MMG 301 Exam 2

Describe the central dogma of molecular biology

Describe the central dogma of molecular biology

The central dogma of molecular biology is that information is transferred from DNA to RNA to protein.

List the types of genetic elements found in bacteria

• Chromosomes: Circular-threadlike structures made of DNA molecules that contains genes, most of the info carried to survive in the BP

• Plasmids: A small ring of DNA that carries accessory genes separate from those of the bacterial chromosomes. Free replicating exogenous pieces of DNA (much smaller than chrom.)

Describe the basic structure of an operon

-Single transcription unit. (multiples genes being transcribed together) -Promoter (starts) -Coding genes: ORF (g1-g2-etc) -Terminator (stops)

Describe how a change in a nucleotide propagates through the various steps of information transfer, especially for frame shifts and changes in key features

Frameshift- causes changes in all amino acid codons downstream of mutations sites. inserts or deletes a

Arrange in order the following DNA features and how the correspond to mRNA and protein.

1. The promoter (-35 and -10)1. promoter

2. any operators

3. +1 of transcription,

4. Shine Dalgarno sequence (RBS)

5. Start codon

6. coding sequence,

7. stop codon,

8. transcriptional terminator

Diagram a gene, including the features need for transcription

List the components of RNA polymerase and the function of both the core enzyme and the holoenzyme

RNAp: Sigma factor + core enzyme core enzyme: can convert DNA to RNA holoenzyme: directs to specific sequences (sigma factor in the holoenzyme directs to specific sequences to make sure transcription starts at the right spot)

Recall the steps of transcription initiation

RNA polymerase attaches to the promoter region of DNA strand, and synthesis begins. Usually the -35 & -10 region.

Illustrate DNA structures that cause transcription termination: Intrinsic termination

when transcription is ended due to stem loop formed by inverted repeat in the RNA followed by a stretch of Uridines

Illustrate DNA structures that cause transcription termination: Rho-dependent termination

Rho protein recognized specific DNA sequences and causes a pause in the RNA polymerase & is a race between polymerase and a rho protein that is bonded to the mRNA

Summarize how advancements in sequencing have led to changes in microbiology

Advancement in sequencing has gone down in price due to the advancement of technology. As new tech is developed, the easier it is to have access to tech, allowing for more people to use it, such as smaller microbe labs. moores law.

Describe the process of determining a bacterial genome or set of genomes, transcriptome and proteome

1. Isolate nucleic acid from a sample 2.Preform sequencing by synthesis and obtain small sequencing reads

2. Assembling small sequences into larger Contigs (align to existing genomes if possible)

3. Use PCR and site specific sequencing to fill in an gaps

4. Analysis (annotation, phylogenetic)

Summarize the process of gene annotation

The identification of protein coding genes within DNA sequences in a database (BLAST) Analysis of genomic sequences to identify protein-coding genes and determine the function of their products.

Indicate how genomics technologies can assist in understanding bacterial evolution

Genomic tech. lets you see the duplicates, and see the evolution happening after the ancestral gene duplicates a couple of times.

Compare the information determined by genomics, metagenomics, transcriptomics and proteomics

-Genomics: comparison of genome sequences -Metagenomics: analyzing microbial communities by genome and/or RNA sequencing -Transcriptomics: study of transcriptomes and their functions -Proteomics: identification and study of proteins of the cell

Describe the function of sigma factors

Initiates transcription of genes. It dissociates from the gene when RNA poly binds to specific part of DNA & unwinds it, reading a single strand. Then the core enzyme will convert the DNA into RNA and the single strand gets longer until it is mRNA

Draw DNA & RNA transcript and illustrate important sequences for recognition of RNAP and ribosomes

Describe how genes may be regulated at various points within gene expression, including non transcriptionally

Molecular process of repression

whatever is being repressed will bind to a repressor protein

that complex will then bind to the operator and block transcription

Molecular process of induction

inducer binds to a repressor protein

the repressor protein is released from the operator site and transcription proceeds

Molecular process of activation

activator protein binds the inducer and genes begin to be transcribed

Determine the state of each regulator of the lac operon given environmental conditions

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Indicate functions and steps of two component systems

1. environmental signal binds to a receptor (sensor kinase)

2. phosphorylation of response regulator

Describe basic mechanism of quorum sensing and why a cell would use QS

quorum sensing is a count of how many cells are similar in the surrounding environment through measuring microbe-excreted chemicals

(a way for cells to sample microbe-excreted chemicals in the environment to measure population density)

Explain non-transcriptional mechanisms of gene regulation

small RNA based regulation: base pair with a region of mRNA

binding to the ribosome binding site or 3' end can either prevent or stimulate translation

riboswitches: mRNA's that can bind small regulatory molecules in a region upstream of the ribosome binding site

Mutant definition

an organism with a heritable change in genome sequence.

