Chromosome: A discrete unit of the genome carrying many genes, composed of duplex DNA and proteins, visible during cell division.
Human genome contains 22 pairs of autosomes and one pair of sex chromosomes.
Homologous chromosomes: Pairs with the same gene sequence, loci, length, and centromere location.
Non-homologous chromosomes: Chromosomes that don't belong to the same pair.
Structural gene: A gene encoding any RNA or polypeptide product other than a regulator. A gene encodes an RNA, which can encode a polypeptide.
Alleles, Loci, and Genetic Recombination
Allele: Alternative forms of a gene at a specific locus on a chromosome.
Locus: The position on a chromosome where a gene resides, occupied by one of the alleles for that gene.
Genetic recombination: Joining of separate DNA molecules into a single molecule through crossing over or transposition.
Each chromosome contains a single, long DNA molecule with sequences of individual genes.
DNA as Genetic Material
In Bacteria and Viruses
Bacterial Transformation: Genetic properties transferred between bacterial strains by extracting DNA from one strain and adding it to another.
Transforming principle: DNA taken up by a bacterium, altering the recipient cell's properties.
Neither heat-killed S-type nor live R-type bacteria can kill mice, but simultaneous injection of both can, similar to live S-type.
Phage Infection: DNA is the genetic material of viruses. When bacteriophage DNA and protein are labeled, only DNA is transmitted to progeny phages upon infecting bacteria.
In Eukaryotic Cells
Transfection: Eukaryotic cells acquire new genetic markers by incorporating added DNA, leading to a new phenotype.
Polynucleotide Chains
Nucleoside: A purine or pyrimidine base linked to the 1' carbon of a pentose sugar.
DNA contains a deoxyribose sugar (2'–H).
RNA contains a ribose sugar (2'–OH).
Nucleotide: A nucleoside linked to a phosphate group on either the 5' or 3' carbon of the (deoxy)ribose.
DNA contains adenine, guanine, cytosine, and thymine.
RNA has uracil instead of thymine.
Successive (deoxy)ribose residues in a polynucleotide chain are joined by a phosphate group between the 3' carbon of one sugar and the 5' carbon of the next.
One end has a free 5' end, and the other has a free 3' end.
DNA Structure
Supercoiling
Supercoiling occurs in “closed” DNA with no free ends.
Supercoiling: The coiling of a closed duplex DNA in space, crossing over its own axis, resulting in a twisted and condensed form.
Double Helix
The B-form of DNA is a double helix of two antiparallel polynucleotide chains.
Nitrogenous bases are flat purine or pyrimidine rings facing inward, pairing via hydrogen bonding to form A-T or G-C pairs.
G-C bonds need higher energy to break than A-T bonds.
Complementary: Base pairs that match up in pairing reactions, A with T (or U in RNA) and C with G.
Nucleic Acid Detection
Non-Specific Methods
Absorption: Nucleic acids absorb light at 260nm for quantitation.
Non-specific stains: Ethidium bromide, SYBR green, detected by instruments or visualization on a gel.
Specific Sequence Detection
Hybridization of a labeled nucleic acid (probe) to complementary sequences.
Probes may be radioactively or fluorescently labeled.
Hybridization requires single-stranded nucleic acid template and probe, dependent on complementarity, buffer conditions, and temperature.
DNA Separation Techniques
Gel electrophoresis: Separates DNA, RNA, or protein fragments by size using an electric current to cause negatively charged DNA to migrate toward a positive electrode.
Genetic Information
Central Dogma: Information cannot transfer from protein to protein or protein to nucleic acid but can transfer between nucleic acids and from nucleic acid to protein.
DNA undergoes replication to copy itself.
DNA undergoes transcription into RNA.
RNA can undergo reverse transcription into DNA (in viruses).
RNA undergoes translation into proteins.
Replication, transcription, reverse transcription, and translation rely on enzymes and specific genetic sequences like polymerases, endonucleases, and exonucleases.
DNA Replication
Semiconservative Replication
The Meselson–Stahl experiment used “heavy” isotope labeling to show that the single polynucleotide strand is the unit of DNA that is conserved during replication.
Semiconservative replication: DNA replication by separation of parental duplex strands, each acting as a template for a complementary strand. Daughter strand sequences are determined by complementary base pairing with the separated parental strands.
Polymerases and Replication Fork
Replication is undertaken by a complex of enzymes that separate parental strands and synthesize daughter strands.
Denaturation: Separation of two DNA strands due to breaking hydrogen bonds between bases.
Renaturation: Reassociation of denatured complementary single strands of a DNA double helix.
