Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4⁺ and REV5⁺ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.     

Fine Structure Mapping in Yeast with Sunlamp Radiation

The X-ray mapping procedure of Manney and Mortimer (1964) is the most widely applicable and convenient method for fine structure analysis in yeast, but suffers the disadvantage that suitable X-ray machines or gamma ray sources are very expensive. Although many other recombinogens are known, none gives a linear dose-response like X-rays and few are as convenient or give as reproducible results. Experiments with Saccharomyces cerevisiae reported in this paper show, however, that the near-ultraviolet radiation emitted by fluorescent sunlamps gives linear dose-response relations, as reproducible results as ionizing radiations, and map distances which correlate highly with those obtained by using (60)Co gamma rays. It is suggested that this convenient recombinogen may be a suitable low-cost substitute for ionizing radiations in fine structure mapping.     

Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-³H]1-N-naphthylphthalamic acid and 3-indolyl-[2-¹⁴C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (K_D at 10^-8mol·l^-1) for one site and low affinity (K_D at 10^-6 mol·l^-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (K_D below 10^-8 mol·l^-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.Abbreviations IAA Indol-3-yl acetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid W.M. was supported in part by an allowance from CSIRO and in part by a fellowship of the Deutsche Forschungsgemeinschaft; he acknowledges the friendly hospitality of the CSIRO Division of Plant Industry. The authors thank R. Hertel (Freiburg) for valuable discussion.     

Auxin transport inhibitors: fluorescein and related compounds

Fluoresceins are shown to be effective inhibitors of indoleacetic acid transport as measured by the receiver agar block technique, eosin having the same order of activity as 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid, with fluorescein less effective. It is suggested that many of their characteristic effects on plants, especially those which involve auxin, are at least partially due to their effects on auxin transport.     

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.     

Biomechanical evaluation of porous biodegradable scaffolds for revision knee arthroplasty

Tibial bone defect is a critical problem for revision knee arthroplasty. Instead of using metallic spacer or cement, biodegradable scaffolds could be an alternative solution. A numerical model of a revision knee arthroplasty was thus developed to estimate the mechanical resistance of the scaffold in this demanding situation. The tibia, scaffold, and prosthesis were represented by simplified parameterised geometries. The maximal gait cycle force was applied asymmetrically to simulate a critical loading. Several parameters were analysed: 1) inter-individual variability, 2) cortical bone stiffness, 3) cortical bone thickness, 4) prosthesis fixation quality, and 5) scaffold thickness. The calculated scaffold strain was compared to its experimental ultimate strain. Among the tested parameters, failure was only predicted with scaffold thickness below 5 mm. This study suggests that biodegradable bone scaffolds could be used to fill bone defects in revision knee arthroplasty, but scaffold size seems to be the limiting factor.