Relate changes in genotype to changes in phenotype

Genotype is a nucleotide sequence of a characteristic whereas Phenotype is the physical/observable characteristic of an organism.

Due to mutation in the genotype, the result of phenotype also changes.

List and show examples of single nucleotide mutations

-Point mutations

• base pair substitutions:

Missense: changes codon so RNA calls for different tRNA

nonsense: Changes to the last codon in a set of 3 (TAG->UAG)

Silent mutation: may change a codon, but usually still ends up being a functioning protein

Explain how and when single nucleotide changes can alter phenotype and when they cannot alter phenotype

silent mutation= no effect on phenotype

missense= resulting in a codon that codes for a different amino acid (faulty protein)

nonsense= results in a premature stop codon (no final product)

Describe a frameshift mutation and its effect on proteins/ phenotype

caused by insertions or deletions

causes changes in all amino acid codons downstream of the mutation site

Identify a second site suppressor mutations and their mechanism of action

Mutations that occur in tRNA recognition can read the mutant mRNA sequence, but insert the correct amino acid.

UAG on the mRNA results from mutation on DNA

UAG is a nonsense (stop) codon and would normally produce a truncated (shortened) protein - But...

A suppressor tRNA inserts a correct amino acid at the UAG codon and normal protein is made

Order the steps of homologous recombination, including the proteins involved in recombination

1. Nick= cuts a single strand of DNA

2. SSB= a single strand binding protein binds to the nick site

3. Strand invasion= Rec A protein, helps mediate strand protein

4. Cross-strand exchange= (holliday junction)

5. resolution= heteroduplex DNA

Distinguish between specialized and generalized transduction, including host DNA that is moved

specialized transduction: only genome DNA that is adjacent to the prophage can be transferred to a recipient cell

generalized transduction: bacterial DNA moved is random

Generalized transduction stages

Lytic cycle: (produces transducing particles): phage comes into donor cell / host chromosome is destroyed / viral or bacterial DNA is packaged into the phage / transducing particles contain donor cell DNA

Transduction: transducing particle enters recipient cell / recipient cell is infected / homologous recombination occurs

Diagram transfer of the F+ plasmid as either an episomeor integrated into the chromosome

1. transfer of a copy of the plasmid DNA only

2. integration into the cell chromosome followed by chromosome transfer. Separate F plasmid: donor cell-F+ recipient cell-F- -> F+ DNA that is transferred is: F Plasmid Integrated F Plasmid: Donor cell- Hfr Recipient Cell- F- + Chromosomal genes DNA that is transferred is: F DNA + Segment of chromosome

Order the major evolutionary events since the cooling of the earth's crust

cooling of the crust

conversion of prebiotic reactions into cellular life

cellular common ancestor diverges into bacteria and archaea branches of life

rise of photosynthetic bacteria in the anoxic earth

oxidation event

multicellular life

Explain how oxygen levels rose in the atmosphere; what is one type of geological evidence for this "great oxidation event"

organisms evolved the ability to use water as a source of electrons- resulting oxygen accumulated in the atmosphere

evidence= iron branding in rock formations

Microbial mats

very thick biofilms

Stromatolites

Layered rock that results from the activities of prokaryotes that bind thin films of sediment together

Describe why molecular sequences are best for determining phylogenetic relationships; what sequence is most commonly used

molecular sequencing shows how organisms are related to each other via genes (mutations, insertions or deletions)

16s rRNA sequence is used

Order the steps in isolating 16S rRNA genes by PCR

1. DNA is isolated from pure cultures of microbes/ entire microbial communities

2. 16s rRNA genes are amplified by PCR using primers

3. PCR reaction products are checked by gel electrophoresis to check for size correctness

Define fitness

ability to survive and reproduce

Core genome

genes shared by all strains of the species

Pan genome

the totality of the genes present in the different strains of a species

Distinguish between vertical and horizontal gene transfer

Vertical gene transfer moves information from mother to daughter cells, in horizontal gene transfer a completely separate cell donates parts of its DNA to a recipient cell

What is genetic drift and how has it been studied in E. Coli populations

-some members of a population reproduce slightly faster than the others -where a fraction of members of a population changes as a result of random sampling. thus sub-populations can change and evolve in different ways.

