a. Complete knowledge about a genome would have to include:

b. A gene is expressed when its product is produced.

c. Genetic analysis involves research on the genomes of several organisms (e.g. E. coli and Homo sapiens).

d. One goal of this work is to develop a genetic map of the genome, showing gene locations, control elements, and what each gene does (Figure 17.1).

e. Genetic mapping is an extension of chromosome mapping (Chapter 16).

f. DNA sequences are now easily determined (Methods 14.1) and the genetic code, providing a translation of codons, is also known (Table 14.1).

g. The current challenge in genetic analysis is knowing both where each gene begins and ends and how the genetic code is used in each organism.

h. Messages in gene sequences are read three bases at a time with a reading frame in the 5' to 3' direction.

i. To know where to begin reading is often a problem, but the code indicates that genes usually begin with the RNA codon UAG, the DNA equivalent of ATG.

j. To find a gene sequence, one therefore looks for an open reading frame (ORF), defined as an ATG sequence that encodes a fairly long sequence of amino acids before reaching a termination codon, either TAA, TAG, or TGA.

k. An ORF could be a gene, but is not known to be such without further experimental evidence.

l. Experimental evidence using mutants provides information about the function of a gene by finding a mutant that either abolishes or alters the gene's function.

m. A marker is such a mutation that is used to map and identify a gene, as it provides an experimental definition of a gene.

n. Gene locations are then found by performing genetic crosses (see Section 16.8) between individuals carrying the markers of interest.

o. A point mutation is a change in a single nucleotide pair and is shown on a map as a point called its site, which establishes the locus of the gene.

a. Mutations, phenomena essential for the evolution of every species, occur spontaneously at random sites in every organism.

b. Mutagens are agents that can induce mutations, and include X-rays, UV light, and various chemicals.

c. X-rays are ionizing radiation, which knocks electrons out of atoms and produces free radicals that are damaging to cells.

d. X-rays often break chromosomes, causing damaging rearrangements of DNA.

e. UV light rearranges the pyrimidine bases in DNA, causing lethal chemical formations that stop replication of the molecule (Figure 17.2).

f. Some of this damage can be repaired by special enzymes.

g. Other enzymes allow damaged areas to be bypassed during replication, but the product is erroneous and a mutation has thus occurred.

h. Chemical agents cause a variety of mutations, including removing amino groups from nucleotides bases (e.g. converting G—C to A—T, Figure 17.3).

i. Chapter 20 further addresses chemical agents and their damaging effects.

a. Bacteria such as E. coli, along with the viruses (phage) that infect them were used to discover valuable information about genetic expression.

b. Specific bacteria mutants were discovered by plating bacteria on various controlled media (e.g. one containing streptomycin) and cloning the cells that survived.

c. Auxotrophic mutants, those that cannot make all of their own components, are useful in genetic analysis, and are found by the plating technique illustrated in Figure 17.4.

d. Section 12.3 reviews auxotrophic mutants.

e. A lethal mutation produces an inviable organism; a conditional lethal mutation (Figure 17.5) causes death only under certain conditions.

f. Conditional lethals are useful for studying mutations, and include temperature-sensitive (ts) and cold-sensitive (cs) mutants that die under certain temperature conditions but otherwise survive.

g. Bacteriophage mutants that are host-dependent (Figure 17.5) have identifiable defects that are useful for mapping, as they reproduce easily in permissive strains of bacteria but not in restrictive strains.

a. Genetic markers can be mapped using simple phage crosses.

b. The smaller the R (recombination rate) value, the better the resolution between two map positions.

c. Since R is the percent of recombinant progeny, increasing the total progeny number can provide a smaller, thus more accurate, R value.

d. In the 1950s, Seymour Benzer used large numbers of rII ("r two") phage T4 mutants to map bacterial mutations with increasingly precise R values (Figure 17.6).

e. A two gene cross involving can grow (C) and no growth (N) E. coli hosts for phage T4 shows four types (two parental types and two recombinant types) of offspring, and provides the R value that represents the distance between the two genes.

f. DNA recombination (Figure 17.7) involves enzyme-driven exchanges of DNA molecules to form heteroduplex structures, in which a segment of double helix has one strand from one parent and another strand from the other parent.

g. Methods 17.1 covers the mapping of deletion mutants, which are missing small segments of DNA.

a. Complementation tests can find the limits, or boundaries, of genes.

b. Two mutants complement each other if one supplies a function that the other cannot.

c. By infecting bacteria with phage carrying complementary mutants, and noting whether growth occurs, mutations can be separated into genes (Figure 17.8).

d. Mutations that define each gene do not mix with each other on a gene map, but are discrete regions.

a. Charles Yanofsky, et al., worked with E. coli tryptophan auxotrophs, and mapped their mutations using complementation tests.

b. Yanofsky's group determined amino acid sequences of the wild type form, and compared them with those of several mutants (Figure 17.9), showing that each mutation correlates with a single amino acid replacement in the protein product.

a. Nirenberg, working with information from experiments by Crick, Brenner, Barnett, and Watts-Tobin, showed that three-base codons encode each amino acid (Chapter 14).

b. Crick, et al., showed that the genetic code has no commas or spaces, so that a shift in the reading frame mechanism (from addition, deletion, or other event) will result in the wrong three bases being read and the wrong polypeptide being produced.

c. A frameshift mutation is one where the reading frame is shifted out of phase, as just described.

