Genetics Midterm 1

Bacteria and Archaea Characteristics

• Mostly unicellular (sometimes multicellular) ***

• Mostly microscopic ***

• Simple cellular organization

• No mitochondria or nucleus

• Do not form complex multicellular organisms

• Small genome with less genes

***Similar with Eukarya

Eukarya Characteristics

• Mostly unicellular (sometimes multicellular) ***

• Mostly microscopic ***

• Complex cellular organization with many organelles

• Have mitochondria & a nucleus

• Can assemble into complex multicellular organisms

• Large genomes with more genes

***Similar to Bacteria and Archaea

Rank the complexity

Archaea, Bacteria, Eukarya, Viruses

Viruses < Bacteria/Archaea < Eukarya

Most life on earth is what?

Most life on Earth is microbial

99% of life on Earth is unknown

Molecular genetics is?

Molecular genetics is a field of study and series of techniques that are trying to understand how differences in the structures or expression of DNA molecules manifest as variation among organisms

Gene define

Gene: Sequence of nucleotides in DNA that is transcribed to produce a functional RNA (protein-coding or non-protein-coding)

Genome define

Genome: All the genetic information of an organisms

What are the two general themes to genes at the molecular levels?

• The genetic functions of DNA flow directly from its molecular structure

• All of the genetic functions of DNA depend on specialized proteins that read the information encoded by DNA sequence

F. Miescher experiment. What was it? What was found?

Extracted DNA for the first time and characterized it.

• Extracted weakly acidic phosphorus rich “nuclein” from the nuclei of human white blood cells.

  • Revealed that its major component was deoxyribonucleic acid

What did Feulgen find?

Stained cells that revealed that DNA is localized almost exclusively within chromosomes

What type of bond joins nucleotides?

Phosphodiester bonds which are covalent bonds

Nucleotides are composed of what?

Sugar/Deoxyribose, a phosphate group, a nitrogenous base

Phenotype define

The observable characteristics of an organism

F. Griffith experiment. What was it? What was found?

• Two forms of bacteria: smooth/virulent (wild type) strain and rough/nonvirulent (mutant type) strain

• Injecting rats with S kills . Injecting rats with R does not kill. Injecting rats with heat-killed S does not kill, control.

• Injecting heat-killed S combined with R kills. Tissue analyzed showed living S recovered

• Shows bacterial transformation exists

Discuss smooth and rough bacteria and how genetic bacterial transformation was a part of Griffith’s experiment

• S has gene which creating coating layer that protects it from immune system

• R has no coding for the gene. This is the genetic info acquired during transformation

Avery, MacLeod, and McCarty. What was it? What was found?

• Identified the transforming principle

• Purified transforming principle combined with different enzymes

  • Showed that Protein, RNA, and Fats were not the reason because when they were removed from the mixture and introduced to R cells, they still became S cells

  • When DNA was removed from the purified transforming principle, the R cells stayed R cells. No transformation occured.

Hershey-Chase Waring blender experiment. What was it? What was found?

• T2 phages grew in a medium of 32P-labeled DNA. Introduce phages to bacteria culture, which inserts 32P DNA into bacteria. Blended briefly to remove protein ghost shells. Phosphorus detected in cells and not supernatant.

• T2 phages grew in a medium of 35S-labeled protein. Introduce phages to bacteria culture, which inserts normal DNA into bacteria. Blended briefly to remove 35S-labeled protein ghost shells. Sulfur detected in supernant and not cells.

  • Concludes that DNA is entering cells not protein

Bacteriophage are what

Viruses that inject bacteria

Life cycle of bacteriophage T2

• Phage attaches to bacterium host

• Phage injects genome into host cell

• Phage DNA replicates, and new phage proteins are made

• Phage particles assemble

• Cell bursts releasing new phages

List experiments in order

• Avery et al.

