Microbial Genetics and Genetic Engineering Notes

Microbial Genetics (Ch. 9)

• Ribosome: Composed of two parts.
• Genetic Engineering (Ch. 10)
• Nonspecific Defenses (Ch. 14)
• Specific Immunity (Ch. 15)
• Exam Details:
  • No lab component.
  • 85-100 points possible.
  • May include Zzna connection.
  • Ribosomal codon relevance.
  • Final test on May 29.
  • Thalidomide (research example).
  • Telomere mutation.

Mutations

• Point Mutations:
  • Missense: Replacement of one amino acid (e.g., 146 amino acids).
  • Nonsense: Codon #40 becomes a stop codon, resulting in only 39 amino acids.
• Frameshift Mutations:
  • Deletion: Example - "The cat ate his big dog."
  • Insertion
• Radiation:
  • Ionizing Radiation: Causes breakage of ribosomes.
  • Non-ionizing Radiation: Diffuse; causes ribosomes to be cut.

Ames Test

• Used to identify mutagens; based on reversions; no lab animals used.
  1. His+: Able to synthesize the amino acid histidine; can grow on a medium with or without histidine.
  2. His-: Unable to synthesize histidine; cannot grow on a medium without histidine, but can grow on a medium with histidine.
• Process:
  • His- bacteria lacking histidine are exposed to a suspected mutagen (direct mutagen).
  • Rat liver extract may be added to simulate mammalian metabolism.
  • If the bacteria grow on media lacking histidine (His+), it indicates a reversion and suggests the substance is a mutagen.
  • Comparison with a control group is essential.
• Thymine-Thymine dimers can undergo light repair.

Genetic Recombination

• Evidence for DNA: Bacterial Transformation.
• Griffith's Experiment (1928):
  • Studied Streptococcus pneumoniae to develop a vaccine.
  • Observed two strains: rough (R) and smooth (S).
  • Experiment 4: Live R bacteria received DNA from dead (heat-killed) S bacteria, leading to capsule formation in R bacteria, making them virulent.
• Transformation: Transfer of a gene from one bacterium to another.
• Homologous Recombination: A piece of DNA is removed, and another is inserted.
• Ampicillin Sensitivity/Resistance:
  • Sensitive: Does not grow on selective medium (ampicillin).
  • Resistant: Grows on selective medium (ampicillin).
• Viruses package any DNA.

Gene Technology (New Genetics in 1980)

• First successful introduction of a human gene (encoding interferon) into a bacterial cell.
• The cell produced interferon at a high rate and divided (cloning).
• This marked the birth of genetic engineering.
• Protein products from recombinant DNA technology:
  • Interferons
  • Insulin
  • Interleukins
  • Tumor Necrosis Factor (TNF)
  • Erythropoietin
  • Tissue Plasminogen Activating Factor (tPA)
  • Hemoglobin
  • Relaxin

Restriction Enzymes (RE)

• Natural enemies of bacteria are bacteriophages (viruses that affect bacteria).
• Bacteria have enzymes that chop up viral DNA upon entry, without harming the bacterial DNA.
• These enzymes are called Restriction Endonucleases.
• RE recognize specific nucleotide sequences within a DNA strand, bind to it, and cleave the strand at a specific place.
• Sequences are typically 4-8 nucleotides long and symmetrical (rotational symmetry).
• Palindrome example:
  • AAGCTT
  • TTCGAA
• Restriction enzyme cuts leaving sticky ends.
  • A GCTT
  • TTCGA A
• Sticky ends can be joined because of base pairing (A-T, C-G).
• Methylases recognize the same bacterial DNA sequences, bind to them, and add methyl (-CH3) groups to the nucleotides, preventing RE recognition.
• Fragments produced by the same enzyme from any DNA source can be joined together, constructing chimeric genomes.
• In an experiment, a frog rRNA gene was inserted into a plasmid in E. coli, which then produced rRNA.

Microbial Genetics Topics

• DNA Structure
• DNA Replication
• Protein Synthesis
  • Transcription
  • Translation
• Mutations
  • Types of Mutations
    • Base Substitution (point mutations)
    • Frameshift Mutations
• Mutagens
  • Chemical
  • Radiation
• Identifying Mutagens (Ames test)
• Genetic Recombination and Transfer
  • Transformation
  • Conjugation
  • Conjugation Hfr
  • Transduction

