The Origin and Evolution of Cells
The Origin and Evolution of Cells 2
Models
Tools of Cell Biology
Tools of Cell Biology 2
Tools of Cell Biology 3
Viruses and Tools
Genetics Review 1
Genetics Review 2
Genetics Review 3
Metabolic Energy and ATP
Electron Transport Chain
Photosynthesis
Mitochondria
More Mito
100
What are Eukaryotic and Prokaryotic cells? What are some 2 differences and similarities between these type of cells?
Shared characteristics include a plasma membrane, cytosol, DNA as genetic material, and ribosomes (organelles involved in protein production)
Prokaryotic Cells: Lack a nucleus, Do not have membrane-bound organelles, have a single, circular chromosome, usually smaller than eukaryotic cells
Eukaryotic Cells: Have nucleus, membrane bound organelles, multiple linear chromosomes, are usually larger than prokaryotic cells
100
What is Transcription and Translation?
Transcription—nucleotide gene sequence is copied from DNA into RNA.
Translation—nucleotide sequence of RNA is used to specify the order of amino acids in a protein
100
What are some important considerations when choosing a model?
- Relevance to system(s) of interest
- Ability to test hypotheses at different levels of complexity
- Ease of use and time required to carry out experiments
-Responsible use of resources
- Cost
100
Define Resolution
Bonus: what is the magnification of modern light microscopes?
ability to distinguish objects separated by small distances— is even more important than magnification. It is determined by the wavelength of light uses and the light-gathering power of the lens(es)
1000x
100
What is Fluorescence resonance energy transfer (FRET)? What is it used to study?
Fluorescence resonance energy transfer (FRET): Two proteins are coupled to different fluorescent dyes.
This is used to study interactions between proteins in a cell.
100
What do northern blots, western blots, and southern blots investigate?
Bonus: How do you know if there is "more" protein in a Western blot.
mRNA, protein, DNA
For Western blots, the darker the “line” in the blot, the more signal there is (in other words, darker=more of that protein in that sample).
100
What are plaques?
In a culture of bacteria on agar, the replication of T4 leads to formation of clear areas of lysed cells (plaques).
100
What is Genotype and Phenotype?
Genotype is the genetic makeup of an individual.
Phenotype is the resulting physical appearance (or other expressed characteristics, such as behavior, blood type, etc.) of the organism
100
What are codons and what does it mean when the genetic code is degenerate?
Bonus: Name a genetic code that does not follow the universal code.
All 64 possible triplets (called codons) were assigned in this way.
The code is degenerate: many amino acids are specified by more than one codon
Mitochondrial genetic code
100
What are plasmid vectors? What do they accomplish?
Fragments of human DNA can be cloned in plasmid vectors: small circular DNA molecules that can replicate independently in bacteria.
Recombinant plasmids with human DNA inserts can be introduced into E. coli, where they replicate along with the bacteria to yield millions of copies of plasmid DNA.
100
What is G when a reaction is spontaneous? Non-spontaneous?
If ΔG < 0, this reaction will proceed in the forward direction.
If ΔG > 0, the reaction will proceed in the reverse direction; B will be converted to A.
100
What is the purpose of Complex 1. Is it a proton pump?
Electrons from NADH enter the electron transport chain at complex I. Yes
100
Give the net reaction of Photosynthesis.
What are the two main components of Photosynthesis.
Bonus Where do they occur?
Light reactions: sunlight energy drives synthesis of ATP and NADPH, coupled to the oxidation of H2O to O2. Thylakoids
Calvin Cycle (dark reactions) : the ATP and NADPH drive synthesis of carbohydrates from CO2. Stroma
100
How many membranes does the mitochondria have?
Bonus: Which membrane has "folds" and what are these folds called?
Bonus: What are the benefits of these folds?
2
The inner membrane has numerous folds (cristae), which extend into the interior (matrix).
Increased surface area means a large amount of inner membrane into which many copies of Complex I, II, III, IV, and ATP synthase can be inserted
100
What are the proteins encoded by the nuclear genes?
•Proteins needed for replication and expression of mitochondrial DNA
•Most proteins needed for oxidative phosphorylation
•All the enzymes involved in mitochondrial metabolism
200
What did Miller's Experiment show?
