Microbial GeneticsUnlocking the Secrets of Heredity

Chapter 8

•Chromosome: discrete cellular structure composed of a neatly packaged DNA molecule

•Eukaryotic chromosomes-DNA wound around histones-located in the nucleus-diploid (in pairs) or haploid (single)-linear appearance

•Prokaryotic chromosomes-DNA condensed into a packet by means of histone-like

proteins-single, circular chromosome

Chromosomes

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A sampling of genes related to obesity in the

human genome• http://www.obesity.chair.ulaval.ca/gene

map.html

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•A closer look at chromosome 1

•Genes are SPECIFIC & DISCRETE segments of DNA

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• A map of E. coli’s ~5000 genes

• Notice it is single & circular

• Does E. coli have 1 or 2 alleles of each gene? How do you know?

• Humans were first thought to function with 100,000 genes and now the number has dropped to ~35,000 genes although this is still a hot topic in research

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DNA is lengthy and occupies a small part of the cell by coiling up into a smaller package.

Fig. 9.3 An Escherichia coli cell disrupted to release itsDNA molecule.

Genome

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•Gene

-a certain segment of DNA that contains the necessary code to make a protein or RNA molecule

•Three categories of genes-structural genes: code for proteins

-genes that code for RNA machinery used in protein production

-regulatory genes: control gene expression

Gene

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Genetic Terms

• Genotype• an organism’s genetic makeup; its entire

complement of DNA

• Phenotype• is the expression of the genes: the proteins

of the cell and the properties they confer on the organism.

• Size, shape, color, environment

• Which one is easier to see?

•Nucleotide: basic unit of DNA structure

• phosphate• deoxyribose sugar• nitrogenous base

•Nucleotides covalently bond to each other in a sugar-phosphate linkage

•Pairing of bases dictated by the formation of hydrogen bonds between bases

The DNA Code

5′

3′5′

5′

3′

DT A

D

CG

DG C

G C

P

P

P

P

P

D

D

A T

P

D

P

DT A

D

CG

P

P

P

P

P

P

G C

A T

5′

3′

4′

2′

1′D

P

P

P

D

P

P

H

H

H

H

H

H

N

NN

N

N

O

O

N

N

H

H

H

HN

N

NN

O

D

D

D

D

D

D

D

D

O

OO

OO

O

O

O

O

OO

OO

Sugar

OHN–H

H–

CH3O

(a)

OH

SugarN–

H–N

Hydrogen bond

N–H

Nature of the double helix- antiparallel arrangement: one side of the helix runs

in the opposite direction of the other

- the order of the bond between carbon on deoxyribose and the phosphate is used to keep track of the direction of the two sides

- one side runs from 5’ to 3,’ and the other side runs 3’ to 5’

- this is a significant factor in DNA synthesis and protein production

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DNA Replication

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DNA Replication• What you need to replicate DNA:

1. Original DNA template (parental chromosome)

2. Nucleotides (Guanine, Cytosine, Adenine, Thymine)

a pool of nucleotides will be free floating in the cytoplasm waiting to be used

3. EnzymesI.e., DNA polymerase (hooks together nucleotides), ligase (ligates)

4. Energy (ATP)

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Replication

• Don’t worry about knowing the leading strand, lagging strand and 5’ and 3’ terminology for the exam. These are more in depth than we will go.

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DNA Replication in Prokaryotes• Certain enzymes

unwind the DNA.• Then, DNA

polymerase can read the parent strand and attach a complementary nucleotide to the new strand of DNA.

• The nucleotides are free in the cytoplasm.

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DNA replication is semi-conservative since each new chromosome will have one “old” and one “new” strand

Transcription

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Transcription (RNA Synthesis)

• What you need to synthesize RNA:1. Original DNA template (parental chromosome) with a promoter site (DNA sequence indicating start site) and a terminator site

2. Nucleotides(G, C, A, U)Ribose (sugar) + phosphate + N base Uracil is substituted for thymine

3. EnzymesI.e., RNA polymerase (hooks together nucleotides)

4. Energy (ATP)

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Transcription

•RNA polymerase: large, complex enzyme that directs the conversion of DNA into RNA

•Template strand: only one strand of DNA that contains meaningful instructions for synthesis of a functioning polypeptide

