遗传学实验技术

The introduction of chromosome banding techniques about 20 years ago has not only provided a very powerful tool for chromosome identification, but has also highlighted structural and functional differences between different chromosomal segments (1). Restriction endonucle ...

In the fully contracted metaphase chromosome, the length of DNA is compacted at least 10,000 times, and many details of chromosome organization cannot be distinguished. Study of the more elongated prophase chromosomes permits the observation of finer details; for example, the number of ba ...

Hematological malignancies encompass a wide variety of diseases. To a certain extent the same basic method can be used to prepare chromosomes from all these diseases, largely because they all yield singlecell cultures relatively easily. Nevertheless, the fact that the different malig ...

The advent of chromosome banding techniques some 20 years ago (1,2) allowed the unequivocal identification of every chromosome in the human metaphase and provided a mapping scheme along each chromosome. Subsequently, a great deal of research has centered on preparing longer chromosom ...

Osmium tetroxide is an evil-smelling chemical used as a fixative in electron microscopy that can also be used to modify thymidine residues within DNA (1,2). The ability of osmium tetroxide to modify DNA is very sensitive to DNA conformation, In particular, osmium tetroxide will attack thymidi ...

A rigorous study of the interactions between DNA and proteins or other molecules requires the accumulation of a wide range of information regarding the nature of such an interaction, Among the parameters on which it is desirable to gain information are: the size and number of the binding site(s) on the ...

The RecA protein of Escherichia coli is essential for genetic recombination and has been extensively characterized (1–3). In vitro, purified RecA protein is able to promote recombination reactions of two types: (i) strand transfer between circular single-stranded DNA (ssDNA) and homo ...

Most transcription activator proteins have three important features that can be probed at the molecular level: They bind to specific sequences near promoters, they can be interconverted between active and inactive forms by covalent or noncovalent modification, and, when bound at targ ...

A type II restriction enzyme purchased from a commercial supplier comes with a specified number of units of enzyme activity. The units of restriction enzyme activity are defined by the minimal amount of enzyme needed to complete the digestion of 1 l.tg of bacteriophage γ DNA in 1 h. These units are usual ...

Type II restriction endonucleases cleave double-stranded DNA at sequence-specific sites typically 4–6 bp in length. Although large DNA molecules (viral DNA, plasmid DNA, and chromosomal DNA) are the physiological substrates for these enzymes, activity is often shown with small synt ...

An increasing number of structural studies are aimed at identifying the principles that govern protein-DNA recognition in gene regulation (1). This work depends on the successful reconstitution of protein-DNA complexes from their purified components. X-ray crystallography and ...

There are a number of proteins involved in DNA replication, recombination, or repair that bind stoichiometrically to single DNA strands of any nucleotide sequence, and which in some cases can also bind to single-stranded RNA. The best known examples are the ssb protein of E. coli, the gene 32 protein of ...

The asymmetric carbon atoms present in the sugars of nucleotides and in all the amino acids (with the exception of glycine) results in nucleic acids and proteins displaying optical activity. Further contributions to the optical activity of the polymers result from their ability to form well ...

Interference studies are just the inverse approach of “footprinting ” experiments. In both types of experiments the effect of a chemical modification of a single base on the binding of a sequence-specific protein is determined, but the experiments differ in the sequence of the binding event a ...

Fluoresence spectroscopy is a useful technique for investigating the interaction of DNA binding proteins with DNA. Generally, use is made of the intrinsic fluorescence of the protein arising from the aromatic amino acids, which is frequently perturbed in a DNA-protein complex (see Chap ...

Changes in the fluorescence emission spectrum of a protein on binding to DNA can often be used to determine the stoichiometry of binding and equilibrium binding constants; in some cases the data can also give an indication of the location of particular residues within the protein. The experimen ...

Bending of DNA by proteins plays an important role in transcription initiation, DNA replication, and recombination. The degree of pro- tein-induced DNA bending is conveniently determined by combin- ing gel retardation techniques with the use of so-called bending vectors (1,2). Bending v ...

Membrane filtration has a long history in the analysis of proteinnucleic acid complex formation, having first been used to examine RNA-protein interactions (1), before being introduced to DNA-protein interaction studies by Jones and Berg in 1966 (2). The principle of the technique is stra ...

In biological systems photoreactive derivatives have been widely applied to study specific interactions of receptor molecules with their ligands by photoaffinity labeling (1–3).

Photochemical crosslinking is a powerful method for studying all types of protein-nucleic acids interactions. In particular, UV-induced crosslinking has been successfully applied to the study of protein-DNA interactions (1). Ultraviolet (UV) light is a zero-length crosslink ...