A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Aprocedure forextracting plasmid DNAfrombacterial cellsis described. Themethodissimpleenough topermit theanalysis bygel electrophoresis of100ormoreclonesperdayyetyieldsplasmid DNAwhichis pureenough tobedigestible byrestriction enzymes.Theprinciple ofthe methodisselective alkaline denaturation ofhighmolecular weightchromosomal DNAwhilecovalently closedcircular DNAremains double-stranded. Adequate pHcontrol isaccomplished without usinga pHmeter.Uponneutralization, chromosomal DNArenatures toformaninsoluble clot,leaving plasmid DNAin thesupernatant. Largeandsmallplasmid DNAshavebeenextracted bythis method.

Arapid alkaline extraction procedure forscreening recombinant plasmid DNA

In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.

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The green fluorescent protein

Although DNA is the carrier of genetic information, it has limited chemical stability. Hydrolysis, oxidation and nonenzymatic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.

Instability and decay of the primary structure of DNA

In vivo medical imaging has made great progress due to advances in the engineering of imaging devices and developments in the chemistry of imaging probes Several modalities have been utilized for medical imaging, including X-ray radiography and computed tomography (x-ray CT), radionuclide imaging using single photons and positrons, magnetic resonance imaging (MRI), ultrasonography (US), and optical imaging In order to extract more information from imaging, “contrast agents” have been employed For example, organic iodine compounds have been used in X-ray radiography and computed tomography, superparamagnetic or paramagnetic metals have been used in MRI, and microbubbles have been used in ultrasonography Most of these, however, are non-targeted reagents

Molecular imaging is widely considered the future for medical imaging Molecular imaging has been defined as the in vivo characterization and measurement of biologic process at the cellular and molecular level1, or more broadly as a technique to directly or indirectly monitor and record the spatio-temporal distribution of molecular or cellular processes for biochemical, biologic, diagnostic, or therapeutic application2 Molecular imaging is the logical next step in the evolution of medical imaging after anatomic imaging (eg x-rays) and functional imaging (eg MRI) In order to attain truly targeted imaging of specific molecules which exist in relatively low concentrations in living tissues, the imaging techniques must be highly sensitive Although MRI, US, and x-ray CT are often listed as molecular imaging modalities, in truth, radionuclide and optical imaging are the most practical modalities, for molecular imaging, because of their sensitivity and the specificity for target detection

Radionuclide imaging, including gamma scintigraphy and positron emission tomography (PET), are highly sensitive, quantitative, and offer the potential for whole body scanning However, radionuclide imaging methods have the disadvantages of poor spatial and temporal resolution3 Additionally, they require radioactive compounds which have an intrinsically limited half life, and which expose the patient and practitioner to ionizing radiation and are therefore subject to a variety of stringent safety regulations which limit their repeated use4

Optical imaging, on the other hand, has comparable sensitivity to radionuclide imaging, and can be “targeted” if the emitting fluorophore is conjugated to a targeting ligand3 Optical imaging, by virtue of being “switchable”, can result in very high target to background ratios “Switchable” or activatable optical probes are unique in the field of molecular imaging since these agents can be turned on in specific environments but otherwise remain undetectable This improves the achievable target to background ratios, enabling the detection of small tumors against a dark background5,6 This advantage must be balanced against the lack of quantitation with optical imaging due to unpredictable light scattering and absorption, especially when the object of interest is deep within the tissue Visualization through the skin is limited to superficial tissues such as the breast7-9 or lymph nodes10,11 The fluorescence signal from the bright GFP-expressing tumors can be seen in the deep organ only in the nude mice 12,13 However, optical molecular imaging can also be employed during endoscopy14 or surgery 15,16

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New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging

The Preparation and Characterization of Ribonucleic Acids From Yeast

Formation of the American Society for Photobiology for meeting the needs of investigators studying the effects of light on man and other organisms is described. The scientific program of the first meeting of the society included discussions of the followiug topics: detrimental effects of excessive exposure to sunlight and artificial uv radiation; repair of uv-induced damage to cells; the roles of light in the human environnnent; photochemistry in photobiology; effects of near uv light; photosynthesis; bioluminescence; light perception without eyes; chronobiology; the ultraviolet world of insects; vision; and solar energy conversion. (HLW)

The Science of Photobiology

Dose dependent decrease in extractability of DNA from bacteria following irradiation with ultraviolet light or with visible light plus dye.

Repair of radiation-induced damage in Escherichia coli. I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation.

A much higher yield of DNA single-strand breaks was obtained in the DNA polymerase-deficient mutant Escherichia coli K-12 pol A1 after a given dose of x-rays than had been found before in Escherichia coli. The increased yield of single-strand breaks was due to the absence of a rapid repair system, which had not been described in Escherichia coli K-12. This absence probably accounts for the x-ray sensitivity of the pol A1 mutant. The rapid repair system can be reversibly inhibited in pol+ cells.

Kendric C. Smith

Papers

The Preparation and Characterization of Ribonucleic Acids From Yeast

The Science of Photobiology

Dose dependent decrease in extractability of DNA from bacteria following irradiation with ultraviolet light or with visible light plus dye.

Repair of radiation-induced damage in Escherichia coli. I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation.

DNA polymerase required for rapid repair of x-ray--induced DNA strand breaks in vivo.