Microbial Genetics

• Dr. Gary Andersen, 913-279-2211• Some slides used with permission from Curtis Smith, KCKCC

• Reference: Chapter 7,8 from (Black, J., 2005)

Basic Units of Genetics

• Genomes – the total of the genetic material in a cell.

• Gene - The unit of heredity for a given genetic trait. The site on a DNA molecule that carries the code for a certain cell function.

• Viruses – 4 or 5 genes, E. coli – 4228 genes, Human ~ 31,000 genes.

A Big Question to Struggle With• Which is more important… nature or nurture?

Genetics or the environment? In determining the characteristics and behavior of an organism?

Nucleic AcidsI. Nucleic acids are located in the nucleoid

of bacteria, and the nucleus of eukaryotes. There are 2 kinds of nucleic acids: RNA & DNA.

Ruptured E. coli cellshowing DNA

DNA

A. CHARACTERISTICS OF DNA

DNA (deoxyribonucleic acid) is made of subunits called nucleotides. Nucleotides are made of 3 components. These 3 components are linked together with a covalent bond.

E. Coli = 4.6 million nucleotide pairs (~1mm)

Corn = 2.5 billion nucleotide pairs

Human = 3 billion nucleotide pairs (2nm wide by 2 meters long)

Significance of DNA Structure

• Maintains the code with high degree of fidelity. (double strand assures accurate replication)

• Provides a method for introducing a high degree of variety. (unlimited variety of sequences possible)

1. COMPONENT 1 - Phosphate

Phosphate group - Phosphate functions as a structural part of nucleic acids.

2. COMPONENT – Ribose Sugar

2, DEOXYRIBONUCLEIC ACID

Ribose - A five carbon sugar that functions as part of the DNA backbone (ie. structural). “2, Deoxy” means without oxygen on the number 2 carbon atom.

3. COMPONENT – Nitrogen Bases

NITROGEN CONTAINING BASES

Function: express genetic information.

composition :

2 PURINES: ADENINE (A) GUANINE (G)

double ring structures

2 PYRIMIDINES: THYMINE(T) CYTOSINE(C)

single ring structures

Nucleotide Base

Composed of one Nitrogen base, one Deoxyribose, and one Phosphate group

Phosphate

Deoxyribose

Adenine(Nitrogen base)

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4 Nucleotides

• DNA Structure

DNA Structure4. DNA is a double helix (there are 2 strands of

DNA) which are intertwined with 5 base pairs per turn.

5. DNA has complimentarity

that is A always bonds with T

and G always with C

6. DNA is always antiparallel. The 2 strands of DNA are always oriented in opposite directions. ( 5’ PO3 end – 3’ OH end)

http://www.umass.edu/microbio/chime/dna/dna53.htm

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DNA Bonds

3-D Image ofDNA

B. RNA

RIBONUCLEIC ACID

Similar to DNA except:

1. RNA is single stranded

2. RNA has a ribose sugar instead of deoxyribose. (Oxygen on #2 C).

3. RNA has URACIL (u) instead of thymine

4. RNA is always shorter than DNA, ~ 1,000 nucleotides in length

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C. FUNCTIONS OF RNA

1. rRNA (ribosomal) - comprises the ribosome (site of protein synthesis). (60% of a ribosome is made of RNA, the rest is protein).

2. tRNA (transfer) carries amino acids to the ribosome during protein synthesis. Also known as the “ANTICODON”

3. mRNA (messenger) - a complimentary strand of RNA equal in size to 1 gene (normally ~1,000 nucleotides). “CODON” - coded info from DNA (bound for the ribosome)

THE CENTRAL DOGMA OF BIOLOGY “Francis Crick – 1956”

There are 3 parts to the flow of information in all cells.

Transcription Translation

DNA -------------mRNA-----protein

Replication

Central Dogma of Biology

DNA REPLICATION1. Where 2 parental strands of DNA are

copied into 2 daughter strands. Rate = 1,000 nucs per seconds without error. This leads to binary fission in bacteria.

Cell Division) = 2 daughter cells2. Each cell receives 1 parental strand and

1 daughter strand. (semiconservative replication)

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As the two replication forks meet, the two new chromosomes separate—each containing one new and one old strand

Replication bonding

• Replication Fork

1. EVENTS IN DNA REPLICATION

a. DNA unwinds using the enzyme DNA Helicase

b. SSBP holds the 2 strands apart (single strand binding proteins)

c. Note: 2 replication forks. DNA replication is considered bi-directional replication.

DNA REPLICTIONCONTINUED

d. Polymerization requires DNA Polymerase (POL III) which is an enzyme that synthesizes 2 nucleotide strands (daughter strands) from 2 parental (templates) strands.

e. DNA exonuclease (POL I) removes any mistaken base pairs.

f. DNA ligase seals any gaps and joins the 2 strands together.

