Harpers

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 397quired for denaturation-renaturation. Segments of DNAwith high degrees of base-pair matching require more energyinput (heat) to accomplish denaturation—or, to putit another way, a closely matched segment will withstandmore heat before the strands separate. This reaction isused to determine whether there are significant differencesbetween two DNA sequences, and it underlies theconcept of hybridization, which is fundamental to theprocesses described below.There are about 3 10 9 base pairs (bp) in eachhuman haploid genome. If an average gene length is3 × 10 3 bp (3 kilobases [kb]), the genome could consistof 10 6 genes, assuming that there is no overlap and thattranscription proceeds in only one direction. It isthought that there are < 10 5 genes in the human andthat only 1–2% of the DNA codes for proteins. Theexact function of the remaining ~98% of the humangenome has not yet been defined.The double-helical DNA is packaged into a morecompact structure by a number of proteins, mostnotably the basic proteins called histones. This condensationmay serve a regulatory role and certainly hasa practical purpose. The DNA present within the nucleusof a cell, if simply extended, would be about1 meter long. The chromosomal proteins compact thislong strand of DNA so that it can be packaged into anucleus with a volume of a few cubic micrometers.DNA Is Organized Into GenesIn general, prokaryotic genes consist of a small regulatoryregion (100–500 bp) and a large protein-codingsegment (500–10,000 bp). Several genes are often controlledby a single regulatory unit. Most mammaliangenes are more complicated in that the coding regionsare interrupted by noncoding regions that are eliminatedwhen the primary RNA transcript is processedinto mature messenger RNA (mRNA). The coding regions(those regions that appear in the mature RNAspecies) are called exons, and the noncoding regions,which interpose or intervene between the exons, arecalled introns (Figure 40–1). Introns are always removedfrom precursor RNA before transport into thecytoplasm occurs. The process by which introns are removedfrom precursor RNA and by which exons areligated together is called RNA splicing. Incorrect processingof the primary transcript into the maturemRNA can result in disease in humans (see below); thisunderscores the importance of these posttranscriptionalprocessing steps. The variation in size and complexityof some human genes is illustrated in Table 40–1. Althoughthere is a 300-fold difference in the sizes of thegenes illustrated, the mRNA sizes vary only about 20-fold. This is because most of the DNA in genes is presentas introns, and introns tend to be much larger thanexons. Regulatory regions for specific eukaryotic genesare usually located in the DNA that flanks the transcriptioninitiation site at its 5′ end (5′ flankingsequenceDNA). Occasionally, such sequences arefound within the gene itself or in the region that flanksthe 3′ end of the gene. In mammalian cells, each genehas its own regulatory region. Many eukaryotic genes(and some viruses that replicate in mammalian cells)have special regions, called enhancers, that increase therate of transcription. Some genes also have DNA sequences,known as silencers, that repress transcription.Mammalian genes are obviously complicated, multicomponentstructures.Genes Are Transcribed Into RNAInformation generally flows from DNA to mRNA toprotein, as illustrated in Figure 40–1 and discussed inmore detail in Chapter 39. This is a rigidly controlledprocess involving a number of complex steps, each ofwhich no doubt is regulated by one or more enzymes orfactors; faulty function at any of these steps can causedisease.RECOMBINANT DNA TECHNOLOGYINVOLVES ISOLATION & MANIPULATIONOF DNA TO MAKE CHIMERIC MOLECULESIsolation and manipulation of DNA, including end-toendjoining of sequences from very different sources tomake chimeric molecules (eg, molecules containingboth human and bacterial DNA sequences in a sequence-independentfashion), is the essence of recombinantDNA research. This involves several uniquetechniques and reagents.Restriction Enzymes Cut DNAChains at Specific LocationsCertain endonucleases—enzymes that cut DNA at specificDNA sequences within the molecule (as opposedto exonucleases, which digest from the ends of DNAmolecules)—are a key tool in recombinant DNA research.These enzymes were called restriction enzymesbecause their presence in a given bacterium restrictedthe growth of certain bacterial viruses called bacteriophages.Restriction enzymes cut DNA of any sourceinto short pieces in a sequence-specific manner—incontrast to most other enzymatic, chemical, or physicalmethods, which break DNA randomly. These defensiveenzymes (hundreds have been discovered) protect thehost bacterial DNA from DNA from foreign organisms(primarily infective phages). However, they are presentonly in cells that also have a companion enzyme whichmethylates the host DNA, rendering it an unsuitablesubstrate for digestion by the restriction enzyme. Thus,