BME 201 Midterm

Currently has hw questions besides 8 cause that hasn't been graded yet (cards up to #37) and lecture notes.

Is it true that all human cells contain the same basic set of membrane enclosed organelles? Are there any examples of human cells without a complete set of organelles?

It is false. Red blood cells do not have a complete set of organelles.

What is the fate of a protein with no sorting signal?

It will permanently remain in the cytosol.

A signal sequence or the translocation of a protein can be genetically characterized. How would you identify a signal sequence in protein? The signalling sequence in question should allow the transferring of the protein into a particular organelle (the nucleus).

1. Modify replication

2. Use PCR to successively “cut off” (refrain from replicating) one amino acid per replication

3. Tag the modified proteins with fluorescence conjugated proteins

4. Observe which proteins end up in cell, the first modified protein to not make it signifies the end of the signal sequence

For a given signal sequence, design an experiment to determine whether it can direct a protein to a particular organelle.

One can use fluorescent labeling (protein) and immunofluorescence (organelle membrane) and fluorescence microscopy to confirm the overlapping of the protein and organelle.

When cells are treated with drugs that depolymerize microtubules, the Golgi apparatus is fragmented into small vesicles and dispersed throughout each cells. When the drugs are removed, cells typically recover and grow normally. After reformation, if observed through electron microscopy, the Golgi apparatus appears perfectly normal. Does this mean the Golgi apparatus has been synthesized anew? If not, what happened?

The Golgi apparatus merely reformed. Microtubules would reform the Golgi apparatus
(similar to vesicles absorbing into a membrane, would have a lower energy cost to reform than rebuild)

(True or False) There is no fundamental distinction between signalling molecules that bind to cell-surface receptors and those that bind to intracellular receptors. Explain.

True

• Both signalling molecules that cause a protein cascade and cell effect

• Diverse forms and function (gas, pH, temp, mech force, proteins, cAMP) => no fundamental difference just from intracellular vs. cell-surface

• Small hydrophobic molecule (intracell) vs. large molecule OR gas vs. protein : second has much greater difference, all signalling molecules, makes the first seem much lesser and not fundamental.

• There is a difference but not a fundamental one

(True or False) The interaction between signaling molecules and receptors is the only way for an animal cell to receive extracellular signals.

False
Mechanical force, gap junction etc. Multiple other ways.

Cells have been cloned from a cDNA library and the made plasmid clones are placed in mammalian cells which do not normally express the receptor the clones may have. The petri dishes of these modified mammalian cells are introduced a radioactive ligand for the receptor. Why don’t all the colonies on the petri dishes transfected with the positive pool have bound radioactive ligands?

The cDNA library is a black box technique. We do not choose the specific cDNA sequence, it is any of the myriad of cDNA that the mRNA of the original cell produces. Only some of the cDNA snippets actually code for the receptor.

Cells have been cloned from a cDNA library and the made plasmid clones are placed in mammalian cells which do not normally express the receptor the clones may have. The petri dishes of these modified mammalian cells are introduced a radioactive ligand for the receptor. The cells are then fixed in ice-cold methanol and the subjected to autoradiography. How would you complete the cloning of the receptor gene so that you have a pure plasmid containing the receptor cDNA?

As the cells have been fixed, they will not grow or divide. The radioactive colonies could be separated and the plasmid removed, then the plasmid could be filtered out and used to create more transfected cells.

Why are growth factors necessary for lab grown tissues and cells?

Growth factors contain the signal molecules that inform cells that they should divide and grow. Without this signal, cells won’t divide or will do so at a much lower rate.

A telephone conversation is analogous to _______ cell signalling.

synaptic (across a small gap, neurons)

Talking to people at a cocktail party is analogous to _______ cell signalling.

paracrine (target near by cell, diffusion through ECM)

A radio announcement is analogous to _______ cell signalling.

endocrine (signal is sent through the bloodsteam)

Talking to yourself is analogous to _______ cell signalling.

autocrine (cell targets itself)

Name several examples of small hydrophobic signal molecules that interact with intracellular signal molecules.

Cholesterol, testosterone, Vitamin D, estradiol

What are the major differences between bacterial and eukaryotic transcription?

Eukaryotic

• Transcription in the nucleus

• Requires multiple proteins to activate the RNA polymerase at the promoter sequence

• Nucleosome packing, more complex chromatin structure

• 3 RNA polymerases

Bacterial / Prokaryotes

• 1 RNA polymerase

• Only requires sigma factor to activate RNA polymerase at the promotor sequence

• Transcription occurs in the cytoplasm

• Transcription and translation are coupled

Function of mRNA

Codes for proteins

Function of rRNA

Components of ribosome

Function of snRNA

Splicing of RNA transcripts

Function of SnoRNA’s

Chemical modification of other RNA’s

(True/False) The s subunit is a permanent component of the RNA polymerase from E. coli, allowing it to initiate at appropriate promoters in the bacterial genome.

