Exam 3 Flashcards
The difference between bright-field and fluorescence microscopy is…
That one views the light passing through the sample and the other actually looks at light emitted from the sample.
One drawback of electron microscopy is that it…
Cannot be used to view living cells
Your lab TA just gave you a culture dish in which he claims there are cells called fibroblasts attached to the bottom. You cannot see anything on the bottom and think the TA is putting one over on you. Which method of microscopy would you use to best and most easily visualize the unstained cells without killing them?
Phase contrast microscopy
Which type of microscopy requires coating the sample with a thin layer of a heavy metal and can produce three-dimensional images of small surface projections from cells?
Scanning electron microscopy (SEM)
Proteasomes act primarily on proteins that have been marked for destruction by the covalent attachment of which small protein?
Ubiquitin
Which of the following statements is NOT true about the differences between liver cells and kidney cells in the same organism?
They contain different genes
After an RNA molecule is transcribed from a eukaryotic gene, portions called ____________ are removed and the remaining ___________ are spliced together to produce an mRNA molecule with a continuous coding sequence.
Introns, exons
Which of the following modifications can be made to an RNA molecule in eucaryotic cells before the RNA molecule becomes a mature mRNA?
Splicing, addition of methylguanosine cap to 5’end, and polyadenylation
Use of this common reporter gene fused to your gene of interest (such as lamins) would allow you to see where the lamins are localized in a living cell and see how the lamin distribution changes during the cell cycle. What is this reporter gene called?
Green fluorescence protein (GFP)
You would like to eliminate or reduce the expression of the intermediate filament vimentin in certain mouse cells you are studying. Which of the following experimental approaches could you potentially use to reduce the gene expression?
Use RNA interference (RNAi) or make a knockout mouse
What is the benefit of phase contrast microscopy?
Ability to see living (or dead) cells without stains
What is the benefit of fluorescence microscopy?
Ability to label specific structures individually and compare fluorescence/distributions
What is the benefit of SEM?
Can see fine details of surfaces (3D)
What is the benefit of DIC?
Ability to see living (or dead) cells without stains and has slightly more 3D than phase contrast
What is the benefit of bright-field?
Can use stains to differentiate and see different cell structures
What is the benefit of TEM?
Better resolution, can see smaller structural details compared to light microscopy
When we say that the gene for a specific type of serotonin receptor is expressed in smooth muscle, what is meant by expressed?
The gene has been transcribed into mRNA
What does it mean to say that a gene is “turned off?”
No mRNA is being transcribed from that gene (it’s present but not expressed)
What would happen to the mRNA if the mRNA wasn’t polyadenylated?
It would likely be degraded more rapidly than if polyadenylated and may not be exported from the nucleus
What does phosphorylation do for RNA processing?
Phosphorylation of the tail of RNA polymerase II allows RNA-processing proteins to assemble there
What does capping do?
Modifies 5’ end of transcript (synthesized first) by addition of an atypical guanine bearing a methyl group after RNA polymerase II has produced some nucleotides
What is polyadenylation?
Provides a newly transcribed mRNA that’s trimmed and another enzyme adds a series of repeated A nucleotides
What is splicing?
Introns removed by spliceosome and exons stitched together with ligase
A major regulator of the cell cycle is a protein called cyclin. Cyclin levels gradually increase before mitosis, and then their levels suddenly drop when mitosis nears completion. Interestingly, mRNA levels for cyclin are constant throughout the cell cycle. What would you expect to be responsible for the rapid drop in cycling levels at the end of mitosis?
Increase in ubiquitin addition to cyclins
Antibodies can be membrane-bound or secreted, depending on whether a stretch of additional amino acids are present or not. What process do you think determines if these amino acids are present?
Alternative RNA splicing
You have identified a gene that is affected by an immune disorder. Describe two experiments you could do that would help you figure out what the gene does.
Three main approaches: See it (where it is localized?), block it (prevent it from functioning and see what happens) , and add it (either overexposes it or express it where it normally isn’t and see how it affects the cell). Specifically, use an antibody and add it to different tissue sections, add GFP and transfect it into cells, knockout mouse or use RNAi in cultured cells, transgenic mice
What cells would you expect to have a high density of cytoplasmic intermediate filaments?
Human skin epithelial cell or nerve cell but not amoeba or plant cell
Why is there an initial lag in the rate of formation of microtubules?
Due to nucleation of microtubules, the process of initially forming a microtubule from the tubulin subunits is slow (slower to build microtubule from scratch than adding tubulin dimers onto existing microtubules
Why does the curve of rate of formation of microtubules level out?
Since concentration of free tubulins begins to decrease, you get to an equilibrium point where the amount of tubulin being removed from microtubules is the same as the amount being added
If you were to measure the rate of formation of microtubules but add purified centrosomes, what part of the new graph would be dissimilar than the normal graph?
The beginning because it will nucleate faster so the curve will rise more quickly
How is cell function regulated?
Regulate activity/conformation of individual proteins by affecting its ability to bind to other molecules and altering levels of individual proteins *Both change in response to extracellular signals
How are proteins regulated?
Transcriptional control (rate), RNA processing control, mRNA transport and localization control (nuclear export), mRNA degradation control (rate), translation control (rate), protein degradation control (rate), and/or protein activity control (protein processing/transport)
Why does an increase in mRNA not necessarily equal an increase in protein?
There’s multiple steps to regulating protein levels
What are some experimental approaches to studying proteins/cells?