• Feulgen

• Griffith

• Hershey-Chase

• Miescher

• Miescher

• Feulgen

• Griffith

• Avery et. al

• Hershey-Chase

Describe the important prime carbons of (deoxy)ribose sugar

1’ is where the connection to the base is

2’ is where you can determine if its oxy or deoxy

• If H = deoxyribose

• If OH = ribose

3’ is where the connection to the next phosphate/nucleotide is

5’ is where the connection to the previous nucleotide is

What are the purines? How many rings?

Adenine and Guanine

Two rings

What are the pyrimidines? How many rings?

Thymine and Cytosine.

One ring.

What is it called when a base is attached to a sugar with no phosphate group?

Nucleoside

DNA polarity

5’ to 3’

5’ has exposed phosphate

3’ has exposed sugar

Discuss Franklin’s X-ray findings

She purified and crystallized DNA so it all aligned on an X-ray picture.

Showed that DNA was helical, but could not tell it was double helical.

Discuss Chargaff’s findings

In all organisms, ratios of A to T and G to C are roughly 1:1

How many hydrogen bonds are shared between G and C?

Three hydrogen bonds

How many hydrogen bonds are shared between A and T?

Two hydrogen bonds

What 3 DNA measurements did Franklin and Wilkins find?

DNA has:

• 20 Å diameter

• 3.4 Å spacing between repeating units

• a helix that undergoes a complete turn every 34 Å

What did Watson and Crick find?

• Strands are antiparallel

• Sugar — phosphate backbone on the outside

• Base pairs in the middle

• Two chains held together by H bonds

• Semiconservative replication

In a double helical strand of DNA, the ratio of AG/TC of one strand is 0.25.

What must be the AG/TC ratio of the whole molecule?

1

In a double helical strand of DNA, the ratio of AG/TC of one strand is 0.25.

What must be the AG/TC ratio of the complementary strand?

4

Discuss B DNA vs Z DNA

B-form DNA, or right-handed DNA is the typical form with a right handed helix and a smooth backbone

Z-form DNA, or left-handed DNA is the less stable version of DNA with a left-handed helix and an irregular backbone, causing the major groove to be larger

What does Z-DNA have a higher concentration of?

GC pairs

How does a DNA carry information

Base sequence

What are the three major chemical differences between RNA and DNA

• Ribose instead of deoxyribose

• Nitrogenous base uracil instead of thymine

• Most RNA is single stranded

True or False?

RNA can form base pairs

True.

RNA can form base pairs within other parts of the same molecuel

What are the three possible models for DNA replication?

Semi-conservation: Watson and Crick

Conservative: Parental stays intact. Both daughter helices are new.

Dispersive: Both strands of both daughter helices contain original and new DNA

Meselson and Stahl experiment. What did they do? What did they find?

Grew E. coli in medium containing ¹⁵N then switched to medium containing ¹⁴N

• When grown in ¹⁵N, there is a low band.

• When switched to ¹⁴N, there is a single band in middle. This proves it is not conservative.

• When grown in the ¹⁴N again, there were two bands. This proves it is not dispersive.

What are the three strict requirements for DNA polymerase action?

Four dNTPs

• For incorporation into chain

• For energy

Single-stranded template

Primer with exposed 3’ hydroxyl

List the parts of DNA replication in order.

Specify the two processes that each part belongs to

1. dsDNA

2. Helicase

3. ssDNA

4. SSBPs

5. Primase

6. Primer

7. Pol III

8. Pol I

9. Ligase

10. Topoisomerase

1-6 is Initiation

7-10 is elongation

Label 1-10

1. dsDNA

2. Helicase

3. ssDNA

4. SSBPs

5. Primase

6. Primer

7. Pol III

8. Pol I

9. Ligase

10. Topoisomerase

List the steps of DNA replication: Initiation

1. Initiator protein binds to the origin of replication

2. DNA Helicase unwinds helix

3. Replication bubble forms with replication fork at each end

4. Single-strand binding proteins keep the helix open

5. Primase synthesizes RNA primers

What is special about the origin of replication

It is a short sequence of specific nucleotides that is AT rich

List the steps of DNA replication: Elongation

1. The correct nucleotide sequence is copied from the template strand to a newly synthesized DNA strand using DNA polymerase II, which catalyzes phosphodiester bonds between nucleotides

2. DNA polymerase I replaces RNA primer with DNA sequence

3. DNA ligase covalently joins successive Okazaki fragments together

Leading stand define

Continuous synthesis in direction of replication fork (5’ to 3’)

Lagging strand define

Discontinuous synthesis of a 3’ to 5’ strand in small 5’ to 3’ segments (Okazaki fragments)

What are the three ways cells ensure accuracy of genetic information?

