MIC 230 Exam 3

True or false: DNA is unstable and can not be detected in the environment for very long

False: DNA is highly stable and can be isolated from the environment after a long time

What is the central dogma

Replication → transcription → translation

Used by all life

Structure of DNA

Made up of nucleotides

Structure of nucleotides

Sugar, phosphate, nitrogenous bases

What holds DNA strands together?

Complementary base pairing (hydrogen bonds)

DNA strands compared to RNA

RNA strands are single-stranded and contain uracil instead of thymine and has two hydroxyl groups

The phosphate is always on what carbon?

5’

The hydroxyl group is always on what carbon?

3’

How does the genome fit into the bacteria cell?

Supercoiling

How does the genome fit into the eukaryotic cells?

DNA wraps around histones

What are some similarities between archaea and bacteria and eukaryotes?

Circular chromosomes and histones

Replication is

semiconservative and antiparallel

Replication happens in what direction?

Template strand is 3’-5’ and polymerase reads it like that, but the new strand is built in a 5’-3’ direction

DNA polymerase

* replicates new DNA strands
* Adds the 5’ phosphate from a new nucleotide to an existing 3’ OH
* Requires a primer (RNA)

What is the issue with the replication fork and what is the solution?

Issue: DNA poly is going away from helicase

Solution: re-prime new unzipped and DNA poly moves to that

Where does replication being in bacteria and archaea chromosomes?

Origin of replication (ORI)

Transcription

Production of RNA from DNA

Does RNA polymerase need a primer?

No

How does RNA poly know where to start?

Recognizes the promoter of a gene

What is the sigma factor (s) and what does it do?

Subunit that recognizes and binds to the promoter, it eventually comes off when transcription starts

Polycistronic

Prokaryote mRNA can have multiple genes transcribed together from a single promoter

Operons

a series of related genes that are co-transcribed on a polycistronic (lac operon)

What are the difference in transcription between bacteria and archaea/eukaryotes?

* Bacteria and archaea both have polycistronic mRNA
* Bacteria and archaea have no introns
* Archaea and eukaryotes have similar RNA poly

Translation

mRNA codes for a protein

What are the necessary parts of translation?

mRNA (template), ribosomes, tRNA, amino acids

Ribosomes

large cellular structure consisting of several proteins

What are the different rRNA ribosomes

5S rRNA = structural

23S rRNA = structural and enzymatic activity

16S rRNA (in small subunit) = binds mRNA, positions ribosome at start position

What are the sizes of the actual bacteria and eukaryote ribosomes?

Bacteria 50S + 30S = 70S

Eukaryotes 60S + 40S = 80S

General characteristis of mutations

* a permanent, HERITABLE change in the genetic material
* random
* effect genotype and can influence phenotype
* are the driving force behind evolution

How do we write genotypes?

use *italics*

*glyA*, *hisC*, *recA*

How do we write phenotype?

capital letters

Gly, His

What are the possible outcomes of a point mutaiton

Missense, nonsense, and silent

What is a missense mutation

Wrong amino acid makes the protein unable to function

What is a nonsense mutation

an early stop codon often is highly detrimental

What is a silent mutation

No change to phenotype

Insertion and deletions alter what?

The reading frame

If the insertion/deletions are not in multiples of 3 what happens?

Frameshift

True or false: Frameshifts are not a big deal

False: they are highly detrimental

What are the two main causes of mutations

spontaneous and induced

What are some causes spontaneous mutations?

* errors during DNA replication
* Alterations of nitrogenous bases

What are some causes of induced mutations?

Chemicals, radiation, sun (UV)

What are chemically induced base analog muations?

Certain things can take the place of nucleotides and cause them to pair with the wrong thing. For example, a thymine can be converted to a 5-bromauracil, which pairs with a G, so what would have been an A-T is now a G-C

What are chemically induced deaminations

The removal of an amino acid group on a nucleotide which changes what it is (purine → pyrimidine)

What are chemically induced intercalators?

Something is put in between the layers of DNA, the effect is that DNA poly stops working when it hits it

What is ionizing radiation induced mutations?

