Molecular Biotechnology 

About: Molecular Biotechnology is an academic journal published by Springer Science+Business Media. The journal publishes majorly in the area(s): Medicine & Gene. It has an ISSN identifier of 1073-6085. Over the lifetime, 2686 publications have been published receiving 67708 citations. The journal is also known as: Applied Biochemistry and Biotechnology, Part B.

The Comet Assay for DNA Damage and Repair: Principles, Applications, and Limitations

Abstract: The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

The staden sequence analysis package

Abstract: I describe the current version of the sequence analysis package developed at the MRC Laboratory of Molecular Biology, which has come to be known as the “Staden Package.” The package covers most of the standard sequence analysis tasks such as restriction site searching, translation, pattern searching, comparison, gene finding, and secondary structure prediction, and provides powerful tools for DNA sequence determination. Currently the programs are only available for computers running the UNIX operating system. Detailed information about the package is available from our WWW site: http://www.mrc-lmb.cam.ac.uk/pubseq/.

Recombinant protein expression in Pichia pastoris.

Abstract: The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.

Methods for construction of adenovirus vectors.

Abstract: Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.

Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites.

Abstract: The function of ancillary domains and modules attached to the catalytic domain of mutidomain proteases, such as the matrix metalloproteinases (MMPs), are not well understood. The importance of discrete MMP substrate binding sites termed exosites on domains located outside the catalytic domain was first demonstrated for native collagenolysis. The essential role of hemopexin carboxyl-domain exosites in the cleavage of noncollagenous substrates such as chemokines has also been recently revealed. This article updates a previous review of the role of substrate recognition by MMP exosites in both preparing complex substrates, such as collagen, for cleavage and for tethering noncollagenous substrates to MMPs for more efficient proteolysis. Exosite domain interaction and movements—“molecular tectonics”—that are required for native collagen triple helicase activity are discussed. The potential role of collagen binding in regulating MMP-2 (gelatinase A) activation at the cell surface reveals unexpected consequences of substrate interactions that can lead to collagen cleavage and regulation of the activation and activity of downstream proteinases necessary to complete the collagenolytic cascade.