It showed that organic molecules could form spontaneously in conditions thought to have existed on early Earth.
200
What do all cells use as a source of energy? What is the full name of this molecule.
adenosine 5′-triphosphate (ATP)
200
Give one example on how highly differentiated cells are important models for studying particular aspects of cell biology
•Muscle cells are a model for studying cell movement at the molecular level.
•Giant neurons are used to study ion transport and cytoskeleton function.
•Red blood cells have been useful in studying the cytoskeleton
200
What is bright-field microscopy? What is the disadvantage of this method?
Bright-field microscopy—light passes directly through the cell (or bounces off the surface of the sample)
Cells are often preserved with fixatives and stained with dyes. As a result, it may be difficult to study living cells.
200
What is Super-resolution microscopy?
In super-res microscopy, only a small random fraction of the probes are fluorescent at any one time. Multiple images are obtained over time such that the individual fluorescent molecules can be resolved from one another and a super-resolution composite image is constructed.
200
What is Real-time PCR? What does it measure?
Real-time quantitative PCR can also be used to measure the expression of a gene at the level of transcription.
200
What are the sources of cells used in tissue culture/cell biology research?
Bonus: What is a primary culture?
Bonus: What is the Hayflick Limit?
Historically, embryos or tumors were frequently used as starting material because they contain rapidly growing cells that often divide without limit. Other sources of stem cells, such as induced pluripotent stem cells, are increasingly used in place of embryonic stem cells. Cells can also be taken from specific tissues/organs, sampled from the environment, etc.
An initial cell culture from tissue is a primary culture.
200
Name all of the LOF mutations and GOF mutations.
Bonus: Give the definition
Loss of function (LOF)
Null mutant – complete absence of gene function
Hypomorphic mutant – partial reduction in gene function
Haploinsufficiency mutant – loss of one copy of gene in diploid
Gain of function (GOF)
Hypermorphic mutant – increased level of normal activity of gene
Neomorphic mutant – novel activity of gene
200
What are restriction endonucleases?
Bonus: what are restriction maps?
Restriction endonucleases: enzymes that cleave DNA at specific sequences.
The order of restriction fragments can also be determined, and maps of restriction sites (restriction maps) generated.
200
What is PCR used for?
Amplify specific sequence of DNA
200
What do cells do to power unfavorable reactions?
Couple it with favorable reactions.
200
What is the purpose of Complex 2. Is it a proton pump?
Complex II receives electrons from succinate, which are transferred to FADH2 (rather than to NADH), and then to coenzyme Q. No
200
What are the two complexes of a photosystem? How are they oriented and what do they do?
A photosystem consists of a reaction-center complex (a type of protein complex) surrounded by light-harvesting complexes (photocenters)
The light-harvesting complexes (pigment molecules bound to proteins) transfer the energy of photons to the reaction center
200
What is cardiolipin?
Mitochondria catalyze synthesis of the phospholipid cardiolipin.
Cardiolipin improves efficiency of oxidative phosphorylation by restricting proton flow across the membrane.
The shape of this molecule is also thought to play a role in the formation of cristae.
200
What are presequences?
What is the Tom Complex?
What is the Tim Complex?
Proteins are targeted to the mitochondrial matrix by amino-terminal sequences (presequences) that are removed by proteolytic cleavage after import.
Presequences bind to receptors on the mitochondria that are part of a protein complex (translocase of the outer membrane; Tom complex).
Proteins are then transferred to another complex in the inner membrane (translocase of the inner membrane; Tim complex).
300
What molecules is hypothesized to be the original genetic information in the first cells?
Bonus: What characteristics of RNA would have allowed it to serve as a simple source of genetic information in the absence of more complex molecular machinery found in modern-day cells?
Bonus: What characteristics of DNA may make it a better solution for storing genetic material in modern cells?
RNA
RNA can serve as a template for its own replication and can catalyze reactions. Consequently, RNA is believed to have been the initial genetic system.
Double stranded - back-up copy. And it tends to be more stable (double strands connected by many hydrogen bonds)
300
What are the two types of Present-day prokaryotes?
Bonus: Which group of bacteria did photosynthesis evolve from?
Archaebacteria—many live in extreme environments.
Bacteria—a large group that live in a wide range of environments.
Cyanobacteria
300
Which model organism are extremely useful for elucidating biochemical pathways?
