Molecular Bio - Comp Exam

Question 1

Describe the structure of DNA.

Answer 1

DNA Structure

• Consists of 2 long polynucleotide chains with 4 types of nucleotide subunits.
• Nucleotides = 5-carbon sugar and nitrogenous base covalently linked via glycosidic bond.
  
  • Sugar in DNA is deoxyribose.
  • Adenine, cytosine, guanine and thymine are bases.
• 3D structure is a double helix.
  
  • 1 turn every 10 bp.
• Antiparallel: each strand’s sequence is complementary to partner.

Question 2

Describe the universal features of cells on earth.

Answer 2

Universal Features of Cells on Earth

• All Cells Store Their Hereditary Information in the Same Linear Chemical Code: DNA.
• All Cells Replicate Their Hereditary Information by Templated Polymerization.
• All Cells Translate RNA into Protein in the Same Way.
• Each Protein Is Encoded by a Specific Gene.
• All Cells Function as Biochemical Factories Dealing with the Same Basic Molecular Building Blocks.
• All Cells Are Enclosed in a Plasma Membrane Across Which Nutrients and Waste Materials Pass.
• Life Requires Free Energy.

Question 3

Describe the structural organization of nucleosomes.

Answer 3

Nucleosomes - Structural Organization

• Digestion with nucleases break down DNA by cutting between nucleosomes and degrading the exposed DNA between nucleosome core particles (linker DNA).
• Each individual nucleosome core particle consists of 8 histone proteins (histone octamer):
  
  • 2 molecules each of H2A, H2B, H3, H4.
  • And 2x stranded DNA that is 147 nucleotide pairs long.
• Linker DNA can be few to 80nt; nucleosomes repeat every 200 nucleotide pairs or so.

Question 4

What is a chromatin?

Answer 4

Nuclear DNA + protein.

Question 5

Describe histone modifications.

Answer 5

Histone Modifications

• Amino acid side chains of histones are subject to a variety of covalent modifications:
  
  • Occurs on the core of the histone as well as the tail.
  • Acetylation of lysines (loosens chromatin structure).
    
    • Added by histone acetyl transferases (HATs); removed by histone deacetylase complexes (HDACs).
  • Mono, di and tri-methylation of lysines:
    
    • Added by methyl transferases; removed by histone demethylases.
  • Phosphorylation of serines.
• Recruitment of these enzymes depends on gene regulatory proteins.
• All are reversible but can persist long after regulatory proteins have disappeared.
• Important consequences for the types of proteins the modified DNA attracts: this determines how/when/if gene expression takes place.
• Example - H3 Tail Modifications to N-terminal Tail:
  
  • Reading histone code involves joint recognition of marks at other sites on nucleosome along with tail recognition.
  • Few meanings are known.

Question 6

Describe DNA packaging.

Answer 6

DNA Packaging

• Eukaryotic DNA is packaged into chromsomes.
• The human genome is 3.2x10⁹ nucleotides distributed over 24 different chromosomes.
• Each chromosome is a single long linear DNA molecule associated with proteins that fold and pack it into compact structure.
  
  • Similar to packing 24 miles of thread into a tennis ball.
  • DNA + protein = chromatin.
• Each human cell contains 2 copies of each chromosome (maternal and paternal homologs).
  
  • except germ cells and RBCs.
• 22 pairs of autosomes and 2 sex chromosomes.
• 46 total chromosomes.

Question 7

Describe the requirements of a chromasome.

Answer 7

Chromosome - Requirements

• A copy must be passed on to each daughter cell at division: requires replication, separation of copies and partitioning to daughter cells.
  
  • DNA replication origin: where duplication of the DNA begins.
  • Centromere: Allows one copy of each duplicated and condensed chromosome to be pulled into each daughter cell when the cell divides. The kinetochore protein complex attaches to the centromere.
  • Telomeres: at the ends of a chromosome, contain repetitive sequences that enable the ends to be efficiently replicated.

Question 8

Describe chromatin remodeling.

Answer 8

Chromatin Remodeling

• Nucleosomes are in a constant state of flux.
  
  • DNA is unwrapped in the nucleosome 4 times per second, and remains unwrapped for 10-50 milliseconds before tightening up again.
• Chromatin remodeling complexes allow further loosening of DNA/histone contact.
• Proteins are related to helicases and are ATP dependent; bind to both protein core and DNA.
• Changes structure of nucleosome temporarily, making DNA less tightly bound.
• Repeated cycles catalyzes nucleosome sliding, making DNA available to other proteins in the cell.

Question 9

Describe nucleosome packing.

Answer 9

Nucleosome Packing

• Forms a dense fibrous structure with diameter of 30 nm.
• Unknown how this fiber is formed.
• Maybe zig-zag model:
  
  • Stacking may be facilitated by histone tails (esp H4).
  • Histone H1 “linker histone” is present in 1:1 ratio with nucleosome cores.
• Histone tails help to condense chromatin:
  
  • Histone tails are largely unstructured, suggesting that they are highly flexible.
  • Tails can form interactions with adjacent nucleosomes.

Question 10

Describe the regulation of chromatin structure.

Answer 10

Regulation of Chromatin Structure

• Certain types of chromatin structure can be inherited.
• Epigenetics: A form of inheritance that is superimposed on the genetic inheritance based on DNA.
• Examples:
  
  • DNA methylation
  • Chromatin structure
  • Histone modification

Question 11

Describe heterochromatin.

Answer 11

Heterochromatin

• Chromatin that is very condensed:
  
  • Stains darkly throughout the cell cycle, even in interphase.
• Thought to be late replicating and genetically inactive.
• Highly concentrated at centromeres and telomeres.
• Contains very few genes; those that are present are resistant to gene expression.
• Position effect: activity a gene depends on position on chromosome:
  
  • Will be silenced if relocated near heterochromatin.
• All the rest is less condensed and known as euchromatin.

Question 12

How do genomic changes occur?

Answer 12

Genomic Change - How They Occur

• Occur as mistakes in DNA replication and repair:
  
  • Rare occurrence: 1/1000 nucleotide pairs is randomly changed in the germ line every million years
  • Movement of transposable elements also play a role.
• Range of changes can occur:
  
  • Base pair substitutions.
  • Large scale rearrangements:
    
    • Duplications
    • Deletions
    • Inversions
    • Translocations

Question 13

Describe human variation in genome evolution.

Answer 13

Genome Evolution - Human Variation

• Human sequences vary 0.1% from one to another:
  
  • Human and chimps differ 1%.
  • Majority of mutation are neither harmful or beneficial.
  • Neutral mutations can become fixed in a population.
• SNPs - single-nucleotide polymorphisms:
  
  • Points in the genome where one group has one nucleotide and another group as another.
  • Variation occurs at a high rate (1% or more).
• CNVs - copy number variants:
  
  • Presence of many duplications and deletions of large blocks of DNA.
  • Some blocks are common and others rare; significance of most is unknown.

Question 14

Describe the importance of high-fidelity DNA replication.

Answer 14

Multicellular Organisms Need High Fidelity Replication

• Germ cells have to have low mutation rates to maintain the species.
• Somatic cells need low mutation rates to avoid uncontrolled proliferation/cancer.

Question 15

Describe proofreading in DNA replication.

Answer 15

DNA Replication - Proofreading

• DNA polymerase makes 1 mistake out of every 109 nucleotides copied, thanks to proofreading:
  
  • First step is just before a new nucleotide is added: enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place.
• Exonucleolytic proofreading:
  
  • Takes place immediately after incorrect bases is added.
  • DNA polymerase requires a perfectly paired 3’ terminus.
  • 3’ to 5’ exonuclease clips off unpaired residues at 3’ primer terminus.

Question 16

Describe DNA replication.

Answer 16

DNA Replication

• DNA polymerase synthesizes DNA by catalyzing the following reaction: (DNA)_{n residues} + dNTP → (DNA)_{n+1 residues} + P₂O₇^4-.
• Template directed—new chain is assembled in a preexisting DNA template that is complementary to the incoming bases.
• Requires separation of the two parental strands.
• Requires dATP, dGTP, dCTP and dTTP.
• DNA polymerase requires a primer with a free 3’ -OH to begin.
• Occurs during DNA synthesis phase (S) which lasts ~8hrs for mammalian cells.
• Chromosomes are replicated to produce two complete copies, joined at centromeres until M phase.
• Replication is activated in clusters (replication units) consisting of 20-80 origins.
• Different regions of each chromosome are replicated in a reproducible order during S phase, depending on chromatin structure.
  
  • Heterochromatin is late-replicating (timing related to packing of DNA in chromatin):
    
    • Example: X chromosomes of females: Almost all of inactive X is condensed into heterochromatin and is replicated late in S phase; the active homolog is less condensed and replicates throughout S phase.
  • Regions of genome with less condensed chromatin replicate first.

Question 17

Describe the proteins involved at the replication fork in DNA replication.

Answer 17

Replication Fork - Proteins

• DNA helicase - unwinds DNA
  
  • Protein with 6 identical subunits that binds and hydrolyzes ATP.
  • This causes conformational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole.
  • Capable of prying apart the helix at rates of 1000 nucleotide pairs/sec.
• Single-stranded DNA binding proteins: bind tightly and cooperatively to exposed SS DNA:
  
  • Help stabilize unwound DNA.
  • Prevent formation of hairpins.
  • DNA bases remain exposed.
• Sliding clamp: Keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered.
• Assembly requires clamp loader: hydrolyzes ATP as it loads the clamp onto a primer-template junction.
• Leading strand: clamp remains associated with DNA polymerase for long stretches.
• Lagging strand: Clamp loader stays close so it can assemble a new clamp at start of each new Okazaki fragment.

Question 18

Describe the reassembly of chromatin after replication.

Answer 18

Reassembly of Chromatin After Replication

• Replication requires not only DNA replication but synthesis and assembly of new proteins.
• Eucaryotes have multiple copies of genes for each histone.
• Histone proteins are synthesized mainly in S phase; amount made is highly regulated to meet requirements.
• For efficient replication, chromatin-remodeling proteins are needed to destabilize DNA-histone interface.
• As replication fork passes through chromatin, histone octamer breaks into:
  
  • an H3-H4 tetramer, distributed randomly to daughter duplexes.
  • 2 H2A-H2B dimers which are released from the DNA.
• Freshly made H3-H4 fills in spaces; H2A/H2B dimers are 1⁄2 old and 1⁄2 new; they are added at random to complete complex.
• This orderly addition requires histone chaperones (chromatin assembly factors).
• Directed to DNA with sliding clamp called PCNA.

Question 19

Describe telomeres.

Answer 19

Telomeres

• End replication problem on lagging strand: no place for RNA primer.
• Bacteria have circular genomes; eukaryotes have telomeres.
• Special sequence GGGTTA at the end of each chromosome repeated ~1000x.
• Enzyme called telomerase replenishes these sequences by elongating parental strand in 5’ to 3’ direction using an RNA template on the enzyme.
• After extension of parental strand by telomerase, replication of lagging strand can be completed by DNA polymerase, using extension as template.
• This mechanism (plus a 5’ nuclease) ensures 3’ end is longer, leaving a protruding SS end that loops back and tucks into the repeat.
• T-loops:
  
  • Structures protect ends and distinguishes them from broken ones that need to be repaired.
  • Shelterin - protective chromosome cap made up of proteins.

Question 20

Describe the types of DNA damage.

Answer 20

DNA Damage

• Mutations are not only caused by mistakes in replication.
• 5000 purine bases are lost every day due to a spontaneous reaction called depurination.
• Spontaneous deamination of C to U occurs at 100 bases/day.
• Can also occur from exposure to reactive forms of O₂ in the cell or chemicals in environment.
• UV radiation from sun can produce a covalent linkage between two adjacent pyrimidines (T-T or C-T):
  
  • Pyrimidine dimers.
• If unrepaired when DNA replicated, these changes lead to either a deletion or a base pair substitution in the daughter strand.

Question 21

Describe the DNA repair pathways.

Answer 21

Question 22

Describe the regulation of homologous recombination.

Answer 22

Homologous Recombination - Regulation

• Accurate repair process can still cause problems for a cell:
  
  • Use of a non-functioning homolog to “repair” the other homolog.
    
    • Loss of heterozygosity:
      
      • Critical first step in cancer development.
      • Rare occurrence.
• Processing of broken ends is coordinated with the cell cycle:
  
  • Nucleases for generating 3’ invading strand are only active in S and G2 phase.
    
    • Ensures a replicated chromosome or sister chromatid will be the most likely template for repair.
  • Prevention of repair in the absence of damage:
    
    • Loading of Rec A on DNA is tightly controlled.
    • Repair proteins dispersed throughout the cell:
      
      • After damage, repair occurs in “factories” or “foci” at the sites of damage.
• Mutations in proteins involved in recombination can cause cancer:
  
  • Brca1 and Brca2 lead to increased rates of breast cancer.
    
    • Brca1 regulates the processing of broken ends of chromosomes.
      
      • Mutations lead to use of non-homologous end-joining process.
    • Brca2 maintains Rad51 (RecA) inactive until it is at site of damage.
      
      • Does not bind to DNA to form invading strand.

Question 23

Describe the repair of double-strand breaks.

Answer 23

DS-Break Repair

• Non-homologous end joining (NHEJ) and homologous recombination (HR) in mammals repair DNA double-strand breaks.
• The process involves:
  
  • End binding and tethering.
  • End processing (removing mismatched or damaged nucleotides and replacing them).
  • Ligation.
• Several human syndromes are associated with dysfunctional NHEJ, including SCID (Severe combined immunodefiency).
• HR requires a homologous section of DNA to act as a template for repair of the damaged/broken fragment.
• HR is more accurate than NHEJ because of the template. The importance of HR is derived from the fact that the mechanism is conserved throughout evolution.

Question 24

Describe Holliday junctions and how they are resolved.

Answer 24

Holliday Junctions

• Structures are present only transiently.
• Resolution - strands of the helices are cleaved by endonuclease (RuvC).
• Resolution has two outcomes:
  
  • Crossing over:
    
    • Rare event.
    • Only 2 cross over events/ chromosome.
  • Gene conversion:
    
    • 90% of Holliday junctions in humans resolve this way.

Question 25

Describe transposons.

Answer 25

Transposons

Question 26

Describe conservative site-specific recombination.

Answer 26

Conservative Site-Specific Recombination

• Mediates rearrangements of other types of mobile DNA elements.
• Break and join two DNA double helices on each molecule:
  
  • Depending on positions and relative orientations of recombination sites, can get:
    
    • DNA integration, DNA excision, or inversion.
  • Differ from transposition:
    
    • Need special sites on each DNA that serve as recognition sites for recombinase.
      
      • Only transposon sequence is required for transposition.
    • Form transient high energy covalent bonds and use this energy to complete DNA rearrangement.
      
      • No covalent protein/DNA intermediate in transposition.
      • Gaps must be filled by DNA polymerase and ligase.
• Difference in outcome is in the relative orientation of DNA sites.
• Many bacterial viruses move in and out of host genome by this mechanism:
  
  • ex. Bacteriophage lambda.

