遗传学实验技术

The development of in vitro transposition technologies have provided many powerful tools for the molecular genetics research laboratory. In this chapter we describe some of these tools with a focus on the Tn5 transposition system. Tn5 technologies are particularly useful because the T ...

A brief survey of the retrotransposition cycle is given with emphasis on intermediary steps and their identification by biochemical analysis. References include some older key publications that may not be easily accessible via electronic databases. Methods for enrichment of viru ...

This chapter provides a simple guide for the computational analysis of transposable element (TE) sequences. Web links are provided for a number of sequence analysis applications, and their potential use in the analysis of TE sequences is briefly described. The level of detail provided is int ...

Detection of novel insertions and the ability to explore heterochromatin are two key goals in the study of mobile elements in Drosophila. The Southern blot analysis of individual flies can prove useful in both types of investigations, alone or in combination with genetic and cytological app ...

The technique of in situ hybridization of DNA probes to Drosophila chromosomes has been initially applied to the salivary gland polytene chromosomes and is now routinely used for mapping single-copy and repetitive DNA sequences, such as transposable elements, to the euchromatic regi ...

In situ hybridization is particularly appropriate for mapping specific DNA sequences on polytene chromosomes of Drosophila and other dipterans. This technique is based on the recognition and binding of one labeled sequence (the probe) to homologous sequences on chromosomes fixed on a ...

Transposable elements (TEs) are ubiquitous components of all living organisms, and in the course of their coexistence with their respective host genomes, these parasitic DNAs have played important roles in the evolution of complex genetic networks. The interaction between mobile DN ...

Experimental transfer of discrete nucleic acids to eukaryotic cells has allowed us to take giant strides in our basic understanding of eukaryotic gene expression and regulation. Viral particles transfer nucleic acid naturally to cells by the process of infection; the process of nucleic ...

Linker scanning mutagenesis is a powerful method with which to assay the contribution of individual DNA sequence elements within a transcriptional control region (1). By replacing discrete segments of DNA with heterologous segments of the same length, the topological and spatial org ...

Obtaining the promoter sequence for a gene when only the cDNA sequence is available can be an arduous task, especially because genomic DNA libraries usually in λ phage vectors need to be screened. An easier method to obtain promoter sequence without the need for libraries is to use the technique of inv ...

Transcription factors usually engage in multiple regulatory interactions with other proteins in their normal promoter or enhancer context. Conventional yeast two hybrid screens, in which a putative protein interaction domain is fused to a heterologous DNA binding domain like that ...

The presence of methylated cytosines (as 5-methylcytosine, 5-MeC) in eukaryotic DNA was established nearly 50 years ago. Nevertheless, the function of methylated nucleotides in DNA has not yet been fully established. They have been proposed to play a role in regulation of gene expression, in g ...

The vast bulk of the genome of eukaryotic cells exists as a highly regular array of nucleosomes. A single nucleosome comprises 146 bp of DNA wrapped around a histone core particle. Arrays of nucleosomes usually exist as condensed chromatin fibers with a nucleosome repeat length of about 200 bp. The D ...

In vitro transcription provides information not necessarily available from transfection assays about the regulatory activity of a promoter transcribed by RNA polymerase II. In the particular application discussed here, when an oligonucleotide duplex containing a recognit ...

Nuclear extracts prepared by appropriate methods contain all of the components for transcription (1). Although nuclear extracts are not defined like transcription systems utilizing purified transcription components (2–4) the extracts can be used to study aspects of promoter reg ...

Perhaps the most common technique used in the study of DNA-binding proteins is the electrophoretic mobility shift assay (EMSA) or gel shift assay. It can be used with crude protein mixtures or purified proteins in studies of, for example, the DNA sequence requirements of binding, kinetics of bind ...

The rabbit reticulocyte lysate (RRL) is a convenient system for in vitro translation of transcription factors (1). By using in vitro-synthesized transcription factors, the time-consuming and often difficult process of protein purification from cellular extracts of in vivo expres ...

The analysis of transcription factors in terms of their structure, function, and mode of interaction with DNA and other components of the transcriptional machinery has been greatly facilitated in recent years by the identification of genes encoding these proteins. Not only has this allo ...

The ability of oligonucleotides to bind double-stranded DNA in a sequence specific manner, forming a DNA triple helix structure, has led to a number of novel approaches in the study of various structural and functional properties of DNA in vitro and in vivo including transcription inhibition ...

Transfections into Schneider’s Drosophila line 2 (abbreviated SL2 or S2) derived from Drosophila embryos (1) have been used to analyze activation properties of mammalian transcription factors (Table 1), to identify activation and inhibitory domains, as well as to investigate speci ...