Which model organism allowed studies to identify many mutations responsible for developmental abnormalities?
Which model organism has similar genes and mechanisms that exist in vertebrates?
E. coli
C. elegans
Drosophila
300
What is Phase-contrast microscopy and differential interference-contrast (DIC) microscopy. What is the advantage of this method?
Phase-contrast microscopy and differential interference-contrast (DIC) microscopy convert variations in density or thickness to differences in contrast that can be seen in the final image.
These can be used for living cells.
300
What is electron microscopy?
Uses beams of electrons. Much greater resolution (0.2 nm) than light microscopy is possible because of the short wavelength of electrons.
300
What are the two methods to label cells via their patterns of mRNA or protein expression. What do they detect?
In situ hybridization > detects mRNA
Immunocytochemistry > detects protein
300
What are permanent or immortal cell lines?
Name the first human cell line.
Stem cells and tumor cells can proliferate indefinitely in culture and are referred to as permanent or immortal cell lines.
The first human cell line (HeLa) was established in 1951 from a cervical cancer biopsy taken from Henrietta Lacks. It became the most widely used cell line for cancer research and other studies, including development of the polio vaccine.
300
What are reporter gene constructs? What is transgenesis?
Bonus: What are brainbow mice?
Reporter gene constructs can be used to label cells that exhibit specific patterns of gene expression.
transgenesis – insertion of (usually engineered) DNA into an organism’s genome.
Brainbow mice – use multiple combinations of different fluorescent labels to generate many distinctly labeled neurons. It’s a useful tool for deciphering neural circuitry of complex brains.
300
What is the central dogma?
Bonus: Name three examples that break this dogma.
DNA → RNA → Protein
RNA be copied into RNA
Not all RNAs are translated into protein
RNA can be reverse transcribed into DNA
Look at slides for more examples.
300
What are the components of a plasmid vector?
-Origin of replication – site where DNA replication begins
-Selection marker (e.g., an antibiotic resistance gene)
-Sequences that can be cut by specific restriction enzymes (restriction enzyme cut site, multicloning site, polylinker)
-May also include a promoter region to drive expression of a transgene
300
Give the net reaction of Glycolysis. Where does it occur? Does it require oxygen?
Bonus: In anaerobic conditions, what happens to the NADH and pyruvate produced to give some energy?
Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O
Cytosol
Does not require oxygen
Converted to lactate or ethanol
300
How are electrons transferred to complex 3? How are electrons transferred to complex 4?
•Electrons are transferred to complex III by Coenzyme Q (ubiquinone).
•Cytochrome c then carries electrons to complex IV (cytochrome oxidase), where they are transferred to O2
300
Where are pigments organized in the chloroplast memebrane?
The photocenters
300
What are porins.
Porins form channels that allow the free diffusion of small molecules.
Bonus: What types of small molecules need to enter or leave the mitochondrion?
300
What does Protein translocation require?
What is the purpose of HSP70?
Protein translocation requires the electrochemical potential established across the inner membrane during electron transport.
Proteins must be unfolded and require Hsp70 chaperones to maintain proper conformation of the protein.
400
What do Lysosomes and peroxisomes do?
Specialized metabolic compartments for the digestion of macromolecules and for various oxidative reactions
400
Define nucleus, mitochondria, chloroplasts, vacuoles, endoplasmic reticulum, Golgi apparatus, and cytoskeleton.
We should know this by now, look at slides if we need answer key.
400
Which model organism has the mutation Casper? What does it do?
Zebrafish (Danio). Makes them clear.
400
What is Fluorescence microscopy?
Bonus: What is the main protein used for Fluorescence?
Bonus: What is DAPI?
A fluorescent dye (fluorophore) is attached to a molecule of interest in fixed or living cells.
The fluorescent dye molecules absorb light at one wavelength and emit light at a different wavelength.
Jellyfish Green Fluorescent Protein (GFP)
Dye that binds to the nucelus. Glows blue
400
What does TEM (Transmission electron microscope) and SEM (Scanning electron microscope) visualize?
TEM (Transmission electron microscope)– to visualize structures within cells.
SEM (Scanning electron microscope) – to visualize surfaces of objects.
400
What can microarrays do?
