Microbial GeneticsAriane Ruby B. Sogo-anMST Biology

Microbial Genetics• Mutation in Bacteria• Genetics Exchange in Bacteria• Recombination and Genetic Engineering

Learning Objectives: 1. Define Mutation. 2. Explain the mechanisms involved in Mutation. 3. Familiarize the processes involved on how Genetic

Information are transferred in Bacteria4. Give the importance of Recombination and Genetic

Engineering

Report Outline• Nucleic Acids• Central Dogma• DNA Replication in Bacteria• RNA Synthesis in Bacteria• Protein Synthesis in Bacteria• Changes in the DNA molecule through Mutation• Transfer of Genetic Information in Bacteria

Genetics• Genetics is a study of Heredity.

• HOW the information contained in Nucleic Acids is expressed?• HOW this type of molecule is duplicated?• HOW this duplicated molecules are transmitted to progeny?

Nucleic Acids• Nucleic Acids are large organic molecules that are found in ALL

cells. • Two Types:

• DNA (Deoxyribonucleic Acid) • directs protein production

• RNA (Ribonucleic Acid)

Nucleic Acids• Nucleic Acids are large organic molecules that are found in ALL

cells. • Two Types:

• DNA (Deoxyribonucleic Acid) • directs protein production

• RNA (Ribonucleic Acid)

Nucleic Acids• Composition:

• Constructed from a string of small molecules called NUCLEOTIDES.

Component of a Nucleotide

Nitrogenous Base

Primary Structure of RNA

Primary Structure of DNA

DNA and RNA

Ribonucleic Acids• RNA are normally single stranded molecules.

• Types: (based on their function)• mRNA (Messenger)• tRNA (Transfer)• rRNA (Ribosomal)

• Look for specific example

Deoxyribonucleic Acids• Double Stranded, with each strand wrapped around the other

in a helical fashion forming a double helix. • Hydrogen bond is specific since A-T (or U in RNA) and G-C

ATCCGGC TAGGCCG

• Molecule is more stable.

Deoxyribonucleic Acids• Determines the characteristics of an organism and maintains

and controls the vital processes of all cells. • How is genetic information expressed?

• Transcription (involves formation of RNA molecule using DNA as a template)

• Translation (consists of the synthesis of a protein using the genetic information in the RNA)

The Central Dogma

Transcription of DNA to mRNA

The Central Dogma• Gene

• The unit of genetic information or hereditary material contained in DNA molecule.

• Sequenced nucleotide in the DNA molecule that codes for RNA molecule and ultimately for the synthesis of a protein.

The Central Dogma• Theory stating that genes guide the synthesis of mRNA and in

turn, directs the order in which amino acids are resembled to form protein.

• Also postulates that a DNA molecule can direct its own replication by giving rise of two identical DNA molecule.

The Central Dogma• Reverse Transcription

• Example: Certain cancer causing viruses (retroviruses) are able to synthesize DNA using RNA as a template.

DNA Replication in Bacteria• Genome – total genetic information in bacteria which consists

of circular DNA molecules found within the cell. • Most of the genome is contained in a single bacterial

chromosome, although smaller pieces of circular DNA called plasmids may also carry a few important genes such as those coding for resistance to microbial drugs.

DNA Replication in Bacteria• The bacterial chromosomes contains most of the genetic

information of bacteria and is attached to the plasma membrane.

• Size of the chromosomes varies from species to species. • Example: (per chromosome)

• Mycoplasma – fewer than 1 M nucleotide base pairs and a genome can code for 1000 proteins.

• E. Coli – 4.5 M nucleotide base pairs that can code for 4500 proteins.

DNA Replication in Bacteria• Both DNA strands are duplicated with each strands functions

as a template that specifies the sequence of bases in the newly formed complementary strand.

• DNA polymerases • Process nucleotides from the cytoplasm that are complementary

to the template and fit them into place. • Parental and New strand = semiconservative.

DNA Replication

DNA Replication• 1. The original double helix molecule.• 2. Helicase enzyme breaks the hydrogen bonds between

complementary base pairs. This unzips the double helix at a position called the replication fork.