Ionizing radiation leads to free radical formation from ionized water that can break DNA strands and damage other macromolecules

What is UV radiation induced mutations?

UV radiation forms pyrimidine-dimers. Distorts shape of DNA, causing problems during replication. The result is often mismatches or deletions

True or false: Mutation frequency is very high

False: mutation frequency is only about 10^-8 per base pair

What are the 4 main repair mechanisms

Photoreactivation, Excision repair, SOS response, Homologous recombination

Photoreactivation

* repairs dimers


* activated by blue light
* photolyase binds to dimer → cuts bond between the dimers → photolyase leaves
* Humans do not do this

Excision repair

* the general response to damage
* basic or nucleotide excision
* multiple proteins and enzymes involved
* endonuclease
* DNA poly
* ligase
* found in all life

SOS response

* occurs in bacteria
* last resort, only occurs if normal repair systems can’t keep up with all of the damage
* RecA (sends SOS signal) cleaves LexA (can no longer prevent transcription), results in production of enzymes for repair
* Issue is that these repair enzymes are “sloppy”
* Goal is just to get genome replicated fast and repair as much as possible so the cell can divide and hopefully survive

Homologous recombination

* used to repair mistakes perfectly
* also used to repair DSB
* involves two double strands that are the exact (or nearly exact) same sequences
* tries to fix original so that it can divide again without error
* crossing over during meiosis is a version of this
* can be used as a template to fix errors/damage
* RecA is an important protein

Reverse mutation

* NOT a repair mechanism
* is a mutation of a mutation

What are the two ways to find/identify mutants?

Selections, scree, and ames test

Selections

* vast majority of cells die
* only cells with specific phenotypes will grow
* Ex. antibiotic resistance
* Ex. selective media

Screen

* most cells grow, but a different phenotype is apparent
* Ex. differential media, MAC, lactose ferm
* Ex. phototroph vs autotroph
* phototroph: parent strain (WT) that does not have additional nutrient requirement
* Autotroph: mutants that require additional nutrients to grow

Ames test

rat liver thing

Transformation

* transfer of free “naked” DNA from environment
* Some bacteria are naturally competent (rare)
* some bacteria we can make competent in lab

What types of genes are exchanged by transformation?

* any gene
* mostly random process
* to be maintained by the recipient cell, the gene must provide an advantage (or at least no disadvantage)

Transduction

transfer of bacterial DNA from one cell to another by a phage

What are the two types of transduction

Generalized and specialized

Generalized transduction

any region (random) of the donor DNA can be transferred. If the new gene provided an advantage it would be selected over

Specialized transduction

only certain genes near the site of insertion of a lysogenic phage can be transferred. Doesn’t pick up a specific gene BUT it does pick up the gene that are next to the viral DNA (this is the “specialized” part)

What experiment was griffith’s?

The one with rough and smooth streptococcus in mice

What was Avery’s experiment?

The one with Streptococcus in mice as well as DNase, RNase, and proteinase

Why is it important that streptococcus is naturally competent?

Cause this allowed Griffith’s and Avery’s experiments to work

Conjugation

Transfer of DNA requiring cell-to-cell contact

first shown in 1946 by Joshua Lederberg, Ester Lederberg, and Ed Tatum

Plasmid is what’s being transferred

Plasmids

* Self replicating extrachromosomal DNA that is usually not essential for growth
* Usually smaller than chromosomes
* May encode beneficial products for specific growth conditions or environments
* Exchanged semi-frequently when it is advantageous

Why do bacteria have plasmids?

* To “streamline” the genome
* To increase the coding capacity of the species
* Selfish genes

What is “streamlining” the genome?

bacterial cells want to divide rapidly so being able to get rid of genes that are not needed all the time is helpful

What are selfish genes

Plasmids that are viral like, which encode for proteins that promote the passage of the plasmid from one cell to another

Why exchange DNA?

