Cell Bio Exam 3 Flashcards
(136 cards)
1
Q
What are the five components of the nucleus?
A
1) Nuclear envelope 2)Nuclear lamina 3)Nucleoplasm 4)Nucleoli 5)Chromatin
2
Q
What is the nuclear envelope?
A
The double membrane surrounding the nucleus. Consists of an outer and inner membrane and is perforated by large nuclear pores. The outer membrane is continuous with the RER and contains ribosomes.
3
Q
What is the nuclear lamina?
A
The cytoskeleton of the nucleus, a fibrous meshwork of proteins on the inner surface of the inner nuclear membrane, made up of a network of intermediate filaments formed from nuclear lamins
4
Q
What is the function of nucleoli?
A
Nucleoli synthesize ribosomes by producing the rRNA molecules and assembling them into ribosomes
5
Q
What is chromatin?
A
The complex of DNA, histones, and nonhistone proteins found in the nucleus of eucaryotic cells. These chromatins make up chromosomes.
6
Q
How is the nuclear envelope shape supported?
A
The nuclear lamina supports the nuclear membrane from the inside. Lamin protein filaments make up the cortical skeleton of the lamina.
7
Q
Describe the role of the lamina in DNA replication.
A
Chromatin attaches to sites on the lamina during DNA replication. Lamina acts as a scaffold to regulate the replication.
8
Q
What are SPECKLES?
A
Microdomains within the nucleus where mRNA is spliced. These are not evenly distributed throughout the nucleoplasm.
9
Q
What are Cajal bodies?
A
These bodies bind to the nucleolus in actively transcribing cells, and may generate snRNPs
10
Q
What are GEMs?
A
Geminis of the coiled bodie. These are associated with Cajal bodies and play a role in snRNP assembly for splicing. They contain SMN (survival of motor neuron) proteins, which are required for snRNP formation.
11
Q
What are PML bodies?
A
Promycrocytic leukemia bodies. They play a role in transcription regulation and show increased expression in lymphoid cancers and other diseases.
12
Q
Which direction do nuclear pores allow movement: IN or OUT?
A
Two of the above are true (LOL). Nuclear pores allow transport in AND out of the nucleus.
13
Q
Describe the size of molecules able to travel through nuclear pores.
A
5kDa molecules can freely pass, 17 kDa molecules take time to pass, and 60 kDa don’t pass at all
14
Q
What shape are nuclear pores seen on a freeze fracture scanning electron micrograph?
A
Nuclear pores are star shaped.
15
Q
Describe the nuclear pore complexes.
A
They are octagons composed of >30 proteins called nucleoporins. They allow movement of small molecules by diffusion and large molecules can be transported by accessory proteins.
16
Q
Describe the different paths of a small and large protein traveling through a nuclear pore.
A
Large molecules (>17kDa) will pass through the central transporter using a nuclear localization signal. Small molecules will diffuse through openings in the spoke-ring assembly
17
Q
What amino acids are most common in central transporters of the nuclear pore?
A
Phenylalanine and glycine (F-G) repeats.
18
Q
How is RNA able to pass out of the nuclear membrane even though it is hydrophillic?
A
RNA forms a ribonuclearprotein (RNP). The protein contains a nuclear export signal (NES) that has 5-6 hydrophobic residues that bind to exportin, this complex can then exit through the nuclear pores into the cytoplasm.
19
Q
How is gene splicing related to the process of nuclear export?
A
Following splicing (Pre-mRNA to mRNA), exon junction complexes (EJCs) are bound to the splice junction. The EJC contains Aly, a protein that can bind to TAP. Once TAP is bound, the mRNA can be exported to the cytoplasm.
20
Q
Describe the mechanism of TAP assisting in mRNA export.
A
TAP forms a heterodimer with p15. TAP/p15 then interacts with the FG repeats of the nucleoporins, guiding the mRNA for export.
21
Q
How are EJCs useful for detecting mRNAs that have premature stop codons?
A
As the ribosomes slides along the mRNA during translation, the EJCs are displaced. If a premature stop codon is reached, then the resulting mRNA will still be partially associated with EJCs. The cell recognizes these mRNAs and destroys them to prevent further translation of the mutated mRNA.
