DNA Structure and Replication

• The genome, a long sequence of deoxyribonucleic acid (DNA), forms the hereditary basis of every living organism.
• The genome provides the complete set of hereditary information for the organism and its individual cells.

DNA Sequence

• The DNA sequence codes for all proteins of an organism.
• Proteins have roles in the development and functioning of an organism-structural, metabolic, regulation, and signal transduction.

Genes

• The genome divides into chromosomes, which are further divided into genes.
• A gene is a DNA sequence encoding an RNA.
• In protein-coding (structural) genes, the RNA encodes a polypeptide.

Bacterial Transformation

• Bacterial transformation provided that DNA is the genetic material of bacteria.
• In bacterial transformation genetic properties transfer from one bacterial strain to another by extracting DNA from the first strain and adding it to the second strain.
• During bacteriophage reproduction, the DNA from the parent phages transmits to the progeny phages produced by infecting bacteria.
• The genetic material of some viruses is RNA instead of DNA.
• Genetic engineering can introduce new traits into cells or organisms through introduction of new DNA.

DNA

• DNA consists of a polynucleotide chain of nitrogenous bases linked to a sugar-phosphate backbone.
• A nucleoside consists of a purine or pyrimidine base linked to the 1' carbon of a pentose sugar.
• A nucleotide is a nucleoside linked a phosphate.
• Consecutive (deoxy)ribose residues on a polynucleotide chain are joined by a phosphate group between the 3' carbon of one sugar and the 5' carbon of the next sugar.
• One end of the chain has a free 5' end and the other end of the chain has a free 3' end.
• DNA has a deoxyribose sugar (2'-H) and RNA has a ribose sugar (2'-OH).
• DNA contains the four bases adenine, guanine, cytosine, and thymine while RNA contains uracil instead of thymine.
• Most DNAs in nature exist as a double helix structure while RNAs exist as a single strand.
• Two strands of DNA join and form a double helix, which can also wind around itself in a process called supercoiling.
• Supercoiling can only occur in closed or linear DNA
• Characterization of a supercoiled molecule of DNA is by its linking number (L) which is the result of the writhing number (w) and twisting number (t).
• The linking number of a particular closed molecule can only be changed by breaking one or both strands.
• The two polynucleotide chains are joined by hydrogen bonding between purine and pyrimidine nitrogenous bases (Guanine binds to Cytosine while Adenine binds to Thymine or Uracil).
• Chargaff's rule states that the proportion of Guanine (G) in DNA is always identical to the proportion of Cytosine (C), describing the composition of that DNA.
• The two polynucleotide chains run antiparallel to each other.
• Each base pair is rotated ~36° around the axis of the helix relative to the next base pair, so ~10 base pairs make a complete turn of 360°.
• In B-DNA, the double helix is said to be "right-handed" and the turns run clockwise as viewed along the helical axis.
• 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, making DNA replication semi-conservative.
• Each strand of a DNA duplex acts as a template for synthesis of a daughter strand.
• A complex of enzymes separates the parental strands at a replication fork and synthesizes the daughter strands; determined by complementary base pairing with the separated parental strands.
• DNA polymerases are the enzymes that synthesize DNA.
• Nucleases are enzymes that degrade nucleic acids, including DNases and RNases, and can be categorized as endonucleases or exonucleases.
• Endonucleases break individual phosphodiester linkages within RNA or DNA molecules, generating discrete fragments; while exonucleases remove nucleotide residues one at a time from the end of the molecule, generating mononucleotides.
• The central dogma of molecular biology states that DNA is transcribed into RNA, which is translated into polypeptides (proteins).
• RNA may be converted into DNA by reverse transcription (as in replication of RNA viruses), but the translation of RNA into proteins is unidirectional and cannot run in reverse.
• Separating the two strands without disrupting the covalent bonds that form the polynucleotides is a crucial property of the double helix.
• Heating causes the two strands of a DNA duplex to separate (denaturation).
• Denaturation reverses under certain conditions (renaturation/hybridization).
• The ability of two single-stranded nucleic acids to hybridize is a measure of their complementarity.