See it, block it, or add it
What is the see it approach?
Use microscopy and other methods (antibodies) to measure protein and mRNA expression to see where, in which cells, where within the cells it is expressed, does its location or expression change depending on signals coming in, or does modification of proteins change it
What is the block it approach?
“Knockout” mice or RNA interference or using inhibitors or dominant negative constructs
What is the add it approach?
Transgenic animals (new gene) or cell transfections with expression vectors
What is a benefit of cultured cells?
Easier to see and manipulate
What does growing cells in culture dishes require?
Salts, amino acids, vitamins, glucose, etc. (anything a normal cell needs to survive) and also requires growth factors found in the bloodstream
What is a primary cell culture?
Individual cells dissociated from tissue (some divide in culture, some do not)
What are immortalized cell lines?
Tumor cells like HeLa or manufactured cell lines that use tumor-promoting chemicals, genes, viruses, etc. that have continual cell division
What can you use to find where proteins are localized?
Light microscopy: Immunofluorescence (use labeled antibodies), labeled toxins, or directly tag (i.e. GFP)
What are some useful techniques to finding proteins in cells?
- ) Fluorescence microscopy
- ) Video and digital microscopy (follow in real time)
- ) Recombinant DNA techniques (i.e. express protein joined to GFP)
What are ways to get proteins into eukaryotic cells?
Transfection of DNA (get plasmid into cells using various methods such as Ca precipitation, lipids, electroporation), microinjection into cells, or recombinant viruses (replication deficient, infectious: express transgene)
The major types of intermediate filaments found in neurons are…
Neurofilaments
Keratins are NOT found in:
Bones
What regulates the formation and breakdown of keratin filaments?
Phosphorylation and dephosphorylation
Which of the following types of intermediate filaments are found in all animal cells?
Nuclear lamina
In most cells the minus ends of microtubules are stabilized by a structure called the _____________, which generally lies next to the nucleus.
Centrosome
A microtubule is said to have a polarity, with a plus end and a minus end, because:
The tubulin subunits are all lined up in the same direction, leaving α−tubulin exposed at one end of the microtubule and β-tubulin at the other.
What is dynamic instability?
The ability of a microtubule to switch back and forth between polymerization and depolymerization.
If GTP hydrolysis occurs on a tubulin molecule at the plus end of a microtubule protofilament before another tubulin molecule is added, what typically happens to the microtubule?
The microtubule depolymerizes
What is a major function of intermediate filaments?
Prevent cells from breaking when under mechanical stretch
What are ways to manipulate gene expression?
Cell transfections or transgenic animals, “knockout” animal with null mutation, site directed mutagenesis (make constitutively active and dominant negative mutants), RNA interference to block or reduce gene expression
What do mutant keratin filaments do?
Cause skin disease because cells without intact filaments rupture and are weak because skin surface is about 85% keratin (mutations in basal layer)
What are functions of the cytoskeleton?
Structure and support, intracellular transport, contractility and motility, and spatial organization (i.e. cell polarity)
How are filaments composed and held together?
Formed spontaneously by subunit polymerization and held together by intermolecular forces (NOT covalent bonds)
What are some characteristics of intermediate filaments?
Only found in vertebrates, have high tensile strength, extensive lateral contacts give filament mechanical strength, and lack polarity
What are the two types of intermediate filaments and what types are within each?
Cytoplasmic: keratin in epithelial cells, vimentin and related in connective tissue, muscle, and glial cells, and neurofilaments in nerve cells; Nuclear: laming in all animal cells
In eukaryotic cells, the cell cortex is made of:
A network of actin filaments
Cilia and flagella bend as a result of…
Sliding of adjacent microtubules that are attached together
Dynein is to movement towards the cell center as _______ is to movement away from the cell center.
Kinesin
In the axons of neurons, vesicles and organelles are transported both towards and away from the cell body. This bi-directional movement is due to
Different motor proteins that move in opposite directions along the same microtubules.
The difference between cilia and flagella is
Cilia are not as long as flagella
In eukaryotic flagellum, the bending of microtubules is driven by:
The motor protein ciliary dynein
Addition of actin monomers to actin filaments is __________ at the __________ end.
Faster; plus
The concentration of actin monomers is high in the cytosol. What keeps these monomers from polymerizing totally into filaments?
The monomers are bound by proteins that prevent their polymerization
What are the steps to formation of microtubules?
Nucleation (slow initial formation that is enhanced at the centrosomes), elongation (rapid tubulin addition of existing microtubules), and steady state (reduced concentration of free tubulin limits further polymerization)
Which end of the microtubule is attached to the centrosome?
Minus
What is the difference between dynamic instability in microtubules and treadmilling in actin?
Dynamic instability is rapid growing and shrinkage in microtubules and treadmilling is when one end grows while the other shrinks and the filament seemingly moves
What are the three types of motor proteins?
Kinesin (plus end away from cell body), dynenin (minus end towards cell body), and myosin (on actin filaments)
What moves motor proteins and where do they move?
ATP hydrolysis; each type in one direction on microtubules that are of the same polarity
What is similar between actin and microtubules in their formation?
They both add more rapidly to the plus end that has ATP bound
What is actin polymerization limited by?
Nucleation and free actin concentration
What is the steady state?
Occurs at critical concentration and free monomer
Why is there preferential addition of G-actin at the plus ends?
Because critical concentration is lower at the plus ends versus minus ends