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Transcription

4 types of RNA can be transcribed:1. Messenger ribose nucleic acid (mRNA)

mRNA (RNA molecule that serves as a message of the protein to be produced)

2. Transfer ribose nucleic acid (tRNA)tRNA (64 different tRNA molecules participate in translation)

3. Ribosomal ribose nucleic acid (rRNA)rRNA (forms the ribosome)

4. Regulatory RNA

•RNA is similar to DNA in terms of its general properties, but its structure is different in several ways

-single-stranded molecule that exists in helical form; can assume secondary and tertiary levels of complexity, leading to specialized forms of RNA (tRNA and rRNA)

-contains uracil (U) instead of thymine; does not change the DNA code because uracil still follows the pairing rules

-contains ribose rather than deoxyribose

The RNAs

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The RNAs• mRNA- message from DNA, single stranded• rRNA- part of ribosome• tRNA- transfers amino acids to ribosome• Regulatory RNAs:

- micro RNAs, anti-sense RNAs, riboswitches, small interfering RNAs

• Primer RNAs: operative in both prokaryotic and eukaryotic cells

• Ribozymes: remove unneeded sequences from other RNAs

Transcription: Initiation

• RNA polymerase recognizes promoter region• RNA polymerase begins its transcription at a special

codon called the initiation codon• As the DNA helix unwinds it moves down the DNA

synthesizing RNA molecule

Transcription: Elongation

• During elongation the mRNA is built, which proceeds in the 5’ to 3’direction (with regard to the growing RNA molecule)

• the mRNA is assembled by the adding nucleotides that are complementary to the DNA template.

• As elongation continues, the part of DNA already transcribed is rewound into its original helical form.

Direction oftranscription

Early mRNAtranscript

Nucleotidepool

Transcription: Termination

At termination the polymerases recognize another code that signals the separation and release of the mRNA strand,or transcript.

Late mRNA transcript

Elongation

Translation

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Translation

• Decoding the “language” of nucleotides and converting/translating that information into the “language” of proteins.

• The nucleic acid “language” is in the form of codons, groups of three mRNA nucleotides.

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Where does translation occur?

• Translation occurs at the RIBOSOME!

• The green mRNA strand is “threaded” through the ribosome.

• The ribosome “reads” the mRNA strand codons with the help of the genetic code and tRNA (next slides)

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tRNA• Decoder molecule which

serves as a link to translate the RNA language into protein language – One site of the tRNA has

an anticodon which complements the codon of mRNA

– The other site of the tRNA has an amino acid attachment site corresponding to a specific amino acid as noted in the genetic code

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Codons• Triplet code that specifies a given

amino acid• Multiple codes for one amino acid

– Degenerate(repetitive) code is good to allow for a certain amount of mutation to occur without having an effect on the amino acid sequence

– 1 Start codon– 3 Stop codons– 64 total possible codons– 20 amino acids

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Translation and the “Genetic Code”

• We use the “genetic code” (at right) to translate mRNA nucleotide sequence (codons) into amino acid sequence which make up proteins.

• The “genetic code” is degenerate which allows for a certain amount of mutation. I.e. UUU and UUC both code for Phe

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• There is one start codon, AUG, that codes for the amino acid methionine.

• There are 3 stop codons, UAA, UAG and UGA that signal the ribosome to stop translation and let go of the polypeptide chain (protein).

Translation and the “Genetic Code”

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Translation• Ribosomes

bind mRNA near the start codon (ex. AUG)

• tRNA anticodon with attached amino acid binds to the start codon

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Translation• Ribosomes move to

the next codon, allowing a new tRNA to bind and add another amino acid

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Translation• Series of amino acids

form peptide bonds

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Translation• Stop codon

terminates translation

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Polyribosomal Complex-a single mRNA is long enough

to be fed through more than one ribosome

-permits the synthesis of hundreds of protein molecules from the same mRNA transcript

-occurs only in prokaryotes, where there transcription and translation both occur in the cytoplasm

-Would you see this in Eukaryotes?

