Microbial Genetics: Recombinant DNA Technology

Microbial Genetics: Recombinant DNA Technology

Terminology

• Genetics is the study of genes, how they carry information, how information is expressed, and how genes are replicated.
• A gene is a segment of DNA that encodes a functional product, often a protein.
• Genome refers to all the genetic material in a cell.
• Genomics involves the molecular study of genomes.
• Genotype represents the genes of an organism.
• Phenotype is the expression of the genes.

Prokaryotic Genomes

• DNA is found in two structures: chromosomes and plasmids.
• Chromosomes are the main portion of DNA localized to the nucleoid area.
• Most bacteria has one circular dsDNA chromosome.
• The chromosome is packaged and folded into loops.
• The chromosome is held in place by associated molecules of protein and RNA.
• Plasmids are additional small, circular DNA
• Plasmids replicate independently of the chromosome
• Plasmids carry information required for replication and cellular traits
• Types of plasmids/factors include:
  • Fertility factors instruct for conjugation.
  • Resistance factors include resistance genes.
  • Bacteriocin factors are genes for toxins against competitor bacteria.
  • Virulence plasmids instruct for structures, enzymes, or toxins that enable a bacteria to become pathogenic

Eukaryotic Genomes

• DNA is found in two locations: Nuclear chromosomes and Extra-nuclear DNA
• Nuclear chromosomes generally have >1 chromosome in their genome
• The Nuclear chromosomes are usually linear and found in the nucleus
• Extra-nuclear DNA is found in mitochondria and chloroplasts
• Extra-nuclear DNA is circular, resemble that of prokaryotes (endosymbiotic theory!)
• Extra-nuclear DNA codes for about 5% of the RNA and polypeptides required for the organelle's replication and function.
• Nuclear DNA encodes for the rest.

Eukaryotic Nuclear Chromosome Packaging

• Nucleosomes are Positively charged histone proteins which stabilize and package negatively charged DNA and are connected by linker DNA
• Chromatin fiber is nucleosomes clumped with other proteins.
• Chromatin fibers fold and organizes into active loosely packed euchromatin and inactive tightly packed heterochromatin
• During nuclear division, duplicated chromatin fully condenses.

Characteristics of Microbial Genomes

Flow of Genetic Information

• Genetic information flows from DNA (genotype) to mRNA (transcription) to polypeptide (translation) to phenotype.

DNA

• DNA is a polymer of nucleotides, which includes Deoxyribose and phosphate group and nitrogenous base
  • The nitrogenous base includes adenine, thymine, cytosine, guanine
• The backbone is deoxyribose-phosphate.

DNA Double Helix

• 2 DNA strands are held together by complementary base pairing, hydrogen bonds between A & T or C & G
• Strands are anti-parallel (One runs 5'-3', the other 3'-5')

DNA Replication

• DNA polymerase add nucleotides in the 5' - 3' direction
• Replication is semi- conservative; each new DNA strand remains associated with one of the parental strands

Characteristics of DNA Replication

• Replication is bidirectional
• Prokaryotic DNA synthesis proceeds in both directions from one origin.
  • There are to sets of enzymes, two RL forks, and two leading/lagging strands
• Eukaryotic chromosomes are large and have thousands of origins per molecule, or replication would take days instead of hours
• Methylation after DNA replication
  • Eukaryotes methylate cytosines
  • Prokaryotes methylate adenine and some cytosines
• Plays roles in gene expression, DNA replication initiation, protecting against viral infection (viral DNA is non-methylated), and mismatch repair, only work on the new unmethylated DNA

Transcription

• Transcription is the synthesis of RNA from DNA using the enzyme RNA polymerase.

Initiation of Transcription

• Polymerase binds to promoter sequence in duplex DNA ("closed complex").
• Polymerase melts duplex DNA near transcription start site, forming a transcription bubble ("open complex").
• Polymerase catalyzes phosphodiester linkage of two initial rNTPs.

Elongation & Termination of Transcription

• Polymerase advances 3' → 5' down template strand using nascent RNA. Melts duplex DNA, adding rNTPs to growing RNA.
• At transcription stop site, polymerase releases completed RNA and dissociates from DNA.