Question 27

Describe meiosis.

Answer 27

Meiosis

• Gametes are haploid.
• Arise from meiosis:
  
  • Involves two cell divisions but one round of DNA synthesis to produce half the number of chromosomes.

Question 28

Describe fertilization.

Answer 28

Fertilization

• Released egg is surrounded by granulosa cells and an ECM rich in hyaluronic acid.
• Capacitated sperm must penetrate granulosa cells:
  
  • Uses hyaluronidase.
• Sperm binds to zona pellucida:
  
  • Acts as a species barrier.
• Zona pellucida induces sperm to undergo acrosome reaction.
  
  • Contents help sperm to tunnel through zona pellucida.
  • Alters sperm so can bind and fuse with plasma membrane of egg.
• Sperm binds egg plasma membrane first by tip and then side.
  
  • Microvilli on egg aide in the process.
• Certain membrane proteins are crucial to binding:
  
  • ZP 1, 2 and 3.
    
    • ZP 2 and 3 form long filaments.
    • ZP 1 cross-links the filaments.

Question 29

Describe gonadal development.

Answer 29

Gonadal Development

• Sry in somatic cells direct differentiation into Sertoli cells instead of follicle cells.
• Sertoli cells secrete anti-Mullerian hormone:
  
  • Suppresses female development.
  • Causes Mullerian duct to regress.
• Induce Leydig cell differentiation in other somatic cells:
  
  • Secrete testosterone (responsible for 2ndary sexual characteristics.
• In absence of Sry:
  
  • Genital ridge becomes an ovary.
  • PGC becomes an egg.
  • Somatic cells differentiate into:
    
    • follicle cells - support cells.
    • theca cells - estrogen-producing cells.

Question 30

Describe the central dogma of biology.

Answer 30

Central Dogma

• DNA is transcribed to messenger RNAs which act as templates for protein synthesis in translation.

Question 31

Describe how cells replicate their hereditary information by templated polymerization.

Answer 31

All Cells Replicate Their Hereditary Information by Templated Polymerization

Question 32

Describe transcription.

Answer 32

Transcription

Question 33

Describe translation.

Answer 33

Translation

• Translation proceeds in three phases:
  
  • Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.
  • Elongation: The tRNA transfers an amino acid corresponding to the next codon. The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain (polypeptide).
  • Termination: When a stop codon is reached, the ribosome releases the polypeptide.

Question 34

Describe proteins.

Answer 34

Proteins

• Nitrogen and carbon containing compounds that consist of large molecules composed of one or more long chains of amino acids that are an essential part of all living organisms.

Question 35

Describe the initiation of DNA replication.

Answer 35

DNA Replication - Initiation

• Prereplication complexes bind an origin of replication. Activated DNA helicases find origins of replication, polymerase and other supporting proteins are added, phosphorylation initiates DNA synthesis at the appropriate time in the cell cycle.

Question 36

Describe short telomere syndromes.

Answer 36

Short Telomere Syndromes

• Short telomere syndromes (STSs) are accelerated aging syndromes often caused by inheritable gene mutations resulting in decreased telomere lengths.
• Short telomere syndromes are multisystem disorders with widespread clinical manifestations.
• Organs with high cell turnover, such as the bone marrow, liver, lungs, and immune system, are commonly affected.
• Key clinical cues to suspect short telomeres in a patient are a personal or family history of premature graying of hair (at age <30 years), unexplained cytopenias, idiopathic pulmonary fibrosis, and cryptogenic cirrhosis.
• Flow cytometry - fluorescence in situ hybridization is the initial screening test, followed by genetic sequencing.
• Treatment requires a multidisciplinary approach.

Question 37

Describe inherited human syndromes resulting from faulty DNA repair.

Answer 37

Question 38

Describe the special translesion DNA polymerases.

Answer 38

Special Translesion DNA Polymerases Are Used in Emergencies

• The cell recognizes that DNA is damaged and replication has stalled.
• Specialized proteins cause the release of the DNA polymerase and bring in the correct translesional polymerase to allow the damaged DNA to be bypassed.
• The polymerase clamp is then reactivated and replication proceeds.
• Great potential for mutation.
• Despite being tightly regulated by a variety of transcriptional and posttranslational controls, the low-fidelity TLS polymerases also gain access to undamaged DNA where their inaccurate synthesis may actually be beneficial for genetic diversity and evolutionary fitness.

Question 39

Describe homologous recombination in crossing over.

Answer 39

Homologous Recombination in Crossing Over

• Crossing over and gene conversion can occur in the same chromosome.
• Multiple opportunities for genetic reassortment.

Question 40

Describe free radicals.

Answer 40

Question 41

Describe the effect of radiation on rates of mutation.

Answer 41

Effects of Radiation on Mutation Rates

• Ionizing radiation (X-rays etc.) dislodges electrons in tissue causing free radicals which often damages DNA.
• UV light induces the formation of pyrimidine dimer: two thymine bases covalently bonded that blocks replication.
• SOS system in bacteria: SOS system allows bacteria cells to bypass the replication block with a mutation-prone pathway.

Question 42

Decribe the interconnected genetic origin of DNA repair deficiency disease.

Answer 42

Question 43

Describe RNA folding.

Answer 43

RNA Folding

Question 44

Describe transcription.

Answer 44

Transcription

• DNA is transcribed by RNA polymerase. The RNA moves stepwise along the DNA unwinding the DNA as the polymerase progresses. The incoming nucleosides (ribonucleoside triphosphate: ATP, UTP, CTP, GTP) supply energy needed for catalysis from their triphosphate bonds.

Question 45

Describe the different categories of RNA molecules.

Answer 45

RNA Molecules - Categories

Question 46

Describe transcription start and stop signals.

Answer 46

Transcription Start and Stop Signals

Question 47

Describe RNA Polymerase II.

Answer 47

RNA Polymerase II

• The TATA box is 25 nucleotides from the transcription initiation site. The protein TBP recognizes and binds to the TATA box providing the initial step to begin transcription.
• A series of additional proteins are added until the entire complex of proteins are assembled and transcription starts.
• The initiation of transcription usually requires multiple activator proteins along with proteins that unwind the chromatin. The activator proteins help coordinate the acquisition of the multiple different proteins needed for transcription initialization. They also play a role in modifying DNA shape which affects the rate of transcription

Question 48

Describe RNA capping.

Answer 48

RNA Capping

• As soon as the mRNA is ~25 nucleotides long it is capped. 3 enzymes work together to:
  
  1. Dephosphorylate the 1st nucleotide,
  2. Add a GMP in reverse linkage (5’ to 5’ instead of 5’ to 3’), and
  3. Add a methyl group to the guanosine.
• The cap allows mRNA to be distinguished from noncoding RNAs and is also important for establishing translation.
• And sometimes another methyl group is added to the ribose of the 1st nucleotide.

Question 49

Describe RNA splicing.

Answer 49

RNA Splicing

Question 50

Compare the sizes of exons to introns.

Answer 50

Exon Size vs. Intron Size

• Exons are much more similar in size among eukaryotes than are introns.

Question 51

Describe mRNA splice site mutations.

Answer 51

mRNA Splice Site Mutations

• mRNA splice site mutations have serious consequences.

Question 52

Describe the generation of the 3’ end of eukaryotic mRNAs.

Answer 52

Generation of 3’ End of mRNA - RNA-Processing Enzymes

Question 53

Describe exosomes.

Answer 53

Exosomes

• The exosome is a protein complex that cleans up damaged RNAs before they leave the nucleus.
• It is rich in RNAases and chops up RNAs for recycling.
• Another definition:
  
  • Exosomes are cell-derived vesicles that are present in many and perhaps all eukaryotic fluids, including blood, urine, and cultured medium of cell cultures.
  • Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. Although the exosomal protein composition varies with the cell and tissue of origin, most exosomes contain an evolutionarily-conserved common set of protein molecules.

Question 54

Describe microRNA.

Answer 54

microRNA

• microRNA are ubiquitous small noncoding RNAs.
• A microRNA (miRNA) is a small non-coding RNA molecule (containing ~ 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression.
• The first miRNA was discovered in the early 1990s. However, miRNAs were not recognized as a distinct and important class of biological regulators until the early 2000s.
• MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing.
• Biogenesis of miRNA is under tight temporal and spatial control.
• Dysregulation of miRNA is associated with many human diseases, particularly cancer and neurodevelopmental disorders.
• Regulation takes place at multiple levels including transcription, Drosha processing, Dicer processing, RNA editing, RNA methylation, uridylation, adenylation, Argonaute modification and RNA decay.

Question 55

Describe snRNPs.

Answer 55

snRNPs

• Biochemical modification of noncoding RNAs is implemented by snRNP (small nucleolar RNAs [snoRNA] + specific proteins).
• The most common modifications are pseudouridylation and 2’-O methylation
• Ribosomal RNAs have more than 100 of each type of modification.
• Spliceosomal RNAs are also modified.
• Evidence is accumulating that mRNAs are also modified in this fashion.

Question 56

Describe the nucleolus.

Answer 56

Nucleolus

• The nucleolus is the site where rRNA is processed and assembled into ribosome subunits. The nucleolus is not membrane bound, but a dense assembly of pre and post processing – rRNA and the proteins needed to assemble the ribosome.

Question 57

Describe the nucleus.

Answer 57

Nucleus

• The nucleus has multiple aggregates including cajal bodies where most snoRNAs are assembled into snRNPs.
• The protein Fibrillarin is present in both the nucleolus and cajal bodies. Identified with an antibody against with a red flourochrome.
• Coilin is a protein found only in the cajal body. Shown here by immunohistochemistry with a flourochrome that appears pink in combination with the flourochrome for fibrillarin (arrows).
• Interchromatin granule clusters (green) contain stockpiles of RNA processing components and are located near sites of transcription.

Question 58

Describe the genetic code.

Answer 58

Genetic Code

Question 59

Describe how tRNA molecules match amino acids to codons in mRNA.

Answer 59

tRNA - Matching to Amino Acids

• tRNA have a complex 3 dimensional structure. Most tRNAs have extensive biochemical modification of nucleotides.
• A. y = pseudouridine D = dihydrouridine.
• B. and C. X ray diffraction projections of tRNA.
• D. tRNA icon
• E. The tRNA sequence, in this case the tRNA-Phe

Question 60

Describe wobble base pairing.

Answer 60

Wobble Base Pairing

• The nucleotide listed in the 1st column can base pair with any of the nucleotides listed in the 2nd column.
• For example when U is at the 3rd codon position it can pair with 3 different nucleotides in the anticodon (I = deamination of A).

Question 61

Describe how incorporation of the correct AA is ensured.

Answer 61

Incorporation of Amino Acids

• 2 adapters help ensure the correct amino acid gets incorporated into a peptide.
  
  1. aminoacyl-tRNA synthetase which helps ensure that the correct amino acid is coupled to the tRNA.
  2. The tRNA is an adapter whose anticodon pairs with the codon in the mRNA to identify the correct amino acid to adds to the growing peptide.

Question 62

Describe the addition of amino acids to peptides.

Answer 62

Translation - Addition of Amino Acids

• The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of the growing peptide and the amino group on the incoming AA.
• The formation of the peptide bond is energetically favored because of growing C terminus has been activated by the covalent attachment of the tRNA.

Question 63

Describe translating an mRNA into a peptide.

Answer 63

Translating an mRNA into a peptide.

• There are 4 steps involved in protein synthesis:
  
  1. New tRNA binds to A site pairing with codon.
  2. Carboxyl end of growing peptide is released from tRNA in P site. Peptide bond formation between the previous AA added and the new one. tRNAs are in P site and A site.
  3. Large subunit moves along mRNA held by small subunit, shifting tRNAs to P and E sites on small subunit
  4. Small subunit moves 3 nucleotides along the mRNA and ejects the tRNA in the E site.
• The entire complex moves 3 nucleotides along the mRNA as a result of the two translocation steps. The ribosome is thus reset and ready for another tRNA-AA.

Question 64

Describe a ribozyme.

Answer 64

Ribozymes

• The Ribosome Is a Ribozyme – RNA capable of enzymatic function.
• 2/3 of a ribosome is RNA. The 3D conformation of an entire ribosome was solved in 2000.
• The structure helped demonstrate that the rRNA is responsible for the overall structure, the ability to position tRNAs and its catalytic activity.
• The primary role of the proteins seems to be to stabilize the RNA core.

Question 65

Describe the initiation of translation.

Answer 65

Translation - Initiation

• The initiation of protein synthesis requires the presence of several translation initiation factors. The poly A tail must be bound by poly A binding proteins that interact with eIF4G to ensure that both ends of the mRNA are intact.
• 2 GTP hydrolysis steps provide the energy needed. The last 2 steps shown here are the ribosome has begun the standard elongation steps we discussed previously.
• A consensus sequence flanking the start codon (ACCAUGG) tells the ribosome to begin translation on this AUG. If the flanking nucleotides differ very much, the ribosome will move to the next AUG. This results in alternative starting points for the protein.

Question 66

Describe stop codons.

Answer 66

Translation - Stop Codons

• The binding of a release factor to an A site containing a stop codon causes termination of translation. The peptide is released and in a series of reactions the ribosomal subunits dissociate and are then ready to initiate translation again.

Question 67

Describe polyribosomes.

Answer 67

Polyribosomes

• Synthesis of a protein takes ~20 seconds to several minutes. Upon completion of translation, the ribosomal subunits are near the 5’ end of the mRNA because of the association of the 5’ and 3’ ends of the mRNA. Thus they can quickly restart translation.

Question 68

Describe antibacterial compounds produced by fungi.

Answer 68

Antibacterial Compounds - Produced by Fungi

• Inhibitors of Prokaryotic Protein Synthesis Are Useful as Antibiotics
• Fungi produce many antibacterial compounds that exploit differences in ribosomal subunits. Bound tRNA is shown in purple and the antibiotic binding sites are shown with the colored spheres.

Question 69

Describe the quality control mechanisms of translation.

Answer 69

Translation - Quality Control Mechanisms

• Quality Control Mechanisms Act to Prevent Translation of Damaged mRNAs – Nonsense Mediated Decay.
• Incorrect splicing can result in premature stop codons. Nonsense mediated decay eliminates mRNAs with premature stop codons. As the mRNA is being transported through the nuclear pore, a ribosome does a test run on it. Exon junction complexes (EJC) are bound at exon junctions and are removed by the moving ribosome. The stop codon should be in the last exon. If the ribosome hits a stop codon while EJCs are still present, the presence of EJCs will activate nonsense mediated decay and the mRNA will be rapidly removed to prevent any additional wasted energy.
• This system may help prevent the translation of mutated genes with premature stop codons that could cause severe damage.

Question 70

Describe molecular chaperones.