False
The s (sigma) subunit only attaches to the polymerase in initiation of transcription (not permanent).

What are the roles of general transcription factors in RNA polymerase II mediated transcription?

General transcription factors allow the polymerase to begin transcription. They aid positioning and pull apart the two strands of DNA in order for transcription to begin.

(True/False) Eukaryotic mRNAs carry 3’ ribosyl OH groups at both their 3’ and 5’ ends.

True, these groups exist on both ends of Eukaryotic mRNA.

(True/False) Since introns are largely genetic “junk”, they do not have to be removed precisely from the pre-mRNA during RNA splicing.

False, introns hold genetic information that can be deciphered. It can be dangerous to leave these introns attached as they can cause the RNA to fail to perform its function and to be read or cause improperly shaped proteins.

(True/False) The 3’ ends of most RNA polymermase II pre-mRNA transcripts are defined by the termination of transcription, which releases a free 3’ end to which a poly-A tail is quickly added.

False, the termination signal is a string of A-T pairs which fold together when the RNA polymerase II copies them, forcing the polymerase off the DNA strand as the U-A base pairs cannot remain bonded against the force of the folded structure.

Transcription can be ordered to terminate by a cleavage of the RNA chain from AAUAAA not a termination signal. A poly-A tail is not added until pre-mRNA processing in the nuclear pore.

What does “export-ready” mRNA mean, and what distinguishes an “export-ready” mRNA from a bit of excised intron that needs to be degraded?

Export-ready mRNA has a cap-binding complex, no hnRNP proteins, and a poly(a) tail and is ready for use and export.

Is it possible to synthesize a protein in vitro?

Yes, it is possible. Much like PCR, where we can amplify a given DNA sequence by adding primers, DNA polymerase, and nucleotides together in a buffered solution, the same principle suggests that protein synthesis should theoretically be possible by combining amino acids, ribosomes, mRNA, and tRNA into solution.

For the RNA sequence below indicate the amino acids that are encoded in the three reading frames. If you were told that this segment of RNA was in the middle of an mRNA that encoded a large protein, would you know which reading frame was used? How so? AGUCUAGGCACUGA

Frame one is used, middle of an mRNA so no start/stop sequences.

Frame 1: Ser-Leu-Gly-Thr

Frame 2: Val-STOP-Ala-Leu

Frame 3: Ser-Arg-His-STOP

Which of the following mutational changes would you predict to be the most deleterious to gene function?

a.) Insertion of a single nucleotide near the end of the coding sequence

b.) Removal of a single consecutive nucleotide near the beginning of the coding sequence

c.) Deletion of three consecutive nucleotides in the middle of the coding sequence

d.) Deletion of four consecutive nucleotides in the middle of the coding sequence

e.) Substituting of one nucleotide for another in the middle of the coding sequence

b.)

It would cause a reading frame shift in the very beginning of the sequence, almost the entire coding of the mRNA would be wrong.

One strand of a section of DNA from E. coli reads: 5’-GTAGCCTACCCATAGG-3’

What would be the mRNA sequence in this region?

RNA polymerase synthesizes from 5’ to 3’. Therefore mRNA should be read from the 3’ to 5’ strand to create a 5’ to 3’ mRNA.

complement read: 3’-CATCGGATGGGTATCC-5’

mRNA strand: 5’-GUAGCCUACCCAUAGG-3’

One strand of a section of DNA from E. coli reads: 5’-GTAGCCTACCCATAGG-3’
How many different peptides could potentially be made from this sequence of RNA? Would the same peptides be made if the other strand of DNA served as the template for the transcription?

Three potential as there are three reading frames, two actual as the second starts with a stop codon.

No, different location for starting codon and opposite bases.

List three examples of epithelial sheets found in the human body

Skin, small intestine, urinary bladder lining

(True/False) Unlike conventional ion channels, individual gap-junction channels remain open continuously once they are formed.

False. Rising calcium levels cause gap junctions to close.

The permeability of gap junctions is regulated by Ca 2+ . Would you expect gap junctions to open or close when the intracellular concentration of Ca 2+ rises? Why is this response advantageous?

Close. If increasing calcium ion concentrations are detected inside the cell, they could be indicative of a compromised plasma membrane, which means that the cell was damaged. It would be harmful to other cells if their contents were allowed to freely flow out through a damaged cell or if the damaged cell allowed cytotoxic elements into other cells.