• 3. There is an abundant supply of nucleotides in the nucleus for the formation of the new polynucleotides.

• 4. Nucleotides base pair to the bases in the original strands.• 5. DNA polymerase joins together the nucleotides together with

strong covalent phosphodiester bonds To form a new complementary polynucleotide strand.

• 6. The double strand reforms a double helix under the influence of an enzyme.

• 7 Two copies of the DNA molecule form behind the replication fork. These are the new daughter chromosomes.

RNA Synthesis in Bacteria • Transcription• Involves the assembly of nucleotides by an enzyme called RNA

polymerase that uses a strand of DNA as its template. • Begins when RNA polymerase binds to the DNA at the

promoter site near the gene to be transcribed. • RNA polymerase travels along the length of the DNA strand

until it reaches a termination site.

Transcription of DNA to mRNA

RNA Synthesis in Bacteria • After mRNA is made, it will be used as a guide to make

proteins.• Ribosomal RNA, after its made, becomes associated with

proteins to form ribosomes. • tRNA are small RNA molecules that are involved in translating

the information in the mRNA into proteins.

MUTATION

The Genetic Code• The start codon is AUG. Methionine is the only amino acid

specified by just one codon, AUG.

• The stop codons are UAA, UAG, and UGA. They encode no amino acid. The ribosome pauses and falls off the mRNA.

Mutation: Base Substitution (Point Mutations)

G

C

Glu

(d) Run-on mutation

G

C

(a) Silent mutation

Mutation: Insertions and Deletions

Figure 8.17a, d

THEBIGCATATETHERAT

THEBIGCBATATETHERAT

Run-on mutationStop codon lost so

protein is extra long

(can also produce nonsense and run-ons)

Summary of Mutation Types

Spontaneous and Induced Mutation

• Spontaneous mutation rate = 1 in 109 (a billion) replicated base pairs or 1 in 106 ( a million) replicated genes. Mistakes occur during DNA Replication just before cell division. This is natural error rate of DNA polymerase.

• Mutagens increase mistakes to to 10–5 (100 thousand) or 10–3 ( a thousand) per replicated gene

Mutagen

• Mutation relevant

• Cause DNA damage that can be converted to mutations.

Physical mutagens

High-energy ionizing radiation: X-rays and g-

rays strand breaks and base/sugar

destruction

Nonionizing radiation : UV light pyrimidine

dimersChemical mutagens

Base analogs: direct mutagenesis

Nitrous acid: deaminates C to produce U

Alkylating agents

Intercalating agentsLesions-indirect mutagenesis

1 Mutaagenesis

Chemical MutagensBase pair altering chemicals (base

modifiers) deaminators like nitrous acid, nitrosoguanidine, or alkylating agents like cytoxan

Base analogues “mimic” certain bases but pair with others - E.g. 5-fluorouracil, cytarabine

Acts like a “C”

cytarabine

cytoxan Nitrous acid

BASE PAIR ALTERING CHEMICALS

Deaminating Agent• *Deaminating agent - Nitrous acid - removes the anime group

from Adenine and Cytosine

• Nitrous acid is a deaminating agent that converts cytosine to uracil, adenine to hypoxanthine, and guanine to xanthine. The hydrogen-bonding potential of the modified base is altered, resulting in mispairing.

Adenine Hypoxanthine Guanine Xanthine

BASE ANALOGS

Alkylating agents• Alkylating agents like EMS/MMS(ethyl/methly methyl

sulphonate) add methyl groups to Guanosine . Bulky attachment to the side groups or bases.

Hydroxilating Agents

• Addition of OH (Hydroxyl Group)

hydroxylamine (HA)

Intercalating Agents• Intercalation agents are compounds that can slide between

the nitrogenous bases in a DNA molecule. • This tends to cause a greater likelihood for slippage during

replication, resulting in an increase in frameshift mutations.• Example (Sodium Azide)

Chemical Frameshift Mutagens Intercalate into DNA

Aflatoxin fromAspergillus fungus growing on corn

Benzpyrene in cigarette smoke

ATGCTAGCCG

ATGC

TAGCCG

ATGCCGTAGCCG

Carboplatin (anti-cancer drug)

Daunarubicin (anti-cancer drug)

Bleomycin (anti-cancer drug produced by

Streptomyces)

Mutation: Ionizing Radiation

• Ionizing radiation (X rays, gamma rays, UV light) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone.