DNA Structure

• DNA = deoxyribonucleic acid: the genetic carrier.
• Double helix made of two strands twisted around each other, oriented in opposite directions.
• Basic unit: deoxynucleotide or nucleotide, made of deoxyribose (sugar), a phosphate group, and a base.
• Four bases: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G).
• All nucleotides have the same sugar and phosphate but differ in the base; hence four different nucleotides.
• P = phosphate, S = sugar (deoxyribose).
• Complementary base pairing: A pairs with T (A=T), and C pairs with G (C=G).
• Example: If a sequence is 5’ A T T C G A G C T 3’, the complementary sequence is 3’ T A A G C T C G A 5’.
• Hydrogen bonds are used in complementary base pairing.
• One strand starts with the 5’ end, and the other ends with the 3’ end; they are oriented in opposite directions.
• Distinction between DNA and RNA:

• Eukaryotic DNA is linear with multiple chromosomes; prokaryotic DNA is circular with one chromosome.
• Some bacterial cells have additional small circular DNA called a plasmid.

DNA Replication

• Replication is the duplication of DNA; the two strands separate after hydrogen bonds break.
• Example:
  • 5’ T A C G A C 3’
  • 3’ A T G C T G 5’
• The two strands separate:
  • 5’ T A C G A C 3’ (old strand)
  • 3’ A T G C T G 5’ (old strand)
• Each strand serves as a template to build the complementary strand.
  • New strands are synthesized based on the old ones.
• The result is two copies of DNA, each with one old and one new strand (semiconservative replication).
• Genetic information flow: DNA → RNA → Protein
  • Transcription: DNA makes RNA.
  • Translation: RNA makes a protein.
• Eukaryotic cells: Transcription in the nucleus, translation in the cytoplasm.
• Prokaryotic cells: Transcription and translation occur simultaneously in the cytoplasm.

Differences Between DNA and RNA

Types of RNA

1. mRNA (messenger RNA): Carries genetic information.
2. rRNA (ribosomal RNA): In ribosomes; moves along mRNA to place amino acids.
3. tRNA (transfer RNA): Transfers amino acids from the cytoplasm to the site of protein assembly.

Protein Synthesis

• Two-step process: Transcription and translation.

Transcription

• Takes place from only one strand of DNA.
• Example:
  • DNA: 5’ A T G G G C A T C C T A G G C A T G T A A 3’
  • 3’ T A C C C G T A G G A T C C G T A C A T T 5’
• mRNA sequence (complementary to the DNA strand): 5’ A U G G G C A U C C U A G G C A U G U A A 3’
• Codons: Sequence of three bases in mRNA that encodes for one amino acid.
• There are 64 codons:
  • One start codon (AUG, encodes methionine).
  • Three stop codons.
  • The rest encode for 20 different amino acids.

Translation

• tRNA is a folded single-stranded molecule of RNA with two ends.
  • One end picks up the amino acid.
  • The other end has an anticodon (complementary to the codon on mRNA).
• This allows tRNA to place the amino acid in the correct position in the polypeptide chain.
• Example: If the codon is AUG, the anticodon is UAC. If the codon is GGC, the anticodon is CCG.

Mutations

• A mutation is a change in the base sequence in a DNA molecule that causes a change in the sequence of amino acids in the polypeptide chain and thus the function of the protein.
• Proteins are end products of genes.

Types of Mutations

• Base Substitution (Point Mutations):
  • Missense: Substitution of one amino acid by another.
    • Example: In sickle cell anemia, valine replaces glutamic acid as the sixth amino acid in hemoglobin.
  • Nonsense: A normal codon changes to a stop codon.
    • Results in a shorter polypeptide chain.
• Frameshift Mutations:
  • Result from deletion or insertion of bases.
  • Nearly always results in a nonfunctional protein because every amino acid after the mutation is different.
  • If a frameshift mutation causes a stop codon to be inserted, the protein will be too short and non-functional.
• Example of Frameshift Mutation
  • Normal mRNA sequence: 5’ A U G G G C A U C C U A G G C A U G U A A 3’
• If a deletion or insertion of one base occurs, it will cause a change in the sequence of bases after the deletion or insertion.

Mutagens

• Mutations can be spontaneous (random) or induced.
• Mutagens (cause mutations) can be chemical or radiation.
  • Radiation:
    • Ionizing (gamma rays and X-rays): Causes breakage in chromosomes.
    • Nonionizing (ultraviolet): Causes thymine dimers.

Identifying Mutagens (Ames Test)

• Used to identify potential mutagens (most are carcinogens) based on their ability to cause back-mutations in bacteria.
• Uses Salmonella typhimurium that lost the ability to synthesize histidine (His-).
• A back-mutation (reversion) occurs when His- mutates to His+.
• His- bacteria grow in a medium with the suspected mutagen. Bacteria are then inoculated on a medium lacking histidine.
• If many colonies grow (His+), the suspected mutagen is indeed a mutagen.

