clostridium taeniosporum, a non-pathogenic anaerobe closely related to the C. botulinum Group II members, was isolated from Crimean lake silt about 60 years ago. Its endospores are surrounded by an encasement layer which forms a trunk at one spore pole to which about 12–14 large, ribbon-like appendages are attached. The genome consists of one 3,264,813 bp, circular chromosome (with 26.6% GC) and three plasmids. The chromosome contains 2,892 potential protein coding sequences: 2,124 have specific functions, 147 have general functions, 228 are conserved but without known function and 393 are hypothetical based on the fact that no statistically significant orthologs were found. The chromosome also contains 101 genes for stable RNAs, including 7 rRNA clusters. Over 84% of the protein coding sequences and 96% of the stable RNA coding regions are oriented in the same direction as replication. The three known appendage genes are located within a single cluster with five other genes, the protein products of which are closely related, in terms of sequence, to the known appendage proteins. The relatedness of the deduced protein products suggests that all or some of the closely related genes might code for minor appendage proteins or assembly factors. The appendage genes might be unique among the known clostridia; no statistically significant orthologs were found within other clostridial genomes for which sequence data are available. The C. taeniosporum chromosome contains two functional prophages, one Siphoviridae and one Myoviridae, and one defective prophage. Three plasmids of 5.9, 69.7 and 163.1 Kbp are present. These data are expected to contribute to future studies of developmental, structural and evolutionary biology and to potential industrial applications of this organism.

Differences in economic opportunities give rise to strong migration incentives, across regions within countries, and across countries. In this paper we focus on responses to differences in welfare benefits across States within the United States. We apply the model developed in Kennan and Walker (2008), which emphasizes that migration decisions are often reversed, and that many alternative locations must be considered. We model individual decisions to migrate as a job search problem. A worker starts the life-cycle in some home location and must determine the optimal sequence of moves before settling down. The model is sparsely parameterized. We estimate the model using data from the National Longitudinal Survey of Youth (1979). Our main finding is that income differences do help explain the migration decisions of young welfare-eligible women, but large differences in benefit levels provide surprisingly weak migration incentives.

Temperature sensitivity of DNA polymerization and growth of a dnaX (Ts) mutant is suppressible at 39 to 40°C by mutations in the initiator gene, dnaA . These suppressor mutations concomitantly cause initiation inhibition at 20°C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX (Ts) . One dnaA (Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA (Cs,Sx) suppression of dnaX , which codes for both the τ and γ subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX (Ts) mutant at the somewhat intermediate temperature of 39 to 40°C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA (Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX (Ts) strains growing at 39 and 34°C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX (Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA (Cs,Sx) dnaX (Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39°C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39°C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38°C. Fourth, the dnaX (Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA (Cos) allele, which causes overinitiation, into the dnaX (Ts) mutant exacerbated its temperature sensitivity.

To define in vivo interactions of Escherichia coli DNA replication components, extragenic suppressors of a dnaZ(TS) mutant were isolated. A temperature-sensitive dnaZ mutant, which is defective in polymerization, was placed at 39 degrees C to select temperature-insensitive revertants. Some of these revertants also were cold sensitive, a phenotypic property that facilitated study of the suppressor. Mapping of the cold sensitivity indicated that some of the suppressor mutations are intragenic but others are located within the initiation gene, dnaA. The dnaA mutations that suppress the dnaZ(TS) defect are designated dnaA(SUZ, CS). The dnaA(SUZ, CS) strains have a defect in DNA synthesis at low temperature that is typical of an initiation defect. These data suggest that the dnaA product, an initiation factor, interacts in vivo with the dnaZ protein, a polymerization factor.

Mutations ts2158 and ts1882, which confer temperature sensitivity of septum formation, map near leu in the region of min 2.0 to 2.1 on the Escherichia coli chromosome. These mutants stop division abruptly and grow as filaments at 42 C; when returned to 28 C, division resumes after about 30 min to produce short cells. The product of the gene defined by these mutations probably is required during all stages of septum formation rather than specifically for initiation of septation. Filaments that formed at 42 C contained incomplete constrictiions (septa). When actively dividing filaments (i.e., those incubated at 28 C until division resumed) were shifted to 42 C a second time, division again stopped abruptly and incomplete constrictions persisted during the incubation at 42 C. Filaments that were subjected to 28 C incubation for a brief time (10, 20, or 30 min) before being shifted again to 42 C did not resume division as would be expected of a strain defective in initiating septation. Mutations ts 1882 and ts2158 and recessive to the ts+ allele, which is consistent with the interpretation that these mutations cause the loss of a function. They did not complement each other and presumably represent one cistron. Mutants carrying ts1882 and ts2158 mutations were compared with a mutant defective in the ftA allele, also known to map near leu.

Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX + and Y +; the shorter F210 carries dnaY +, but not X +. Lambda transducing phages that carry dnaX + or Y + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

Lambda tp mutants, selected for their ability to form turbid plaques on lon hosts, overproduce repressor. The tp1 and tp2 mutations have been located within (or adjacent to) the cIII gene. The tp1 mutation reduced late gene expression, as measured by endolysin synthesis (in the absence of functional cI repressor) and progeny phage yield. The tp4 mutation was mapped in the cY-cII region, and complementation tests indicated that tp4 affects the diffusible product of the cII gene. The tp4 mutation also reduced progeny production, but did not markedly affect endolysin synthesis.

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the gamma and tau subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the gamma and tau subunits of holoenzyme. To characterize biochemically the tau subunit, for which there has been no activity assay, the dnaZX gene was fused to the beta-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (tau) were replaced by beta-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained beta-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the beta-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the gamma subunit.

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli . A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH + is carried on the F′ 13 episome and is dominant over the dnaH − mutation.

Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants. Those suppressed strains which concomitantly became cold sensitive were chosen for further study. Intragenic suppressor mutations, which caused cold-sensitive defects in DNA polymerization, were located in dnaZ by transduction with lambda dnaZ+ phages. Extragenic suppressor mutations were mapped within the initiation gene dnaA. These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells. The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo. A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal [whether the strain was dnaZ+ or dnaZ(Ts)].