• Nucleotide excision repairs mutations

X-rays and Gamma Rays Cause Breaks in DNA

Ionizing Radiation: UV

• UV radiation causes thymine dimers, which block replication.

• Light-repair separates thymine dimers

• Sometimes the “repair job” introduces the wrong nucleotide, leading to a point mutation.

Figure 8.20

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Genetic Transfer

• Horizontal Gene Transfer Among Prokaryotes• Horizontal gene transfer

• Donor cell contributes part of genome to recipient cell• Three types

• Transformation• Transduction• Bacterial conjugation

© 2012 Pearson Education Inc.

Bacterial Sexual Processes• Eukaryotes have the processes of meiosis to reduce

diploids to haploidy, and fertilization to return the cells to the diploid state. Bacterial sexual processes are not so regular. However, they serve the same aim: to mix the genes from two different organisms together.

• The three bacterial sexual processes:• 1. conjugation: direct transfer of DNA from one bacterial cell to

another.• 2. transduction: use of a bacteriophage (bacterial virus) to

transfer DNA between cells.• 3. transformation: naked DNA is taken up from the environment

by bacterial cells.

Transformation• We aren’t going to speak much of this process, except to

note that it is very important for recombinant DNA work. The essence of recombinant DNA technology is to remove DNA from cells, manipulate it in the test tube, then put it back into living cells. In most cases this is done by transformation.

• In the case of E. coli, cells are made “competent” to be transformed by treatment with calcium ions and heat shock. E. coli cells in this condition readily pick up DNA from their surroundings and incorporate it into their genomes.

Figure 7.33 Transformation in Streptococcus pneumoniae-overview

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Conjugation• Conjugation is the closest analogue in

bacteria to eukaryotic sex.• The ability to conjugate is conferred by the

F plasmid. A plasmid is a small circle of DNA that replicates independently of the chromosome. Bacterial cells that contain an F plasmid are called “F+”. Bacteria that don’t have an F plasmid are called “F-”.

• F+ cells grow special tubes called “sex pilli” from their bodies. When an F+ cell bumps into an F- cell, the sex pilli hold them together, and a copy of the F plasmid is transferred from the F+ to the F-. Now both cells are F+.

• Why aren’t all E. coli F+, if it spreads like that? Because the F plasmid can be spontaneously lost.

Figure 7.35 Bacterial conjugation-overview

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F plasmid Origin oftransfer

Conjugation pilus Chromosome

F+ cell F– cell

Donor cell attaches to a recipient cell withits pilus.

Pilus may draw cells together.

One strand of F plasmid DNA transfersto the recipient.

F+ cell F+ cell

Pilus

The recipient synthesizes a complementarystrand to become an F+ cell with a pilus; thedonor synthesizes a complementary strand,restoring its complete plasmid.

Figure 7.36 Conjugation involving an Hfr cell-overview

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Donor chromosome

Pilus

Pilus

F+ cell

Hfr cell

F+ cell (Hfr)

F plasmid

F– recipient

Part of F plasmidDonor DNA

F plasmid integratesinto chromosome byrecombination.

Cells join via aconjugation pilus.

Portion of F plasmid partiallymoves into recipient celltrailing a strand of donor’sDNA.

Conjugation ends with piecesof F plasmid and donor DNAin recipient cell; cells synthesizecomplementary DNA strands.

Donor DNA and recipientDNA recombine, making a recombinant F– cell.

Incomplete F plasmid;cell remains F–

Recombinant cell (still F–)

Hfr Conjugation• When it exists as a free plasmid,

the F plasmid can only transfer itself. This isn’t all that useful for genetics.