22
Q
What is a nuclear localization signal?
A
NLS: sequeence that targets cytoplasmic proteins to the nucleus. This sequence binds IMPORTIN α. These sequences are rich in Lysine (K) or other basic residues.
23
Q
What protein does IMPORTIN α interact with once the NLS binds to it?
A
NLS-IMPORTINα interacts with cytoplasmic IMPORTIN β. This complex binds to one of the filaments of the nuclear pore and can then be transported into the nucleus.
24
Q
Once the IMPORTIN-protein complex is in the nucleus, what protein does it bind to?
A
Ran-GTP. This association causes the IMPORTIN to release the protein.
25
How do the IMPORTINs leave the nucleus once the protein is released?
RanGTP-Importinβ leaves through the nuclear pore, and then binds to a cytoplasmic a GTPase (RanGAP1) causing the release of Importinβ. Importinα leaves by binding to Exportin and then leaving through the pore.
26
Where is [Ran-GTP] high? Where is [Ran-GDP] high?
[Ran-GTP] is high in the nucleus. [Ran-GDP] is high in the cytoplasm.
27
What is the difference between heterochromatin and euchromatin?
Heterochromatin is tightly packed chromatin in which genes are not expressed. Euchromatin is loosely packed and genes are actively transcribed.
28
Describe the structure of a nucleosome.
Nucleosomes are made of histone proteins that form octamers over which 146 base pairs of supercoiled DNA wind. The are rich in basic amino acids (lysine and arginine) because their positive charges can interact with the backbone of the DNA molecule.
29
What are the heterodimers that make up the histone octamer?
2 H2A-H2B dimers, and 2 H3-H4 dimers
30
Which histones are most highly conserved?
Those that make up the octamer: H2A, H2B, H3 and H4. H1 is less conserved
31
What type of bonds connect histones with DNA?
Ionic bonds between the positively charged amino acids and the negatively charged phosphates of the DNA backbone.
32
Where are regulatory sequences of histones found?
On the tails that extend out of the nucleosome. These amino acids are important for epigenetic effects.
33
What is the diameter of one nucleosome?
10nm
34
What is the diameter of the fiber that forms when the 10nm nucleosomes associate?
A 30nm fiber forms.
35
What is the larger fiber that forms from 30nm fibers?
80-100nm supercoiled loops form. These attach to a scaffold with multiple proteins.
36
What are the major methods of histone modification?
(1) Acetylation: causes looser winding, greater transcription (2) Methylation: tighter winding, transcriptional repression (3) Phosphorylation: may reduce affinity of histone for DNA , loosening the chromatin (4)Ubiquitination: promotes methylation (5)Sumoylation: negatively regulates gene transcription in yeast (6)Proline isomerization: allows for methylation
37
What enzyme activity is seen in many transcriptional coactivatiors?
Histone acetyl transferase (HAT)
38
What is CBP?
A coactivator, Creb-binding protein, that acetylates histones to loosen DNA
39
What molecule is recruited following acetylation of histones?
A chromatin remodeling complex called SWI/SNF that slides the histone proteins to expose naked DNA that contains the TATA box
40
Describe the overall process of gene activation.
1) A transcriptional activator binds to GRE and recruits a coactivator. 2)The coactivator acetylates and loosens the DNA from the histones 3)A chromatin remodeling complex is recruited, exposes TATA box 4)Transcription factor binds to TATA box 5)Further acetylation opens up DNA even more 6)Initiation complex assembled and RNA polymerase binds, transcription begins
41
What are the 4 models for the actions of histone remodeling proteins?
1)Nucleosome slides, exposing element 2)Histone octomer changes conformation 3)Histone subunits are exchanged (swi/snf-related) 4)Histone leaves the DNA (FACT complex)
42
What is the FACT complex?
A histone chaperone that both destabilizes and restores nucleosomal structure. Centromeric histone remodeling in yeast
43
What are HDACs?
Histone deacetylases. Important for transcriptional repression causing the DNA to be too tightly coiled to transcribe.
44
What is an example of a transcription repressor protein in higher organisms?
SMRT/N-CoR: Silencing mediator of retinoid and thyroid receptors/nuclear co-repressor.