Answer 70

Molecular Chaperones

• Molecular Chaperones Help Guide the Folding of Most Proteins.
• Many energetically favorable pathways are available for protein folding.
• Cells use additional proteins “chaperones” to ensure that peptides fold into the correct functional conformation or to refold them if they get damaged (for example after a heat shock).
• Heat shock proteins (e.g. hsp70 family) recognize hydrophobic stretches of amino acids on the surface of a protein (generally these should be found on the interior of a protein) and bind to them along with hsp40 proteins using ATP hydrolysis to drive the reaction. Hsp40 dissociates and ATP rebinds inducing hsp70 dissociation. The process is repeated multiple times which helps the target protein to refold into the correct structure.

Question 71

Describe the signals for protein quality control.

Answer 71

Protein Quality Control - Signals

• Exposed Hydrophobic Regions Provide Critical Signals for Protein Quality Control.
• Usually an exposed section of hydrophobic amino acids indicates a protein is misfolded. If not corrected the result is usually accumulation of protein aggregates that can be harmful.
• Diseases of proteopathy include prion diseases, Alzheimer’s and Parkinson’s.

Question 72

Describe a proteasome.

Answer 72

Proteasome

• If the protein rescue process does not induce correct folding, the cell needs to eliminate the protein prior to aggregation. The proteasome searches for misfolded proteins in the nucleus and cytoplasm, ingests them and degrades them so that the AAs can be recycled. The cap (blue) binds damaged proteins that have been ubiquitylated, unfolds them (using ATP) and feeds them into the inner cylinder which is lined with proteases facing the inner chamber.
• Only marked proteins can enter to prevent the random destruction of correctly folded proteins. Special proteins (E3) recognize misfolded proteins and add multiple ubiquitin peptides to the misfolded peptide. In this case the ubiquitin peptides are all linked at lysine 48 which is the key identifier of a damaged protein.
• The cap has multiple functional domains:
  
  1. Ubiquitin receptor
  2. Unfoldase
  3. Ubiquitin hydrolase

Question 73

Describe regulated destruction of proteins.

Answer 73

Proteins - Regulated Destruction

• Protein lifespan is regulated by signals that direct the protein to the proteasome
• Activating specific E3 ligases that target a protein for destruction.
• Degradation signals within the protein can be induced leading to ubiquitylation and subsequent destruction.

Question 74

Describe the many steps from DNA to protein.

Answer 74

DNA to Protein

Question 75

Describe how transcriptional misregulation leads to disease.

Answer 75

Transcriptional Misregulation Leads to Disease

• Abstract: The gene expression programs that establish and maintain specific cell states in humans are controlled by thousands of transcription factors, cofactors and chromatin regulators. Misregulation of these gene expression programs can cause a broad range of diseases. Here we review recent advances in our understanding of transcriptional regulation and discuss how these have provided new insights into transcriptional misregulation in disease.
• Figure 1:
  
  • Transcriptional regulation:
    
    • A. Formation of a pre-initiation complex. Transcription factors bind to specific DNA elements (enhancers) and to coactivators, which bind to RNA polymerase II, which in turn binds to general transcription factors at the transcription start site (arrow).
    • B. Initiation and pausing by RNA polymerase II. RNA polymerase II begins transcription from the initiation site, but pause control factors cause it to stall some tens of base pairs downstream.
    • C. Pause release and elongation. Various transcription factors and cofactors recruit elongation factors,
    • D. Chromatin structure is regulated by ATP-dependent remodeling complexes that can mobilize the nucleosome, allowing regulators and the transcription apparatus increased access to DNA sequences.
    • E. Transcriptional activity is influenced by proteins that modify and bind the histone components of nucleosomes. Some proteins add modifications (writers), some remove modifications (erasers) and others bind via these modifications (readers). The modifications include acetylation (Ac), methylation (Me), phosphorylation (P), sumoylation (Su) and ubiquitination (Ub).
    • F. Histone modifications occur in characteristic patterns associated with different transcriptional activities. As an example, the characteristic patterns observed at actively transcribed genes are shown for histone H3 lysine 27 acetylation (H3K27Ac), histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 79 dimethylation (H3K79me2) and histone H3 lysine 36 trimethylation (H3K36me3).
• Figure 2:
  
  • Master transcriptional regulators and reprogramming factors.
  • Transcription factors that have dominant roles in the control of specific cell states and that are capable of reprogramming cell states when ectopically expressed in various cell types.

Question 76

What are the 3 major types of intracellular transport?

Answer 76

Transport

• Gated transport:
  
  • Transport between nucleus and cytosol through nuclear pore complexes (active transport and free diffusion).
• Transmembrane transport:
  
  • Membrane protein translocators directly transport specific proteins from cytosol across an organelle membrane.
• Vesicular transport:
  
  • Membrane-enclosed transport intermediates move proteins between various compartments via vesicles.

Question 78

What do sorting signals do in a cell?

Answer 78

Protein Sorting Signals

• Protein transfer/transport to various compartments guided by sorting signals.

Question 79

Describe protein sorting signals.

Answer 79

Protein Sorting Signals

• Stretch of amino acids, typically 15-60 residues long.
• Localized on N or C terminus or within protein sequence.
• Multiple scattered sequences in protein may form signal patch.
• Signal sequences are both necessary and sufficient for protein targeting.
• Physical properties of sequence (e.g., charge, hydrophobicity) more important than actual sequence.
• If removed, the sorting signal can be removed by signal peptidase.
• Signal sequences are recognized by complementary receptors.

Question 80

Describe nuclear pore complexes.

Answer 80

Nuclear Pore Complexes

• Perforate nuclear envelope in eukaryotic cells.
• Molecular mass ~125 mill Da.
• Composed of 30 different proteins or nucleoporins.
• Arranged in octagonal symmetry with one or more aqueous pores.
• Each nuclear envelope has 3000-4000 NPCs.
• Transport molecules in both directions.
• Passive diffusion of small molecules and facilitated transport.
• Transport facilitated by binding of particles to fibrils extending from NPC.

Question 81

What determines whether a molecule freely diffuses through NPC or is transported via active transport?

Answer 81

Size.

Question 82

Describe nuclear transport.

Answer 82

Nuclear Transport

• Gated, bidirectional, and selective.
• Proteins needed in the nucleus are imported from the cytoplasm where they are synthesized.
  
  • Histones, DNA & RNA polymerases, topoisomerases, gene regulatory proteins.
• tRNA and mRNA molecules synthesized in nucleus and exported to cytosol.

Question 83

Describe the process of nuclear import?

Answer 83

Nuclear Import

• Nuclear import receptors (NIRs) recognize the nuclear localization sequence (NLS).
• Each receptor recognizes a subset of cargo proteins.
• NIRs are soluble cytosolic proteins that bind to NLS on protein and to NPC proteins present on fibrils that extend into cytoplasm.
• NPC proteins have phenylalanine glycine (FG) repeats which serve as binding sites for import receptors.
• Receptors plus cargo traverse NPC by binding, dissociating, and re-binding to adjacent FG repeats.
• Cargo released inside nucleus and NIR return to cytoplasm.

Question 84

Describe the process of nuclear export.

Answer 84

Nuclear Export

• Nuclear export works similar to import but in opposite direction.
• Relies on nuclear export signals (NES) on molecules that need to go out of nucleus.
• Need complementary nuclear export receptors (NER).
• NER binds to cargo present in nucleus and NPC proteins.
• Binding, dissociation and re-binding facilitates transport.
• Cargo released into cytoplasm.

Question 87

What are nuclear localization signals (NLS)?

Answer 87

Nuclear Localization Signals

• NLS are sorting signals that direct molecules to nucleus.
• Short sequences rich in the positively charged AAs lysine and arginine.
• Located on many different sites on protein.
  
  • Form loops or patches on surface.
• Result in selective import of proteins into the nucleus.
• Highly specific, changing one Lys to Thr prevents transport into nucleus.

Question 88

Describe mitochondrial signal sequences.

Answer 88

Mitochondria - Signal Sequences

• N terminal and internal signal sequences.
• Signal sequence for matrix proteins is best understood.
• Positively charge residues cluster on one end and uncharged hydrophobic on the other end to form amphiphilic alpha helix.
• Specific receptor proteins recognize this configuration rather than precise sequence.
• Multi-subunit protein complexes called protein translocators mediate translocation.

Question 90

Describe the translocase of the outer membrane (TOM).

Answer 90

Mitochondria Translocators - TOM

• Present in the outer membrane.
• Required for import of all nuclear encoded proteins.
• Inserts them in outer membrane.
• 2 components:
  
  • Receptors for mitochondrial precursor proteins.
  • Translocation channels.

Question 91

Describe translocase of the inner membrane (TIM).

Answer 91

Mitochondria Translocators - TIM

• Usually considered present in both inner and outer membrane, new evidence suggests only present in inner membrane.
• 2 components:
  
  • Receptors for mitochondrial precursor proteins.
  • Translocation channels.
• 2 TIM complexes:
  
  • TIM 22
    
    • Mediates the insertion of a specific subclass of proteins (e.g., ATP, ADP and Pi transporter).
  • TIM 23
    
    • Transports soluble proteins into matrix and helps insert membrane proteins in inner membrane.

Question 92

Describe the sorting and assembly machinery in mitochondria.

Answer 92

Mitochondria Translocators - SAM

• Translocates and inserts/folds beta barrel proteins in the outer membrane.

Question 93

Describe the OXA complex in mitochondria.

Answer 93

Mitochondria Translocators - OXA

• Mediates insertion of proteins synthesized in mitochondria.

Question 94

Describe ER signal sequence.

Answer 94

ER - Signal Sequence

• ER signal sequences vary in AA sequence.
• Have 8 or more non-polar AA at its center.
• ER signal sequence guided to ER membrane by 2 components:
  
  • Signal recognition particle (SRP)
    
    • Rod shaped, made of 6 different polypeptides bound to a single small RNA molecule, with large hydrophobic pocket lined by methionines.
    • Cycles between ER membrane and cytosol and binds to ER signal sequence.
    • Pocket can accomodate hydrophobic signal sequences of different size, shape, and sequence.
  • SRP receptor.

Question 95

Describe the process of co-translational translocation across ER.

Answer 95

ER - Co-Translational Translocation

• SRP wraps around larger ribosomal subunit.
• One end binds to ER signal sequence of emerging protein and other end to elongation factor binding site.
• Transiently blocks protein synthesis giving protein time to enter the ER membrane.
• SRP-ribosome complex binds to SRP receptor present in ER membrane.
• Interaction brings the assembly to a translocator.
• SRP and receptor released and protein translocated across the ER membrane into the lumen.

Question 96

List the translocators in the mitochondrial membrane.

Answer 96

Mitochondria - Translocators

• Translocase of the Outer Membrane (TOM)
• Translocase of the Inner Membrane (TIM)
• Sorting and Assembly Machinery (SAM)
• OXA complex.

Question 97

Describe vesicular transport.

Answer 97

Vesicular Trasport

• Proteins and other biomolecules are transported via transport vesicles.
• Vesicles bud off from primary compartment and fuse with the next one.
• Different shapes and sizes: small sperical, large irregular or tubular.
• Contents of vesicle called cargo.
• Transport is directional.

Question 98

What are the 3 types of vesicular transport systems.

Answer 98

Vesicular Transport Systems

• Biosynthetic-secretory.
• Endocytic pathway.
• Retrieval pathway.

Question 99

What are the 3 types of coated vesicles?

Answer 99

Coated Vesicles - Types

• COPI
• COPII
• Clathrin

Question 100

Describe COPI coated vesicles.

Answer 100

Coated Vesicles - COPI

• Mediates transport from Golgi cisternae.

Question 101

Describe COPII coated vesicles.

Answer 101

Coated Vesicles - COPII

• Mediates trasport from ER.

Question 102

Describe clathrin coated vesicles.

Answer 102

Coated Vesicles - Clathrin

• Mediate transport from Golgi apparatus and from plasma membrane.
• Each clathrin subunit: 3 large and 3 small polypeptide chains that form a 3-legged structure called triskelion.
• Triskelions assemble into a basket-like structure of hexagons and pentagons that form coated pits on the cytosolic side of membrane.
• Adaptor proteins form a second layer between the cage and membrane.
• Trap various transmembrane proteins including receptors that capture soluble cargo inside vesicle.

Question 109

What is the role of Rab and SNARE in vesicle targeting?

Answer 109

Vesicle Targeting - Rab and SNARE

• Specificity in targeting is acheived by surface markers on vesicles and complimentary receptors on target membrane.
• Rab proteins direct vesicle to specific spots on target membrane.
• SNARE proteins mediate fusion of vesicle with membrane.

Question 110

Describe lysosomes.

Answer 110

Lysosomes

• Membrane enclosed compartments filled with hydrolytic enzymes.
• Heterogeneous - derived from late endosomes.
• Important for intracellular digestion of macromolecules.
• About 40 types of enzymes: proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases.
• Require acidic environment and proteolytic cleavage for optimal activation.
• Lysosomal membrane protects cell against its enzymes.
• Transporters in membrane pump out end products of digestion.
• Vacuolar ATPase pumps H⁺ into lysosomes to maintain the acidic pH and to drive transport of small metabolites.

Question 111

What are the 3 pathways materials are delivered to lysosomes?

Answer 111

Lysosomes - Material Delivery

• Endocytosis
• Phagocytosis
• Autophagy

Question 112

How are proteins destined for lysosomes sorted?

Answer 112

Lysosome - Protein Sorting

• Lysosomal hydrolases have the sorting signal mannose-6-phosphate (M6P) attached to them in the CGN.
• M6P receptors in TGN recognize the sugar.
• Receptors bind to hydrolases and to adaptor proteins in assembling clathrin coats.
• Packaged into clathrin-coated vesicles that bud from TGN.
• Contents delivered into endosomes and then to lysosomes.

Question 113

Describe sub-cellular fractionation.

Answer 113

Sub-Cellular Fractionation

• Tissue: Mechanical blending
• Homogenate: Suspension of different cell types.
• Centrifugation to seperate different cell types, based on size & density.
• Lysis of cells: osmotic shock, ultrasonic vibration, mechanical blending, forcing through small orifice.
• Ultracentrifugation: separate organelles.

Question 114

What is column chromatography?

Answer 114

Column Chromatography

• Separation of molecules by column chromatography.
• A solution containing a mixture of different molecules, is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose.
• Large amount of solvent is passed slowly through the column and collected in separate tubes as it emerges from the bottom.
• Various components travel at different rates through the column and are fractionated into different tubes.

Question 115

What are the matrices for column chromatography?

Answer 115

Matrices for column Chromatography

• Ion-exchange chromatography
  
  • Insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge.
• Gel-filtration chromatography
  
  • Small beads that fill the matrix are inert but porous.
  • Molecules that are small enough to penetrate the beads are delayed and travel more slowly.
• Affinity chromatography.
  
  • Insoluble matrix is covalently linked to specific ligand, such as an antibody or enzyme, that will bind to a specific protein.
  • Bound proteins are then eluted by dissociating the antibody-antigen complex with concentrated salt solutions or solutions of high or low pH.

