Clone-a population of cells that are genetically identical
Genome-all the genes present in a cell or virus; procaryotes normally have one set of genes (haploid) whereas eucaryotic microbes usually have two sets (diploid)
Genotype-the specific set of genes an organism possesses
Phenotype-the collection of characteristics of an organism that an investigator can observe
DNA as the Genetic Material
Griffith (1928) demonstrated the phenomenon of transformation: nonvirulent bacteria could become virulent when live, nonvirulent bacteria were mixed with dead, virulent bacteria
Avery, MacLeod, and McCarty (1944) demonstrated that the transforming principle (the material responsible for transformation to virulence in Griffithís experiments) was DNA
Hershey and Chase (1952) showed that for the T2 bacteriophage, only the DNA was needed for infectivity; therefore, they proved that DNA was the genetic material
Over the past decades the relationship between DNA, RNA, and protein has been established
DNA is the genetic material of cells; DNA is precisely copied by a process called replication
A gene is a DNA segment that encodes a polypeptide, an rRNA, or a tRNA
Genes are expressed when the information they encode is transcribed, forming an RNA molecule complementary to the original DNA template
mRNA molecules direct the synthesis of proteins; the decoding of the mRNA information occurs during a process called translation
Nucleic Acid Structure
DNA Structure
DNA is composed of purine and pyrimidine nucleosides that contain the sugar 2¢-deoxyribose and are joined by phosphodiester bridges
DNA is usually a double helix consisting of two chains of nucleotides coiled around each other
The purine adenine (A) on one strand of DNA is always paired with the pyrimidine thymine (T) on the other strand, while the purine guanine (G) is always paired with the pyrimidine cytosine (C); thus, the two strands are said to be complementary
The two strands are not positioned directly opposite one another; therefore, a major groove and a smaller minor groove are formed by the double helix backbone
The two polynucleotide chains are antiparallel (i.e., their sugar-phosphate backbones are oriented in opposite directions)
RNA structure
RNA differs from DNA in that it is composed of the sugar ribose rather than 2¢-deoxyribose
RNA differs from DNA in that it contains the pyrimidine uracil (U) instead of thymine
RNA differs from DNA in that it usually consists of a single strand that can coil back on itself, rather than two strands coiled around each other
Three different kinds of RNA exist: ribosomal (rRNA), transfer (tRNA), and messenger (mRNA); they differ from one another in function, site of synthesis in eucaryotic cells, and structure
The organization of DNA in cells
In procaryotes, the DNA exists as a closed circular, supercoiled molecule associated with basic (histonelike) proteins
In eucaryotes, the DNA is more highly organized; it is associated with basic (histone) proteins and is coiled into repeating units known as nucleosomes
DNA Replication
Pattern of DNA synthesis
DNA replication is semiconservative: each strand of DNA is conserved, but the two strands are separated from each other and serve as templates for the production of another strand (according to the base-pairing rules discussed earlier)
Replication forks are the areas of the DNA molecule where this strand separation occurs and the synthesis of new DNA takes place
A replicon consists of an origin of replication and the DNA that is replicated as a unit from that origin
The bacterial chromosome is usually a single replicon
Small closed circular DNA molecules, such as plasmids and some virus genomes, replicate by means of a rolling-circle mechanism
The large linear DNA molecules of eucaryotes employ multiple replicons to efficiently replicate the relatively large molecules within a reasonable time span
Mechanism of DNA replication-as observed in E. coli
DnaA protein binds to the origin of replication
Helicases unwind the two strands of DNA and as they do so topoisomerases (e.g., DNA gyrase) relieve the tension caused by the unwinding process
Single-stranded DNA binding proteins (SSBs) keep the single strands apart
Primases synthesize a small RNA molecule (approximately 10 nucleotides) that will act as a primer for DNA synthesis
DNA polymerase III synthesizes the complementary strand of DNA according to the base-pairing rules; on one strand (the leading strand), synthesis is continuous, while on the other (the lagging strand), a series of fragments are generated by discontinuous synthesis; a multiprotein complex called a replisome organizes all of these processes
DNA polymerase I removes the primers and fills the gaps that result from the RNA deletion
DNA ligases join the DNA fragments to form a complete strand of DNA
DNA replication is extraordinarily complex; at least 30 proteins are required to replicate the E. coli chromosome
