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263 Cards in this Set

  • Front
  • Back
What two fundamental properties do cancer cells share?
Unregulated Cell Proliferation and Metastic Spread
What genomic alterations are associated with cancer?
Single-nucleotide substitutions to large-scale chromosomal rearrangements, amplifications, and deletions
Where do most cancers derive from?
A single abnormal cell
How does age relate to cancer?
Cancer is an incidence of age and drastically increases with age
How do primary cells behave in culture?
They require growth factors and only survive a limited number of cell divisions
What does it mean that primary cells in culture can become "established"?
The cells can be grown indefinitely and their characteristics are changed and continue to change in culture
How does the transformation of a cell affect its adherent properties?
The adherent properties are lost including anchorage dependence, serum dependence, and density-dependent growth inhibition (contact-inhibition).
How does cancer generally occur?
It is a multi-step process requiring multiple mutations.
What does age-related cancer indicate?
That cancer develops from the accumulation of several mutagenic events.
What did the experiment involving transgenic mice carrying oncogenes indicate about cancer?
That tumors in vivo involve more than one genetic defect. As more and more oncogenes were inserted into the mice, the percentage of tumor-free mice decreased rapidly.
What does tumor progression rely upon?
Successive rounds of mutation followed by natural selection.
How does a tumor develop?
Through repeated rounds of mutation and proliferation, giving rise eventually to a clone of fully malignant cancer cells.
What happens at each step of tumor development?
A single cell undergoes a mutation that enhances cell proliferation, so that its progeny becomes the dominant clone in the tumor.
What does proliferation of the clone provide the tumor?
Hastens occurrence of the next step of tumor progression by increasing the size of the cell population at risk of undergoing an additional mutation.
What drives the process of tumorignesis?
The accumulation of mutation and genetic alterations that progressively release the cell from the normal controls on cell proliferation and malignancy.
What do cancer cells show higher than normal rates of?
Mutation, chromosomal abnormalities, and genomic instability.
Mutator Phenotype
The high level of genomic instability in cancer cells.
What gross defects does genomic instability manifest itself in?
Translocations, aneuploidy, chromosome loss, DNA amplification, and chromosomal deletions.
Which level of genetic instability is the most successful in terms of tumor formation?
Those with the "optimum" level of genetic instability. Those with too much are generally to unstable to function.
Epigenetics
Factors that affect gene expression in a heritable way but do not alter the nucleotide sequence of DNA
How does epigenetic changes affect tumor progression?
Changes such as DNA methylation and histone acetylation modify gene expression and may potentially lead to tumor progression
What is an example of epigenetic changes affecting tumor progression?
The DNA repair genes MLH1 and BRCA1 are transcriptionally silenced in certain cancer cells.
Can epigenetic changes create selective advantages to tumor cells?
Yes, many mutations that make cells cancerous alter the proteins that determine chromatin structures.
What are the chances for a single cell randomly selected from a cancer to create a new tumor?
Rare, flow cytometry has revealed that cancer cells are heterogeneous and only some cells can generate new tumors.
How did cytometry reveal this?
Cells grown from a tumor were treated with a dye (taken up by the cells) and then analyzed by flow cytometry. About 0.4% of the cells pump out the dye and are only lightly stained. Only lightly stained cells could proliferate extensively and form tumors.
How are new stem cells produced?
They are self renewing.
How are cells organized in a normal adult cell lineage?
In a hierarchy. Rare, slowly dividing stem cells produce cells that proliferate extensively (the "transit amplifying cells") and eventually into specialized cells of that lineage.
Can cancer stem cells differentiate into transit-amplifying cells?
Yes, but the reverse process does not occur.
Which group of cells are the minority and which are the majority of neoplastic cells?
Cancer stem cells represent the minority while the transit-amplifying cells represent the majority.
What are the two steps toward metastasis?
Loss of contact inhibition and loss of adherence to substrate.
How do cancer cells react to contact inhibition?
Cancer cells, unlike most cells, usually continue to grow and pile up on top of one another after they have formed a confluent monolayer.
What causes cancer cells to loss adherence to substrates?
Cytoskeletal changes, changes in expression of adhesion molecules, and increased expression of proteases resulting in degradation of extracellular matrix.
What is angiogenesis and how does it relate to metastasis?
The formation of new blood vessels. Cancer cells may also begin to express angiongenic factors such as vascular endothelial growth factor (VEGF).
What cells are incorporated in tumors?
Tumors consist of many cell types, in addition to the cancer cells, including fibroblasts, vascular epithelial cells, smooth muscle cells, and leukocytes.
