生物化学技术

This chapter describes a DNA extraction method that can be used both on freeze-dried leaves and on fresh leaves, and is based on the method of Saghai-Maroof et al. (1), modified by David Hoisington and Jack Gardiner at the University of Missouri at Columbia (personal communication). The scale of the ext ...

Labeled nucleotides (radioactive or fluorescent) can be incorporated efficiently into double-stranded DNA by nick translation. Nick translation works by using DNase and DNA polymerase I enzymes. DNase cuts one strand of the DNA, exposing 5′-phosphoryl and 3′-hydroxyl (OH) termini DN ...

End-labeling is a rapid and sensitive method for radioactively, or nonisotopically, labeling DNA fragments and is useful in visualizing small amounts of DNA. End-labeling can also be used to label fragments at one end. All the enzymes employed are specific to either the 3′ or 5′ termini of DNA and will co ...

The purpose of this chapter is to illustrate how to obtain information on DNA and protein sequences from databases. This is most conveniently achieved using a Web browser (Netscape or Internet Explorer). This chapter is loosely based on a course given by the author at the University of Hertfordsh ...

The polymerase chain reaction (PCR) has rapidly become a standard laboratory technique. With the continuous development of PCR technology, there has been a growing need for PCR product quantitation. High-performance liquid chromatography (HPLC) is well accepted as a quantitative t ...

The polymerase chain reaction (PCR) is now commonly used in laboratories involved in research studies and clinical diagnostic work (1,2). A major advantage of PCR combined with reverse transcription (RT-PCR) is that it can be used to amplify and detect rare mRNA within a specimen. However, conve ...

The identification of individual chromosomes is of great importance in cytogenetics, in order to detect aneuploidies or chromosomal rearrangements associated with genetic diseases. This can be achieved by several techniques based either on the intrinsic staining properties of ...

The technique for labeling chromosomes by annealing an oligonucleotide DNA primer to the denatured DNA of chromosome preparations on glass slides and extending it enzymatically in situ with the incorporation of labeled nucleotides was first described by Koch et al. in 1989 (1). Since then, t ...

The human CYP1 family consists of at least three proteins, CYP1A1, CYP1A2, and CYP1B1 (1), CYP1A1 is absent or present at very low levels in human liver (2, 3), but its expression is readily detectable in lung (4, 5). By contrast, CYP1A2 is constitutively expressed in human liver (2, 3) and is absent in lung (4, 5). CYP1 ...

CYP2A6 has been isolated and purified to apparent homogeneity from human liver (1, 2). The level of CYP2A6 protein expressed in liver is low (∼4% of total hepatic P450 content) (3), although substantial intermdividual variation exists (3–5). This P450 is primarily a hepatic protein, as suggested by ...

CYP2B6 protein has been isolated and purified from human liver (1, 2). In a panel of 60 individual human liver microsome samples, this P450 accounts for

CYP2C8 is a major CYP2C protein expressed in human liver 1–4. Considerable interindividual differences (-20-fold) have been observed in hepatic CYP2C8 content (5) and a blmodal dlstrlbutlon in CYP2C8 protein amounts m a panel of human liver mlcrosomes has been reported (6). Experiments with ...

CYP2C 19 is one of at least three CYP2C enzymes expressed in human hver (1, 2), although the abundance of this P450 is generally less than that of etther CYP2C8 or CYP2C9 (3). Considerable interindividual differences occur in hepatic CYP2C19 content (1, 2), primarily owmg to a genetic polymorphism in th ...

CYP2C9 is a major CYP2C enzyme expressed in human liver (1–3). Recent experiments with primary cultures of human hepatocytes have suggested that CYP2C9 protein expression can be increased in cells treated with a P450 inducer such as phenobarbital, dexamethasone, or rlfampin (rifampic ...

CYP2D6 is an enzyme that is subject to genetic polymorphism 1. This P450 is absent in individuals with the poor metabolizer CYP2D6 phenotype 2, 3), resulting in a substantially reduced capacity to metabolize a large number of clinically useful drugs, including metoprolol, propafenone, hal ...

CYP2E1 is expressed in adult (1, 2) and fetal (3) human liver in addition to extrahepatic tissues such as lung and placenta (4). Treatment of primary cultures of human hepatocytes with ethanol induces CYP2E1 protein (5) and this is consistent with the finding that hepatic CYP2El protein (6) and mRNA (7) a ...

At least two cytochromes P450 belonging to the CYP3A subfamily may be expressed in adult human liver (1), CYP3A4 and CYP3A5. A third enzyme, CYP3A7, is expressed in human fetal liver (2). The CYP3A enzymes account for an estimated ~30% of total human cytochrome P450 content in adult liver (3), although lar ...

The human CYP4.411 gene encodes a P450 protein that has been isolated from human kidney (1, 2) and liver (3) and purified to apparent homogeneity. CYP4All mRNA is present in greater abundance in kidney than in liver, whereas it is absent in lung (3). Various fatty acids, including lauric acid (2–4), are subst ...

CYP7A1, which is the only CYP7A subfamily P450 tdentified to date (1), catalyzes cholesterol 7α-hydroxylation, the first, and rate-limiting, step in the converston of cholesterol to bile acids. CYP7Al has been Isolated from human liver and purified to apparent homogeneity (2). Rodent-mod ...

The progress of science is frequently the result of the mtroductlon of new types of instruments, and new instruments bring with them new techmques that must be mastered if one is to optimize new methods when seeking answers to specific questlons. The hlstory of cytochrome P450 (P450) research is in ...