Showing papers by "Michael McClelland published in 1988"

KGB: a single buffer for all restriction endonucleases.

Abstract: Most recommended restriction buffen contain Na and d\". However, in bacteria the most abundsnt intracelhilar cation and anion are usually potassium and gM îruK*. respectively (1). Furthermore, restriction endoaocleases cleave DNA in potassium glutamate (KGbj) over a much broader concentration range than they do in N»Q (2). These facts encouraged as to investigate the possibility that we could use KGlu in a chloride-free buffer and achieve normal level] of activity for all restriction endonuclease*. We have tested fifty-five restriction endonucleases for their ability to cleave DNA in a series of KGlu buffers (KGB, see Table 1) and compared the level of activity with that found under conditions recommended by the vendors (New England BioJabs, Boehringer Mannheim Biochem. and International Biotech. Inc.). Assays were performed u partial digests (0.2 units per ng of DNA in 30 ul for 30 min.) and as overnjfht digestions with excess enzyme to ensure that no lost of specificity (star activity) occurred. Most restriction endonudeases, potymerases and Ugase showed broad KGta concentration optima and aD enzymes functioned in 100 raM KGlu (IX KGB). Reducing agent was not normally required Some enzymes worked well in concentrations of KGlu over 400 mM (data not presented). KGB can be used to simplify laboratory prccedms tnrtnrting double digesa, DNA cleavage followed by end-labeling, or me digestion of DNA embedded in agarose prior to pulsed field gel electrophoresw DNA in KGB can be phenol extracted and ethanol precipitated using sandard protocols.

Recognition sequences of Type II restriction systems are constrained by the G+C content of host genomes

Abstract: I show that the recognition sequences of Type II restriction systems are correlated with the G + C content of the host bacterial DNA. Almost all restriction systems with G + C rich tetranucleotide recognition sequences are found in species with A + T rich genomes, whereas G + C rich hexanucleotide and octanucleotide recognition sequences are found almost exclusively in species with G + C rich genomes. Most hexanucleotide recognition sequences found in species with A + T rich genomes are A + T rich. This distribution eliminates a substantial proportion of the potential variance in the frequency of restriction recognition sequences in the host genomes. As a consequence, almost all restriction recognition sequences, including those eight base pairs in length (Not I and Sfi I), are predicted to occur with a frequency ranging from once every 300 to once every 5,000 base pairs in the host genome. Since the G + C content of bacteriophage DNA and of the host genome are also correlated, the data presented is evidence that most Type II "restriction systems" are indeed involved in phage restriction.

Torsional Stress, Unusual DNA Structures, and Eukaryotic Gene Expression

Abstract: Torsional stress in DNA plays a significant role in regulating gene expression in prokaryotes. It has been known for more than a decade that different promoters show a differential response to the level of in vivo supercoiling in E. coli (Smith et al., 1978), and extensive studies indicate that the in vivo level of supercoiling itself is tightly regulated (Menzel and Geliert, 1983). In E. coli and other bacteria the amount of torsional stress is sufficient (Sinden et al., 1980) to favor the formation of locally untwisted alternate DNA structures. The energetic cost of forming these from the normal unconstrained DNA double helix is more than compensated for by relief of some of the excess torsional tension in the DNA (Cantor and Efstratiadis, 1984).