45
How do repressor proteins function?
They recruit HDACs and Methyltransferases to silence the genes
46
Where does methylation occur on a cytosine?
On carbon #5 of cytosines that are usually next to guanines. This is referred to as a CpG island. In promoter regions, cytosines on both strands are methylated leading to gene silencing
47
How is dsRNA formed?
Repetitive non-coding DNA is transcribed in both directions leading to the formation of double-stranded RNA
48
What is the function of Dicer?
It cleaves dsRNA into smaller pieces that can be matched based on complementarity to euchromatin
49
What is the function of histone methyltransferase?
HMT is an enzyme that methylates K9 of H3 once the small RNA binds to euchromatin. The methylated K9 is then recognized by HP1, which begins the conversion to heterochromatin.
50
Describe telomeres.
A highly repetitive sequence at the ends of each chromosome that forms a "cap" by folding of a 3' overhang that is left over from the DNA replication. These structures allow cells to determine their age because telomeres shorten with each cell division, eventually ceasing to divide when the telomeres are too short.
51
What enzyme elongates telomeres?
Telomerase, which is a reverse transcriptase. This enzyme has a piece of RNA that is complementary to the repetitive DNA of the telomere. It provides a place for DNA polymerase to begin replication.
52
What is epigenetics?
When the change in gene expression or phenotype is NOT the result of a change in DNA sequence, but rather changes in chromatin structure, histone modifications, DNA modifications, X-inactivation ... etc.
53
What are centromeres?
Repetitve DNA called α-satellite DNA that has 171bp repeats. This structure recruits the kinetochore which binds to microtubules during cell division.
54
What epigenetic alterations occur after fertilzation of an egg?
The diploid genome is stripped of its methylation
55
What is the relationship between differentiation of cells and methylation?
As cells become differentiated, more and more of the DNA is 5-Cytosine methylated
56
Are primordial germ cells completely methylated, or completely unmethylated?
Completely unmethylated.
57
What is Dnmt1?
A methyltransferase that methylates daughter strands after every DNA replication.
58
What is an imprinted gene?
One that is silenced (methylated) only if from the male of female parent. These genes will remain methylated during the embryonic demethylation process. Purpose is unknown, but only occurs in mammals.
59
What are replication foci?
Locations within the nucleus where replication takes place. The nuclear lamina is though to be involved with creating these foci.
60
Is newly replicated DNA wound around histones IMMEDIATELY?
Yes. Newly synthesized DNA immediately has the "beads on a string" appearance indicative of nucleosome structures.
61
Do histones have chaperone proteins?
Yes. These chaparones may function in cooperation with chromatin remodeling enzymes. These may also cause the non-random inheritance of core histones during DNA replication and cell division
62
What are the two models for the non-random distribution of histones to daughter strands?
(1) (H3H4)2 tetramers are randomly distributed to daughter strands, but H2A/H2B dimers disassociate and randomly associate with old or new tetramers (2) (H3H4)2 tetramers are separated by histone chaperones and each H3/H4 dimer is assigned to a different daughter strand.
63
How does DNA damage occur?
Errors in replication, UV light, alkylating agents, body heat, free radicals, ionizing radiation. Occurs in ~10,000 bases per day
64
What are the effects of unrepaired DNA damage?
Mutations can cause tumors or heritable changes. Abnormal transcription, chromosomal translocations, and deletions can also occur.
65
What is the difference between the rate of mutation in germ cells versus the rate of mutation in somatic cells?
germ cells: 1 base per 85 million bases is mutated. somatic cells: thousands of bases are damaged every day
66
What are reactive oxygen species?
Free radicals that are produced by cellular respiration. Peroxisomes carry out reactions that generate hydrogen peroxide, which is normally degraded by an enzyme called catalase.
67
What is the most common result of a mutagen?
Alkylation of the base. Ex: occurs in burnt meat
68
What are nitrosoureas?
A class of chemicals, R-NO3 and urea that is used in chemotherapy. They are lipophilic, and can therefore cross the blood brain barrier, but they also damage the lung.
69
What is depurination?
A break in the base-sugar bond causes the loss of thousands of adenines and guanines every day.
70
What is deamination?