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Introns and Exons

Eukaryotic mRNAs code for just one protein, unlike bacterial mRNAs, which often contain information from several genes in series

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Transcription and translation in eucaryotes

• Similar to procaryotes except– AUG encodes for a different form of

methionine– Transcription and translation are not

simultaneous (since eucaryotes have a nucleus----transcription occurs in the nucleus, translation occurs ?)

– Eucaryotes must splice out introns to achieve a mature mRNA strand ready to go to the ribosome.

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How are genes regulated?• Cells regulate genes in 3 major ways:

1. Feedback inhibition– The end-product inhibits the pathway (similar to a

thermostat….when it reaches the desired temperature it turns off)

2. Enzyme induction– If a substrate is present, the enzyme for the substrate is

induced.

3. Enzyme repressiona. If a nutrient is present, the enzyme to make it is

repressed.b. If a nutrient is absent, the enzyme to make it is

turned on.

-only found in bacteria-coordinated set of genes-all regulated as a single unit-either inducible or repressible

Operons

lac Operon

lac Operon

Phase Variation

• Bacteria turn on or off a complement of genes that leads to obvious phenotypic changes

• Phenotype is heritable! • Most often traits affecting the bacterial cell

surface

• Examples: - Neisseria gonorrhoeae: production of attachment

fimbriae- Streptococcus pneumoniae: production of a capsule

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What if a gene changes?• Mutation=a change in the sequence of

DNA

• Effects of mutations• none-->no change in a.a. sequence or….

• Good-->new aa. Seq-->antibiotic resistance– Increases variability in the gene pool

• Bad-->new aa. Seq-->mutate active site of enzyme

• For humans, cancer is the product of a combo of bad mutations.

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Types of Mutations• Point Mutation

• put the cat out--->puc the cat out• put the cat out--->put

• Frameshift (reading frame of mRNA shifts)

• put the cat out--->put hec ato ut• Deletion• Addition• Duplication

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The Effects of Base Substitution

(a point mutation) • When a base is substituted in

DNA the mutation may have 2 effects:– Changes the amino acid– Does not change the amino acid– Why doesn’t a mutation always

change the amino acid sequence? Because the genetic code is degenerate and has amino acids that may be coded for by different codons. (I.e., AAA and AAG both code for phenylalanine)

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The Effects of Frameshift Mutations

• The addition, deletion or insertion of one or more nucleotides drastically changes the amino acid sequence.

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Mutation Rates• Normal Mutation Rate=1/1 million per gene

– Mutations are constantly occurring since our enzymes are not 100% perfect …These are called spontaneous mutations and increase in occurrence as we age….when do we get cancer?

• Mutagen=certain chemicals or radiation that bring about mutations.

• Mutagen Mutation Rate= 1/1000-1/100,000 per gene (10-1000X the normal rate)

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Repair of mutations involves enzymes recognizing, removing, and replacing the bases.

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Mutagen Examples • 5-Bromouracil and

acridine are 2 mutagen examples that can “insert” themselves in DNA and cause errors in DNA replication, transcription and translation.

• Notice how similar in structure mutagens can be. There is just one change to thymine that can have dire consequences

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Thymine Dimers Caused by Radiation

• Radiation, such as X-rays and UV rays, can cause dimers to form in DNA.

• Thymine dimers can interfere with DNA replication, transcription and translation.

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What is the connection to cancer?

• Cancer is a genetic disease. It is the consequence of a change in DNA sequence.

• Carcinogen=substance that causes cancer

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What is the connection to cancer?

• Are mutagens also carcinogens?

• The Ames Test uses bacteria to identify possible carcinogens by looking for mutations to occur. Once a mutagen is identified, it is tested in animals to test if it is a carcinogen.

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The Ames test is used to screen environmental and dietary chemicals for mutagenicity and carcinogenicity without using animal studies.

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Is there another way for the genetic makeup to

change?• Yes-->Genetic Recombination

• Effects of genetic recombination– increase diversity in gene pool– may cause cancer

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When and where does genetic recombination

take place?

• During meiosis of human gametes

• In bacteria, occurs when DNA is transferred between bacteria.