Question 116

What are restriction endonucleases?

Answer 116

Restriction Endonucleases

• Enzymes isolated from bacteria that cut DNA at specific sites.

Question 118

What happens in a ligase reaction?

Answer 118

Ligase Reaction

• “Glues’ DNA ends together.
  
  • Much easier with compatible cohesive ends.

Question 119

How can DNA be cloned using bacteria?

Answer 119

DNA Cloning in Bacteria

• DNA fragment to be cloned is inserted in plasmid that has been engineered to serve varius functions i.e. carry and replicate manipulated gene products.

Question 120

When analyzing DNA, what makes analysis by electrophoresis different from SDS-PAGE?

Answer 120

DNA Analysis

• Agarose gel used instead of polyacrylamide gel.
• DNA is already negatively charged, SDS not needed to add negative charge.

Question 121

Describe cloning vectors.

Answer 121

Cloning Vectors

• Plasmids have been engineered to serve various functions i.e. carry and replicate manipulated gene products.

Question 122

Describe SNPs.

Answer 122

SNPs

• You are 99.9% the same as the person next to you.
• There are 3 million base pair differences between you and the person sitting next to you.
• SNP variants can be neutral, pathogenic, or predisposing.

Question 123

In development of an embryo, what are the four phases of development?

Answer 123

Embryo Development - Phases

1. Proliferation
2. Specialization
3. Interaction
4. Movement

Question 124

What does it mean to say that homologous proteins are functionally interchangeable?

Answer 124

Homologous Proteins

• Basic machinery for development is similar for all organisms.
• Homologous proteins are functionally interchangeable.

Question 125

Describe the process of gastrulation.

Answer 125

Gastrulation

• Blastula consists of a sheet of epithelial cells facing the external medium.
• This sheet gives rise to ectoderm.
  
  • Ectoderm is precursor of nervous system and epidermis.
• Part of epithelial sheet becomes tucked into the interior giving rise to endoderm.
  
  • ​Endoderm is the precursor of gut, lung and liver.
• Group of cells move into the space between ectoderm and endoderm giving rise to mesoderm.
  
  • Mesoderm is precursor of muscles and connective tissue.

Question 127

How is development controlled?

Answer 127

Development Program

• Instructions for producing a multicellular animal is contained in non-coding regulatory DNA associated with each gene.
• DNA contains regulatory elements that serve as binding sites for gene regulatory proteins.
• Regulatory DNA defines the sequential program for development.
• Coding sequences in DNA similar in most organisms but non-coding sequences make one organism different from another and provide uniqueness.

Question 128

Describe how development decisions impact cell fate.

Answer 128

Development Decisions and Cell Fate

• Cells make developmental decisions long before they show any outward signs of differentiation.
• “Determined” - cells that are fated to develop into a specialized cell type despite changes in environment.
• “Completely undetermined” - cells that can change rapidly due to alterations in environment.
• “Committed” - cells that have some attributes of a particular cell type but can change with environment.

Question 129

Describe inductive signaling.

Answer 129

Inductive Signaling

• Most important environmental cues are signals from neighboring cells.
• Induction of a different developmental program in select cells in a homogenous group leading to altered character - inductive signaling.
• Few cells closest to the source take on induced character - signal is limited in time and space.
• Types of signals:
  
  • Short range: cell-cell contacts.
  • Long range: substances that can diffuse through the extracellular medium.

Question 130

What are morphogens?

Answer 130

Morphogens

• Morphogen - a long range inductive signal that imposes a pattern on a field of cells.
• Exerts graded effects by forming gradients of different concentrations.
• Each concentration can direct the target cells into a different developmental pathway.
• Gradient formed by:
  
  • Localized production of an inducer that diffuses away from its source.
  • Localized production of an inhibitor that diffuses away from its source and blocks the action of a uniformly distributed inducer.
• Morphogens need an ‘on’ and ‘off’ system.
• Antagonists or extracellular inhibitors bind to the signal or its receptor and block interaction.

Question 133

What is gastrulation?

Answer 133

Gastrulation

• Transformation of the blastula, a hollow sphere of cells, into a layered structure with a gut.

Question 134

What are the phases of neural development?

Answer 134

Phases of Neural Development

• Phase 1 - different cell types (neurons, glial cells) develop independently at widely separate locations in embryo according to local program and are unconnected.
• Phase 2 - axons and dendrites grow out along specific routes setting up a provisional but orderly network of connections between various parts of the nervous system.
• Phase 3 - continues into adult life, connections are adjusted and refined through interactions with distant regions via electric signals.

Question 135

Describe the origin of the nervous system.

Answer 135

Nervous System - Origin

• Neurons are produced in association with glial cells (provide supporting framework and nutrition).
• Both cell types develop from ectoderm from a common precursor.
• CNS (brain, spinal cord, and retina) derived from neural tube.
• PNS (nerves, sensory neurons) derived from neural crest.

Question 136

Describe the molecular mechanisms of neuronal migration.

Answer 136

Neuronal Migration - Molecular Mechanisms

• A typical immature neuron has a cell body, a long axon and several short dendrites.
• Axon and dendrites not distiguishable at first.
• Tip of axon/dendrite has an irregular, spiky enlargement called growth cone.
• Growth cone crawls through surrounding tissue, trailing the axon or dendrite behind.
• Growth cone has the engine and steering apparatus that directs the process along the right path.
• One of the growth cones starts migrating fast, becomes dominant, and develops axon-specific proteins - this will form axon.

Question 137

Describe growth cones.

Answer 137

Growth Cones

• Growth cone behavior is dictated by its cytoskeletal machinery.
• Growth cone throws out filopodia and lamelopodia.
• Monomeric GTPases Rho and Rac control the assembly/disassembly of actin filaments, which control movement of growth cone.
• Growth cones withdraw cells from unfavorable surfaces and steer them towards favorable ones where they persist for longer time.

Question 138

Describe the migration of growth cones.

Answer 138

Growth Cones - Migration

• Growth cones travel towards target cells along predictable routes.
• Exploit two major cues to find their way:
  
  • Extracellular matrix environment - sensed by receptors present on membrane.
  • Chemotactic factors - released by neighboring cells. Attractive or repulsive.

Question 140

Describe the mechanism of commissural neuron guidance.

Answer 140

Mechanism of Commissural Neuron Guidance

• First stage depends on secretion of netrin by cells of the floor plate.
  
  • Binding of netrin to its receptor causes opening of TRPC (Transient receptor potential C) channels.
  • Allow entry of extracellular Ca²⁺.
  • Leads to activation of machinery for extension of filopodia and movement of growth cone.
  • Non-commissural neurons in neural tube do not have netrin receptors, so do not migrate towards floor plate.
• Midline cells secrete Slit.
  
  • Slit receptor Roundabout present on commissural neurons.
  • Slit repels growth cones and blocks entry to the midline.
  • Growth cones become sensitive to another repulsive signal called semaphorin.
  • Trapped between 2 sets of repellants, growth cones travel in a narrow track.

Question 142

Describe activity-dependent synaptic remodeling such as in retinal/tectal neurons.

Answer 142

Activity-Dependent Synaptic Remodeling (Retinal/Tectal Neurons)

• Each axon initially branches widely and makes multiple synapses with target cell.
• Profusion of weak synapses.
• Network subsequently trimmed by elimination of synapses and retraction of axon branches.
• Accompanied by sprouting of axons to develop denser distribution of synapses that survive.
• Synaptic remodeling dependent upon 2 rules that create spatial order:
  
  • Axons from cells in different regions of retina compete for tectal neurons.
  • Axons from neighboring sites which are excited at same time cooperate/collaborate to retain and strengthen synapses with tectal neurons.
• Activity-dependent synaptic remodeling depends on electrical activity and synaptic signaling.

Question 146

Describe the use of chemotactic factors in the migration of growth cones.

Answer 146

Growth Cone Migration - Chemotactic Factors

• Secreted by cells, act as guidance factors at strategic points along path.
• May be attractive or repulsive.
• Examples:
  
  • Netrin
  • Slit
  • Semaphorin

Question 148

What regulates which growth cones synapse and where?

Answer 148

Neurotrophic Factors

• Axonal growth cones reach eventual target cells.
• Halt, communicate and make synapses with target cells.
• Signal from target tissue regulate which growth cones synapse and where.

Question 149

Describe preprogrammed production of cerebral cortex layers.

Answer 149

Programmed Production of Cerebral Cortex Layers

Question 150

Describe the characteristics of stem cells.

Answer 150

Stem Cells - Characteristics

• Not terminally differentiated.
• Can divide without limit.
• Ability to renew themselves.
• Following division each new cell has ability to remain stem cell or become differentiated into a different cell type.
• Undergo slow division.

Question 151

What does totipotency mean?

Answer 151

Developmental Capacity - Totipotency

• Ability of cell to give rise to all cells of an organism, including embryonic and extra-embryonic tissues.
• Example: zygote

Question 152

What does pluripotency mean?

Answer 152

Developmental Capacity - Pluripotency

• Ability of a cell to give rise to all cells of the embryo and subsequently adult tissues.
• Example: embryonic stem cells.

Question 153

What does multipotency mean?

Answer 153

Developmental Capacity - Multipotency

• Ability of a cell to give rise to different cell types of a given lineage.
• Example: adult stem cells.

Question 154

Describe the maintenance of stem cells.

Answer 154

Stem Cells - Maintenance

• A steady pool of stem cell population maintained by:
  
  • Asymetric division - creates 2 cells, one with stem cell characteristics and another with the ability to differentiate.
  • Independent choice - Division makes 2 identical cells but the outcome is stochastic and/or influenced by environment.

Question 155

Describe the layers and cells of the epidermis.

Answer 155

Epidermis - Layers and Cells

• Layers from deep to superficial:
  
  • Basal cell layer:
    
    • Attached to basal lamina and contains only dividing cells in epidermis.
  • Prickle cell layer:
    
    • Cells have numerous desmosomes that attach tufts of keratin filaments.
  • Granule cell layer:
    
    • Forms boundary between inner metabolically active strata and outer dead epidermis cells.
  • Squame layer:
    
    • Flattened dead cells, densely packed with keratin but no organelles.

Question 156

Describe the renewal of the epidermis.

Answer 156

Epidermis - Renewal

• Continuous wear and tear needs constant repair.
• Self-renewing process:
  
  • Basal cells divide with some maintaining the basal layer and others move to layers above.
  • Cells move through prickle cell layer, granule cell layer, etc.
  • Change in gene expression at each step of differentiation, acquiring phenotype appropriate for that layer.
  • Cells start undergoing partial degradation, losing nucleus and other organelles.
    
    • Depends on partial activation of apoptotic machinery.
  • Time from ‘birth’ of cell in basal layer to shedding from surface is 1 month.

Question 157

Describe the regulation of epidermal stem cells.

Answer 157

Epidermal Stem Cells - Regulation

• Regulation helps to control size of stem cell population.
• Contact with basal lamina controls numbers of stem cells (most important signal).
• Maintenance of contact preserves stem cell potential.
• Loss of contact triggers terminal differentiation.
• Stem cell markers not well known.
• Proliferative potential of stem cells directly correlates with expression of beta-1 subunit of integrin (helps mediate adhesion to basal lamina).
• Clusters of cells with high levels of integrin found near the basal lamina and in bulge of hair follicle.

Question 165

What factors govern the renewal of the epidermis?

Answer 165

Renewal of Epidermis - Governing Factors

• Rate of stem cell division.
• Probability that one daughter cell will remain a stem cell.
• Rate of division of transit amplifying cells.
• Timing of exit from basal layer and the time the cell takes to differentiate and be sloughed away from surface.

Question 166

Describe olfactory neurons.

Answer 166

Olfactory Neurons

• Humans ~40 million olfactory neurons.
• Bipolar neurons with a dendrite facing the extracellular environment (interior space of the nasal cavity) and an axon that travels along the olfactory nerve to the olfactory bulb in the brain.
• Many tiny hair-like cilia protrude from the dendrite.
• Supporting cells present in between neurons - hold neurons in place and separate them from one another.
• Basal cells in the epithelium - cells in contact with basal lamina.
• Sensory surfaces of epithelium kept moist and protected by a layer of fluid (mucus).

Question 167

Describe olfactory receptors.

Answer 167

Olfactory Receptors

• The free surfaces of cilia have odorant receptor proteins (olfactory receptors).
• A type of G protein coupled receptor.
• Odorant receptor genes - 1000 in dog, 350 in humans.
• Each neuron expresses only one of these genes enabling the cell to respond to only one class of odorant (organic smell molecules).
• Structural features of odorant recognized by the receptor.
• All olfactory neurons respond by a common mechanism.
• A given olfactory receptor can bind to a single class of odor molecules (may include a variety of odor molecules).
• Affinity variable depending on how well the ligand binds to the receptor.
• Activated olfactory receptor in turn activates an intracellular G-protein (G_olf).
• Activates adenylate cyclase in the plasma membrane which results in an influx of sodium and calcium into the cell.
• This influx of positive ions causes the neuron to depolarize, generating an action potential.

Question 168

Describe the function of olfactory receptors.

Answer 168

Olfactory Receptors - Function

• Action potentials relayed via the axon to the brain.
• Relay stations in brain called glomeruli.
• Located in olfactory bulbs one on each side of the brain.
• 1800 glomeruli/bulb in mouse brain.
• Although olfactory neurons expressing the same odorant receptor are located in different places on the olfactory epithelium, their axons converge on the same glomerulus.

Question 170

Describe embryonic stem cells.

Answer 170

Embryonic Stem Cells

• Pluripotency well established.
• Good growth properties in cell culture (in vitro).
• Have ability to differentiate into a wide range of cell types if provided the right set of culture conditions.
• Develop into different cell types with characteristics appropriate for that site (even germ cells).
• Capable of proliferating indefinitely in culture with unrestricted developmental potential.
• Unlike the zygote, ES cells are incapable of generating a full organism.

Question 171

Describe the derivation of embryonic stem cells.

Answer 171

Embryonic Stem Cells - Derivation

• Derived from the blastocyst stage of embryo.
• When put back in blastocyst they can integrate well with the embryo.

Question 173

What is Somatic Cell Nuclear Transfer (SCeNT)?

Answer 173

Somatic Cell Nuclear Transfer (SCeNT)

• Combines cloning methods with embryonic stem cell technology to produce cells which are custom-made for patient.
• Steps:
  
  • Nucleus taken from somatic cell of patient and injected into oocyte of a donor replacing the oocyte nucleus.
  • Blastocyst generated from this hybrid oocyte and ES cells isolated.

Question 174

What are Induced Pluripotent Stem Cells (iPSC) and how are they developed?