1. Redundancy

2. Precision and proofreading of cellular replication enzymes

3. DNA repair enzymes

Discuss how DNA replication occurs in circular bacterial chromsomes

Replication proceeds in two directions from a single origin replication

Unwinding DNA creates supercoiled DNA ahead of replication fork, which DNA topoisomerase relaxes by cutting the sugar phosphate backbone between nucleotides

Synthesis continues bidirectionally until replication forks meet and it is cut into two daughter chromosomes

Mutation define

Heritable changes in the base pair sequence of DNA

Forward mutations define

Changes in wild-type allele to a different mutant allele

Common —> Rare

A⁺ —> a or b⁺ —> B

Reverse mutations define

Changes a mutant allele back to wild-type

Rare —> Common

a —> A⁺ or B —> b⁺

Substitution define

Replacement of a base by another base

What are the two types of substitution? Define both

Transversion: Purine replaced by pyrimidine, or a pyrimidine replace by a purine

Transition: Purine replaced by Purine, or a pyrimidine replace by a pyrimidine

Insertion define

Block of one or more base pair added to DNA

Deletion define

Block of one or more base pairs lost from DNA

Point mutations define

Point mutations affect one or a few base pairs, altering one gene at a time

What are the four types of point mutations

Transitions (Substitution)

Transversion (Substitution)

Deletions

Inserations

True or False?

Spontaneous mutations occur at a high rate

False.

Spontaneous mutations occur at a very low rate

True or False?

Only a small fraction of mutations affect gene function.

True.

Only a small fraction of mutations affect gene function.

What are the ranges of mutation rates per gene?

10^-9 < x < 10^-3

What are the two things that can effect mutation rates

• Differences in gene size

• Susceptibility of particular genees to various mutagenic mechanisms

Why does human sperm have higher rate of mutation than human eggs?

Human sperm goes through many more replications. Male germ cells undergo mitosis continually.

What is more rare? Forward mutations or revertants? Why?

Revertants because the mutation needs to be exactly the same spot. Many mutations distrupt gene function, while only a few restore function

Luria and Delbruck experiment. What did they do? What did they find?

A fluctuation test to show that mutations in bacteria occur spontaneously through examining the origin of bacterial resistance to phage infection.

Had bacteria grow and then eventually introduced to a selective agent/bactericide

Hypothesis 1: Resistance is a physiological response to a bactericide. If this is true then there would be a small consistent amount of colonies

Hypothesis 2: Resistance arises from random mutations. If this is true, then there would be more resistant colonies, but inconsistent numbers

Results show more resistant colonies with inconsistent numbers. Therefore, resistance arises from random mutations

What does replica plating verify

Replica plating verifies that bacterial resistance is the result of preexisting mutations

How does replica plating verify that bacterial resistance is the result of preexisting mutations

1. Grow master plate with no penicillin in medium, then press onto velvet.

2. Press a plate with penicillin onto the velvet cloth

3. Only penicillin-resistant colonies grow. On replications they grow in the same position

Mutations occur as the result of random processes and occurred before exposure to penicillin

What are the five natural processes that can cause DNA damage?

• Depurination/depyrimidation

• Deamination

• Oxidation

• Cosmic/X Rays

• UV Rays

Do natural processes cause DNA damage or DNA base sequence error?

DNA damage

What are the two types of DNA replication processes that can cause DNA base sequence error?

Trinucleotide repeats

Tautomers

Do DNA replication errors cause DNA damage or DNA base sequence error?

DNA base sequence error

What are the types of mutagens that can cause DNA damage/base sequence error?