Mutations

• Mutations -changes in the sequence of DNA-may occur spontaneously or may be induced by mutagens.
• Point mutations (a mutation that changes a single base pair) can be caused by the chemical conversion of one base into another or by errors that occur during replication.
  • The most common point mutations are transitions (the substitution of one pyrimidine by the other or of one purine by the other) and transversions (a purine is replaced by a pyrimidine or vice versa).
• Insertions and/or deletions can result from the movement of transposable elements.
  • An insertion (or deletion) within a coding region usually eliminates the activity of the gene because it may alter the reading frame, such an insertion/deletion is called a frameshift mutation.
• Effects of some mutations can be reversed.
  • A point mutation can revert either by restoring the original sequence (true reversion) or by gaining a mutation elsewhere in the gene that compensates for the point mutation (second-site reversion). -Suppression mutation: the second mutation in another gene can reverse the phenotype of first mutation, unlike second-site reversion where both the first and second mutations occur in the same gene.
  • An insertion can revert by deletion of the inserted sequence (second-site reversion).
  • A deletion of a sequence cannot revert in the absence of some mechanism to restore the lost sequence.
  • Mutations that inactivate a gene are called forward mutations and their effects are reversed by back mutations.
• The frequency of mutation at any particular base pair is statistically the same, except for hotspots, where the frequency is increased by at least an order of magnitude.
  • Spontaneous mutations may occur at hotspots, and different mutagens may have different hotspots.
  • Chemical modification of one of the four standard bases can result in the presence of a modified base in the DNA: 5-methylcytosine encourages spontaneous mutation, generating a hotspot.
  • "Slippery sequence" is a type of hotspot: a homopolymer run or region where a very short sequence (one or a few nucleotides) is repeated many times in tandem, which can either increase or decrease in length during replication of the sequence.
• Some very small hereditary agents do not encode polypeptide, but consist of RNA (viroids) or protein (prions) with heritable (infectious) properties.