Answer 174

Induced Pluripotent Stem Cells (iPSC)

• Somatic cells can be reprogrammed to form iPSC by exposing them to defined, limited sets of transcription factors (genes for stem-ness).
• Reprogramming adult cells:
  
  • Better if adult cells could be converted into ES-like cells by manipulating gene expression directly.
  • Biochemical comparisons of ES cells with other cell types suggest some candidate genes.
  • Key determinants of ES cell character are a set of 4 gene regulatory proteins: Oct4, Sox2, Klf4, and Myc.
  • When injected into fibroblasts they form ES-like cells including the ability to differentiate into other cell types.
  • Yield low.

Question 176

Describe the regeneration of olfactory neurons.

Answer 176

Olfactory Neurons - Regeneration

• Individual olfactory neurons survive for only a month.
• Neural stem cells residing among the basal cells in the olfactory epithelium generate replacements for the lost neurons.
• Basal stem cells in contact with basal lamina divide and differentiate into olfactory neurons.
• Odorant receptor proteins help in axonal guidance and allow the growth cone to migrate to and establish connection with correct glomerulus in olfactory bulb.
• Regeneration of olfactory receptor cells is one of the only few instances of adult neurogenesis in the CNS.
• Has raised considerable interest in dissecting the pathways for neural development and differentiation in adult organisms.

Question 179

What are the different sources of human embryonic stem cells?

Answer 179

Human Embryonic Stem Cells - Alternative Approaches to Obtaining

1. Somatic Cell Nuclear Transfer (SCeNT)
2. Induced Pluripotent Stem Cells (iPSC)

Question 180

What is signal transduction?

Answer 180

Signal Transduction

• Extracellular signaling molecules bind to receptors in target cells to initiate a chain of events.
• Cell-to-cell communication.
• Must traverse from outside of cell to inside of cell.
• Important in development of organism and coordination of metabolism.
• Required for multi-cellular organisms.

Question 182

How does signal transduction work?

Answer 182

Signal Transduction

• Extracellular signaling molecules bind to receptors in target cells to initiate a chain of events referred to as signal transduction.
• Induce 2 types of response:
  
  • Change in activity or function of enzymes or proteins in cell (FAST RESPONSE).
  • Change in amounts of proteins by change in expression of genes (SLOW RESPONSE).

Question 183

What is endocrine signaling?

Answer 183

Cell Communication - Endocrine Signaling

• Long distance signaling.
• Freely diffusible signals.
• Long lasting (long half-life) - takes time to find target through circulatory system.

Question 184

What is paracrine signaling?

Answer 184

Cell Communication - Paracrine Signaling

• Acts locally on cells nearby.
• Not as freely diffusible.
• Short-lived.
• Example:
  
  • NO - produces cGMP (a 2nd messenger), a small molecule can interact with other proteins and regulate activity.
    
    • Influences muscle relaxation in circulatory system.

Question 185

What is synaptic signaling?

Answer 185

Cell Communication - Synaptic Signaling

• Acts locally.
• Short-lived.
• Uses neurotranmitters.

Question 186

What is autocrine signaling?

Answer 186

Cell Communication - Autocrine Signaling

• Cells respond to their own signals or those released by same type of cell.
• Signals cell growth, division, maturation.
• e.g. growth factors in CA cells.

Question 187

What is direct cell signaling?

Answer 187

Cell Communication - Direct Cell Signaling

• e.g. immune cells.
• Ag-presenting cells to T cells.

Question 188

What can signals effect?

Answer 188

Signal Transduction

• Cytoskeletal changes.
• Membrane changes.
• Protein translation changes.
• Cell cycle changes.

Question 189

What is a G-protein?

Answer 189

Cell Communication - G-Protein

• Heterotrimeric G proteins are guanine nucleotide-binding proteins that assist in transmitting signals to intracellular targets.
• Consist of 3 subunits - alpha, beta, gamma.
• Receives input from receptor and impacts effector.

Question 190

What is an effector?

Answer 190

Cell Communication - GPCR Effector

• Generates cAMP, an important second messenger that mediates cellular responses to a variety of ligands.

Question 191

What are the steps in GPCR signal relaying?

Answer 191

Cell Communication - GPCR Signal Sequence

• Ligand binds to receptor.
• Conformational change occurs in receptor.
• Receptor binds to G protein.
• Receptor acts as a GEF.
• Conformation of G-alpha is changed, GDP released and GTP binds.
• G-alpha becomes active and can bind to and activate effector.
• Effector molecule then creates second messenger (which in this case adenylyl cyclase catalyzes cAMP).
• Eventually, GTP on G-alpha is hydrolyzed to GDP (occurs after certain amount of time).
• G-alpha returns to inactive step to be recycled through process again.

Question 193

How does cholera effect the body?

Answer 193

Cholera

• Modifies G protein by keeping G-alpha in the active form indefinitely.
• Makes pathway always active.
• Pump Cl- and water out of cell in intestine and causes severe diarrhea.

Question 194

How does cAMP, as a second messenger, cause a biological response from target proteins?

Answer 194

Cell Communication - cAMP

• Activates a cAMP-dependent protein kinase (PKA), which phosphorylates proteins, either activating or inactivating them.
  
  • PKA contains 4 subunits, 2 catalytic and 2 regulatory.
  • cAmp molecules bind to the regulatory subunits.
  • Releases active C-subunits.
• Can also activate cAMP-gated cation channels.

Question 196

What are receptor tyrosine kinases?

Answer 196

Cell Communication - Receptor Tyrosine Kinases (RTKs)

• Enzyme linked receptors.
  
  • Enzyme domain is in the cytoplasmic tail of the integral membrane protein.
  • Respond to growth factors and mediate signals.
• 3 domains:
  
  • Extracellular
  • Transmembrane
  • Cytoplasmic (tyrosine kinase domain)

Question 197

Describe the function of receptor tyrosine kinases.

Answer 197

Cell Communication - RTK Function

• Growth factor binds to extracellular domain.
• Conformational change.
• Dimerization of 2 receptors.
• Autophosphorylation of tyrosines on cytoplasmic domain.
• Cytoplasmic domain acts as scaffold and recruits other proteins.
• Binds to proteins with SH2 (src homology) domain.
  
  • i.e. Grb2 (also has SH3 domain)
• SH3 binds to prolines in son of sevenless (SOS)
• SOS (a GEF) binds to Ras (small monomeric G protein - small GTPase)
• Cascade:
  
  • Ras -> MKKK (Raf) -> MKK (Mek) -> MK (Erk) -> nucleus ->increase gene transcription
• Uncontrolled gene transcription = CA

Question 199

Describe JAK-STAT receptor function.

Answer 199

Cell Communication - JAK-STAT Receptor

• Ligand binds to Janus kinases (JAKs).
• JAKs dimerize and phosphorylate each other.
• Receptor binds and phosphorylates Signal Transducer and Activators of Transcription proteins (STATs).
• STATs separate from receptor, dimerize, enter nucleus, and bind to DNA causing transcription.
• Example: erythropoeitin

Question 203

What is a G-protein-coupled receptor?

Answer 203

Cell Communication - G-Protein-Coupled Receptor

• Composed of 3 parts:
  
  • Extracellular domain - binds ligand.
  • Transmembrane domain - anchors receptor.
  • Cytoplasmic domain - associated with G-protein.
• 3 subunits: alpha, beta, gamma.
• AKA 7 transmembrane receptor.
• Affect olfaction, sight, taste.

Question 207

What is adenylyl cyclase?

Answer 207

Cell Communication - Adenylyl Cyclase

• Generates cAMP as secondary signal that goes on to interact with its target proteins to cause a biological response.

Question 210

How does PKA regulate proteins?

Answer 210

Cell Communication - PKA

• Regulates proteins by phosphorylating them.
• Phosphorylation can:
  
  • Change conformation of protein by the addition of 2 negative charges.
  • Form part of a structure that other proteins recognize.
  • Cause activation or inactivation.
  • Cause alteration of intracellular localization of target proteins.
  • Cause alterations in abundance of target proteins.

Question 213

What are JAK-STAT receptors?

Answer 213

Cell Communication - JAK-STAT

• More direct route for impacting transcription.

Question 215

Describe serine-threonine receptor function.

Answer 215

Cell Communication - Serine-Threonine Receptor

• Ligand binds to receptors (Type I and Type II).
• Type II phosphorylates sites on Type I.
• Type I then phosphorylates R-Smads (receptor specific Smad such as Smad1 and Smad2).
• R-smad complexes with Co-Smad and migrates to nucleus.
• Example: iron metabolism - hepcidin hormone.

Question 216

Describe the cAMP targets.

Answer 216

cAMP Targets

• cAMP interacts with it’s target proteins to cause a biological response.
• cAMP activates cAMP-dependent protein kinase (PKA) - 4 subunits.
• Inactive PKA: 2 catalytic subunits & 2 regulatory subunits.
• Binding of 2 cAMP molecules to regulatory subunits of tetramer → release of active C subunits.

Question 217

What happens if a signal is not turned off?

Answer 217

Errant Signaling

• A point mutation in codon 12 results in a change from Gly to Val and constitutively activates Ras (Ras-G12V).
• Mutations that result in constitutively activate Ras are frequently found in human cancer.

Question 218

What differenciates cell types?

Answer 218

Cell - Structure and Function

• All cells contain the same genetic material.
• Differentiation depends on differences of gene expression.

Question 219

What are some differences in the ways cells express proteins?

Answer 219

Different Cell Types - Different Proteins

1. Common proteins (housekeeping proteins), i.e. glucose metabolism.
2. Specifically limited proteins, i.e. hemoglobin.
3. Typical human cell expresses 30-60% of its 21,000 structural genes (protein coding) but level of gene expression varies - fingerprint expression profiles e.g. microarrays or RNA Seq.
4. Other factors post transcription include: alternative splicing (dystrophin gene), post translational modification.

Question 220

What are the 6 locations where gene expression is controlled?

Answer 220

Gene Expression - Locations of Control

1. Transcriptional control
2. RNA processing control
3. RNA transport and localization control
4. Translation control
5. mRNA degradation control
6. Protein activity control

Question 222

Describe DNA motif recognition.

Answer 222

DNA Motif - Recognition

• Regulatory proteins associate with, recognize specific DNA sequence, and bind to bases in the major groove.
• Major groove presents a specific face for each of the specific base pairs.
• Surface of protein is extensively complementary to the surface of the DNA region to which it binds.
• Contacts made with the DNA involve 4 possible configurations:
  
  • Possible H-bond donors.
  • Possible H-bond acceptors.
  • Methyl groups.
  • H atom.

Question 223

Describe DNA binding motifs.

Answer 223

DNA Binding Motifs

• Gene regulation requires:
  
  • Short stretches of DNA of defined sequence - recognition sites for DNA binding proteins.
  • Gene regulatory proteins - transcription factors that will bind and activate gene.
• Recognition sequences for regulatory proteins e.g. GATA1: TGATAG
• Logo: ex. TAATTGC
• Recognition sequences can be proximal or distal to first exon (few base pairs - 50kb away).

Question 224

Describe the helix-turn-helix DNA binding motif.

Answer 224

DNA-Binding Motifs - Helix-Turn-Helix

• Most simple and common DNA-binding motif.
• 2 alpa-helices connected by short chain of AA that make the “turn” (turned at fixed angle).
• Longer helix = recognition module - DNA-binding module - fits in major groove.
• Side chains of AA recognize DNA motif.
• Symmetric dimers: bind DNA as dimers.

Question 225

Describe the zinc finger domain DNA-binding motif.

Answer 225

DNA-Binding Motifs - Zinc Finger Module

• DNA-binding motif includes Zn atom.
• Binds to major groove.
• Zn finger domains found in tandem clusters.
• Multiple contact points.

Question 226

Describe the Leucine zipper DNA-binding motif.

Answer 226

DNA-Binding Motif - Leucine Zipper

• 2 motifs:
  
  • Type I:
    
    • 2 alpha helical DNA binding domain.
    • Grabs DNA like clothes pin.
    • Activation domain overlaps dimer domain.
    • Interactions between hydrophobic AA side chains (leucines)
  • Type II:
    
    • Dimerizes through leucine zipper region (homo- / hetero-).
    • Interactions between hydrophobic AA side chains (leucines).
    • Leucine residue every 7 AA down one side of alpha-helix in dimerization domain: forms zipper structure.

Question 227

Describe the helix-loop-helix DNA-binding motif.

Answer 227

DNA-Binding Motifs - Helix-Loop-Helix

• Consists of a short and a long alpha-chain connected by a loop.
• Can occur as homodimers or heterodimers.
• Three domains or modules to this protein:
  
  • DNA binding
  • Dimerization
  • Activation

Question 228

How does DNA looping and a mediator complex affect activation?

Answer 228

Transcriptional Activation

• DNA looping and a mediator complex allow the gene regulatory proteins to interact with the proteins that assemble at the promoter.
• Mediator serves as an intermediary between gene regulatory proteins and RNA polymerase II.

Question 229

How can RNA stability regulate gene expression?

Answer 229

Post-Transcriptional Regulation of Gene Expression - RNA Stability

• Most mRNAs have 1/2-life 30 mins (Globin 1/2-life is 10 hours).
• Poly-A tail confers stability and gradual shortening by an exonuclease acts as a timer.
• Once reduced to 25 nt, 2 pathways converge leading to degradation.
  
  1. Decapping - 5’ cap removed and exposed mRNA degraded from 5’ end.
  2. mRNA degraded from 3’ end through poly-A tail and into coding region.

Question 230

How can protein activity control regulate gene expression?

Answer 230

Post-Transcriptional Regulation of Gene Expression - Protein Activity Control

• Post translational modifications required by proteins to be functional.
  
  • Phosphorylated
  • Glycosylated
  • Bind to other subunits or partners.
  • Modified by enzymes (i.e. thrombin cuts fibrinogen to form fibrin).
• Proteins must fold into their 3-D conformations.
• Molecular chaperones help proteins fold.
  
  • Heat shock proteins (Hsp60 & Hsp70)
• Bind co-factors.

Question 231

How can protein degradation regulate gene expression?

Answer 231

Post-Transcriptional Regulation of Gene Expression - Protein Degradation

• Ubiquitin identifies unfolded or abnormal proteins for destruction.
• Ubiquitin binding process:
  
  • E1 ubiquitin activating enzyme links ubiquitin to cysteine side-chain.
  • Transfered to E2 ubiquitin conjugating enzyme (with accessory protein E3 ubiquitin ligase).
    
    • E3 activated by phosphorylation, ligand binding, or protein subunit addition.
  • Transferred to lysine side chain of proteins with degradation signal.
    
    • Degradation signal activated by phosphorylation, unmasking of signal by protein dissociation, or creation of destabilizing N-terminus.
• Ubiquitin chain is recognized by proteasome.
• Aberrant protein destroyed.

Question 233

What are the 4 phases of the cell cycle?

Answer 233

Cell Cycle - 4 Phases

• S phase - DNA synthesis
• M phase - separate chromosomes and divide cells.
• GAP phases - allow more time for growth:
  
  • G₁ - between M & S
  • G₂ - between S & M

Question 234

What are the 3 major transition checkpoints in the cell cycle?