Cytosines converted to uracil by losing their amine (NH2) group.
71
Describe the effect of a deamination after 1 replication, and then after 2 replications.
The U (in place of a C) begins paired with G. After one replication, the U will pair with A. Then next replication will put a T in place of the U. So, the overall mutation in the long run is a G:C pair being replaced by an A:T pair.
72
What does exposure to ultraviolet radiation usually cause in DNA?
Pyrimidine-dimers. Adjacent pyrimidines (T or C) form a covalent bond. These mutations inhibit polymerases and arrest replication
73
What can cause double stranded breaks in chromosomes?
Ionizing radiation, chemicals, reactive oxygen species, replcation errors, nucleases, programmed breaks...
74
How many genes are involved in DNA repair?
130 genes are involved in DNA repair.
75
What are the general steps to DNA repair?
(1)Recognition of defect (2)Excision/removal of abnormality (3)Polymerization to fill in the gap (4)Ligation of sugar-phosphate backbone
76
How do the transcription-coupled pathway and the global pathway differ for detecting pyrimidine dimers?
In the manner of recognition of damage. Transcription coupled repair recognizes damage when the RNA polymerase is stalled. In the global pathway, the protein XPC scans the chromosome and binds to the dimer.
77
Describe the process of excision of pyrimidine dimers from DNA strands.
(1) DNA strands are separated around the dimer by XPB (5') and XPD (3') (2)Incision: the phosphodiester bonds are broken in a section of the DNA around the dimer by XPG(5') and XPF(3'). (3) Excision: the region around the damaged DNA is removed (4)DNA polymerase fills the gap (5) a ligase reforms the phosphodiester bond
78
What is Xeroderma pigmentosum?
A genetic condition in which the nucleotide excision repair mechanisms are malfunctioning. Pyrimidine dimers cannot be corrected. These patients cannot be exposed to sunlight without heavy sunscreen protection. Most die of skin cancer.
79
What is base excision repair?
A process for removing individual damaged bases. Ex: de-aminated cytosine to uracil
80
What is the evolutionary rationale for not having uracil in DNA?
Because cytosine is relatively instable, deaminated cytosine (uracil) can be recognized as damaged cytosine in DNA
81
Describe the steps in base excision repair.
(1) Glycosylase removes damaged nitrogenous base leaving the phosphodiester bond intact (2)Hole is called apurinic/apyrimidinic (AP) site (3)AP endonuclease cuts the phosphodiester bond (4)DNA polymerase β removes the sugar leaving behind free 3'OH (5)Polymerase can add cytosine back into damaged strand (6)DNA ligase seals the phosphodiester backbone
82
Describe the steps in mismatch repair.
(1)MLH2 recognizes bulges (2)MLH1 then scans the DNA duplex until it finds a nick, indicating newly copied strand of DNA (3)Newer strand of DNA is removed (4)DNA Polymerase re-copies the template
83
What pathology is associated with mutations in MLH1/2 genes?
(1) Hereditary nonpolyposis colorectal cancer (HNPCC), an autosomal dominant familial form of colon cancer. (2)Muir-Torre syndrome: a subtype of HNPCC that causes skin cancers, and sometimes breast and urogenital cancers
84
Are heterozygotes for mutations in MLH1/2 genes likely to develop cancer?
Yes. They will develop benign tumors, which will become malignant following loss of heterozygosity mutations.
85
What is the worst type of DNA damage?
double stranded breaks
86
What is non-homologous end joining?
A method to ensure proper realignment of broken chromosomes when there is no sister chromatid available. Ku binds to the exposed broken ends and then recruits a kinase, DNA-PKcs. The phosphodiester bond is then sealed by DNA ligase
87
What is homologous recombination repair?
If double stranded break occurs during/after the S1 phase, then a sister chromatid exists. Kinases slow down the cell cycle, and the damaged strand of DNA invades the homologous region of the sister chromatid. The sister chromatid serves as a template for an accurate copy of the DNA sequence.
88
What happens when DNA repair fails?
Mutations occur. Any change in DNA sequence relative to the reference sequence is considered a mutation.
89
What is the difference between a mutation and a polymorphism?
Mutations produce visible, maladaptive, phenotypes. Polymorphisms produce no observable phenotype changes.
