Analysis of Gene Expression

Introduction

Monitoring Gene Expression

Reporter Genes for Monitoring Gene Expression

Easily Assayable Enzymes as Reporters

Light Emission by Luciferase as a Reporter System

Green Fluorescent Protein as a Reporter

Gene Fusions

Deletion Analysis of the Upstream Region

Locating Protein Binding Sites in the Upstream Region

Location of the Start of Transcription by Primer Extension

Location of the Start of Transcription by S1 Nuclease

Transcriptome Analysis

DNA Microarrays for Gene Expression

Serial Analysis of Gene Expression (SAGE)

Introduction

Gene expression may be examined in a variety of ways, both at the level of individual genes and, increasingly in recent years, at the level of the whole genome. By analogy with genomics, the sum total of an organisms RNA transcripts are sometimes referred to as the transcriptome. Here we will first consider individual genes and then cover approaches to screening expression of large numbers of genes simultaneously. This is known as transcriptome analysis and, with proteomics and metabolomics (see Ch. 26), makes up the area of functional genomics. Of the plethora of newly coined terms ending in -ome, perhaps the nicest is the "unknome" proposed by Mark Gerstein of Yale University. This consists of the large proportion of genes with no currently known function!

Gene expression is measured by monitoring the RNA or protein products that are made.

Monitoring Gene Expression

Expression of most genes results in their transcription to give RNA followed by translation of the RNA to give the final gene product, protein. In addition there are a few genes that produce non-coding RNA (such as tRNA or rRNA) and so have RNA as the final gene product. Although housekeeping genes are needed all the time, most genes are expressed only under certain environmental conditions or in particular tissues or at certain stages of the developmental cycle, as discussed in Chapters 9 and 10. Measurement of gene expression means estimating the level of gene product synthesized. Since most genes vary in expression under different conditions it is necessary to measure the level of gene expression under a variety of conditions.

It is possible to monitor gene expression directly by measuring the levels of protein or RNA. Proteins may be detected by running cell extracts on polyacrylamide gels or by antibody-based assays. If the protein is an enzyme the enzyme activity may be assayed. Direct detection and assay of proteins as gene products is deferred until Ch. 26, which covers proteomics.

Here we will consider the monitoring of gene expression at the transcriptional level. The transcriptional expression of a gene may be estimated by measuring the level of mRNA directly.This may be done by hybridization (Northern blotting) using a DNA probe specific for the sequence of the gene under investigation. Hybridization has already been discussed in Ch. 21. The use of fluorescent probes has greatly increased the sensitivity of Northern hybridization; nonetheless, for accurate measurement of the expression of individual genes under many different conditions, using gene fusions with reporter genes is preferable.

Genes whose products are convenient to assay are used as "reporters".

Reporter Genes for Monitoring Gene Expression

Genes that are used in genetic analysis because their products are easy to detect are known as reporter genes. They are often used to report on gene expression, although they may also be used for other purposes, such as detecting the location of a protein or the presence of a particular segment of DNA.

Suppose that a DNA molecule, such as a cloning plasmid, has been inserted into a new bacterial host cell or a transgene has been inserted into the chromosome of a new animal host. Antibiotic resistance genes are often used to monitor whether the DNA is indeed in the intended location. Thus antibiotic resistance genes may be regarded as reporter genes (Fig. 25.01). As already discussed in Ch. 22, after transformation of the plasmid into the target cells, they are treated with the antibiotic. Those functional genomics The study of the whole genome and its expression reporter gene Gene that is used in genetic analysis because its product is convenient to assay or easy to detect transcriptome The total sum of the RNA transcripts found in a cell, under any particular set of conditions

Reporter Genes for Monitoring Gene Expression 695

Bacterial cells

Transform

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