Answer 234

Cell Cycle - Transition Check Points

• Checkpoint I: START - cell commits to cell cycle entry and chromosome duplication (also called restriction point).
  
  • If conditions are not conducive to mitosis, cell cycle will not proceed.
  • Once started, cells will continue cell cycle even if conditions change.
• Checkpoint II: G₂/M - chromosome alignment on spindle in metaphase.
• Checkpoint III: metaphase-to-anaphase transition - trigger sister chromatid separation and cytokinesis.

Question 235

Describe Cdks.

Answer 235

Cell Cycle - Cdks

• Cyclin dependent kinases (Cdks) govern cell cycle, phosphorylate proteins downstream to activate them and regulate cell cycle events.
• Causes cyclical changes in phosphorylation of substrates that regulate cell cycle events.
• Activities of Cdks rise and fall during cell cycle.
• Levels of Cdks remains constant.

Question 236

Describe cyclins.

Answer 236

Cell Cycle - Cyclins

• Proteins that regulate Cdks.
• Levels vary and cycle during the cell cycle (hence name ‘cyclins’).
  
  • Expression controls what step of the cell cycle the cell is in (cyclin present -> cyclin-Cdk complexes formed triggering cell cycle events).
• Cdks are dependent on cyclins - must be bound to cyclin to have protein kinase activity.
• Direct Cdks to their specific target.

Question 237

What are the 4 classes of cyclins?

Answer 237

Cyclins - 4 Classes

1. G₁/S cyclins:
   
   • Start cell cycle
   • Activates Cdks in late G₁, helping trigger progression through START.
   • Levels drop in S phase.
2. S cyclins:
   
   • Stimulates DNA duplication by binding Cdks after progression through START.
   • Levels remain high until mitosis.
3. M cyclins:
   
   • Initiate mitosis by binding to Cdks that stimulate entry into mitosis at G₂/M checkpoint.
   • Removed at mid-mitosis.
4. G₁ cyclins:
   
   • Govern activity of G₁/S cyclins, controlling progression through START checkpoint.

Question 238

What are the 4 Cdks?

Answer 238

Cdk - 4 Types

• 4 different Cdks that form cyclin-Cdk complexes:
  
  • G₁/S-Cdk
  • S-Cdk
  • M-Cdk
  • G₁-Cdk

Question 239

Describe the formation and activation of the cyclin-Cdk complex.

Answer 239

Cyclin-Cdk Complex - Formation & Activation

• Cdk is inactive without cyclin being bound. The active site is blocked by a region of the protein called the T-loop.
• Binding of cyclin causes T-loop to move out of active site, partially activating Cdk.
• Phosphorylation of Cdk at T-loop, by Cdk activating kinase (CAK), fully activates enzyme (“cave site”).

Question 240

Describe the regulation of Cdk activity by an inhibitor.

Answer 240

Cdk - Regulation by Inhibitor

• Wee1 - a kinase inhibits M-Cdk by phosphorylating the “roof” site, inhibiting the M-Cdk complex (ensures lots of primed M-Cdk by end of G₂).
  
  • Cdc25 - a phosphatase that dephosphorylates the “roof” site of M-Cdk, reactivating the M-Cdk complex to initiate mitosis.
  • Double-circuit positive feedback ensures fast M-Cdk activation.
    
    • Activated M-Cdk activates more Cdc25.
    • Cdc25 inhibits Wee1.
• p27 - a Cdk Inhibitory Protein (CKI) that binds to both Cdk and cyclin to inactivate complex.
  
  • Primarily used for control of G₁/S-Cdks & S-Cdks early in cell cycle.

Question 241

Describe APC/C and its role in the transition from metaphase to anaphase.

Answer 241

APC/C - Transition from Metaphase to Anaphase

• APC/C is a ubiquitin ligase that initiates progression from metaphase to anaphase in mitosis.
• Levels rise in mid-mitosis.
• Adds ubiquitin to:
  
  • Securin - protects cohesin protein linkages that hold sister chromatid pairs together in early mitosis by inhibiting separase.
• Cyclins
  
  • Activated by Cdc20.
  • Targets S-cyclins and M-cyclins.
  • Leads to addition of polyubiquitin to M-cyclin in M-Cdk complex.
    
    • Cyclins destroyed, Cdks dephosphorylated.
  • In anaphase S-cyclins destroyed.

Question 242

What are the various DNA-binding domain structural motifs?

Answer 242

DNA-Binding Domain Structural Motifs

• Helix-turn-helix
• Zinc finger motif
• Leucine zipper
• Helix-loop-helix
• Homeodomain
• Beta-sheet

Question 243

What is apoptosis?

Answer 243

Apoptosis

• Programmed cell death, or cell death under physiological conditions.
  
  • Used to eliminate unwanted cells.
  • Important for removal of abnormal, non-functional, potentially dangerous cells.
    
    • Lymphocytes after destroying and ingesting microbes.
    • Cells with DNA damage that cannot be repaired.
  • Used to maintain correct organ size (cell division = cell death).
• Clean form of cell death:
  
  • Tightly controlled and regulated.
  • No spread of damage or initiation of inflammatory process.
• Most common cell death.

Question 244

How is apoptosis critical for development?

Answer 244

Apoptosis - Critical for Development

• Removes unwanted cells.
• Sculpts hands, feet, etc, during embryonic development.
• Examples:
  
  • Tail of tadpole.
  • Development of mouse paw.

Question 247

What are caspases?

Answer 247

Apoptosis - Caspases

• Caspase = cysteine aspartyl specific protease.
  
  • Cysteine in active site.
  • Cleaves proteins at aspartic AA residues.
• Proteases that mediate intracellular proteolytic cascade of apoptosis.
• Activation of caspases is key event.
• 2 Classes:
  
  • Initiater caspases - activates multiple procaspases into executioner caspases.
  • Executioner caspsases - destroys actual targets:
    
    • Cleaves downstream proteins.
    • Cleaves inactive endonuclease.
    • Targets cytoskeleton.
    • Attacks cell adhesion proteins
    • Cells roll up into ball.

Question 248

What are procaspases?

Answer 248

Apoptosis - Procaspases

• Initial, inactive, precursor of caspases.
• Activated by protease cleavage.
• Procaspases cleaved at specific sites to form a large and small subunit which form a heterodimer.

Question 250

Describe the intrinsic apoptotic pathway.

Answer 250

Apoptosis - Intrinsic Pathway

• Cells activate apoptosis from inside cell in response to injury, DNA damage and lack of oxygen, nutrients, or extracellular survival signals.

1. Translocation of cytochrome c from the intermediate space of mitochondria is key event.
2. Released into cytosol and will bind to Apaf1 (procaspase-activating adaptor protein).
3. Apaf1 forms apoptosome which activates caspase-9.
4. Caspase-9 activates downstream exectutioner caspases - caspase-3 (common to both pathways).

Question 251

Describe the Bcl2 family of proteins.

Answer 251

Bcl2 Family of Proteins

• Regulate intrinsic pathway.
• Bcl2 controls release of cytochrome c into cytosol (Bcl = B cell lymphoma)
  
  • 2 types:
    
    • Anti-apoptic (pro-survival) - blocks release of cytochrome c (example: Bcl2)
    • Pro-apoptotic - promotes release of cytochrome c.
  • 4 distinct Bcl homology domains (BH1-4)
    
    • Proapoptotic proteins include BH-123 or BH-3 only.

Question 252

Describe the BH123 protein.

Answer 252

Apoptosis - BH123

• Pro-apoptotic - triggers the intrinsic pathway.
• Become activated.
• Form aggregation in mitochondrial outer membrane.
• Induce release of cytochrome c.
• Apoptosome formed by binding to Apaf1

Question 253

Describe Bcl2.

Answer 253

Apoptosis - Bcl2 Proteins

• Anti-apoptotic proteins.
• Includes Bcl2 and Bcl-XL (Both BH1234)
• Mainly located on cytosolic surface of outer mitochondrial membrane.
• Prevent apoptosis by binding to pro-apoptotic proteins (e.g. BH123) and prevent aggregation into active form.
• Can be inhibited by BH3-only protein.

Question 254

Describe the BH3-only protein.

Answer 254

Apoptosis - BH3-Only Protein

• Pro-apoptotic.
• Activated BH3-only protein is cytosolic.
• Translocates to mitochondria after apoptotic signal activates it.
• Inhibits anti-apoptotic Bcl2 protein from inhibiting aggregation of BH123, to release cytochrome c.

Question 255

Describe IAPs.

Answer 255

Inhibitors of Apoptosis (IAPs)

• Bind and inhibit caspases.
• Some IAPs add ubiquitin to caspases to mark them for destruction by proteasome.
• IAPs block apoptosis by binding to caspases.

Question 256

What are anti-IAPs?

Answer 256

Anti-IAPs

• Pro-apoptotic
• If there is apoptotic stimuli or apoptosis signals, this triggers the release of anti-IAPs from mitochondria to block the activity of IAPs.
• Allowing executioner caspases to be activated.

Question 262

What are 4 types of gene expression controls, other than post-transcriptional and post-translational?

Answer 262

Other Controls of Gene Expression

• Coordinated expression of genes.
• Decision for specialization.
• Methylation and genomic imprinting (which genes get expressed or repressed from mom and dad).
• X-chromosome inactivation.

Question 272

What is condensin?

Answer 272

Condensin

• Five subunit protein complex.
  
  • Related to cohesin.
  • Contains 2 SMC subunits (structural maintenance of chromosomes).
  • Contains 3 non-SMC subunits.
• Forms a ring-like structure and uses ATP to promote compaction and resolution of sister chromatids.

Question 275

Describe the phenotype of apoptosis.

Answer 275

Apoptosis - Phenotype

• Overall shrinkage of cell volume and nucleus.
• Loss of adhesion to neighboring cells.
• Formation of blebs on surface.
• DNA fragmentation.
• Cytoskeleton collapses.
• Nuclear envelope disassembles.
• Rapid engulfment of dying cell by phagocytosis.

Question 276

What are the biochemical characteristics of apoptosis?

Answer 276

Apoptosis - Biochemical Cahracteristics

• DNA fragmentation visible on agarose gel.
  
  • Endonucleases cleave DNA in linker regions of nucleosomes.
• Cytochrome C released from mitochondria is a marker of apoptosis.

Question 279

Describe the caspase cascade.

Answer 279

Apoptosis - Caspase Cascade

• Machinery always in place.
• Irreversible.
• 2 pathways:
  
  • Internal:
    
    • Stimuli - DNA abnormalities.
    • Mitochondrial dependent.
  • External:
    
    • Stimuli - removal of survival factors and proteins of tumor necrosis factor family.
    • Mitochondrial independent.

Question 280

Describe cancer.

Answer 280

Cancer

• It takes time to accumulate the mutations necessary for cancer to develop.
• Graph: Incidence colon cancer vs. age.

Question 281

Describe the genetic causes of cancer.

Answer 281

Cancer - Genetic Causes

• Two broad types of mutations in cancer.
• Mutations of genes that regulate cell proliferation.
• Overactivity mutations: gain of function – oncogenes – involves single mutation event and activation of gene causing proliferation (dominant) “The Gas Pedal.”
• Underactivity mutations: loss of function – tumor suppressor genes - involve genes that inhibit growth. Mutation event: one gene – no effect; second mutation causes problems (recessive) “The Brake Pedal.”

Question 282

Describe the activation of oncogenes.

Answer 282

Activation of Oncogenes

• These mutations are dominant, only one allele needs to be present for activity.

Question 283

Describe tumor suppressor genes.

Answer 283

Tumor Suppressor Genes

• Tumor suppressor genes generally encode proteins that inhibit cell proliferation.
• 2 major categories of tumor suppressor genes:
  
  1. Proteins that normally restrict cell growth and proliferation.
  2. Proteins that maintain integrity of the genome

Question 284

Describe retinoblastoma.

Answer 284

Retinoblastoma

• Two forms of retinoblastoma: hereditary form and sporadic form.
• 40% of retinoblastoma is familial in which both eyes are affected (tumors).
• 60% of retinoblastoma is sporadic (no family history) – single tumor one eye.

Question 285

Describe hereditary form of Rb.

Answer 285

Rb - Hereditary Form

• Loss of function or deletion of one copy of Rb in every cell - because defect is inherited.
• These cells are predisposed to be cancerous.
• But have one good copy of Rb gene.
• Somatic event occurs – eliminates one good copy and tumor forms.
• Loss of heterozygosity.

Question 286

Describe the sporadic form of Rb.

Answer 286

Rb - Sporadic Form

• Non-hereditary
• Non-cancerous cells fine – no mutation of Rb.
• Cancerous cells have both copies of Rb mutated.
• Two hit hypothesis – first Rb gene obtains mutation then need second mutation Rb.
• Familial: already have one mutation – predisposed to cancer.
• Sporadic – 2 normal Rb genes, one hit then 2nd hit so more rare than hereditary form.
• Rb protein regulator of cell cycle.

Question 287

Describe the inactivation or loss of tumor suppressor genes.

Answer 287

Tumor Suppressor Genes - Inactivation or Loss

• These mutations are recessive, both copies of the gene (alleles) need to be inactive/lost.

Question 288

Describe the polyp in the tumor progression of colon cancer.

Answer 288

Tumor Progression - Polyp

• Polyp is precursor of colorectal cancer.
• Slow disease progression: 10 years.
• Cut off polyp – cure.
• If left alone, malignant tumor develops from adenoma (polyp).

Question 289

Describe the common mutations in colorectal cancer.

Answer 289

Colorectal Cancer - Common Mutations

• Important loss is Apc mutation: loss of function – Apc is a tumor suppressor gene.
• 40% of colorectal cancers have point mutation in K-Ras.
• 60% inactivating mutation of p53.

Question 290

What are the functions of the cytoskeleton?

Answer 290

Cytoskeleton - Functions

• Represents bones of the cell.
• Important in organization of cell.
• Maintains correctly shaped cells.
• Insures cells are properly structured internally.
• Moves cell.
• Re-arranges the cellular compartment.
• Supports plasma membrane.
• Provides the mechanical strength - resistance to the stress without being ripped apart.
• Pulls chromosomes apart during cell division.
  
  • Defect can lead to chromosome # abnormality.
• Splits dividing cells during cell division.
• Guides intracellular traffic of organelles.
• Vesicles move around by using cytoskeleton as a sidewalk.
• Cells like sperm need to swim - cytoskeleton acts as motor.
• WBCs and fibroblasts need to crawl.
• Muscle cell contraction.

Question 291

What happens when cytoskeleton formation or regulation goes wrong?

Answer 291

Disease occurs.

Question 292

Describe the function of the cytoskeleton in RBCs.

Answer 292

RBC - Cytoskeleton Function

• Responsible for shape of cell (biconcavity).
• Gives strength to the cells.
  
  • RBCs once released from bone marrow, go through 1/2 mil passages in the circulation.
  • Must be flexible and strong enough to squeeze through capillary passages.
  • Must resist shear forces of heart contraction.
• If defective, leads to Hereditary Spherocytosis.
  