• However, sometimes the F plasmid can become incorporated into the bacterial chromosome, by a crossover between the F plasmid and the chromosome. The resulting bacterial cell is called an “Hfr”, which stands for “High frequency of recombination”.

• Hfr bacteria conjugate just like F+ do, but they drag a copy of the entire chromosome into the F- cell.

Transduction• Transduction is the process of moving bacterial DNA

from one cell to another using a bacteriophage.• Bacteriophage or just “phage” are bacterial viruses. They

consist of a small piece of DNA inside a protein coat. The protein coat binds to the bacterial surface, then injects the phage DNA. The phage DNA then takes over the cell’s machinery and replicates many virus particles.

• Two forms of transduction:• 1. generalized: any piece of the bacterial genome can be

transferred• 2. specialized: only specific pieces of the chromosome can be

transferred.

Figure 7.34 Transduction-overview

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Bacteriophage

Phage injects its DNA.

Phage enzymesdegrade host DNA.

Phage DNA

Host bacterial cell(donor cell)

Bacterial chromosome

Phage with donor DNA(transducing phage)

Cell synthesizes newphages that incorporatephage DNA and, mistakenly,some host DNA.

Transducing phage

Transducing phageinjects donor DNA.

Recipient host cell

Donor DNA is incorporatedinto recipient’s chromosomeby recombination.

Transduced cell

InsertedDNA

General Phage Life Cycle• 1. Phage attaches to the cell

and injects its DNA.• 2. Phage DNA replicates,

and is transcribed into RNA, then translated into new phage proteins.

• 3. New phage particles are assembled.

• 4. Cell is lysed, releasing about 200 new phage particles.

• Total time = about 15 minutes.

Why do chromosomes undergo recombination?

Deleterious mutations would accumulate in each chromosome

Recombination generates genetic diversity

Recombination

ABCDEFGhijklmnoPQRSTUVWXYZabcdefgHIJKLMNOpqrstuvwxyz

ABCDEFGHIJKLMNOPQRSTUVWXYZabcdefghijklmnopqrstuvwxyz

Mitotic and meiotic recombinationRecombination can occur both during mitosis and meiosis

Only meiotic recombination serves the important role of reassorting genes

Mitotic recombination may be important for repair of mutations in one of a pair of sister chromatids

Recombination mechanisms

Best studied in yeast, bacteria and phage

Recombination is mediated by the breakage and joining of DNA strands

Benefits of recombination • Greater variety in offspring: Generates new combinations of

alleles• Negative selection can remove deleterious alleles from a

population without removing the entire chromosome carrying that allele

• Essential to the physical process of meiosis, and hence sexual reproduction• Yeast and Drosophila mutants that block pairing are also

defective in recombination, and vice versa!!!!

Genetic Engineerin

g

What is genetic engineering???

Genetic engineering: is the artificial manipulation or alteration of genes.

Genetic Engineering involves:• removing a gene (target gene) from one organism• inserting target gene into DNA of another organism• ‘cut and paste’ process.

Some important terms!!!

Recombinant DNA: the altered DNA is called recombinant DNA ( recombines after small section of DNA inserted into it).

Genetically Modified Organism (GMO): is the organism with the altered DNA.

Genetic Engineering breaks the species barrier!!!

• Genetic engineering allows DNA from different species to be joined together.

• This often results in combinations of DNA that would never be possible in nature!!! For this reason genetic engineering is not a natural process.

• If DNA is transferred from one species to another the organism that receives the DNA is said to be transgenic.

Genetic engineering breaks the species barrier!!!

• Examples of cross-species transfer of genes:

- a human gene inserted into a bacterium- a human gene inserted into another animal- a bacterial gene placed in a plant

Alternative names for genetic engineering:

• Genetic Manipulation

• Genetic Modification

• Recombinant DNA Technology

• Gene Splicing

• Gene Cloning

Tools used in genetic engineering!!!• Source of DNA: Target (foreign) DNA – DNA taken from one organism to be placed into the DNA of a second organism.

• A cloning vector: Special kind of DNA that can accept foreign DNA and exactly reproduce itself and the foreign DNA e.g. Bacterial plasmid (loop of DNA found in bacteria).