DNA Recombination Events•Recombination

-an event in which one bacterium donates DNA to another bacterium called a recombinant

-end result is a new strain different from both the donor and the original recipients

-depends on the fact that bacteria have plasmids and are adept at interchanging genes

-provide genes for resistance to drugs and metabolic poisons, new nutritional and metabolic capabilities, and increased virulence and adaptation to the environment

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Vertical vs. Horizontal Gene Transfer

• Vertical gene transfer=• Genes/DNA passed from an organism to

its offspring

• Horizontal gene transfer=• Genes/DNA transferred between

organisms

• Which type do humans have?

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Types of Genetic Transfer in Bacteria

Conjugationtransfer of plasmid via sex pilus~conjugal visit b/t bacteria

plasmid=“mini-chromosome carrying extra genes”-circular and self-replicating

Transformationgenes transferred from one bacterium to another as “naked” DNA in solution

TransductionDNA transferred from donor to recipient cell inside a virus that infects bacteria(Bacteriophage/phage)

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Plasmids

• self-replicating, gene containing circular pieces of DNA

• 1-5% the size of bacterial chromosome• “mini-chromosome”

• Bacteria can store up many different plasmids for their use & can transfer these to other bacteria.

– I.e. antibiotic resistance genes, toxin production, etc.

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Antibiotic Resistance (R) Plasmid

• Some plasmids can carry many antibiotic resistance genes.

• When bacteria collect many plasmids (they can possess more than one) and these plasmids have many antibiotic resistance genes, a “superbug” may originate.

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Mechanism of Conjugation• A donor cell (called the F+ cell in

this case) contains a F (fertility) plasmid.

• A conjugation pilus forms and the donor cell transfers a copy of the F plasmid to the recipient.

• Now, both cells have a F plasmid

• What would happen if the F plasmid was really an R (antibiotic resistance) plasmid?

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Conjugation• Transfer of plasmid DNA from a F+ (F

factor) cell to a F- cell • An F+ bacterium possesses a pilus • Pilus attaches to the recipient cell and

creates pore for the transfer DNA

Chromosomes

F factor (plasmid)

F Factor Transfer

Transfer of the F factor, or conjugative plasmid

Donor F+

Recipient F–

Bridge madewith pilus

F factorbeing copied

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Conjugation

• High frequency recombination (Hfr) donors contain the F factor in the chromosome

Bridgebroken

Donatedgenes

Partial copyof donorchromosome

Integration ofF factor intochromosome

Pilus

DonorHfr cell

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Transformation

• “Naked” DNA fragments of one disintegrating cell are close to another live cell.

• Some cells have the ability to “pick up” naked DNA fragments and “insert” or recombine the DNA into their own DNA

• The new recombinant cell now has its original DNA, plus some new DNA from the disintegrating cell.

• What if genes a or b were genes for penicillinase (an antibiotic resistance gene)?

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Transformation

• Nonspecific acceptance of free DNA by the cell (ex. DNA fragments, plasmids)

• DNA can be inserted into the chromosome

• Competent cells readily accept DNA, luckily not all bacteria can become competent just like not all bacteria form spores or flagella, etc.

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DNA released from a killed cell can be accepted by a live competent cell, expressing a new phenotype.

Fig. 9.25 Griffith’s classic experiment in transformation

Bacterial transformation

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Mechanism of Transduction

• When you think of Transduction, think virus mediated gene transfer

• The virus is able to kill the initial bacterial cell.

• When the cell lyses, the viral particles which have picked up DNA from the original cell now insert that DNA into a new cell.

• The new cell may or may not insert the new DNA sequence into its chromosome.

• Transduction can be a problem when the red DNA codes for an antibiotic resistance gene.

• Can you see how antibiotic resistance can be transferred?

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Transposons• Transposons=

• small segments of DNA that can move (be transposed) from one region of a DNA molecule to another.

• “jumping genes”• not a “mini-chromosome”, just a linear segment of DNA

that can jump within one chromosome/plasmid or between them.

– Involved in• changes in traits such as colony morphology,

pigmentation, and antigenic characteristics• replacement of damaged DNA• intermicrobial transfer of drug resistance (in bacteria)

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Transposons

• Some genes can “jump” from chromosome to plasmid, from plasmid to plasmid or from plasmid to chromosome,

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Genes & Evolution

• Genes are continually altered due to mutation, recombination, and transposition

• These changes increase genetic diversity of the gene pool and then natural selection acts on diverse populations to ensure survival in many different habitats.