RNA Processing in Eukaryotes

• A gene composed of exons and introns is transcribed to RNA by RNA polymerase
• Processing Involves ribozymes and proteins in the nucleus to remove the intron-derived RNA and splice together the exon-derived RNA into mRNA
• After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis

Translation

• Translation is where mRNA is translated into polypeptides/proteins using ribosomes
• mRNA is read in triplet nucleotides called codons
• Translation of mRNA begins at the start codon: AUG
• Translation ends at a stop codon: UAA, UAG, UGA

Translation Continued

• Components needed to begin translation come together
• On assembled ribosome, a tRNA carrying the first amino acid is paired with the start codon on the mRNA. A tRNA carrying the second amino acid approaches. Met CAC
• the place on the ribosome where the first tRNA sits is called the P site. In the A site next to it, the second codon of the mRNA pairs with a tRNA carrying the second amino acid.
• The first amino acid joins to the second by a peptide bond, and the first tRNA is released.
• The ribosome moves along the mRNA until the second tRNA is in the P site, and the process continues.
• The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide.
• When the ribosome reaches a stop codon, the polypeptide is released.
• Finally, the last tRNA is released, and the ribosome comes apart; the released polypeptide forms a new protein.
• As a ribosome moves down mRNA the start codon (AUG) becomes available to another ribosome = polyribosome
• Numerous identical polypeptides are translated simultaneously from a single mRNA molecule

Control of Transcription

• Constitutive expression: Some genes are expressed at all times.
  • Constantly transcribed and translated, playing a persistent role in cell's phenotype
  • Integral plasma membrane proteins and Enzymes for glycolysis show constitutive gene expression
• Regulated expression: Some genes are expressed only when needed.
  • Protein synthesis uses a lot of energy, so cell conserves energy
  • Prokaryotes and eukaryotes regulate protein synthesis most common way is to stop transcription)
  • Two kinds of enzyme regulation: induction and repression

Operon

• Operon consists of a promoter (P), an adjacent operator (O), and a series of structural genes that code for proteins.
• the operon is regulated, induced or repressed, by the protein product of the regulatory gene (I) located elsewhere
• Inducible operon is not usually transcribed, and must be activated by inducers
• Repressible operon is transcribed continually until deactivated by repressors

The Lactose Operon: An Inducible Operon

• Controlled by regulatory gene that is constantly TS & TL to produce repressor
• Repressor protein binds to DNA at Operator, prevents RNA polymerase from moving beyond the promoter
• When there is no lactose or lactose runs out: there is no inducer so repressor is active
• When lactose becomes available: Lactose acts as an inducer, changes the repressor protein structure (making it inactive and unable to bind DNA), so transcription occurs

The Tryptophan Operon: A Repressible Operon

• When trp is absent from cell's environment: repressor is inactive, therefore structural genes are transcribed & translated so the 5 enzymes needed to synthesize trp are made
• When trp is present in cell's environment trp acts as a corepressor and inhibits it's own synthesis

Mutation

• A mutation is a change in the genetic material
• Mutations may be neutral, beneficial, or harmful. and are caused by a mutagen or occur naturally:
  • A mutagen is an agent that causes mutations
  • Spontaneous mutations occur: In the absence of a mutagen and result from errors in replication, repair, and recombination.
• Mutagens are physical or chemical agents:
  • Ionizing radiation (X rays and gamma rays) energize electrons in atoms, produce ions that can react with the structure of DNA, breaking covalent bonds in DNA backbone.
  • Nonionizing radiation (UV light) causes adjacent thymine bases to covalently bond to one another (thymine dimers) preventing hydrogen bonding with adenine nucleotides in complementary strand which distorts backbone and prevents normal Replication & Transcription.

Kinds of Mutations

• Silent mutation: base substitution but no change to AA resulting in the same amino acid.
• Missense mutation: base substitution that results in an AA change, producing a different amino acid.
• Nonsense mutation: base substitution resulting in early stop codon.
• Frameshift mutation: Insertion or deletion of one or more bases

Thymine Dimers

• UV radiation causes thymine dimers.
• Light-repair uses DNA photolyase, a visible light activating enzyme, that breaks bonds between adjoining thymine nucleotides and restores original DNA sequence.

Mechanisms of DNA Repair

• Several repair mechanisms have evolved in bacterial cells to minimize damage to the DNA.
• Thymine dimer repair: light and dark repair use different enzymes.
• Base-excision Repair: total excision of an incorrect nucleotide within segments.
• Mismatch Repair: enzymes remove a segment with an incorrect base and DNA polymerase fills in the gap

Identifying Mutants

• Distinguishing mutant from wild type cells by observation or testing for altered phenotypes
• Developed methods to recognize mutants amidst wild type neighbors
• Two kinds of selection: positive and negative selection
• Positive (direct) selection detects mutant cells because they grow or appear different and eliminate wildtype phenotypes
  • Grow bacteria on plate containing penicillin isolates penicillin-resistant colonies
• Negative (indirect) selection: detects mutant cells b/c they don't grow. Uses the Replica Plating procedure to isolate an auxotroph
  • an organism with nutritional requirements that differ from wild type phenotype
  • A mutant bacteria has lost the ability to synthesize tryptophan because of its auxotrophic properties, must acquire it from the environment

