Chapter 2 Technologies for Mapping DNA CONTENTS Page Organization and Function of Genetic Figuresu Information . 21 Figure Page What Is a Genome? . 21 2-1. The Structure of DNA . 22 How Are Genomes Organized? . 21 2-2. Replication of DNA . 22 What Is the Genetic Code? . 21 2-3. Gene Expression . 23 How Big Is the Human Genome? . 24 2-4. Number of Human Gene Loci How Does the Human Genome Compare Identified From 1958 to 1987 . 24 to Other Genomes?. 24 2-5. Separation of Linked Genes by Why Does Hereditary Information Crossing Over of Chromosomes Change? . 25 During Meiosis . 26 Genetic Linkage Maps . 26 2-6. Detection of Restriction Fragment Linkage Maps of Restriction Fragment Length Polymorphisms Using Length Polymorphisms . 28 Radioactively Labeled DNA Probes. ., 29 RFLP Mapping . 28 2-7. Somatic Cell Hybridization . 31 When Is a Map of RFLP Markers 2-8. Chromosome Purification by the Complete? . 29 Flow Sorter . 32 Low-Resolution Physical Mapping 2-9. DNA Cloning in Plasmids. 36 Technologies . 30 2-10. Separation of Intact Yeast Somatic Cell Hybridization . 30 Chromosomes by Pulsed Field Chromosome Sorting . 31 Gel Electrophoresis. 38 Karyotyping . 32 2-11. Constructing a Library of Clones Chromosome Banding . 33 Containing Overlapping In Situ Hybridization . 33 Chromosomal Fragments . 38 Other Methods for Mapping Genes . 34 2-12. Making a Contig Map . 39 High-Resolution Physical Mapping 2-13. DNA Sequencing by the Sanger Technologies . 3S Method . 45 Cloning Vectors as Mapping Tools . 35 2-14. DNA Sequencing by the Maxam and Physical Mapping of Restriction Enzyme Gilbert Method . 46 Sites . ... ....4.... 37 2-15. Automated DNA Sequencing Using Physical Mapping of Nonhuman Fluorescently Labeled DNA. ........................ 48 Genomes . 41 Tables Strategies for Physical Mapping of the Table Page Human Genome . 43 2-1. The Genetic Code . 23 DNA Sequencing Technologies . 44 2-2. Haploid Amounts of DNA in Various Automation and Robotics in Mapping and Organisms . 25 Sequencing . 46 2-3. Amount of Genome Sequenced in Chapter 2 References . 48 Several Well-Studied Organisms . 47 Chapter 2 Technologies for Mapping DNA ORGANIZATION AND FUNCTION OF GENETIC INFORMATION what Is a Genome? structure. In multicellular organisms, DNA is gen- erally found as two linear strands wrapped around The fundamental physical and functional unit each other in the form of a double helix. A DNA of heredity is the gene. Genetics is the study of strand is a polymeric chain made of nucleotides, the patterns of inheritance of specific traits. The each consisting of a nitrogenous base, a deoxyri- chemical bearer of genetic information is deoxy- bose sugar, and a phosphate molecule (figure Z- ribonucleic acid (DNA). The DNA of multicellular 1). The arrangement of nucleotides along the DNA organisms such as insects, animals, and human backbone is called the DNA sequence. There are beings is associated with protein in highly con- four nucleotides used in DNA sequences: adeno- densed microscopic bodies called chromosomes. sine (A), guanosine (G), cytidine (C), and thymi- A single set, or haploid number, of chromosomes dine (T). The two strands of DNA in the helix are is present in the egg and sperm cells of animals held together by weak bonds between base pairs and in the pollen cells of plants. All body cells, of nucleotides. In nature, base pairs form only or somatic cells, carry a double set, or diploid num- between A’s and T’s and between G’s and C’s. The ber, of chromosomes, one originating from each size of a genome is generally given as its total parental set, The entire complement of genetic number of base pairs. material in the set of chromosomes of a par- ticular organism is defined as its genome. A full genome of DNA is regenerated each time a cell undergoes division to yield two daughter How Are Genomes organized? cells. During cell division, the DNA double helix unwinds, the weak bonds between base pairs Long before genetic material was identified as break, and the DNA strands separate. Free nucleo - DNA, maps of genes on chromosomes were con- tides are then matched up with their complemen- structed, and many of the details of transmission tary bases on each of the separated chains, and of genes from generation to generation were elu- two new complementary chains are made (figure cidated [Judson, see app, A]. The gene for color- 2-2). In human and other higher organisms, DNA blindness, for example, was assigned to the hu- replication occurs in the nucleus of the cell. This man X chromosome in 1911 (80), about 40 years DNA replication process was first proposed in before the discovery of the structure of DNA. In 1953 by Francis H.C. Crick and James D. Watson fact, it has been known for nearly a century that (19,73,74). the genetic material: ● has a