Genes Encode RNAs and Polypeptides

• Each chromosome consists of a linear array of genes and each gene resides at a particular location on the chromosome, (the genetic locus).
• In organisms with two sets of chromosomes, one of each chromosome pair, each gene is inherited from each parent (one is the paternal allele and the other is the maternal allele).
• Genetic linkage is the tendency for genes on the same chromosome to remain together in progeny instead of assorting independently.
• A typical gene is a stretch of DNA encoding one or more forms of a single polypeptide chain, also known as the "one gene-one enzyme hypothesis.”
  • However, some proteins contain more than one polypeptide (thus more than one gene is required for its production) resulting in the term the "one gene-one polypeptide" hypothesis.
  • Note that some genes do not encode polypeptides, they encode structural or regulatory RNAs.
• Recessive to the wild-type allele is indicative of most mutations that damage gene function because if an organism contains one wild-type allele and one mutant allele, the wild-type allele can direct production of the enzyme and the organism can still function.
• A mutation in a gene affects only the product (polypeptide or RNA) encoded by the mutant copy of the gene and does not affect the product encoded by any other allele.
  • The complementation test determines whether two recessive mutations are alleles of the same or in different genes.
  • Failure of two mutations to complement (produce wild-type phenotype) means that they are alleles of the same gene.
• A null mutation completely eliminates gene function (usually because the gene is deleted) and is lethal when homozygous or hemizygous if the gene is essential to the organism's survival.
  • Testing whether a gene is essential requires a null mutation (one that completely eliminates its function).
• Mutations that impede gene function are called loss-of-function mutations and are recessive; where mutations that cause genes to acquire a new function are called gain-of-function mutations and are dominant.
• Silent mutations indicate mutations without apparent phenotypic effect.
• Different variants of the same gene are called multiple alleles allowing for the possibility of heterozygotes representing any pairwise combination of alleles, including heterozygotes with two mutant alleles.
• A locus may have several alleles with no individual allele when a unique wild-type allele is not necessary for any particular locus.
  • A situation in which there are multiple functional alleles in a population, is described as a polymorphism (the human ABO blood group system is an example).
• Genetic recombination generation of new combinations of alleles at each generation in diploid organisms.
  • During meiosis, corresponding segments between the two homologous copies of each chromosome "cross over” exchanging chromosomal material and generating recombinant chromosomes that are different from the parental chromosomes.
  • Crossing over occurs at a chiasma (the point of synapsis between homologs) involving two of the four chromatids.
  • Single strands in the region of the crossover exchange their partners, resulting in a branch, creating a stretch of heteroduplex DNA in which the single strand of one duplex is paired with its complement from the other duplex.
  • The formation of heteroduplex DNA requires the sequences of the two recombining duplexes to be close enough to allow pairing between the complementary strands.
  • The frequency of recombination between two genes is proportional to their physical distance; and that the probability that a crossover will occur within any specific region of the chromosome is more or less proportional to the length of the region.
  • Recombination between genes that are very closely linked is rare while those that are very far apart has frequency of recombination which is not proportional to their physical distance because recombination happens so frequently.
• The genetic code is read in triplet nucleotides called codons, which do not overlap and are read from a fixed starting point, denoted by an initiation codon (AUG).
  • A genuine functional gene must have an open reading frame: a continuous stretch of codons from the start codon (usually AUG) to the stop or termination codon that can be translated into a functional polypeptide.
  • Three termination codons (UAG, UAA, or UGA) that indicate where translation of a polypeptide stops exist.
  • A reading frame that cannot be translated into a polypeptide because termination codons occur frequently is said to be closed, or blocked.
• Insertion or deletion mutations of individual bases cause a shift in the triplet sets after the site of mutation; these are frameshift mutations, which are more toxic than point mutations because of the frequent destruction of the function of the gene.
• Combinations of mutations that together insert or delete three bases (or multiples of three) do not change the reading of the triplets beyond the last site of mutation, but can however, insert or delete amino acids from the polypeptide.
• A polypeptide is colinear if_the sequence of nucleotides in the gene exactly corresponds to the sequence of amino acids in the polypeptide, as is the case in bacterial and bacteriophage genes.
  • If a polypeptide contains N amino acids, the gene encoding that polypeptide contains 3N nucleotides.
• Bacterial genes are typically expressed by transcription into messenger RNA (mRNA) and then by translation of the mRNA into polypeptide.
  • Messenger RNA (mRNA) is transcribed through the same complimentary base pairing process used in DNA replication (the antisense, or template, strand).
• The process by which information from a gene is used to synthesize an RNA or polypeptide product is called gene expression.
• The coding region is the part of an mRNA molecule that contains a sequence of nucleotides corresponding with the sequence of amino acids in the polypeptide.
• The important stage in RNA processing is splicing, in which introns (regions that do not carry coding information) are removed as well as exons (regions that do carry coding information) are spliced together.
  • Yielding results in an mRNA that is colinear with the polypeptide product.
• Translation is accomplished by a complex apparatus that includes the ribosome (composed of ribosomal RNA) and transfer RNA, which carries the appropriate amino acid to the growing polypeptide, as directed by codons.
• All gene products (RNA or polypeptides) are trans-acting, therefore acting on any copy of a gene in the cell, while a site on DNA that regulates the activity of an adjacent gene is said to be cis-acting.
• Gene expression can be inactivated either by a mutation in a control site or by a mutation in a coding region.
  • Cis-acting mutations only affect the coding region to which it connected, but does not affect the ability of the homologous allele to be expressed.
  • Trans- acting mutations can prevent both alleles from being expressed.

Explore DNA properties, replication, and the central dogma. Understand supercoiling, nucleotide content, and antiparallel strands. Investigate enzymes cleaving phosphodiester bonds and the flow of genetic information.