Modern rapid genome sequencing methods have allowed detailed analysis of genomes that was never before available.

Explain how comparative genomics reveal genes important for causing disease

by comparing pathogenic and nonpathogenic strains of bacteria

In a diagram of the individual components of viruses; including nucleic acid, capsomeres and capsids and envelope, assemble these into a complete virus; where does the envelope originate from

the envelope originates from a membrane and a phospholipid bilayer

Order the steps in the life cycle of a virus that causes lysis of its host cell

1. viron attaches to cell

2. viral DNA enters the cell

3. synthesis of viral nucleic acid and protein inside the cell

4. assembly and packaging of DNA into new viruses

5. cell lysis and release of new virions

What is a virus "titer" and how is it determined; how is a plaque formed on an agar plate

titer= concentration of virus

a plaque is formed from the lysis of single infected cells

Describe how are T4 viral components made precisely at the time they are needed, what is "packaging"?

T4 bacteriophages are lytic; viral components made by different genes at different times so they are available. Packaging is the process of which DNA is guided into the phage head

Distinguish the lytic and lysogenic paths that an E. coli cell can take after it is infected with bacteriophage lambda

lytic= follows vertical gene transfer with a virus

lysogenic= follows horizontal gene transfer

How do viruses efficiently use of their limited genome size

efficient use of genetic material from overlapping genes

(1 piece of DNA encodes for multiple proteins)

Genetic drift in human health example

Infectious Diseases- different generations have varying arrays of immunity to infectious diseases due to genetic drift.

Know the possible outcomes of infection by certain animal viruses and what "transformation" is

latent infection

persistent infection (slow release of viruses)

transformation: converting normal hose cells into tumor cells

What are oncogenes

a gene which in certain circumstances can transform a cell into a tumor cell

Order the steps of the retrovirus life cycle, what type of genome, dsDNA intermediate, why are protease, reverse transcriptase, and fusion inhibitors helpful in treatment of HIV

1. entry and uncoating of the retrovirus

2. reverse transcriptase

3. viral DNA enter the nucleus and goes into host chromosome

4. transcription of proviral DNA produces viral mRNA

5. translation of viral mRNA forms viral proteins, then released from the host cell

Replication enzyme is a distinguishing feature of retroviruses; what are the three major gene regions in retroviruses

gag: core + capsid structural proteins

pol: reverse transcriptase (RNA to DNA), integrase (to integrate into host genome)

env: envelope proteins

Explain how halophilic Archaea maintain water balance while living in high salt conditions

create compatible solutes which equalize water activity between the inside and outside of the cell

How do some Archaea generate ATP energy from light

some archaea use bacteriorhodopsin which allow archaea to get energy directly from light by creating a proton motive force

What are bacteriorhodopsin and retinal

bacteriorhodopsin is a protein

protons in bacteriorhodopsin are moved outside by retinal

What are methanogens and understand what the methanogenesis pathway does

methanogens are anaerobes that reduce carbon to methane

What are the different ether lipids of Archaea; what are the major types of lipids

membranes are made of ether linked lipids

phytanyl or biphytanyl chains are linekd to glycerol phosphate by an ether bond

How some members of the Archaea important for the global nitrogen cycle; what adaptation is important for its survival

Thaumarchaeota: oxidize ammonia to nitrite

its adaptation is an ability to survive survive with very low nutrient levels

How does Nanoarchaeum live; what does it derive from its host

it only exists on the surface of host cells

it is totally dependent on its host cell for nutrients

What habitats is Sulfolobus found in; be able to state in general terms how it gets its energy

found in very hot an acidic environments

obtains energy from respiration (oxidation of sulfur)

What the challenges to organisms living at high temperatures

protein destabilization DNA can denature ATP stability is reduced

Describe the adaptations of proteins and DNA so they can function in high temperature habitat

proteins can have a second structure stabilized by hydrophobic cores

protein folding

ionic bonds between peptide chains

Be able to diagram the step of retroviral (and other enveloped viruses) release from host cell by budding

Explain the endosymbiosis theory and what evidence supports it

mitochondria, hydrogenosomes and chloroplasts contain their own circular genomes that are replicated from pre-existing organelles

(eukaryotic chromosomes contain genes from bacteria)

