生物化学技术

When using cells of mammmalian origin, the amount of biological material available for analytical purposes is often limited (e.g., ~l05 cells/sample, cor responding to few mg tissue). Since most intermediary metabolites are found in a concentratron range of ~50-500 μmol/g in a wide variety of t ...

The guanine nucleotides (rtbo and deoxyribo) serve many important roles in biosynthesis and btological control. They are involved in the synthesis of enzymes (1), and are allosteric regulators for such enzymes as glutamate dehydrogenase (2) and acetyl-CoA carboxylase (3). They regula ...

The “adenylate control hypotheses” proposed by Atkinson from UCLA (1) was further developed into the adenylate energy charge (AEC) concept in the 1960s (2, 3) and thereafter codified (4). This concept arose from making an analogy between the adenme nucleotide pool of ATP, ADP, and AMP, and a chemical s ...

Firefly luciferase has been widely used as a model substrate to study folding (1–3) and renaturation (4–6) of protein because of its rapid and sensitive bloluminescenct activity. Although normally localized in peroxisomes, luciferase folds to the native state on expression in bacteria, ...

Laboratories like the American Type Culture Collection (Rockville, MD) have been using freeze-drying or lyophilization techniques as a means of preserving microorganisms for more than 50 years. There are numerous variations of the method, and elaborate equipment is available to imp ...

The discovery and characterizatton of c&acting promoter regions and transacting regulatory proteins is often aided by use of reporter gene fusions In bacterial systems, frequently used reporters include 1acZ (β-galactostdase), phoA (alkaline phosphatase), cam (chloramphe ...

Transcriptional reporter systems have been extensively used to measure gene expression. Facile measurement of a reporter signal enables the study of gene expression in cases where protein product quantification is problematic. Reporters, such as /gbgalactostdase, provide co ...

The union of transposon mutagenesis and reporter gene fusion technologies has created a very powerful tool for analyzing gene expression. Transposable elements, such as phage Mu and Tn5, are able to move within a DNA molecule or from one DNA molecule to another, and can randomly integrate into the b ...

Bacterial mutagenesis assays have been used as preliminary screens for the evaluation of chemicals because they are rapid, simple, and are correlated with carcinogeneity in humans (1). The activation of bacterial DNA repair systems (recently reviewed; 2,3) can be used as a measure of mutage ...

The heat-shock response is the coordinated induction of a set of protems in response to a variety of cellular stresses, including elevated temperature (1). Several of the induced proteins function to reactivate or degrade denatured proteins, which are the signal initiating the response. T ...

In studies concerning drug—receptor interaction, the fundamental need is the knowledge of theevents that take place when a drug and a receptor combine. The current knowledge of microbial cell structure at the molecular level is restricted so that very often unsatisfymgly indirect meth ...

There are many reasons for testing cell viability. Simple tinctorial assays, such as trypan blue exclusion, have their place, but luminescence assays based on the detection of adenosine triphosphate (ATP) are particularly useful, since it is possible to detect the ATP present in fewer than 20 c ...

Phagocytes form an essential defence against microbial infection and have an important role in debridement following tissue injury. In human subjects, there are essentially two classes of phagocyte: polymorphnuclear leukocytes (PMNL) and mononuclear phagocytes, both deriv ...

This chapter consists of two methodologically rather divergent topics: the construction of a metal sensor bacterial strain and the measurement of bioavailable metal using such a strain. The constructton part IS written assummg that readers are famthar with basic recombinant DNA tech ...

Aquatic environments are continually being subjected to a wide variety of contaminants from industrtal, agricultural, and municipal sources. These contaminants include bacterial and parasitic pathogens, as well as toxic organic and inorgamc chemical compounds.

This chapter provides basic instructions in the use of genetically engineered Escherichia coli strains that luminesce in response to the presence of oxidants. These strains carry plasmid dertvatives of pUCD615 (1) in which Vibrio fischeri 1uxCDABE is driven by selected promoters of ge ...

One of the major routes of human exposure to mercury is by the consumptron of contammated fish and shellfish. Mercury, in the form of methyl mercury (MM), accumulates in these biota by biomagnification through the aquatic food chain, to concentrations orders of magnitude higher than its levels in ...

In several chapters in this book (Chapters 7, 8, 12, 13, 17–22), as well as m other instances (1–10), bacterial constructs were presented that respond by light emission to different environmental stresses. Such strains can serve as very useful tools for the study of bacterial responses to stress, but al ...

In recent years, several molecular methods have been developed for tracking genetically engineered microorganisms (GEMS) in environmental samples (1–3) The majority of the methods are based on monitoring of bacteria tagged with a marker gene, which provides the bacteria with a unique p ...

Catabolism of organic compounds has been extensively described in the literature (1). Many different compounds can be utilized as carbon and energy sources by bacteria, including aliphatics and aromatics, both man-made and natural. Very often these compounds will be broken down to useab ...