structure that is maintained in stable What Is the Genetic Code? form, ● is able to serve as a model for replicas of itself, Most genes carry an information code that speci- ● has an information code that can be ex- fies how to build proteins. Proteins are an essen- pressed, and tial class of large molecules that function in the ● is capable of change or variation. formation and repair of an organism’s cells and tissues. Proteins can be components of essential Each of these features can be described in molecu- structures within cells, or they can carry out more lar terms based on the structure and function of active roles in the overall function of a particular DNA. cell type. Included in this important class of To know how DNA controls cell function, and molecules are hormones such as insulin, antibod- ultimately the structure and function of an en- ies to fight cellular infections, and receptors on tire organism, it is necessary to understand its the cell’s surface for modulating interactions be- 21 tween a particular tissue and its surroundings (68). increase the rate of the biochemical processes that Enzymes are a specialized group of proteins that take place in metabolism. Proteins are long chains of smaller molecules, Figure 2-1 .—The Structure of DNA called amino acids, that fold into the unique struc- tures necessary for protein function. The infor- Phosphate Molecule mation for generating proteins of specific amino / Deoxyribose Sugar Molecule // Nitrogenous , 0 When DNA replicates, the original strands unwind and sewe as templates for the building of new, complementary strands. The daughter molecules are exact copies of the parent, each daughter having one of the parent strands. SOURCE: Office of Technology Assessment, 1988. 23 acid sequences is found in the genetic code-a code ribonucleic acid (mRNA). The structure of based on sequences of nucleotides that are “read” ribonucleic acid (RNA) is very similar to that of in groups of three (table 2-I). Genetic informa- DNA. Figure 2-3 illustrates the major steps in gene tion is transmitted from DNA sequences to pro- expressi’on, namely: tein via another large molecule called messenger Table 2.1.—The Genetic Code Codon Amino Acid I Codon Amino Acid Codon Amino Acid Codon Amino Acid Each codon, or triplet of nucleotides in Uuu Phenylalanine Serine UAU Tyrosine UGU Cysteine Ucu RNA, codes for an amino acid. Twenty Uuc Phenylalanme Ucc Serine UAC Tyrosine UGC Cysteine different amino acids are produced from UUA Leucine UCA Serine UAA stop UGA stop a total of 64 different RNA codons, but UUG Leucme UCG Serine UAG stop UGG Tryptophan some amino acids are specified by more Cuu Leucme Ccu Proline CAU Histidine CGU Arginine than one codon (e.g., phenylalanine is Cuc Leucine ccc Proline CAC Histidine CGC Arginine specified by UUU and by UUC). In addition, CUA Leucine CCA Proline CM Glutamine CGA Arginine one codon (AUG) specifies the start of a CUG Leucme CCG Proline CAG Glutamine CGG Ar~inine protein, and three codons (UAA, UAG, and UGA) specify the termination of a protein. AUU Isoleucine ACU Threonine MU Asparagine AGU Serine Mutations in the nucleotide sequence can AUC Isoleucine ACC Threonine MC Asparagine AGC Serine change the resulting protein structure if AUA Isoleucine ACA Threonme AAA Lysine AGA Arginine the mutation alters the amino acid speci- AUG Methionine ACG Threonine AAG Lysine AGG Arginine fied by a codon or if it alters the reading (start) frame by deleting or adding a nucleotide. Aspartic acid GUU Valine GCU Valine GAU GGU Glycine U=uracil (thymine) A =adenine GUC Valine GCC Alanine GAC Aspartic acid GGC Glycine C=cytosine G =guanine GUA Valine GCA Alanine GAA Glutamic acid GGA Glycine GUG Valine GCG Alanine GAG Glutamic acid GGG Glycine SOURCES: Office of Technology Assessment and National Institute of General MedicalSciences, 198S. Figure 2“3.—Gene Expression I CYTOPLASM In the first step of gene expression, messenger RNA (mRNA) is synthesized, or transcribed, from genes by a process somewhat similar to DNA replication. In higher organisms, this process takes place in the nucleus of a cell. In response to certain signals (e.g., association with a particular protein), sequences of DNA adjacent to, or sometimes within, genes control the synthesis of mRNA. Protein synthesis, or translation, is the second major step in gene expression. Messenger RNA molecules are known as such because t hey carry messages specif ic to each of t he 20 different amino acids that make UP proteins. Once synthesized, mRNAs leave the nucleus of the cell and go to another cellular compartment, the cytoplasm, where their messages are t rans- lated into the chains of amino acids that make up proteins. A single amino acid is coded by a sequence of three nucleotides in the mRNA, called a codon.