The rate of DNA synthesis is 750 to 1,000 base pairs per second in procaryotes, and 50 to 100 base pairs per second in eucaryotes
The Genetic Code
For polypeptide-coding genes, the DNA base sequence corresponds to the amino acid sequence of the polypeptide (colinearity)
Establishment of the genetic code-each codon that specifies a particular amino acid must be three bases long for each of the 20 amino acids to have at least one codon; thus the genetic code consists of 64 codons
Organization of the code
Degeneracy-many amino acids are encoded by more than one codon
Sense codons-61 codons that specify amino acids
Stop (nonsense) codons-three codons (UGA, UAG, UAA) that do not specify an amino acid, and that are used as translation (protein synthesis) termination signals
Wobble-describes the somewhat loose base pairing of a tRNA anticodon to the mRNA codon; wobble eliminates the need for a unique tRNA for each codon because the first two positions are sufficient to establish hydrogen bonding between the mRNA and the aminoacyl-tRNAs
Gene Structure
Gene-a linear sequence of nucleotides that is within the genomic nucleic acid molecule, and that has a fixed start point and end point
Encodes a polypeptide, a tRNA, or an rRNA
Has controlling elements (e.g., promoters) that regulate expression of a gene; may be considered as part of the gene itself, or they may be considered as separate regulatory sequences
With some exceptions, genes are not overlapping
The segment that encodes a single polypeptide is also called a cistron
In procaryotes-coding information is normally continuous although some bacterial genes are interrupted; in eucaryotes-most genes have coding sequences (exons) that are interrupted by noncoding sequences (introns)
Genes that code for proteins
Template strand-the one strand that contains coding information and directs RNA synthesis
Promoter-a sequence of bases that is usually situated upstream from the coding region; serves as a recognition/binding site for RNA polymerase
Recognition site-site of initial association with RNA polymerase (35 bases upstream of transcription initiation site)
Binding site (Pribnow box)-sequence that favors DNA unwinding before transcription begins (approximately 10 bases upstream of transcription initiation site)
Consensus sequences-idealized base sequences found most often when comparing the sequences of different bacteria
Leader sequence-a transcribed sequence that is not translated; contains a consensus sequence known as the Shine-Dalgarno sequence, which serves as the recognition site for the ribosome
Coding region-the sequence that begins immediately downstream of the leader sequence; starts with the template sequence 3¢TAC5¢, which gives rise to mRNA codon 5¢AUG3¢, the first translated codon (specifies N-formylmethionine in bacteria, methionine in archaea and eucaryotes)
Trailer region-nontranslated region located immediately downstream of the translation terminator sequence and before the transcription terminator
Regulatory sites-sites where DNA-recognizing regulatory proteins bind to either stimulate or inhibit gene expression
Genes that code for tRNA and rRNA
tRNA genes-promoters, leaders, coding regions, and trailer regions are found; noncoding regions are removed after transcription; more than one tRNA may be made from a single transcript; the tRNAs are separated by a noncoding spacer region, which is removed after transcription
rRNA genes-have promoters, leaders, coding regions and trailer regions; all rRNA molecules are transcribed as a single large transcript, which is cut up after transcription, yielding the final rRNA products
Mutations and their Chemical Basis
Mutation-a stable, heritable change in the genomic nucleotide sequence
Mutations and mutagenesis
Mutations can alter phenotype
Morphological mutations-result in changes in colony or cell morphology
Lethal mutations-result in death of the organism
Conditional mutations-are expressed only under certain environmental conditions
Biochemical mutations-result in changes in the metabolic capabilities of a cell
1) Auxotrophs-cannot grow on minimal media because they have lost a biosynthetic capability; require supplements
2) Prototrophs-can grow on minimal media
Resistance mutations-result in acquired resistance to some pathogen, chemical, or antibiotic
Mutations can arise spontaneously or can be induced by a mutagen
It is widely held that spontaneous mutations occur randomly and are then selected; however, one hypothesis holds that some mutations are directed or adaptive mutations that may be the result of hypermutation followed by selection of favorable mutations
Spontaneous mutations
Arise occasionally in all cells without exposure to external agents; they are often the result of errors in replication
Errors in replication can occur due to tautomeric shifts, which cause base pair substitutions; other errors can lead to frameshifts