d. A suppressor mutation is one that inserts a base near the site of a deletion, and which shifts the frame back in phase for the rest of the DNA segment being read.

e. Every mutation can be classified arbitrarily as left-shift or right-shift (L or R) and, in general, any L should suppress any R mutation if their sites are close enough.

f. One test of this rule shows that, when three L or three R mutations are combined, the resulting triple mutant behaves like the wild type.

a. Tatum and Lederberg found that recombinant E. coli could be produced by combining two auxotrophic mutants to make a prototroph (Figure 17.10).

b. Recombination occurs when two parent cells come into contact during conjugation (Figure 17.10).

c. Plasmids, small extra circles of DNA in the bacterial cell (Figure 17.11), initiate conjugation and transfer copies of themselves into other cells.

d. The F (fertility) factor is a transferable plasmid (Figure 17.12) that confers conjugation upon a cell by producing pili on the cell surface of an F+ cell.

e. Episomes (Figure 17.13) are a class of plasmids that are either independent DNA molecules, or are integrated into the bacterial chromosome through crossing over.

f. Cells with an integrated F factor (Figure 17.14) are called Hfr for High frequency of recombination.

a. Jacob and Wollman followed the transfer of the Hfr chromosome in crosses of Hfr and F- strains.

b. Their results showed that the bacterial genes occur in a definite sequence and that they are transferred into the F- cells in that sequence.

c. Time-of-transfer experiments are used to map the bacterial chromosome (Figure 17.15).

d. Jacob and Wollman thus showed that the bacterial chromosome is circular before anyone had seen it using electron microscopy.

a. A variety of genetic factors exist and can invade the genomes of most organisms.

b. Viruses are particles that can only multiply within a cell by subverting the cell's genetic apparatus.

c. Each virus particle is a virion consisting of a nucleic acid and a protein coat called a capsid (see text art).

d. Most viruses reproduce in a lytic cycle, where the phage genome is injected into a bacterial cell, subverts the cell's genetic apparatus, fills the cell with new phage, and lyses the cell.

e. Some phage go into a noninfectious prophage state; the cells harboring these phage are said to be lysogenic, and they can grow and multiply normally (see text art.)

f. Phage that can establish lysogeny are called temperate phage.

g. Lysogeny results from a control system that keeps the phage genes quiescent; these phage can be induced to enter a lytic cycle by agents such as UV light (see text art).

h. Temperate phage that can enter lytic cycles are often responsible for outbreaks of human illness:

i. Some viruses can only replicate their DNA in a lytic cycle.

j. Some of these viruses have single-stranded genomes that enter a cell and set up a circular replicative form (RF) similar to a plasmid (see text art).

k. The F factor is a transmissible plasmid that can pass copies of itself into other cells, and is an episome.

l. Sidebar 17.1 describes resistance factors, whose genes confer resistance to antibiotics.

m. Some plasmids are virulence factors that make their hosts pathogenic.

n. Other plasmids produce colicins, toxic agents that kill certain other bacteria.

o. Transposons are small DNA elements that insert themselves into plasmids or into genomes of other organisms.

p. Transposons encode enzymes that promote recombination of the transposons themselves with other DNA molecules (see text art).

q. Insertion sequences are bacterial DNA sequences that can insert themselves into a genome and activate the gene in which they are inserted.

r. Barbara McClintock studied transposable elements in maize, which have receptor elements and regulator elements.

s. All these elements make possible the horizontal transmission of genetic information, which, when added to the vertical transmission of information due to inheritance, gives a new dimension to the mechanisms of evolution.

a. Norton Zinder found that bacterial cells could exchange genetic material even though they were not in contact and conjugating.

b. Virions were found to be carrying some DNA from host cells to other bacterial cells in a mode of exchange termed transduction.

c. Generalized transducers (Figure 17.16) can carry any bacterial genes, and any other piece of DNA, from one cell to another.

d. Specialized transducers (Figure 17.16) can carry only a few bacterial genes near their point of integration in the bacterial chromosome.

e. Transducing phage are thus commonly used for mapping small regions of bacterial chromosomes.

a. Restriction endonucleases in cells attack and degrade phage DNA that is from a different cell.

b. An endonuclease is an enzyme that breaks DNA in the interior of the molecule, rather than at the ends.

c. Each enzyme recognizes and cuts a particular sequence of nucleotides, and the sequences are always palindromic.

d. Every cell has modification enzymes that recognize such palindromic sequences and methylate them to protect them from nucleases.

e. These discoveries added great potential to genetic research using endonucleases.

a. DNA molecules cut with the same endonucleases will have identical ends, most of which are "sticky" for one another.

b. In the early seventies, Chang, Berg, and Cohen realized that any two pieces of DNA that have been cut with the same enzyme can be recombined, and recombinant DNA methodology began.

c. A "target DNA" molecule of interest can thus be inserted into, or recombined with, the DNA of a recipient host cell.

d. Figure 17.17 shows a classic mouse cell host system.

e. The steps to recombining fragments of DNA include the following:

f. The bacterial colonies that result from this process are clones, derived from a single bacterium.

g. A gene library is a set of such clones, containing different fragments of target DNA.

h. A shotgun experiment is one like this, where a specific target gene has not been preselected.

i. This technology has expanded so that genes from plants and animals can be cloned into bacteria, other microorganisms, or cells in tissue culture.