90
Describe the process of complimentation testing for confirming xeroderma pigmentosum.
Transfect patient's cells with WT XP genes (XPA through XPG). Expose the cells to UV radiation. Incubate and observe for viability. The patient cells will only proliferate in plates containing the transfected copy of their mutated gene. So cells from a patient with XPA mutation will only proliferate on the XPA+ plate, but none of the others.
91
What is the Ames test?
The first step in assessing the mutagenicity of a compound. It uses a strain of Salmonella that contains a point mutation in the histidine gene. A potential mutagen is incubated with the Salmonella on a less than minimal histidine plate. Cells that are able to grow and produce colonies, revertants, are taken as evidence that the questionable compound has induced back mutations in the his-gene restoring its function.
92
How long does the cell cycle take?
Depends on the cell's identity. Embryonic cells complete a cycle every 30 minutes. Liver cells take 3 months.
93
What stage are most somatic cells arrested in?
Go. These cells are said to have exited the cell cycle.
94
What are Cdks?
Cyclin-Dependent Kinases. These enzymes control the cell cycle. Ex: MPF (maturation-promoting factor). The concentration of cyclins reach a threshold level and then MPF kinase is activated.
95
During what cell cycle stage is MPF kinase activity active?
Kinase activity of MPF is synchronous with start and end of mitosis.
96
What are the two major transition points in the cell cycle?
START and G2-M. START is initiated when G1 cyclins activate the cdk activity. G2-M is initiated when mitotic cyclins activate cdk. This process of swapping cyclins controls transitions in the cell cycle.
97
Describe Cdk activity in early G1
In early G1, there is little Cdk activity
98
Describe Cdk activity in late G1
Cdk4 and Cdk6 inactivate retinoblastoma (Rb) proteins by phosphorylation. EF2 is released, which activates transcription of Cyclins E and A, allowing the cell cycle to progress.
99
What is the role of Cyclin E-Cdk2?
It promotes entry into the S phase. Forms pre-replication forks during early G1, but replication doesn't begin until the S phase.
100
What is the function of cyclin A?
Required for S phase and passage into G2
101
What is the function of cyclin B?
Required for entry into the M phase. CyclinB-Cdk1 will phosphorylate lamins causing them to dissociate and the nuclear envelope breaks down.
102
In G2 phase, where is CyclinB1 found?
In the cytoplasm
103
In mitosis, where is CyclinB1 found?
In the nucleus, where it is phosphorylated
104
What are cell cycle checkpoints?
Pauses in the cell cycle that occur in order for molecular feedback mechanisms to occur and determine whether cells should progress from one stage of the cell cycle to the next. These mechanisms often involve Cdk inhibitors.
105
What are ATR and ATM?
Molecular sensors that can stop the cell cycle.
106
Describe the mechanism of ATR stopping the cell cycle.
NER creates regions of ssDNA which attracts ATR. ATR begins a phosphorylation cascade that ultimately inhibits Cdc25 phosphatase activity, thus preventing the G2-M transition. The cell is thus stuck in G2.
107
Describe the mechanism of ATM stopping the cell cycle.
ATM initiates a phosphorylation cascade that stabilizes p53 and results in transcription of p21, a Cdk inhibito. The cell cycle is thus stopped in G1.
108
What are the steps of mitosis?
Prophase, prometaphase, metaphase, anaphase, telophase
109
What is cytokinesis?
Cytoplasmic division and membrane separation.
110
What are the major events in prophase?
(1)Chromosomes condense to visible chromatids joined by centromere (2)Cytoskeleton is disassembled and mitotic spindle is assembled (3)Golgi complex and ER fragment, nuclear envelope disperses
111
What are the proteins in the scaffold for condensing chromatins?
Topoisomerase II, Condensin, Cohesin
112
Describe the mechanism of Cohesin and Condensin in chromosome condensation.
Cohesin holds replicated sister chromatids together and is a target of Cdks. Condensin, made of complexes of Smc proteins) forms rings around supercoiled DNA and promotes compaction.
113
What is the kenetochore?
The protein structure which binds to the centromere of each chromatid. Microtubules attach here for mitosis. Contains motor proteins required for chromosome separation. Involved in the mitotic checkpoint.