  • RBCs spherical not bi-concave.
  • Fragile
  • Causes hemolytic anemia which can be severe and lethal.

Question 293

What are the 3 main types of filaments?

Answer 293

Cytoskeleton - Types

• Actin filaments.(like Mardi-Gras beads)
• Microtubules (slinky of life)
• Intermediate filaments (like girders in building)

Question 294

Describe actin filaments.

Answer 294

Cytoskeletal Filaments - Actin Filaments

• Two stranded helical polymers of the protein actin.
• Subunits are compact, globular and self-associate using a combination of end-to-end and side-to-side interactions.
• Flexible structures 5-9 nm in diameter.
• Determine shape of cell’s surface and are necessary for whole-cell locomotion, secretion, endocytosis.
• Can form cell surface projections that help move cells over solid substrate:
  
  • Lamellipodia - flat protrusive veils.
  • Filopodia or microvilli, spiky bundles.
  • Catalyzed by two types of regulated factors:
    
    • ARP complex
    • Formins

Question 295

Describe microtubules.

Answer 295

Cytoskeletal Filaments - Microtubule

• Forms long, hollow, tube-like structure (25nm).
• Made of tubulin subunits that are compact and globular.
• One end attached to a single microtubule-organizing center called a centrosome.
• Determine the positions of membrane-enclosed organelles.
• Directs intracellular transport.
• Make up centrioles and mitotic spindle.
• Found in cilia and flagella.
• Lack of function in cilia = cystic fibrosis.

Question 296

Describe intermediate filaments.

Answer 296

Cytoskeletal Filaments - Intermediate Filaments

• Rope-like fibers (10nm)
• Formed by smaller elongated, fibrous subunits from large heterogeneous family.
• Extend across cytoplasm to provide mechanical strength.
• Span from one cell-cell junction to another to strengthen the epithelial sheet.
• Staggered side to side binding of filaments, forming rope-like structures, allowing filaments to tolerate bending and stretching
• Allows formation of hair and fingernails.

Question 297

Describe the dynamic nature of actin.

Answer 297

Actin - Dynamic and Adaptable

Question 298

Describe the dynamic nature of microtubules.

Answer 298

Microtubule - Dynamic and Adaptable

Question 300

Describe actin polymerization.

Answer 300

Actin - Polymerization

• Actin monomer contains a binding site for ATP or ADP.
  
  • ATP-actin polymerizes
  • ADP-actin depolymerizes
• Actin filaments consist of two protofilaments that twist around each other in a right hand helix.
• Held together by lateral contacts.
• Arranged head-to-tail to generate structural polarity (plus and minus ends).
  
  • Plus end - fast-growing or fast-shrinking
  • Minus end - slow-growing or slow-shrinking.
• Flexible and easily bent.

Question 301

Describe microtubule polymerization.

Answer 301

Microtubules - Polymerization

• Tubulin is a hetero-dimer of alpha and beta tubulin with non-covalent bonds.
• Both have binding site for GTP.
• Tubulin subunits form helical assemblies.
• Self-associate, using a combination of end-to-end and side-to-side interactions.
  
  • Form stiff, hollow, tube made of 13 proto-filaments aligned parallel with alternating alpha-beta subunits.
• Tubulin is polar:
  
  • Plus end - fast growing and shrinking.
  • Minus end - slow growing and shrinking.

Question 302

What must occur prior to the formation of a large filament?

Answer 302

Nucleation - usually requires random collision of 3 subunits.

Question 306

Describe spectrin.

Answer 306

Accessory Proteins - Spectrin

• Attaches to membrane.
• Provides durability and stability in RBCs.
  
  • RBCs circulate 1/2 million times.
  • Tight capillary spaces.
• If defective, fragile RBCs result causing hemolytic anemia (Hereditary Spherocytosis).

Question 307

Describe hereditary spherocytosis.

Answer 307

Hereditary Spherocytosis

• Hemolytic anemia characterized by spherical and fragile RBCs that lyse and release Hgb.
• Most common hemolytic anemia in people of North European Descent (incidence 1/2000).
• Clinical Presentation: hemolysis, anemia, splenomegaly.
  
  • Ranges from mild to severe anemia and can be fatal.
• Caused by mutations in genes for the erythrocyte membrane skeleton of RBCs.

Question 308

What causes hereditary spherocytosis?

Answer 308

Hereditary Spherocytosis - Cause

• Erythrocyte membrane skeleton confers property of durability and stability to RBCs.
  
  • Failure of the EMS leads to mis-shapen and fragile RBCs.
• EMS is dependent upon:
  
  • Ankyrin - attaches spectrin to Band 3 in PM.
  • Spectrin - filaments of the EMS.
  • Band 3 - membrane bound protein that is bound by ankyrin which also binds spectrin.
  • Band 4.2
• Deficiency in any of these proteins can cause fragility.

Question 309

Describe spectrin’s architecture.

Answer 309

Spectrin - Architecture

Question 310

What testing can be used to identify protein defficiencies or mutations significant for hereditary spherocytosis?

Answer 310

Hereditary Spherocytosis - Testing

• RBC EMS Protein Gel
• Osmotic fragility test

Question 311

Describe the osmotic fragility test.

Answer 311

Osmotic Fragility Test

• A few drops of blood placed into slightly hypotonic saline.
• Water rushes in by osmosis.
• Normal cells will swell but not break.
• HS cells will swell and, because they are fragile, will break releasing Hgb.
• Can also be done using increasingly hypotonic solution to develop osmotic fragility curve.

Question 313

Describe the variation of actin filament life span.

Answer 313

Actin Filament - Life-Span

• Intestinal cells - must last a few days, actin filaments are replaced every 48 hours.
• Actin bundles in hair cells of inner ear must last a lifetime.

Question 317

What is listeria?

Answer 317

Listeria

• Pathogenic bacteria that invade your intestinal cells.
• Ubiquitous in soil.
• Found on:
  
  • Unwashed lettuce, animal products, dairy and meats.
• Worst case 1988 outbreak from hotdogs.

Question 318

Describe the Listeria infection.

Answer 318

Listeria - Infection

• Causes serious infection.
• Symptoms:
  
  • HA
  • Stiff neck
  • Confusion
  • Loss of balance
  • Convulsions
  • Fever and muscle aches
• Treatment requires antibiotics.
• Attaches to receptors on enterocytes.
• Enters and replicates in intestinal cells.
• Moves using actin and accessory proteins such as Arp2/3.
  
  • moves at approx. 0.2 micrometers per second.
  • Leaves actin track (comet trails)
  • Bacterial surface causes local nucleation of actin filaments by presenting ActA (activating factor).
  • Cofilin makes branched actin disassemble.
• Causes problems by smashing through organelles.

Question 319

Describe the occurence of Listeria infection.

Answer 319

Listeria - Infection

• More likely to develop food poisoning if immunologically impaired or immunocompromised.
• Pregnant women 10x more likely to get Listeria infection.
• About 1600 Listeria infections in US annualy.
• Most require hospitalization.
• 1 in 5 die.
• Infection in pregnancy can cause miscarriage, stillbirth, or newborn death.
  
  • Fetal loss - 20%
  • Newborn death - 3%

Question 326

To what extent do each of the cytoskeletal proteins contribute to Hereditary Spherocytosis?

Answer 326

EMS Mutations in HS

• Spectrin/Ankyrin defect - 63%
• Band 3 defect - 22%
• Protein 4.2 defect - 3%
• Other defects - 2%
• No known defect - 10%

Question 327

What is the difference between Duchenne and Becker muscular dystrophy?

Answer 327

Muscular Dystrophy

• Duchenne - complete absence of cytoskeletal protein dystrophin.
• Becker - dystrophin protein made but abnormal.

Question 328

Describe Duchenne muscular dystrophy.

Answer 328

Duchenne Muscular Dystrophy

• Most common fatal neuromuscular disorder.
• Severe, progressive muscle degeneration.
• Loss of ability to walk - in wheelchair by 12 years of age.
• Loss of lung and cardiac function.
• Scoliosis
• Death 20-30 years of age from respiratory failure or cardiomyopathy.
• No cure.

Question 329

Describe Duchenne muscular dystrophy treatment.

Answer 329

Duchenne Muscular Dystrophy - Treatment

• No medical treatment that can significantly alter course of disease.
• Maintain patient’s general health.
• Improve quality of life.
• Gluccocorticoids (prednisone) slow the decline in muscle strength but the effect is relatively short (18-36 months) and do not alter the overall clinical course of disease.

Question 330

Describe the Duchenne Muscular Dystrophy outcome.

Answer 330

Duchenne Muscular Dystrophy - Outcome

• Relentless
• Only supportive measures available:
  
  • Physiotherapy
  • Physical aids: wheelchair dependent at 12.
  • Respiratory assistance.
• Many patients now live into 20’s.
• Abnormal hearts and/or respiratory failure causes death.

Question 331

Describe the genetic factors of Duchenne Muscular Dystrophy.

Answer 331

Duchenne Muscular Dystrophy - Genetics

• One of the most common neuromuscular genetic diseases in man (1/3500 male births)
• X-linked recessive.
• Caused by dystrophin gene mutations.
• Largest gene known: produces 427 kDa protein; 14 kb RNA; 79 exons.
• Genetic defect present at birth but does not show symptoms until about 3 years of age (most DMD boys not recognized at birth).

Question 332

Describe the dystrophin protein.

Answer 332

Dystrophin Protein

• Main function is to provide structural stability to the muscle cell membrane during cycles of contraction and relaxation.
• Localized to inner surface of muscle membrane.
• Loss results in destabilization of entire dystrophin-glycoprotein complex.
• 4 functional domains:
  
  • N-terminus
  • Long spectrin-like repeat domain (the cytoskeletal portion)
  • Cysteine rich and C-terminus domains:
    
    • Bind to syntrophin proteins (linking proteins)
    • Bind to dystroglycans/sarcoglycans (dystrophin-associated glycoprotein complex) - transmembrane proteins

Question 334

What is the clinical presentation of a Duchenne musular dystrophy patient?

Answer 334

DMD - Clinical Presentation

• Dystrophic myopathy = progressive muscle degeneration with loss of functional muscle tissue over time with resulting weakness.
• Onset early in childhood.
• Mean age of Dx: 4y 10m.
• Wheelchair by 12-13y.
• Slow walking & generalized weakness.
• Elevated CK in blood (50-100x normal, greater than 1,000 U/L)
• Necrosis of muscle fibers occurs with replacement of fat or connective tissue.
• Leads to pseudohypertrophy: replacement of muscle with adipose and fibrous CT (example: enlarged calves).
• Changes in stature/movement:
  
  • Waddling run/walk.
  • Difficulty in climbing steps.
  • Use of handrail to pull.
  • Walk on tiptoes.
  • Lordosis (excessive inward curvature of spine).
  • Kyphosis (curvature of upper back(humpback))
  • Scoliosis
  • Frequent falls.
  • Gower maneuver (walking hands up legs to stand).

Question 335

What testing can be used to identify Duchenne muscular dystrophy?

Answer 335

DMD - Testing

• Electromyography (EMG) - records electrical activity of muscles.
  
  • Complex repetitive discharges (CRDs) - abnormal spontaneously firing action potentials associated with membrane instability.
    
    • Hallmark abnormality in dystrophinopathies.
• Blood work - elevated CK (50-100x normal)
• Muscle biopsy - histology: proteins
• DNA gene deletion studies - PCR
• Quantitative dystrophin analysis - western blot
• Prenatal DX using fetal DNA (amniocentesis, Chorionic villus sampling (CVS))
• Preimplantation genetics - in IVF, blastomeres can be tested prior to embryo transfer.

Question 336

In the distrophin gene, what are the deletion hotspots?

Answer 336

Dystrophin - Deletion Hotspots

• Exons 3-19 and 42-60

Question 338

What mutations lead to muscular dystrophy?

Answer 338

Genetic Mutations

• Large deletions - 50-70%
• Small deletions, insertions, nt changes (stop codons) - 25-30%
• Large duplications - 5-10%
• BMD associated with in-frame deletions:
  
  • Dystrophin protein of abnormal size.
• DMD associated with frameshift mutations:

Question 340

Describe Becker muscular dystrophy.

Answer 340

Becker Muscular Dystrophy

• Milder form than Duchenne.
• Incidence 1/18,000.
• Similar symptoms.
• Loss of walking after 16y.
• Muscle pain, dilated cardiomyopathy.
• Increased workload on left ventricle leads to enlargement and potentially failure and death.

Question 341

Describe strategy for the use of retinal dystrophin.

Answer 341

Human Retinal Dystrophin

• Retinal dystrophin (DP260):
  
  • Similar to muscle dystrophin but smaller in size (260kDa vs 427kDa)
  • Has 3 of the functional domains of muscular dystrophin (spectrin, Cys, C-term)
  • Mini-dystrophin (instead of micro-dystrophin)
  • Endogenous in 30% of DMD patients.
  • No immunogenicity issue.
• Dystrophin gene has many products driven by different promotors:
  
  • Every cell contains complete gene, thus potentially the ability to express other forms.
• Expression of retinal dystrophin is driven by muscle creatine kinase (MCK) promoter/enhancer.
  
  • If it can be triggered in muscle cells, retinal dystrophin can function in place of regular dystrophin.
• Has been shown effective in mice.

Question 342

Compare DMD & BMD.

Answer 342

DMD vs. BMD

• Both X-linked recessive.
• DMD:
  
  • Worse than BMD.
  • Incidence:
    
    • 1/3 of case are de novo mutations.
    • 2/3 of cases are familial (female carrier).
  • Complete lack of dystrophin.​
  • Do get frameshifts leading to stops
  • Wheelchair-bound by 12yo.
  • Average life span: 25y.
• BMD:
  
  • Variability in symptoms.
  • No frameshifts: protein made.
  • Some dystrophin, but abnormal in quantity and size.
  • Male still walking at 16yo.
  • Average life span: 45y.

Question 344

Describe the pathophysiology of muscular dystrophy.

Answer 344

Muscular Dystrophy

• Muscle membrane susceptible to mechanical injury.
• Injury to sarcolemma.
• Ca²⁺ influx/oxidative stress.
• Degeneration of fibers.
• Cycles of degeneration/regeneration.
• Irreversible necrosis/loss of fiber.
• Replacement of fiber by fat & CT.

Question 345

What are some projected treatment strategies for DMD?

Answer 345

DMD - Treatment Strategies

• Growth factors:
  
  • Myostatin inhibitors = muscle cell growth (more muscle).
    
    • Hypothetically could work, but would make more defective muscle.
• Gene therapy:
  
  • Replace full-length dystrophin gene.
    
    • Problem - too large for adeno-associated virus.
  • Utrophin (similar to dystrophin gene not on X chromosome).
    
    • Try to over express, does not work.
  • Microdystrophins.
    
