microbial genetics microbiology ar
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Microbial GeneticsTRANSCRIPT
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