114
Describe the structure of the kinetochore.
Kinetochores have an inner plate attached to the centromere heterochromatin, and an outer plate that binds to motor proteins that interact with microtubules
115
What are some of the proteins associated with the outer plate of the kinetochore?
Cytoplasmic dynein (motor to - end), CENP-E (a kinesin, motor to +end), Depolymerase (a kinesin that promotes depolymerization of microtubules)
116
What happens to centrioles when DNA replication begins?
They are duplicated when Cyclin E is phosphorylated. They then move to opposite ends of the cells
117
What event marks the end of prophase?
Breakdown of the nuclear envelope (NEBD) occurs when lamins are phosphorylated
118
What are the major events of prometaphase?
(1)Chromosomal microtubules attach to kinetochores (2)Chromosomes move to spindle equator
119
True or false: each sister chromatid's kinetochore captures a microtubule from a different spindle pole
True. This allows the sister chromatids to be separated during mitosis.
120
What is congression?
Centering the chromosomes by an unknown cellular mechanisms. Each microtubule that is captured by the kinetochore must become the same length in order to center each chromosome.
121
What are the major events of metaphase?
Chromosomes are aligned along the metaphase plate, attached by chromosomal microtubules to both poles
122
What are the different types of microtubules contained in the mitotic spindle?
(1)Astral microtubules:centered around centrosome, extend into cytoplasm (2)Kinetochore (chromosomal) microtubules: connect centrosome to kinetochore (3)Polar microtubules: centrosome to just past the chromosome
123
During metaphase, are the microtubules immobile?
No. They are in a constant state of exchanging tubulin subunits, so they appear immobile.
124
What are the major events of anaphase?
(1)Centromeres split and chromatids separate (2)Chromosomes move to opposite spindle poles (3)Spindle poles move farther apart
125
What is the anaphase promoting complex?
APC: large protein complex that ubiquinates other proteins, marking them for proteosomal degradation. APC is active during mitosis. It targets different proteins depending on its binding partner. APC-Cdh1 allows exit from mitosis. APC-Cdh20 allows anaphase to occur when active.
126
What is the role of csecurin?
Securin is an anaphase inhibitor that is ubiquinated by APC-Cdc20. Ubiquitination causes repease of separase, which cleaves cohesin and allows chromatids to separate, thus beginning anaphase
127
What does APC-Cdh1 ubiquinate?
Mitotic cyclins, thus allowing exit from mitosis, and also prevents re-entry into mitosis from interphase
128
What is the function of Mad2?
It binds to kinetochore proteins and delays anaphase until all of the chromosomes are on the metaphase plate. It binds Cdc20 until the kinetochore captures a microtubule. Cdc20 is released and associates with APC leading to the beginning of anaphase
129
What happens in anaphase A?
The tubulin subunits are no longer added to the plus end at the kinetochore, so the spindle fiber gets shorter. Sister chromatids are slowly pulled apart.
130
What happens during anaphase B?
The spindle elongates due to lengthening of the polar microtubules
131
What are the major events of telophase?
(1)Chromosomes cluster at opposiite spindle poles. (2)Chromosomes become dispersed (3)Nuclear envelope assembles around clusters (4)Golgi and ER reform (5)Daughter cells formed by cytokinesis
132
What happens to mitotic proteins during telophase?
They are quickly destroyed and the cell returns to interphase conditions
133
Describe the process of cytokinesis.
The plasma membrane invaginates at the site of metaphase plate, perpendicular to the direction of chromosomal separation. Extra membrane is brought to the cleavage furrow by vessicles that fuse with the invaginating membrane
134
What is a midbody?
Some microtubules of the spindle remain briefly as a bridge between daughter cells. The midbody is removed through a process called abscission.
135
What initiates the cleavage furrow?
A ring of actin and bipolar myosin II called the contractile ring. This ring contracts in order to pinch off the daughter cells from each other. The astral microtubules may be involved in this process.
136
During cytokinesis, where is actin found? Where is myosin found?
Actin is seen around the cortical areas of the two daughter cells, and also concentrated in the cleavage furrow. Myosin is found only in the cleavage furrow.