Base replacement, Alkylation, Hydroxylation, Insertion, Deamination

What is depurination/depyrimidation

• Hydrolysis of a purine/pyrimidine base

• A random base will be added by polymerase

What is Deamination

• The removal of an amino group from C, turning it into U

How do Cosmic rays and X-rays affect DNA

Breaks the sugar-phosphate backbone of DNA which can lead to deletion

How does UV light affect DNA

Causes adjacent thymines to form abnormal covalent bonds (thymine dimers), which can lead to substitution, and if there is enough UV damage to the point where the cell cannot replicate, it initiates cell death

how does oxidative damage affect DNA?

Oxidizes G into GO, which pairs with A instead of C (damage), leading to mutant T-A after replication

How does DNA polymerase help proofread

There is a proofreading part of the molecule, called 3’-to-5’ exonuclease, which recognizes and excises mismatches

How does Base tautomerization result on DNA base sequence error?

Each base has two tautomers. The tautomers match with the incorrect base:

C# matches with A

G# matches with T

T# matches with G

A# matches with C

How do Trinucleotide repeats result in DNA base sequence error

• DNA polymerase slips which causes a loop to form. This causes an expansion and contraction of triplet repeats on the next round of replication.

• 20 human disease genes have unstable trinucleotide repeats

  • Fragile X syndrome

    • Males more affected because only one X chromosome. Also number of repeats increases each generation

  • Huntington Disease

H. J. Muller. What experiment was done? What was found?

Used x-ray dose to increase mutation rate above naturally occurring level in male flies giving them a bar eye

• Crosses X-ray exposed males with bar-eyed females to prepare. All females have this genotype. One X with X-ray, one X with bar-eyed

• Mates the female with a wild-type male

• Wild-type is normal eye; Mutant is normal eye

Hypotheses:

• If F1 female has an X-ray-induced recessive lethal on X, then all non-bar-eyed males will die since they only have one X chromosome

• If F1 female has no X-ray-induced recessive lethal on X, then there will be equal amounts of bar-eyed and non-bar-eyed males

Mutagen Base replacement: What is it? What happens?

Base analog, 5-bromouracil. One tautomer pairs with A, the other pairs with G

Base analogs define

Chemical structure almost identical to normal base

Mutagen Hydroxylation: What is it? What happens?

Hydroxylating agent adds an -OH group to a base

Hydroxylated C now pairs with A instead of G

Mutagen Alkylation. What is it? What happens?

Alkylating agents add ethyl or methyl groups to bases

Modified G pairs with T instead of C

Mutagen Deamination. What is it? What happens?

Deaminating agents, like nitrous acid, remove amine (-NH₂) groups

Changes cytosine to uracil, which pairs with A instead of G

Modifies adenine to hypoxanthine which pairs with C instead of T

How are Deamination different when it is a natural process vs. a mutagen?

Both result in the removal of an amino group, but mutagen deamination required nitrous acid, while natural process just occurs naturally

Mutagen Insertion. What is it? What happens

An intercalator is placed between bases

This can lead to insertion or deletion during replication

What is an intercalator?

A molecule that is similar to base pairs and is able to slip between base pairs in DNA

What is a somatic mutation?

It can occur anywhere in body outside of germ cells. Occurs in non-germ cells

• Not heritable

• Can affect survival of an individual

• Can lead to cancer

Ames Test. What is it? What is it for?

• Test to see if a chemical would cause mutations and therefore might cause cancer

• Has mutant bacteria with three mutations:

  • His^- (cannot produce histamine), DNA repair mechanisms have stopped, and cell wall is more permeable

• Exposed with rat liver enzymes and the potential mutagen/carcinogen

• Plated onto medium without histadine

  • If there are many bacterial colonies, then there are many His^- → His⁺ revertants, that means it is probably a mutagen

• Control just has His^- mutant and rat liver enzyme

  • There will be few bacterial colonies because there are few His^- → His⁺ revertants

Why do you use rat liver enzyme during the Ames test?

You use rat liver enzyme to simulate the enzymes that human livers have that would be able to fight against toxins