Genetic Recombination and Transfer

• Genetic recombination: Exchange of genetic material between different organisms.

  • Containing genetic material from two or more sources of DNA.
  • The new DNA is called recombinant DNA.

• Homologous recombination: Nucleotide sequences are exchanged between similar or identical molecules of DNA.

• The donor cell DNA is integrated into the recipient cell’s DNA by homologous recombination.

• Conjugation: A plasmid or other genetic material is transferred by a donor to a recipient cell via a direct connection.

• Contact is required between F+ and F- cells for conjugation to occur.

• The F plasmid codes for making F pilus (F+).

• In conjugation, the donor chromosome is transferred as single-stranded DNA.

• In conjugation of HFr cell and F- cell, NOT the entire genome of the HFr cell is usually transferred to the recipient cell.

• When F+ cells are mixed with F- cells, eventually all the cells will become F+.

• Transduction: Transfer of a gene from a bacterium to a bacterium via a virus.

Translation Process Overview

• Initiation of translation begins with the formation of an initiation complex that includes:
  • Smaller ribosomal subunit
  • First amino acid + tRNA (transfer RNA)
  • Messenger RNA (mRNA)
• Larger ribosomal subunit joins the complex
• Initiation factors play a crucial role

Structure of the Ribosome:

• The 70S ribosomes consist of two critical binding sites for tRNA:
  • P site (Peptidyl site): This site holds the tRNA carrying the growing polypeptide chain.
  • A site (Acceptor site): This site is where the next amino acid-tRNA binds, matching the mRNA codon.

Steps in Translation:

1. The initiating tRNA carrying the first amino acid binds to the P site of the ribosome.
2. A tRNA that recognizes the subsequent codon in the mRNA enters the A site with the second amino acid.
3. The ribosome catalyzes the formation of a peptide bond between the amino acids in the P site and the amino acid in the A site.
4. The ribosome moves forward along the mRNA by one codon, during which the first tRNA (now empty) is released.
5. A new tRNA carrying the next amino acid enters the A site.

Elongation of the Polypeptide Chain:

• Occurs through repeated cycles of tRNA binding.
• The amino acid from tRNA in the P site is connected to the amino acid of the tRNA in the A site, effectively lengthening the polypeptide.
• This process continues until the ribosome encounters a stop codon.

Termination of Translation:

• The elongation process ends when a stop codon is reached (a codon that does not correspond to any amino acid).
• Upon reaching the stop codon, the ribosomal subunits disassemble, and the newly synthesized polypeptide chain, along with the mRNA, are released.

DNA Damage Repair Mechanisms

• DNA Damage by Radiant Energy: Radiant energy, such as ultraviolet (UV) irradiation, causes damage to DNA.
• UV Irradiation: Can lead to the formation of covalent bonds between adjacent thymines on the same strand of DNA, creating thymine dimers.
  • Interferes with replication and transcription processes.

DNA Repair Mechanisms

• Most cells possess mechanisms to repair damage to DNA.

Light Repair (Photolyase)

• Enzyme called photolyase breaks the covalent bonds between thymine dimers, reversing the damage.
• Requires visible light to function (light provides the energy for bond cleavage); light repair occurs when there is light.

Excision Repair (Dark Repair)

• Enzyme excises the damaged segment from a single strand of DNA.
• Called excision or dark repair (does not require visible light).
• DNA polymerase replaces the damaged nucleotides with new, undamaged nucleotides.
• DNA ligase then forms the final phosphodiester bond to complete the repair.

DNA Transformation Process

• Involves the transfer of naked DNA into a recipient cell.

Steps in DNA Transformation

1. Initial binding: Double-stranded donor DNA binds to specific receptors on the surface of a competent cell.
2. DNA Degradation and Entry: One strand of the donor DNA is degraded by nucleases, and the remaining single-stranded donor DNA enters the cell.
3. Homologous Recombination: The single-stranded donor DNA pairs with a homologous region on the recipient DNA through a breakage-reunion mechanism and is integrated into the recipient genome.
4. Mismatch Repair: If there are differences between the nucleotide sequences, the mismatch repair system is activated and removes either the donor or the recipient strand, replacing it with the complementary sequence.

Selection of Transformants

• Cells are plated on selective media, allowing only the transformants to grow.
• Mismatch Repair System: activated when there are differences between the nucleotide sequences of the donor recipient DNAs.
• Either the donor or the recipient strand is removed and replaced with the complementary sequence.
• Selective media are used to plate cells; only the transformants (cells that have successfully incorporated the donor DNA) will grow.