(True/False) Integrins are the cell surface molecules which mediate cell-cell interaction.

False. Integrins are transmembrane receptors that connect the ECM and cytoskeleton. They affect cell-cell interaction but are NOT on the cell surface.

Describe the structure of ECM and its function in maintaining the architecture of an organ.

The ECM (extracellular matrix) holds cells together and in place, a scaffold for tissues and organs. It is made of proteins and polysaccharides. ECM provides mechanical support, influence to differentiation, cell shape, and cell movement and integrates cells into tissues.

Mesenchymal cells tend to have a bipolar shape. Why?

Mesenchymal cells are typically bipolar because they have a leading lamellipodium and a trailing edge. The lamellipodium is composed of actin cytoskeleton which the cell uses to move. Mesenchymal cells. as opposed to epithelial cells, are more independent and move in the ECM 

Cytosol

Consists of a bit more than ½ the cell volume. Site of protein synthesis and degradation.

Mitochondria

0.5 to 1 micrometer, ATP generator, signalling, differentiation, cell death, cell cycle and growth

What are the three types of protein transport?

1. Gated - cytosol → nucleus through nuclear pores, selective gates, small molecules can freely diffuse

2. Transmembrane - cytosol → mit or ER lumen, protein goes through membrane-bound protein translocator (unfolds to pass through)

3. Vesicular - proteins transported in vesicles, ER → Golgi, compartment to compartment

Almost all proteins begin being synthesized on __________ in the ________.

Ribosomes, cytosol

Signal sequence

continuous stretch of amino acid sequence in a protein, removed by a signal peptidase after sorting

Signal Patch

Discontinuous stretch of amino acids that becomes meaningful after folding. Generally are not removed, signal where the protein should go.

GPI Anchor

• Found on eukaryotic plasma membrane proteins

• Exists in yeast and is added to the C-terminus along with a secretion signal peptide to the N-terminus

• Binds proteins to the cell membrane

Communication between cells is mainly done by _________ __________ molecules which bind to ________ normally on the cell surface activating an ___________ __________ pathway using intracellular signalling proteins which targets _______ proteins.

extracellular signal

receptors

intracellular signalling

effect

List a few types of effect proteins

1. Gene regulatory

2. Ion channel

3. Metabolic

4. Cytoskeletal

What is the main molecule talked about in class that was consistently used as an example?

Insulin

Extracellular signal molecules often act at very (HIGH/LOW) concentrations and the receptors that recognize them usually bind with (HIGH/LOW) affinity.

LOW concentrations (<10^-8 M)

HIGH affinity (>10^8 L/mol)

Ligand

An extracellular signal molecule

In order to reach intracellular receptor proteins a signal molecule must be…

small and hydrophobic

Endocrine Vs. Synaptic Signalling

Endocrine

• relies on diffusion & blood flow → slow

• diluted hormones, reacts at low concentrations

• not precise in time nor space

Synaptic

• Fast & precise

• high local concentrations, low receptor affinity

• neurotransmitters are quickly removed from the synaptic cleft (precise)

The type of cell signalling thought to be partially responsible for the speed of early development which involves one type of cell is…

autocrine

Gap Junction

Narrow water filled channel between cells

Closes with high Ca2+ levels

Allow exchange of small signalling molecules (ions & cAMP) symmetrically

Involved in raising blood glucose (slide 7 lecture note 2)

True/False: One specific signal molecule will only cause one result.

FALSE

the same signal molecule causes different effects in different cells or in different environments

Which strand of DNA is used as a template for RNA? (#’→#’)

3’→5’

RNA is synthesized #’→#"‘ ?

5’→3’

RNA Aptamer

Specially folded RNA molecule that can target any molecule

mRNA

Majority of genes code for these, specifies amino acid sequence of proteins

What are snRNA, rRNA, snoRNA, and tRNA?

sn = small nuclear, direct splicing of pre-mRNA to make mRNA

r = ribosomal, form basic ribosome structure and catalyze protein synthesis

sno = small nucleolar, process and chemically mod rRNA’s

t = transfer, adaptors for mRNA and AA that select amino acids and hold them in place on a ribosome

Bacterial termination signal

A-T pairs with a two-fold symmetric structure (hairpin, wedges open polymerase)

RNA Polymerase II

Eukaryotic, transcribes most genes, protein & snRNA & snoRNA

Svedberg Unit (S)

non-metric sedimentation unit (40S and 60S for ribosomes)
10^13 seconds

What complex determines which RNA pieces to keep? (distinguishes export ready mRNA from introns and pre-mRNA)

nuclear pore complex

Transcription

DNA → RNA

Translation

RNA → Protein

Codon

Group of three consecutive nucleotides

Why is the genetic code redundant?