Tools Used in Genetic Engineering

Restriction Enzymes: - are special enzymes used to cut the DNA at specific places.

- different enzymes cut DNA at specific base sequences known as a recognition site. For example

i) One restriction enzyme will always cut DNA at the base sequence: GAATTC. ii) Another restriction enzyme only cuts at the

sequence: GATC.

- If DNA from two different organisms is cut with the same restriction enzyme the cut ends from both sources will be complementary and can easily stick together.

Restriction enzymes

DNA 1

DNA 2

Tools used in Genetic Engineering

DNA Ligase: enzyme which acts like a glue sticking foreign DNA to DNA of the cloning vector.

• will only work if DNA from the two DNA sources has been cut with the same restriction enzyme i.e. sticky ends of cut DNA will be complementary to each other.

Please note diagram illustrating use of restriction enzymes and DNA Ligase in production of recombinant

DNA Fig. 19.6 pg. 195

Process of Genetic Engineering

Five steps involved in this process:1. Isolation

2. Cutting

3. Insertion (Ligation)

4. Transformation

5. Expression

Note: The following example will explain how a human gene is inserted into a bacterium so that the bacterium can produce human insulin.

Process of Genetic Engineering1. Isolation:

• Removal of human DNA (containing target gene).• Removal of plasmid (bacterial DNA) from

bacterium.

2. Cutting: • Both human DNA and plasmid DNA are cut with

the same restriction enzyme.• Normally plasmid has only one restriction site

while human DNA will have many restriction sites.Please note diagram 19.7 pg. 196

Process of Genetic Engineering

Insertion:• means that target gene is placed into the DNA of

the plasmid or cloning vector.• cut plasmids are mixed with human DNA sections

allowing the cut ends to combine.

TransformationExpression

Applications of Genetic Engineering

You must know three applications: one involving a plant, one animal and one for a micro-organism.

Plants: Weed killer-resistant crops• many types of crop plants have bacterial genes added

to them.• these genes make the plants resistant to certain weed

killers (herbicides).• this means that the weed killers kill the weeds but do

not affect the transgenic plants.

Applications of Genetic Engineering

Animals: There is a growing trend to experiment with inserting human genes into the DNA of other mammals. The transgenic animals formed in this way will then produce a human protein and secrete it into their milk or even into their eggs.

Applications of Genetic Engineering

Animals: Sheep produce human clotting factor• A human gene has been inserted into the DNA of

sheep.• This allows the adult sheep to produce a clotting

chemical needed by haemophiliacs to clot their blood – produced in the milk of the sheep.

Pharming: is the production of pharmaceuticals by genetically modified animals i.e. sheep, cows, goats etc.

Pharming – using animals to make pharmaceuticals

Applications of Genetic Engineering

Micro-organisms: Bacteria make insulin• The human insulin gene has been inserted into a

bacterium (E-coli).• This allows the bacterium to produce insulin for use

by diabetics.

Ethical Issues in Genetic Engineering

GMO’s as a food source:Outlined below are some fears associated with the use of GMO’s as a food source:

• Cannibalism: – eating an animal containing a human gene is a form of cannibalism.- feeding GMO’s containing human genes to

animals that would later be eaten by humans.• Religious reasons: – eating pig genes that are inserted

into sheep would be offensive to Jews and Muslims.• Offensive to vegetarians/vegans: – eating animal genes

contained in food plants cause concern.

Ethical Issues in Genetic Engineering

Animal Welfare:• There is serious concern that animals will suffer as a

result of being genetically modified.• use of growth hormones may cause limb deformation

and arthritis as animals grow.

Ethical Issues in Genetic Engineering

Genetic Engineering in Humans:

The following issues are a cause for concern:• If tests are carried out for genetic diseases, who is

entitled to see the results?• Tests on unborn babies – could this lead to abortion if

a disease is shown to be present?• Insurance/lending companies – will they insist on

genetic tests before they will insure/lend money to a person?

• Need for legal controls over the uses to which human cells can be put.

• Development and expansion of eugenics.

END OF REPORT