• DNA Replication Enzymes at Work

• Steps in Replication

Replication of DNA cont’d• http://www.ncc.gmu.edu/dna/repanim.htm

THE CENTRAL DOGMA OF BIOLOGY

There are 3 parts to the flow of information in all cells.

Transcription Translation

DNA -------------mRNA-----protein

Replication

B. TRANSCRIPTION1. 2nd part of the central dogma of biology2. 1st step in gene expression (i.e.protein

synthesis).3. The cells genetic plan contained in DNA is

transcribed into a complimentary base sequence called messenger RNA (mRNA).

4. The region of DNA that produces or serves as a template for mRNA is called a gene. A gene normally consists of around 1,000 base pairs. It is the smallest segment of DNA that codes for mRNA.

TRANSCRIPTION CONTINUED5. RNA polymerase is the enzyme

responsible for making mRNA

Transcription continued

7. Example:

DNA A T G C C G DNA T A C G G C mRNA A U G C C G 8. mRNA is a blueprint of DNA or a

transcript or code.9. One code word consists of three letters.

• http://www.ncc.gmu.edu/dna/mRNAanim.htm

Animation of Transcription

C. TRANSLATION1. Translation is the 3rd part of the central dogma of

biology (2nd step in gene expression or protein synthesis).

2. After transcription, the coded information in mRNA is translated into an enzyme (protein).

3. This process takes place on the ribosome. Note that the ribosome is made of rRNA and protein.

Translation Graphic

TRANSLATION CONTINUED

4. tRNA STRUCTURE

tRNA utilizes the information in mRNA to determine the sequence of amino acids in a protein. tRNA has a cloverleaf shape. The amino acid end binds one specific amino acid in the cytoplasm. The anticodon end pairs

with the codon on mRNA.

Transfer RNA Structure

TRANSLATION CONTINUED

3. The mechanics of translation Initiation; mRNA bumps into the small

subunit and triggers the two ribosomal subunits to bind together. The first tRNA anticodon (UAC) carrying the amino acid methionine hydrogen bonds with the codon AUG on mRNA.

TRANSLATION CONTINUED

b. Elongation – The second tRNA binds to the second code word on mRNA. A peptide bond forms between the two amino acids. The first tRNA leaves, and the enzyme translocase moves the ribosome down one code word of mRNA at a time. This repeats ~ 300X.

TRANSLATION CONTINUED

C. In termination, one of three possible stop codons is reached. The last tRNA falls away and the two ribosomal subunits fall apart.

d. The Genetic Code

61 sense codons for 20 amino acids

3 nonsense (or stop codons)

64total codonsPg 180 (Black, J., 2005)

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The Genetic Code

Steps in Protein Synthesis

Steps in Protein Synthesis

Steps in Protein Synthesis

Steps in Protein Synthesis

Steps in Protein Synthesis

Protein Synthesis

• http://www.ncc.gmu.edu/dna/ANIMPROT.htm

Translation - Animation

Translation Animation - http://www.wehi.edu.au/wehi-tv/dna/movies/Translation.mov.gz

e. Translation Blockers

1. Streptomycin – (SM) blocks assembly of the ribosome during initiation.

2. Chloroamphenicol – (CA) blocks peptide bond formation during elongation.

3. Tetracycline – TC – blocks the 2nd site on the ribosome during elongation.

4. Erythromycin EM – blocks translocase during elongation.

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Gene Regulation

• How can genes be turned off and on?

• Examples from E. coli– Inducer – example is lactose (lac operon), pg

187 of (Black, J., 2005)– Repressor – argenine (arg operon), pg 187-188

of (Black, J., 2005)

Induction - Lac operon

Repression - Trp operon

III. 5 Ways of Creating Genetic Diversity in Bacteria

A. Mutations

B. Transformation

C. Conjugation

D. Transposition

E. Transduction

A. Mutations

1. Changes in the nucleotide sequence usually due to an error in DNA replication. These occur naturally

at low levels (also known as spontaneous mutations); or by the effects of chemical agents called mutagens; or by physical agents like radiation.

Results of Mutations

2. Most mutations are neutral - they have no effect on the polypeptide. Some mutations result in a less active product; Less often an inactive product; Very few mutations are beneficial. However, these would be passed on!

Types of Mutations

3. Point mutations - a one base change in DNA. There are 3 types:

a. silent mutations - single base substitution in the 3rd base nucleotide position of a codon. This results in NO change in amino acid. Note that the first 2 letters of the genetic code are the most critical.

b. missense mutations - single base substitution in 1st or 2nd base nucleotide position. This results in a changed amino acid. A change in one amino acid usually will have little effect depending on where in the polypeptide it occurs.

c. nonsense mutations - single base substitutions that yield a stop codon. Note: there are 3 nonsense codons in the genetic code = NO PROTEIN

4. Frame Shift Mutations - the addition or deletion of 1 or more bases. These are due to powerful mutagens; chemical or physical.

a. Chemical mutagens - (used in research to study mutagenesis). There are 3 kinds of chemical mutagens.