• “wobble” - tRNA can tolerate 3rd nucleotide mismatches

• there is more than one tRNA for many amino acids

What are the three places ribosomes can be found and their purpose?

1. Rough Endoplasmic Reticulum

2. Cytoplasm

3. Nuclear Envelope

Complex catalytic machine that assembles proteins, organelle

How are ribosomes made?

Subunits assembled at the nucleus (rRNA & ribosomal proteins); subunits are exported to cytoplasm where they join together
“Eukaryotic ribosomal subunits are assembled at the nucleus,
when newly transcribed and modified rRNAs associate with ribosomal
proteins, which have been transported into the nucleus after their
synthesis in the cytoplasm. The two ribosomal subunits are then
exported to the cytoplasm, where they join together to synthesize
proteins.”

Ribosome subunits function

Small = framework for tRNAs

Large = catalyzes formation of peptide bonds to link amino acids into polypeptide chain

Methionine

Encoded by a signal codon (AUG)

Carried by initiator tRNA
Start, always created first

Binds to small subunit

Leaking scanning

Method for getting multiple proteins from one RNA

Requires 5’-ACCAUGG-3’ instead of just AUG

Bacterial ribosome

Readily assembles directly on start codon, Shine-Dalgarno sequence start, polycistronic (many proteins from same mRNA)

Stop Codons

UAA, UAG, UGA

Selenocystein

21st amino acid, in bacteria, archaea, eucaryotes. Se atom in place of S atom of cysteine, enzyme producer.

Antibiotics often inhibit which process in prokaryotes?

Protein synthesis

What components are necessary for cell free protein synthesis?

• Template DNA

• ATP/energy

• RNA polymerase

• Aminoacids & tRNA

• Enzymes & substrates for energy regeneration

• Translation factors

Extracellular Matrix

complex network of proteins and polysaccharide chains that cells secrete

Cell-cell adhesions tie the _________ of neighboring cells together.

cytoskeletons

What are the 4 types of cell junctions

1. Anchoring - tethered to cytoskel, transmit stresses

2. Occluding - seal gaps between cells, epithelia sheets

3. Channel-forming - link cytoplasms of adjacent cells

4. Signal-Relaying - allows signals to pass across plasma membranes of cells (neurons)

What is the common function of all epithelia?

Serving as selective permeability barriers, requires occluding junctions. Tight junctions fulfill this role in vertebrates

FRET

Fluorescent proteins have an emission and absorption wavelengths. There are some fluorescent proteins whose emission wavelength is the same the the absorption wavelength of another fluorescent protein. With this, it can be determined if two fluorescent tagged things of interest are interacting by the strength and color of fluorescence.

Forster resonance energy transfer

Most cells in animal tissues communicate with adjacent cells through _______ junctions.

Gap

Bateriophage (phage)

virus that infects and replicates within bacteria, single stranded covalently closed genome.

Transduction

insertion of DNA

Process of Phage Display

1. Gene insertion

2. Protein display

3. Library Creation

4. Target exposure

5. Binding

6. Isolation of wanted ligand

7. Repopulation of wanted modified phage

Epitope

Part of a biomolecule that is the target of an immune response, site of antigen where antibody binds

In Vitro Tissue Engineering

biofabricate tissues from cells or manufacture cellular products

In Vivo Tissue Engineering

alteration of cell growth and function

Autologous cells

Donor → donor, same cells, same immunologic

Allogeneic cells

donor → recipient, same species, different immonologically, commonly used

Syngeneic cells

genetically identical donor (homozygous twin)

Xenogeneic cells

Donor → different species, pig → human

Induced pluripotent stem cells (iPSCs)

somatic (body) cells made into stem cells using OCT4, SOX2, NANOG, & LIN28, can differentiate into advanced derivatieves of all three primary germ layers

What are the fourth and fifth dimensions recognized in tissue engineering?

time and force

Apoptosis

programmed cell death

Necrosis

murder. Cell died from outside, not planned, causes

Matrix Metalloproteinase (MMP)

Enzymes, degrade ECM

ECM

extracellular matrix, contains ½ of proteins in the body, major signalling system

made of fibers (collagen), proteoglycans, and adhesive glycoproteins

What are the types of epithelia stucture?

1. Simple (1 layer)

2. Stratified (many layers)

3. Columnar (l o n g)

4. Squamous (squashed, flat and thin)

5. Cuboidal (cube! proportional)