Microarrays can compare expression of multiple genes between two samples. This allows you to determine which genes are expressed in one sample but not another, which genes are expressed at higher or lower levels in one sample than in the other, and which genes are expressed at equivalent levels in both samples.
400
What do viruses consist of? How do they reproduce?
What Bacteriophage infects E. coli?
What did retroviruses first demonstrate?
Viruses consist of DNA or RNA surrounded by a protein coat. They reproduce by infecting host cells and usurping the cellular machinery to produce more virus particles.
Bacteriophage T4
Retroviruses first demonstrated the synthesis of DNA from RNA templates.
400
Which experiment showed that DNA replication is semiconservative?
Meselson and Stahl grew E. coli in medium labeled with the heavy isotope 15N.
The heavier DNA could be separated from light DNA (with 14N) by equilibrium ultracentrifugation in a CsCl solution.
400
Name the purpose of RNA polymerase, mRNA, rRNA, and tRNA.
RNA polymerase catalyzes synthesis of messenger RNA (mRNA) from a DNA template.
Ribosomal RNA (rRNA) is a component of ribosomes, sites of protein synthesis.
Transfer RNAs (tRNAs) serve as adaptor molecules that align amino acids along the mRNA template.
400
What are the 5 types of vectors used for larger fragments of DNA?
1.Cosmid vectors contain bacteriophage λ sequences
2. Bacteriophage P1 vectors allow recombinant molecules to be packaged in vitro into P1 phage particles
3. P1 artificial chromosome (PAC) vectors are introduced directly as plasmids into E. coli.
4. Bacterial artificial chromosome (BAC) vectors are derived from a naturally occurring plasmid of E. coli (the F factor).
5. Yeast artificial chromosome (YAC) vectors have yeast origins of replication and other sequences that allow them to replicate as linear chromosome-like molecules in yeast cells.
400
What has to happen to pyruvate before it enters the TCA? What coenzyme has to be there?
Pyruvate undergoes oxidative decarboxylation in the presence of coenzyme A (CoA-SH), forming acetyl CoA.
400
What is the purpose of Complex 5?
BONUS: What are the two components of this complex and their purpose?
ATP is generated as protons move with the concentration gradient through complex V, which is ATP synthase.
ATP synthase consists of two components:
F0 forms a spinning channel through which protons pass.
F1 spins and harvests the free energy by catalyzing the synthesis of ATP.
400
Which component of the light reactions generates NADPH. Which component generate a proton gradient that drives ATP production?
Photosystem I generates NADPH, which is required for converting CO2 to carbohydrates.
Photosystem II and cytochrome bf complex generate a proton gradient that drives synthesis of ATP.
400
What is mitochondrial fusion and fission? What is the purpose of both?
Mitochondrial fusion allows exchange of genetic material.
Fission is important in distribution of mitochondria between daughter cells at cell division, and in facilitating transport of mitochondria to areas of high energy demand.
400
What are presequences cleaved by?
Presequences are cleaved by matrix processing peptidase (MPP), and the polypeptide is bound by other Hsp70 chaperones that facilitate proper folding so the protein can function in the matrix.
500
Unicellular Eukaryotes. Give simple name and scientific if possible
What is the simplest eukaryote?
Which unicellular eukaryote is a ciliated protozoan?
Which unicellular eukaryote have chloroplasts and carry out photosynthesis?
Which multicellular algae represents an evolutionary transition from single cells to multicellular organisms.
Which amoeba alternates between unicellular and multicellular forms depending on the availability of food?
The simplest eukaryotes are the yeasts, s. cerevisiae
Paramecium
Green alga Chlamydomonas
Volvox
Dictyostelium discoideum
500
Describe endosymbiosis. Give the evidence that supports this theory.
This theory holds that ancestors of modern prokaryotic cells were engulfed by and were maintained inside the ancestors of modern eukaryotic cells, eventually leading to the development of membrane bound organelles.
Mitochondria and chloroplasts are similar to bacteria in size and reproduce by dividing
Both contain their own DNA and translation machinery
Their ribosomes are more similar to bacterial ribosomes than eukaryotic cytoplasmic ribosomes
500
Name 3 model organisms and give 2 advantages for each of them. Give me scientific name and common name.
Bonus: Name all 8 and do the same.
Look at slides for answers.
