All genes have to be expressed in order to function. The first step in expression istranscription of the gene into a complementary RNA strand (Figure 11.1a). For somegenes—for example, those coding for transfer RNA (tRNA) and ribosomal RNA (rRNA)molecules—the transcript itself is the functionally important molecule. For other genes,the transcript is translated into a protein molecule.

To understand how a gene is expressed, the RNA transcript must be studied. Inparticular, the molecular biologist will want to know whether the transcript is a faithfulcopy of the gene, or whether segments of the gene are missing from the transcript(Figure 11.1b). These missing pieces are called introns and considerable interest centerson their structure and possible function. In addition to introns, the exact locations of thestart and end points of transcription are important. Most transcripts are copies not onlyof the gene itself, but also of the nucleotide regions either side of it (Figure 11.1c). Thesignals that determine the start and finish of the transcription process are only partlyunderstood, and their positions must be located if the expression of a gene is to be studied.In this chapter we will begin by looking at the methods used for transcript analysis.These methods can be used to map the positions of the start and end points fortranscription and also to determine if a gene contains introns. Then we will brieflyconsider a few of the numerous techniques developed in recent years for examininghow expression of a gene is regulated. These techniques are important as aberrations ingene regulation underlie many clinical disorders. Finally, we will tackle the difficultproblem of how to identify the translation product of a gene.

Studying the RNA transcript of a geneOver the years a variety of techniques have been devised for studying RNA transcripts.Some of these techniques merely detect the presence of a transcript and give someindication of its length, others enable the start and end of the transcript to be mappedand the positions of introns to be located.

11.1.1 Detecting the presence of a transcript and determiningits nucleotide sequence

northern hybridization

11.1.2 Transcript mapping by hybridization between geneand RNA

The problems with incomplete cDNA synthesis mean that other, more direct methodsare needed to identify the start point of a transcript. The most informative of thesemethods are based on examination of the hybridization product formed between acloned gene and its RNA.Nucleic acid hybridization occurs just as readily between complementary DNA andRNA strands as between single-stranded DNA molecules. If a hybrid is formed betweena DNA strand, containing a gene, and its mRNA, then the boundaries between thedouble- and single-stranded regions will mark the start and end points of the mRNA(Figure 11.4a). Introns, which are present in the DNA but not in the mRNA, will “loopout” as additional single-stranded regions.Now consider the result of treating the DNA–RNA hybrid with a single-strandspecificnuclease such as S1 (p. 46). S1 nuclease degrades single-stranded DNA or RNApolynucleotides, including single-stranded regions at the ends of predominantly doublestrandedmolecules, but has no effect on double-stranded DNA or on DNA–RNAhybrids. S1 nuclease will therefore digest the non-hybridized single-stranded DNA