Distinguish between primary endosymbiosis and secondary endosymbiosis

Primary= when a eukaryotic cell engulfs and absorbs a prokaryotic cell

secondary= eukaryotic cell engulfs and absorbs the product of primary endosymbiosis, which is another eukaryote

Describe the three major energy production organelles of Eukaryotes; how does each create energy

mitochondria= aerobic respiration

hydrogenosome= oxidation of pyruvate to hydrogen, CO2 and acetate

chloroplast= photosynthesis

Know the key features of fungi

cell walls of chitin

achlorophyllous chemoorganotrophs (no chlorophyll)

usually filamentous (molds/ branches filaments)

Fungi roles in nature

help with decomposition and mineralization of organic carbon

Be able to describe the three types of mycoses

superficial= Fungal infection of only surface layers (skin/ hair)

Subcutaneous/ Invasive Mycoses= Fungal infection under the skin (underlying tissues)

Systematic Mycoses= Fungal infection that affects internal organs.

What major product is made by the fungus Penicillium notatum

penicillin

Briefly review key features of a eukaryotic cell from slide 1 but do not memorize all the details

fungi role in nature

-major contributors to the decomposition and mineralization of organic carbon. -dominate microbial biomass in soil

Fungi major impacts on humans and ecology

• many are pathogens of crop plants and animals

• major cause of food spoilage -can be a food source

• some types are beneficial symbionts of plant roots -biotechnology-useful metabolites (creating medicine from them--ex penicillin)

Populations

A group of related organisms. Illustrated when an individual cell grows and divides into a population of cells.

Guilds

Populations that do related metabolism (populations that converting food into energy similarly.)

Energy inputs in a lake ecosystem

sunlight

organic carbon

Allochthonous carbon (enters the ecosystem from outside)

reduced inorganic compounds

Communities

groups of organisms that do different things.

Species richness

number of different species present

Methods used to determine microbial community composition; what staining methods are used for microscopic analysis

(usually done using culture independent methods)

microscopic analysis= staining methods (stain DNA and show live vs. dead cells)

genetic analysis= uses PCR or sequencing

Live and dead stain (Viability)

live/dead stains

uses mixtures of fluorescent dyes

(additional counterstain is usually added to stain dead cells red)

What is Fluorescence In Situ Hybridization (FISH) used for

used to detect specific organisms in a population using a fluorescent molecule that attaches to specific sequences

Order the steps for the basic method of Fluorescence In Situ Hybridization (FISH)

1. synthesize oligonucleotide specific for the organism you're looking for

2. chemically attach a fluorescent dye molecule to the oligonucleotide

3. treat sample containing cells to make membranes permeable to the oligonucleotide and dye

4. examine sample using fluorescence microscopy. Where the oligonucleotide has correctly based paired are fluorescent cells

Review what culture-independent methods are and the examples shown in the module; what are the two types of culture-independent analysis that can be done without using DNA sequencing

viability stains (live/dead) and FISH

community sampling

???

What are phylochips and what are they used for

DNA microarrays can be designed using oligonucleotides with sequences useful for phylogenetic analysis

doesn't require DNA sequencing

What is hybridization of DNA

Base pairing ????

Species abundance

proportion of each species within a community (not a lot of stuff in a space vs. a lot of stuff in a space)

Habitats

Large environment that contains different guilds.

Ecosystem

microbial communities that interact with communities of microorganisms and the physiochemical environment.

Staining methods

The use of microscopy and fluorescent dyes that stain DNA used on environmental cells. The stains are used in fluorescent microscopy of environmental samples to visualize both dead (red) and live cells.

What happens in each stage of biofilm development

1. Few cells attach to surface

2. colonization: growth and extracellular polysaccharide is produced

3. development

4. dispersal

How biofilms interact with humans

form on contact lenses

form inside water pipes

can form on prosthetic devices

Explain how Pseudomonas aeruginosa is associated with cystic fibrosis, and how Dornase can help

uses quorum sensing to establish lung infections in patients with cystic fibrosis.

At sufficient cell density, quorum sensing triggers biofilm formation in airways and lungs.

Biofilms contain cells anchored by protein, polysaccharides, and DNA (treatment=enzyme that cuts DNA)

dornase selectively cleaves DNA and reduces viscosity of mucus in the lungs

Microscope Analysis

A type of culture-independent method of determining microbial composition that USES STAINING + NUCLEIC ACID PROBES.