Nucleic Acid Hybridization
Heating causes two strands of a DNA duplex to separate. The melting temperature (T_m) is the midpoint of the temperature range for denaturation.
Complementary single strands can renature or anneal when the temperature is reduced.
Denaturation and renaturation/hybridization occur with DNA–DNA, DNA–RNA, or RNA–RNA combinations.
Hybridization can be intermolecular or intramolecular.
In situ hybridization: Hybridization of a probe to intact tissue to locate its complementary strand, used to identify locations of DNA sequences or RNA.
Mutations
All mutations are changes in the sequence of DNA, occurring spontaneously or induced by mutagens (carcinogens).
Types of Mutations
Point mutation: Changes a single base pair, caused by chemical conversion of one base into another or errors during replication. May or may not be deleterious.
Diseases: Tay-Sachs, sickle cell anemia, color blindness.
Transition: Interchange of pyrimidine (C \leftrightarrow T) or purine (A \leftrightarrow G) bases (e.g., G-C \rightarrow A-T).
Transversion: Replaces a purine with a pyrimidine or vice versa (e.g., A-T \rightarrow T-A).
Functional Categorization of Point Mutations
Nonsense mutation: Results in a premature stop codon.
Missense mutation: Changes the codon so that a different amino acid is encoded.
Silent mutation: Does not affect the amino acid sequence.
Insertion/Deletion Mutations: Add or remove nucleotides; can cause frameshift, typically more harmful than point mutations. Can result from movement of transposable elements.
Insertion: Addition of material from one chromosome to a nonhomologous chromosome.
Duplications: Multiple copies of a chromosomal region.
Inversion: Segment of a chromosome is reversed.
Translocation: Exchange of genetic material between nonhomologous chromosomes.
Structural abnormalities are a hallmark of many cancers.
Reversion of Mutations
Reversion: Forward mutations alter gene function, and back mutations reverse their effects. Point mutations reversed. Insertions can revert by deletion, but deletions cannot revert.
Second-site reversion: A second mutation suppressing the effect of a first mutation within the same gene.
Suppression: A mutation in a second gene bypasses the effect of mutation in the first gene.
Genes and Polypeptides
One gene: one polypeptide hypothesis: A gene is responsible for the production of a single polypeptide.
Most mutations damage gene function and are recessive to the wild-type allele.
Recessive mutations result from loss of function. Dominant mutations result from a gain of function.
Testing if a gene is essential requires a null mutation (completely eliminates its function).
Recombination
Recombination results from crossing over that occurs at a chiasma during meiosis, involving two of the four chromatids.
Recombination occurs by breakage and reunion, proceeding via an intermediate of heteroduplex DNA that depends on the complementarity of the two DNA strands.
Genetic Code
The genetic code is read in triplet nucleotides called codons; each codon codes for an amino acid. Codons are nonoverlapping and read from a fixed starting point. The genetic code is degenerate or redundant.
Mutations that insert or delete individual bases cause a shift in the triplet sets (frameshift mutations). Combinations inserting or deleting three bases (or multiples of three) insert or delete amino acids without changing the reading of triplets beyond the site of mutation.
Reading Frames
Usually only one of the three possible reading frames is translated; the other two are closed by frequent termination signals.
Open reading frame (ORF): A sequence of DNA consisting of triplets that can be translated into amino acids, starting with an initiation codon and ending with a termination codon.
Closed (blocked) reading frame: A reading frame that cannot be translated because of termination codons.
Unidentified reading frame (URF): An open reading frame with an undetermined function.
Gene Expression
A typical gene is expressed by transcription into mRNA and translation of mRNA into polypeptide. Each mRNA includes an untranslated 5' region (5' UTR or leader), a coding region, and an untranslated 3' UTR or trailer.
In eukaryotes, a gene may contain introns not represented in the polypeptide product.
RNA processing: Modifications to RNA transcripts, including alterations to the 3' and 5' ends and removal of introns by splicing.
Exon: Any segment of an interrupted gene represented in the mature RNA product.
Transcription occurs in the nucleus, and translation occurs in the cytoplasm.
Ribosomes and tRNA
Ribosome: A large assembly of RNA and proteins that synthesizes polypeptides under direction from an mRNA template.
Ribosomal RNAs (rRNAs): A major component of the ribosome.
Transfer RNA (tRNA): The intermediate in polypeptide synthesis that interprets the genetic code. Each tRNA molecule can be linked to an amino acid, and has an anticodon sequence complementary to a triplet codon representing the amino acid.