    • BMD shows that large portion of spectrin-like repeats can be deleted from dystrophin and still be functional.
    • Works, but issue with immunogenicity.
• Exon skipping:
  
  • Deletions cause frame shift mutations, skipping exon would put reader back in frame and generate BMD-like dystrophin.
• Nonsense (stop) codon read-through:
  
  • Works…but problem with immunogenicity.
• All approaches have problems.

Question 346

Describe mitochondria.

Answer 346

Mitochondria

• Maternally inherited - mitochondria in eggs.
• Self replicate, segregate during cell division by chance.
• Rate of mtDNA mutation is higher than nuclear genes.
• Provide cellular energy in the form of ATP for the cell by:
  
  • ETC
  • Oxidative phosphorylation.
• Have their own DNA / genome.
  
  • Contains 37 genes for 13 proteins and 24 parts of the machinery to make proteins (rRNA and tRNA).
  • Replicates and gets passed on to new mitochondria.
• If mtDNA mutation occurs, a mixture of normal mitochondria and mutant mitochondria occurs in one cell - called heteroplasmy.
  
  • Need certain level of aberrant mitochondria vs. normal mitochondria for disease to occur.
  • Threshold effect of mutant mitochondria are required for disease manifestation.

Question 348

What are mitochondrial myopathies?

Answer 348

Mitochondrial Myopathies

• A muscle disease caused by mitochondrial dysfunction.
• Characteristics of mitochondrial disorders: clinical variability and age related progression of disease.
• 4 major types of Mitochondrial Genetics Diseases:
  
  • MERRF: myoclonus epilepsy with ragged red fibers (tRNA mutation).
  • MELAS: mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (tRNA mutation).
  • KSS: Kearns-Sayre Syndrome (mtDNA rearrangements).
  • CPEO: chronic progressive external opthalmoplegia (mtDNA rearrangements).
• LHON - Leber Hereditary Optic Neuropathy - blindness in late adolescence, muscle not affected (SBP substitution in mtDNA coding for Complex I).

Question 349

What are mitochondrial myopathies with ragged red fibers?

Answer 349

Mitochondrial Myopathies - Ragged Red Fibers

• Mitochondrial myopathy where aggregates of abnormal mitochondria form red sarcolemmal blotches called Ragged Red Fibers.

Question 350

Describe mutant segregation of mitochondria.

Answer 350

Mitochondria - Mutant Segregation

• Miotochondria segregate, during cell division, by chance.
• Ratio of mutant mitochondria in daughter cells is random and may or may not meet the threshold needed for disease.

Question 351

What are the clinical characteristics of mitochondrial myopathies?

Answer 351

Mitochondrial Myopathies - Clinical Characteristics

• Muscle weakness
• Excercise intolerance
• Lactic acidosis
• Neurological signs: mitochondrial encephalopathies.
• Other associated abnormalities:
  
  • Vomiting, seizures, dementia
  • Stroke-like episodes
  • Ptosis
  • Opthalmoplegia (paralysis of extraocular muscles)
  • Blindness
  • Cardiomyopathy

Question 352

Describe mitochondrial inheritance.

Answer 352

Mitochondrial Inheritance

• Passed from mother to offspring.

Question 354

Describe MERRF.

Answer 354

MERRF - Myoclonus Epilepsy with Ragged Red Fibers

• Clinical features:
  
  • Myoclonus - involuntary jerking of muscle, often first symptom.
  • Myoclonic epilepsy
  • Ataxia (lack of coordinated muscle movements).
  • Ragged Red Fibers (muscle tissue)
  • Seizures, dementia.
• 90% caused by 2 mutations of tRNA^Lys.
• Mutations:
  
  • 85% due to A to G mutation in the mtDNA tRNA^Lys gene at nucleotide position 8344.
  • 5% due to G to C at position 8356 in tRNA^Lys mtDNA gene.

Question 356

Describe MELAS.

Answer 356

MELAS

• Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes.
• Clinical presentation:
  
  • Seizures
  • Stroke-like episodes of hemiparesis (weakness on one side of body).
  • Blindness
  • Headaches
  • Anorexia
  • Recurrent vomiting
  • Lactic acidosis
  • Ragged Red Fibers
• AOO: 2-10 years old (60%)
• Mutation:
  
  • Typically caused by A3243G mutation in tRNA^Leu.

Question 357

Describe mitochondrial genetics in disease.

Answer 357

Mitochondrial Genetics in Disease

• Heteroplasmy: mixture of normal and abnormal mitochondria.
• Threshold - if cells carry too many mutant mitochondria - disease results.
• Need a mutation to occur AND a certain percentage of the mitochondria to be aberrant.
• Some tissues require more energy than others - e.g. neural and muscle tissue.
• Neurological and muscle tissues affected by mitochondrial genetic diseases.
  
  • Brain/CNS, heart, skeletal muscle.

Question 358

Describe CPEO.

Answer 358

CPEO

• Chronic Progressive External Opthalmoplegia
• Mild to moderate mitochondrial myopathy.
• Clinical presentation:
  
  • Ptosis
  • Red Ragged Fibers observed in skeletal muscle.
• Mutation:
  
  • mtDNA rearrangements.

Question 359

Describe LHON.

Answer 359

LHON

• Leber Hereditary Optic Neuropathy
• Mitochondrial mutation that only affects optic nerve.
• No muscle involvement.
• Clinical presentation:
  
  • Acute or subacute, bilateral, central vision loss.
    
    • Onset and progression typically rapid.
    • Initially affects one eye, but eventually both eyes affected at same time.
  • Degeneration of the retinal ganglion cell layer and optic nerve.
• AOO: 20-30 years old.
• Mutation:
  
  • Single base pair substitutions of mtDNA coding genes of Complex I proteins.
  • No tRNA or mtDNA rearrangements.

Question 361

Describe mitochondrial mutations.

Answer 361

Mitochondria - Mutations

• 2 major divisions:
  
  • Point mutations in mtDNA tRNA genes lead to MELAS and MERRF.
  • mtDNA genome deletions and rearrangements lead to KSS and CPEO.

Question 363

Describe the role of heteroplasmy and MERRF.

Answer 363

Heteroplasmy - MERRF

• Characteristics have clinical variability and age related progression of disease due to heteroplasmy.
• Example: MERRF - there is a genotype/phenotype correlation.
  
  • 25yo - 95% mutant tRNA^Lys has severe clinical presentation.
  • 25yo - 85% mutant tRNA^Lys is normal and healthy, but develops disease later in life.

Question 365

Describe Kearns-Sayre Syndrome.

Answer 365

Kearns-Sayre Syndrome

• Clinical presentation:
  
  • Retinitis pigmentosa (degenerative eye disease leading to blindness)
  • At least one of the following:
    
    • Cardiac conduction abnormality.
    • Cerebelar ataxia.
    • Cerebral spinal protein level above 100 mg/dL.
  • May present with optic atrophy, hearing loss, dementia, seizures, cardiomyopathy, lactic acidosis.
  • Red Ragged Fibers seen in skeletal muscle.
• AOO: before 20 years old
• Mutation:
  
  • 85% of KSS due to mtDNA rearrangements including duplicated mtDNA, deleted mtDNA and insertions.
  • 200 different deletions.

Question 366

Describe erythrocytes.

Answer 366

Erythrocytes

• Typical person has 25 trillion RBCs.
• Last 120 days.
• Old, damaged RBCs are removed from the circulation and they need to be constantly replaced.
  
  • Produce 2.4 million RBCs per second.
• Can carry 500-1,000+ L of O₂ per day, depending on activity.
• Hb occupies 33% of the volume of an RBC and 90% of the cell’s dry weight.
• 2 lbs of Hb in normal person.

Question 367

Describe erythropoiesis.

Answer 367

Erythropoiesis

• 65% of Hgb is synthesized before the extrusion of the nucleus.
• Reticulocyte makes remaining 35% of Hgb.
• In reticulocytes, 95% of all protein synthesis is globin synthesis.

Question 368

Describe tissue switching in erythropoeisis.

Answer 368

Erythropoeisis - Tissue Switching

Question 370

Describe the structure of hemoglobin.

Answer 370

Hemoglobin - Structure

• Multi-subunit protein (tetramer):
  
  • 2 α-globin chains
  • 2 β-globin chains
• 8 helical segments to a globin subunit and they are labeled A-H.
  
  • AAs are named according to the helical segment and AA# in that segment (e.g. F8 His is 8th residue in F helix which is 6th segment).
• Heme:
  
  • Prosthetic group - a non-amino acid group of a protein.
  • Nestled in a hydrophobic crevice of the protein chain.
  • One per subunit.
  • Gives blood its color.
  • 75% of globin chain forms α-helix.
  • Has Fe atom that carries O₂.
• 4 protoporphyrin IX rings.

Question 372

Describe the function of hemoglobin.

Answer 372

Hemoglobin - Function

• Best molecule, due to structure, for transporting O₂ from the lungs to tissues.
• “Hb is a lung miniature - breathing as it allows the body to respire.”

Question 374

Describe hemoglobin switching.

Answer 374

Hemoglobin Switching

• Not fully clear how hemoglobin switching occurs.
• Switch from HbF to HbA is closely related to gestational age - time controlled.
  
  • Premature infants switch later after birth to HbA than full-term babies.

Question 375

Describe the types of hemoglobins.

Answer 375

Hemoglobins - Types

• 3 types:
  
  • Embryonic:
    
    • Expressed in yolk sac but not after 8 weeks gestation.
    • Embryonic Hb (ζ2ε2)
  • Fetal:
    
    • Hbf made predominantly in liver (and bone marrow)
    • In fetus, 90-95% HbF until 34-36 weeks gestation.
    • HbF (α2γ2)
  • Adult:
    
    • HbA (α₂β₂) production starts after birth.
    • Reaches adult levels at 1y.
• 4 types of globin chains:
  
  • α
  • β
  • δ
  • γ
• Forms:
  
  • HbA (α₂β₂) - 97% (predominant form).
  • HbA2 (α₂δ₂) - 3%
  • HbF (α₂γ₂) - 0.5%

Question 376

Describe HbF.

Answer 376

Hemoglobin - HbF

• Fetus requires Hb that has a higher affinity for O₂ than mothers Hb so that O₂ can pass from mother to fetus.
• HbF (α₂γ₂)
• Does not bind well to 2,3-BPG:
  
  • Allowing increased affinity for O2.
  • Keeping HbF mostly locked in R form.

Question 377

Describe the types of α and β Hb chains.

Answer 377

Hemoglobin - Chains

• α-like chains:
  
  • ζ (zeta) embryonic
  • α (alpha)
• β-like chains:
  
  • ε (epsilon) embryonic
  • γ (gamma) fetal
  • δ (delta)
  • β (beta)

Question 378

Describe the globin genes.

Answer 378

Hemoglobin - Globin Genes

• Chromosome 11 - β globin genes:
  
  • ε, γ^g, γ^a, δ, β
  • γg = glycine 136
  • γa = alanine 136
• Chromosome 16 - α globin genes:
  
  • ζ, α₂, α₁

Question 380

Describe sickle cell anemia and HbF.

Answer 380

Hemoglobin - Sickle Cell and HbF

• Hbs - E->V substitution at position 6 in β globin.
  
  • Causes polymerization.
  • Sickle shaped RBCs impede circulation - hemolytic anemia.
  • Pain, organ damage, strokes, increased infections, etc.
• Research involving HbF expression is ongoing.
• Currently using hydroxyurea to induce HbF and address inflammation, but this is a chemotherapeutic agent.

Question 381

Describe DNA-only transposons.

Answer 381

DNA-only Transposons

• Exist only as DNA in their movement.
• Predominate in bacteria (largely responsible for the spread of bacterial resistance).
• Use cut-and-paste transposition or replicative transposition.
• DNA only transposons contain:
  
  • Gene encoding transposase.
  • Sequences recognized by the enzyme necessary for movement.

Question 382

How do retrovirus-like transposons move?

Answer 382

Retrovirus-like transposon transposition steps:

• Entire transposon is transcribed by host
  
  • RNA transcript contains reverse transcriptase enzyme
    
    • Translated by host cell
• Reverse transcriptase makes a DS DNA copy of RNA molecule via hybrid DNA/RNA intermediate.
• DS DNA integrates into site on chromosome using integrase, encoded by element.
• Integrase cuts one strand at each end of the viral sequence
• Each exposed 3’ OH ends attacks a phosphodiester bond of target DNA.
• This inserts the viral DNA into target, leaving gaps to be filled/ligated.
• Leaves short repeats on each side of integrated DNA segment.

Question 383

How do nonretroviral transposons move?

Answer 383

Transposition by Nonretroviral Transposons

• Endonuclease and reverse transcriptase bind to L1 RNA
• Endonuclease nicks the target DNA at insertion point
  
  • Releases 3’ OH to serve as primer in reverse transcription step.
• SS DNA copy of L1 directly liked to target DNA.
• Insertion of DS DNA copy of L1 at target site.

Question 384

How can conservative site specific recombination be used to turn genes on and off?

Answer 384

Conservative site specific recombination can be used to turn genes on and off by removing or reinserting segments containing the genes of interest.

Question 386

What are nonretroviral transposons?

Answer 386

Nonretroviral Transposons

• Comprise a large portion of our genome
  
  • Repeated sequences are mutated and truncated nonretroviral transposons
• Most are immobile but few retain ability to move.
  
  • L1 element (LINE or long interspersed nuclear element)
• Require endonuclease and reverse transcriptase to move.
  
  • Do not encode the enzymes
  • Use enzymes from other transposons
• Make up 40% of the human genome.

Question 388

What are the 5 phases of prophase?

Answer 388

5 phases of prophase:

• Leptotene - homologs begin to condense/pair.
• Zygotene - homologs pair and synaptonemal complexes form.
• Pachytene - Synapsis is complete; crossing over occurs.
• Diplotene - Synaptonemal complex begins to break down; homologs begin to separate but remain attached at chiasmata.
• Diakinesis - Reach maximum condensation; separation of homologs and transition stage into metaphase.

Question 391

What are retrovirus-like transposons?

Answer 391

Retrovirus-like transposons

• Resemble retroviruses, but lack a coat.
  
  • Move in an out of chromosomes the same way but unable to leave the resident cell.

Question 393

What are transposons?

Answer 393

Transposons are:

• Mobile genetic elements also called transposable elements, “selfish DNA” and “jumping genes”
• Specialized segments of DNA that move from one position in the genome to another.
• Range in size from 100s to tens of thousands of nt pairs.
  
  • Each with unique set of genes and encodes enzyme that catalyzed movement of transposon.
• Can provide benefits to the cell (i.e. antibiotic resistance in bacteria)
• Can produce genetic variation or spontaneous mutations.
• No sequence homology required - can insert anywhere in the genome.
• Move infrequently: 1 in every 10⁵ cell divisions in bacteria.
• Transposase - enzyme encoded by the transposon itself
  
  • Acts on specific DNA sequence on each end of the transposon
  • Allows insertion into a target DNA site.