Claudio Vásquez

Plasmid curing in Thermus thermophilus and Thermus flavus

and keywords not received 1. I~RODUCTION 2. MATERIALS AND METHODS The elimination of plasmid DNA from plasmidcarrying host strains, or curing, has been described for several species of mesophilic bacteria. Techniques for this purpose involve growth of the strains at elevated temperatures [1], or in the presence of sodium dodecyl sulphate 121, intercalating drugs [3,4], inhibitors of DNA replication [5,6] or transcription [7], and the formation of protoplasts [8,9]. In contrast, curing of extremely thermophilic ~croorga~sms has not been reported so far in the literature. 2.1. Cells and growth conditions Thermus thermophilus HB8 was obtained from the American type culture collection (ATCC 2’7634). Therm~~av~ AT-62 was a generous gift of Dr T. Saiki, Japan. Cells were grown under conditions described in [ 131. 2.2. Plasmid elimination Authors in [lo] isolated several circular deoxyribonucleic acid molecules from 4 different strains belonging to the genus Thermus. We have character&& and cloned in Escherichia coli two of such molecules: pTT8 (6.0 MDa) (we had referred to this plasmid as pTT1; however, following the nomenclature proposed in [lo], hereinafter we shall call it pTT8) from Thermus thermophilus HB8 [ll]; and pTF62 (6,8 MDa) from Thermus fravus AT-62 [12]. To cure the strains from their plasmids, cultures were started with an initial concentration of 5000 cells/ml and were left to grow to stationary phase in the presence of the curing agent as indicated in table 1. Cells were then plated in 2% agar plates containing growth media, but devoid of the curing substance, and colonies were screened for the presence of pTT8 or pTF62 by el~rophoresis in agarose gels. Since pVV8 could not be visualized using direct colony screening, its presence had to be scored by electrophoresis of total DNA preparations from T. thermophilus HB8 in agarose gels. Cell lysates were prepared as in [ 11,121. Several agents known to promote curing of mesophilic bacteria, including ethidium bromide, acridine orange, rifampicin and novobiocin, were tested with T. thermophilus HB8 and T. j?avus AT-62. Of these, only tire gyrase inhibitor novobiocin was effective in eliminating pTT8 and pTF62 from their host strains, although at a low frequency, 2.3. Agarose gel electrophoresis Agarose (I%, Seakem) vertical slab gels were run in 50 mM Tris-HCl, 40 mM sodium acetate and 1 mM EDTA (pH 8.0). Gels were stained with ethidium bromide (1 pglml) and photographed under ultraviolet light. In the course of these experiments, a second plasmid (pVV8, 47 MDa) present in T. thermophilus HB8 was identified. 2.4. Electron microscopy DNA was prepared for electron microscopy as in [14] using pBR322 as a standard. Photographs of Published by ELwvier Science Publishers B. V. 00145793/83/

Conditions affecting DNA cleavage by TthI at a TthI endonuclease—dam methylase overlapping sequence

3.0

La Casa Errázuriz de le Corbusier: cronología del proyecto

The Escherichia coli dam gene codes for a sitespecific DNA methylase which methylates adenine in the sequence GATC at the N6 position of the purine ring [ 11. The effect of this modification on the action of several restriction enzymes whose recognition sites are equal or include the methylated sequence depends on the endonuclease itself. For example, the enzymes MboI, Bell and DpnII do not cleave a sequence containing GAme6TC [2-41, while DpnI requires the presence of 6MeAde in GATC sites in order to cleave the DNA [4]. On the other hand, isoschizomers Sau3A1, FtluEI and PfaI actively hydrolyze sequences that have undergone adenine methylation [S-7]: Sau3A1, however, is inhibited by the methylation of cytosine of the indicated sequence [8]. Backman has recently reported that methylation of adenine residues can also render DNA resistant to cleavage when there is an overlap between the recognition sequence of a restriction enzyme and that of the dam methylase [9]. One of such cases is represented by the sequence F

Impact of Shape, Occupation and External Parameters in the Overall Thermal Performance of Office Buildings in Santiago, Chile

i

La fachada ventilada

present in pBR322 at 1126 base pairs from the f?cuRI site 1101, in which the TaqI (TCGA) [ II] and dam (GATC) recognition sequences overlap. Since this latter phenomenon may lead to erroneouis observations when mapping genomes or during sequencing experiments, we have decided to characterize it further. Working with the enzyme TthI, an isoschizomer of TaqI isolated from Thermus thermophilus HB8 [ 12,131, we have found conditions that either increase or decrease susceptibility to DNA cleavage at the TthILdam methylase overlapping sequence.

La luz en la obra de Le Corbusier

Resumen es: La Casa Errazuriz, que no llego a ser construida, fue el unico proyecto de Le Corbusier en Chile. Los documentos en que se basa este articulo han sido ...

El programa arquitectónico en las bases de un concurso

espanol?Que tipo de fachada es mas e?ciente en terminos energeticos para un clima mediterraneo como el de Santiago? Este articulo presenta un analisis de e?ciencia energetica en edi?cios de o?cinas construidos en Santiago entre 2005 y 2011. Las conclusiones, eso si, distan bastante de lo que vemos en la actualidad: los edi?cios con fachadas mixtas (muros y vanos) resultaron ser mas e?cientes que aquellos con fachadas vidriadas. EnglishWhat kind of facade is the most energy e§cient for a Mediterranean climate like Santiago? This article presents an energy e§ciency analysis in o§ces built in Santiago between 2005 and 2011. The conclusions are far from what we see today: buildings with mixed facades (walls and windows) are more e§cient than glazed facades.

Solar and Lighting Transmission through Complex Fenestration Systems of Office Buildings in a Warm and Dry Climate of Chile

Office buildings in Santiago do not respond adequately to their climate context, which brings issues of discomfort at indoor spaces or high energy consumption in order to achieve comfortable conditions. The paper intends to establish certain relationships between several parameters registered on different office buildings in Santiago in order to address the issue about how different variables related to design and operation of the buildings impact on its overall performance. This is carried out via Principal Component Analysis of selected parameters registered inside several floors within representative office building types present in the city. Preliminary findings suggest that internal conditions hold no relation with external parameters due to the reliance on active air-conditioning systems; or with shape parameters registered on-site. On the other hand it is possible to identify occupation as a key parameter on workspace thermal performance and overheating as a relevant issue to consider at design stages.

Electron microscopy mapping of Escherichia coli RNA polymerase-binding sites on plasmids from thermophilic bacteria

A traves de una serie de mediciones realizadas en el contexto de la investigacion aplicada, este articulo presenta el ventajoso desempeno que presenta el sistema de fachadas ventiladas para la construccion en madera en la zona central de Chile.