The Ames Test: Identifying Possible Chemical Carcinogens

• Uses mutant Salmonella that are histidine auxotrophs (his-)
• Mix his- mutants and liver extract with a suspected mutagen
  • The liver extract simulates body conditions that turn innocuous chemicals into mutagens
• Spread treated bacteria on medium lacking histidine
• If the suspected substance causes mutations, some will reverse the effect of the original mutation, and produce revertant cells (his+) that have the ability to synthesize histidine
• Presence of colonies reveals that the substance is mutagenic

Mechanisms of Genetic Transfer between cells

• Vertical gene transfer occurs during reproduction between generations of cells.
  • It Passes genes to the next generation.
• Horizontal gene transfer transfers genes between cells of the same generation.
  • Donor cells contribute part of its genome to a recipient cell.
  • Three types exist: Transformation, Transduction, and Conjugation
• Once inside a cell, a transposon can jump between different DNA molecules through transposition.

Genetic Transfer Type - Transformation

• Competent cells take Naked DNA from environment
  • Comes from bacteria death due to the host cells' lysis, results in environmental DNA release
• Cells become competent under certain environmental conditions or when able to bind short pieces of DNA to envelope and import into the cell
• Competence is when there is stationary phase of bacterial growth (induced by high cell density and/or nutritional limitation)
• The DNA fragments are usually derived from another bacterium of species and is homologous to the resident chromosome

Genetic Transfer Type - Transduction

• DNA is transferred from one cell to another via a replicating bacteriophage virus
  • known as Transducing phage
• Gene transfer between bacteria also mediated by bacteriophages, acts as vector (bring foreign DNA into cell).
• After integration of DNA into chromosome, phage not only packages DNA but also portion of donor bacterium's DNA
  • generalized transduction: Bacterial DNA may be randomly incorporated into viral DNA and transferred into another bacterial cell and occurs during lytic phage cycle.
• specialized or restricted transduction: Specific bacterial gene(s) can be transferred via bacteriophage into another bacterial cell
  • occurs when the prophage excises imprecisely from the chromosome and bacterial genes lying adjacent to the prophage are included in the excised DNA
• Lytic cycle: cell produces new phages and is destroyed by lysis of the membrane.
• Lysogenic cycle: cell reproduces normally, copying the viral DNA.

Genetic Transfer Type - Conjugation

• Bacteria containing a fertility plasmid (F+) form a conjugation pilus which attaches to a recipient (F-) and transfers plasmid genes to recipient
  • Responsible for conjugation (sex) pilus and transfer of plasmid
• The Adhesin molecule on the surface of donor cell is used by Gram +ve bacteria instead of a sex pilus

Recombinant DNA Technology

• Biotechnology is the use of microorganisms, cells, or cell components to make a product
  • Examples: Foods, antibiotics, vitamins, and/or enzymes
• Recombinant DNA technology is defined as insertion or modifications of genes to produce desired proteins
  • Intentionally modifying genomes by natural/artificial processes,
• Three Goals:
  • Eliminate undesirable phenotypic traits in humans, animals, plants & microbes
  • Combine beneficial traits of two or more organisms to create valuable new organisms
  • Create organisms that synthesize products that humans need

Products of Recombinant DNA in Medicine

Genetic Engineering Methodology

• Isolate plasmid, containing DNA with a gene of interest
• Enzymatically cleave DNA into fragments.
• Isolate fragment with the gene of interest.
• Insert gene into plasmid.
• Insert plasmid and gene into bacterium.
• Culture bacteria to Harvest copies of gene to insert into plants or animals/harvest proteins coded by gene
  • process can Eliminate undesirable phenotypic traits, Create beneficial combination of traits and/or Produce vaccines, antibiotics, hormones, enzymes

Generating Recombinant DNA

• mRNA for human growth hormone (HGH) is used, utilizing reverse transcription for cDNA for HGH. A Restriction enzyme cleaves AAGCTT at a Restriction site in the plasmid (vector), resulting in Sticky ends -Ligase then is used with a now recombinant plasmid. The recombinant plasmid is Introduced into bacteria and forms a Bacterial chromosome, the recombinant plasmid is inoculated from AAGCTT HGH AAGCTT on plates that Utilize media containing antibiotics as a 4th step. Bacteria with a plasmid/HGH gene survive, b/c resistant to resistance genes

Q&A

• Q1: The answer is A, conjugation. Conjugation involves a sex pilus and the transfer of plasmids carrying transposons, which aligns with the question's description of antibiotic resistance spread.
• Q2: The answer is B, transduction. Transduction directly involves the integration of a toxin gene into a bacterial genome during lysogenic conversion, fitting the question's scenario.
• Q3: The answer is B, bacteria are protected from viruses. Restriction enzymes recognize and cut foreign DNA like that from viruses, protecting the bacteria.