* DNA can be used as a nutrient source
* “Fresh” DNA can be used for repair mechanisms (especially via homologous recombination)
* DNA transfer allows for the exchange of genetic material

What are the effects of DNA transfer allowing for the exchange of genetic material

* New genes can be put into a genome
* Increased variability
* Rapid evolution (not small mutations that are slowly selected on, multiple genes being transfered at once)
* This happens ALL OF THE TIME

Genetic engineering

* Use the transfer of DNA
* Techniques for manipulating the nucleic acid of an organism
* Bacterial genetics is the foundation of modern molecular genetics
* Call this “cloning” because bacteria are clones
* GMOs are based on tech developed from or in bacteria

Plasmids as cloning vectors

* Replicated independent of the genome to multicopy per cell
* Small, easy to isolate DNA, simple to transform into hots (gene → plasmid → host)
* Contains selectable markers

Properties of cloning vectors

* ORI (origin of replication)
* MCS (multiple cloning site)
* Selectable marker
* Stably maintained in host

Why is ORI important when it come to cloning

If you want to put a plasmid in E. coli the plasmid has to have ORI that E. coli will recognize

What is the MCS

spot in the plasmid where we put foreign DNA

Molecular cloning

isolation and incorporation of a piece of DNA into a vector so it can be manipulated and replicated

What are the three main steps of molecular cloning

1. Isolation and cut source DNA with restriction enzymes OR generate fragments by PCR
2. Insertion of DNA fragment into cloning vector (plasmid), the DNA ligase covalently connects DNA fragments together through a phosphodiester bond
3. Introduction of cloned DNA into host organism

Recombinant DNA

DNA that we generate/modify through molecular techniques and use to put into an organism

Restriction enzymes

cuts DNA at a specific base sequence (palindromes), this leaves complementary ends that can be ligated back together in research specific recombinants

Applications

Medicine, drugs, antibiotics, vaccine

Genetically modified organisms

Research of gene function (making stuff glow)

Most crops are already already GMO

CRISPR

Bacterial immune system that specifically cuts phage DNA inside an infected cell

Basics of viruses

Acellular genetic element that cannot replicate independently of a living host cell.

Size of viruses

Significantly smaller than prokaryotes

Range from .02 - 3 um

First discovered because they passed through filters that caught bacteria

Are viruses alive?

Order = yes

Process energy (metabolism) = No, but they can influence host metabolism

Regulation = yes (can control gene expression and how/what proteins are made)

Response to environment = yes (can control gene expression and how/what proteins are made)

Growth and development = No

Reproduction = No, they do have a lot of central dogma machinery but they’re always missing ribosomes

Evolutionary adaptation = yes

Components of all viruses

* Made up of a viral genome + protein
* protein = capsid (surrounds viral nucleic acid)
* nucleocapsid (basically the viral particle) = capsid + nucleic acid
* Shapes = helical or icosahedral

Some viruses

* Some contain an envelope that surrounds the nucleocapsid
* Some contain other proteins/enzymes
* DNA or RNA poly
* Tegument proteins
* Specific examples of enveloped proteins

Tegument proteins

* space between capsid and envelop, called tegument, contains them
* can be virus or host derived
* delivered to host cell at same time as virus
* allows virus to take over cell and get it to do what it wants

Give an example of enveloped proteins

Spike protein on corona virus

Viral genome

all are nucleic acids, most are linear genomes, can be DNA or RNA

Steps of viral replication

Attachment

Entry

Synthesis

Assembly

Release

Growth curve of viruses

1 step because viruses are released almost simultaneously so the latent period is very long (eclipse and maturation) while the burst (assembly and release) is quick and at the end

Plaque assay

Titer = number of units per volume of fluid

Plaque assay = dilute virus sample → mix with cells in cell culture → look for zones of clearing called plaques

Plaque forming unit = one viable viral partical

Have to plate the host cell first then virus

Bacteriophage

Viruses that infect bacteria

Virulent phages

phage replication followed by host lysis

Temperate phages

Can integrate its DNA into the host DNA OR kill the host by lysis by choosing a lysogenic and a lytic pathway upon infection

Lytic cycle

phage genes expressed → excised from genome if previously lysogenic → kills host