Transition-substitution of one purine for another, or of one pyrimidine for another
Transversion-substitution of a purine for a pyrimidine or vice versa
Frameshift-often the result of the deletion of nucleotides, which results in an altered codon reading frame
Lesions in the structure of DNA can cause spontaneous mutations (e.g., loss of a nitrogenous base)
Insertion of DNA segments (e.g., insertion sequences and transposons) can cause spontaneous mutations
Induced mutations-caused by mutagens that damage DNA or alter its chemistry
Base analogs are incorporated into DNA during replication and exhibit base-pairing properties different from the bases they replace
Specific mispairing occurs when a mutagen changes a baseís structure and thereby alters its pairing characteristics (e.g., alkylating agents)
Intercalating agents, which become inserted between the stacked bases of the helix, distort the DNA and thus induce single nucleotide pair insertions or deletions that can lead to frameshifts
Many mutagens (e.g., UV radiation, ionizing radiation, some carcinogens) can severely damage DNA so that it cannot act as a replication template; this would be lethal without the repair mechanisms to restore the DNA; however, the repair mechanisms are error prone, which also leads to mutations
The expression of mutations
Forward mutation-a conversion from the most prevalent gene form (wild type) to a mutant form
Reversion-a second mutation that makes the mutant appear to be a wild type again
Back mutation-conversion of the mutant nucleotide sequence back to the wild type sequence
Suppressor mutation-a reestablishment of the wild type phenotype by a second mutation that overcomes the effect of the first mutation; can be in the same gene or a different gene, but does not restore the original sequence
Point mutations-affect only one base pair and are more common than large deletions or insertions
Silent mutations are alterations of the base sequence that do not alter the amino acid sequence of the protein because of code degeneracy
Missense mutations are alterations of the base sequence that result in the incorporation of a different amino acid in the protein; at the level of protein function, the effect may range from complete loss of activity, to no change in activity at all
Nonsense mutations are alterations that produce a translation termination codon, which results in premature termination of the protein during synthesis; location of the mutation within the protein will determine the extent of change in function
Frameshift mutations are insertions or deletions of one or two base pairs that thereby alter the reading frame
Mutations can also occur in regulatory sequences and in tRNA and rRNA genes; all can give observable phenotypes
Detection and Isolation of Mutants
Mutant Detection
Visual observation of changes in colony characteristics
Auxotrophic mutants (i.e., those which have lost the ability to synthesize a particular end product and which therefore require its presence in the growth medium) can be detected by replica plating on media with and without the growth factor; mutants are those growing with the factor but not without it
Mutant selection-achieved by finding the environmental condition in which the mutant will grow but the wild type will not (useful for isolating auxotrophic revertants and resistance mutants)
Carcinogenicity testing
Many cancer-causing agents (carcinogens) are also mutagens
Tests for mutagenicity are used as a screen for carcinogenic potential
The Ames test is a widely used mutagenicity test; it detects an increase in reversion of special strains of Salmonella typhimurium from histidine auxotrophy to prototrophy after exposure to a potential carcinogen
DNA Repair
Excision repair
Corrects damage that causes distortions of DNA (e.g., thymine dimers, apurinic or apyrimidinic sites, damaged or unnatural DNA)
The damaged area is excised, producing a single-stranded gap, and then the gap is filled in by DNA polymerase I and DNA ligase
Removal of lesions-reverses damage without removing and replacing bases; although this process is error free, it is limited to the repair of certain kinds of damage (e.g., photoreactivation to remove thymine dimers)
Postreplication repair-mismatch repair system detects mismatched base pairs in newly synthesized DNA; these are removed and replaced by the action of DNA polymerase I and DNA ligase
Recombination repair
Restores DNA that has damage in both strands by recombination with an undamaged molecule, if available (this frequently occurs in rapidly dividing cells where there is another copy of the chromosome not yet parceled out to a daughter cell)
SOS repair is a type of recombination repair; it is used to repair excessive damage that halts replication; it is an error-prone process that results in many mutations