500
What is Fluorescence recovery after photobleaching (FRAP)? What is it used to study?
A cell region with GFP-labeled protein is bleached by high-intensity light. Used to study rate of protein movement in living cells.
500
What is Differential centrifugation?
Bonus: What is Immunoprecipitation?
Bonus: What is Density-gradient centrifugation?
Differential centrifugation was developed in the 1940s and 1950s to separate cell components on the basis of size and density.
Immunoprecipitation can be used to “pull down” (isolate) specific proteins that bind to an antibody or other protein of interest.
Density-gradient centrifugation—a type of fractionation in which organelles are separated by sedimentation by centrifuging through a gradient of a dense substance, such as sucrose.
500
What is Deep sequencing: RNA-Seq?
What is Chromatin immunoprecipitation-sequencing (ChIP-Seq)?
Deep sequencing: RNA-Seq can be used to compare gene expression between different samples (such as different tissues or developmental timepoints).
Chromatin immunoprecipitation-sequencing (ChIP-Seq) can be used to determine which genes certain regulatory proteins (such as transcription factors) are acting on.
The DNA fragments associated with the precipitated complexes are purified from the proteins and sequenced. These sequences can be compared with the genome maps to discover the precise locations of the genes these proteins may be regulating.
500
What is Culture media? What are 5 things that are included in a culture media?
Culture media provides an environment in which the cell can maintain equilibrium, take in nutrients, and release waste products, and grow.
Includes: Salts, glucose, amino acids, serum, growth factors, antibiotics, pH indicator, buffer.
500
Describe the "transforming principle" and the experiment that found this principle.
Evidence that DNA is the genetic material first came from experiments with the bacterium that causes pneumonia (Pneumococcus).
A “transforming principle” was responsible for inducing the genetic transformation of one strain of the bacteria to another. Lysate from pathogenic bacteria couldbe added to non-pathogenic bacteria, resulting in transformation.
The transforming principle was later identified as DNA when it was shown that the activity of the transforming principle is abolished by enzymatic digestion of DNA, but not by digestion of proteins or RNA.
500
What is in vitro translation? What components are needed for this translation?
Translation in a bottle.
Such in vitro translation systems contain ribosomes, amino acids, tRNAs, enzymes, and synthetic mRNA with known base sequences.
500
Describe Gene Targeting by CRISPR/Cas
Plasmids expressing a guide RNA (gRNA) with sequences homologous to the target gene and the Cas9 nuclease are introduced into cells with or without a mutated copy of the target gene.
In the presence of a mutated gene copy, homology-directed repair replaces the broken target gene with the mutated copy. In the absence of a mutated gene copy, non-homologous end joining reseals the broken DNA, frequently introducing INDELs that
render the gene nonfunctional.
500
Give the net reaction of the Citric Acid Cycle.
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O →
CoA + 2 CO2 + 3 NADH + 3 H+ + FADH2 + GTP
500
Create a whole diagram of the Electron Transport Chain :D. We can just does this together tbh lol.
500
What are the three phases of the Calvin Cycle?
What catalyzes the first phase of the Calvin Cycle?
The Calvin cycle has three phases
1. Carbon fixation (catalyzed by Rubisco – super important enzyme for carbon fixation)
2. Reduction
3. Regeneration of the CO2 acceptor (RuBP)
500
What does the mitochondrial genomes code for?
Bonus: Which complexes involved in the electron transport chain are encoded by the mitochondrial genome? Which are not?
Most mitochondrial genomes encode only a few proteins that are essential for oxidative phosphorylation.
They also encode all the rRNAs and most of the tRNAs needed for translating the protein-coding sequences.
In humans, all the proteins that comprise Complex II are encoded by nuclear genes. Complexes I, III, IV, and ATP synthase include both nuclear-encoded and mtDNA-encoded proteins.
500
What do internal import signals cause?
What is the Oxa1 translocase?
Some proteins with multiple transmembrane domains have internal import signals instead of presequences.
After translocation across the outer membrane, they are bound by Tim9-Tim10 chaperones, which bring them to Tim22. The protein is transferred laterally into the inner membrane.
They are synthesized on ribosomes in the mitochondrial matrix and targeted to the Oxa1 translocase in the inner membrane.
They exit Oxa1 laterally to insert into the inner membrane.