1. alkylating agents. Adds alkyl group, CnH(2n+1) Ex. formalin, nitrogen, mustard, and ethylene oxide (reacts with G changing it to bind with T).

2. base analogs. Mimics a nitrogen base. Ex. AZT is a modified sugar that substitutes for T. Ex. 5 - bromouracil binds with A or G.

3. intercalating agents. Inserts into DNA and pushes bases apart. Ex. AFLATOXIN - a chemical produced by peanut and grain molds. The mold is Aspergillus flavus (fungus).

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b. Physical mutagens:

1. nonionizing radiation - Causes the formation of T= T dimers. UV light @ 260 nm.

2. Ionizing radiation - damages DNA by causing the formation of “free radicals” leading to mutations. 3 Ex. X-rays. Gamma rays from radioactive fallout penetrates the body. Alpha rays from inhaled dust containing radioactive fallout.

B. TRANSFORMATION

The passage of homologous DNA from a dead donor cell to a living recipient cell. Occurs in Streptococcus pneumoniae. When S. pneumo dies the DNA can be absorbed by a living S. pneumo and recombined into the chromosome. The gene for capsule formation is obtained in this way, as is a gene for penicillin resistance. Discovered in 1929 by Fredrick Griffith.

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• Transformati

on Graphi

c

Griffith’s Transformation Experiment

C. CONJUGATION 1. A “mating” process between a donor F+

(bacteria with fertility factor =plasmid) and an F- recipient cell. 2. Occurs in Gram - enteric bacteria like

E.coli3. Discovered in 1946 by Joshua Lederberg

and Edward Tatum.4. Plasmids carry genes that are nonessential

for the life of bacteria. Ex. gene for pili (sex pilus). Ex. plasmid replication enzymes. Ex. Medical Problem: R-Factor = antibiotic resistance!

Conjugation continued

“Normal - Sex” plasmid transfer (usually ~20 of 100 genes).a. Requires a sex pilusb. F + bacteria transmits a copy of the plasmid

to F- bacteria. This converts the F- cell into an F + cell. Medical Problem: The R factor (antibiotic resistance) on the F factor is transmitted! http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/f.html

6. Hfr (High Frequency Recombination)a. Hfr- bacterial plasmid integrates into the

chromosome. b. Medical Problem: Hfr antibiotic resistance

genes are passed during binary fission (every time the cell divides). Therefore, antibiotic resistance spreads very rapidly!

c. When Hfr mate with F – bacteria, only the bacterial genes cross NOT plasmid genes. Genetic diversity results in this case due

to recombination. http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/hfr.html

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D. TRANSPOSITION p 285

1. Transposons (jumping genes) are big chunks of DNA that randomly excise and relocate on the chromosome.

2. Transposons were discovered in 1950 by Barbara McLintock in corn.

3. Causes antibiotic resistance in Staph. aureus, the famous methicillin

resistant Staphlococcus aureus (MRSA) strain!

E. TRANSDUCTION the transfer of genetic material from

donor bacteria to recipient bacteria via a transducing agent (virus!). Bacterial viruses are called bacteriophage.

1. Discovered in 1952 by Zinder & Lederberg.

2. Two kinds of transduction: generalized and specialized.

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2. Generalized transduction: Starts with the LYTIC CYCLE where a T- even phage (Fig. 8.5 pg 210) infects E.coli killing the host cell, and synthesizing 2,000 copies of itself. The T-even phage randomly packages bacterial DNA in a few defective phages. Once a T –even phage infects another E. coli, this genetic information can be recombined into the host cell without causing the lytic cycle. New genetic information is thereby transduced from one bacteria to another.

• Generalized Transduction

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Generalized Transduction

Specialized Transduction3. Specialized transduction

Lambda phage infects E.coli. The phage does not lyse the cell immediately. Instead it integrates into chromosome of the bacteria as a prophage and remains dormant. This is called the LYSOGENIC CYCLE. Phage genes are replicated and passed to all daughter cells until the bacteria is under environmental stress, from lack of nutrients, etc. Then phage gene will excise from the nucleoid and enter the LYTIC CYLE taking one adjacent gene for galactose metabolism.

Specialized Transduction Cont.

The gal transducing phage (lambda) makes ~ 2,000 copies of itself with the gal gene, and infects other E.coli. When gal integrates into the nucleoid of other E. coli, it may provide these bacteria with a new capacity to metabolize galactose.

Specialized Transduction

Graphic

Comparison of Bacteriophage

3. Comparison of bacteriophage transduction in E.coli.

Generalized Specialized

T even phage lambda phage

lytic cycle lysogenic

random packaging specific gal gene

End of Slides