How do cancer cells affect stromal cells?
Cancer cells secrete cytokines and growth factors that act on stromal cells directing them to create a favorable environment for the tumor.
How do tumors evade elimination?
Down-regulating major histocompatiblity proteins and producing factors that suppress immune function such as TGFbeta and Fas Ligand.
What are chemokines?
Hormones that tumors secrete that recruit leukocytes that provide support for the tumor.
What are TAMs?
Tumor Associated Macrophages that produce growth factors and other proteins that support tumor growth.
How do chemokines affect T cells?
They cause regulatory T cells to suppress effector T cells (such as cytotoxic T cells).
What are the nine features that make a cancer cell successful?
1. They disregard the external and internal signals that regulate cell proliferation.
2. They avoid apoptosis.
3. They circumvent programmed limitation to proliferation by escaping replicative senescence (stabilize their telomeres).
4. They induce help from normal cells in their local environment.
5. They induce angiogenesis.
6. They are genetically unstable.
7. They escape from their home tissues (they are invasive).
8. They survive and can proliferate in foreign sites (they metastasize).
9. They evade the immune system.
What are tumor initiators?
Agents which cause DNA damage.
What are tumor promoters?
Agents that do not directly damage DNA, but act to promote tumor formation.
What are some examples of tumor initiators?
Chemical carcinogens. Viruses. Radiation (UV, ionizing radiation).
What is mitogenic?
Substances that encourage cells to undergo cell division.
How do mitogenic substances affect tumor progression?
They can act as tumor promoters?
How does the tumor promoter affect tumor progression?
It expands the population of mutant cells, thereby increasing the probability of tumor progression by further genetic change.
What are some genetic changes that result in oncogenesis?
1. Deletion or point mutation in coding sequence.
2. Gene amplification.
3. Chromosome rearrangement.
How does deletion or point mutation in coding sequence affect oncogenesis?
May produce a hyperactive protein made in normal amounts.
How does gene amplification affect oncogenesis?
Normal protein greatly overproduced.
How does chromosome rearrangement affect oncogenesis?
1. Nearby regulatory DNA sequence causes normal protein to be overproduced.
2. Fusion to actively transcribed gene greatly overproduces fusion protein; or fusion protein is hyperactive.
How do oncongenic mutations affect the function of the cells?
It results in both gain and loss of function.
What is an example of an oncongenic mutation that results in a gain of function?
A single mutation event that results in an activating mutation that enables the oncogene to promote cell transformation. The activating mutation causes overactivity of the cell.
What is an example of an ocongenic mutation that results in a loss of function?
First, a single mutation even that inactivates the tumor supressor gene; however, because humans are diploid, the second chromosome allows the tumor supressor gene to still be transcribed. A second mutation event inactivates the second copy of the gene in the other chromosome. These two inactivating mutations functionally eliminate the tumor suppressor gene, promoting cell transformation.
What are many inherited cancers caused by?
Defects in genes that control DNA repair, such as chronic myelogenous leukemia (CML), xeroderma pigmentosum, and hereditary nonpolyposis colorectal cancer (HNPCC)
What does CML involve and how does it cause cancer?
It involves the Philadelphia chromosome (white blood cells). Results in chromosomal translocation.
How does xeroderma pigmentosum cause cancer?
It results in defective nucleotide excision repair.
How does hereditary nonpolyposis colorectal cancer (HPCC) cause cancer?
It results in defective mismatch repair enzymes.
What is a specific example of translocation in CML?
Tanslocation between chromosome 22 and 9 joins the BCR gene (break point cluster region) on chromosome 22 to the ABL gene from chromosome 9. This results in the fusion of a protein that has the amino terminus of the BCR protein joined to the carboxyl terminus of the ABL tyrosine protein kinase. The ABL kinase domain becomes inappropriately active, driving excessive proliferation of a clone of hematopoietic cells in the bone marrow.
Why have drugs that specifically block the function of the BCR-ABL kinase able to be synthesized?
Because the mutation resulting in CML was known and the BCR-ABL protein has been characterized.
Which drug has been synthesized to block the BCR-ABL kinase?
STI-571 (AKA Gleevec)
How does Gleevec work?
Gleevec sits in the ATP-binding pocket of the tyrosine kinase domain of BCR-ABL and thereby prevents BCR-ABL from transferring a phosphate group from ATP onto a tyrosine residue in a substrate protein. This blocks onward transmission of a signal for cell proliferation and survival.
How are the regulation pathways of growth and differentiation affected in cancer cells?
In normal cells, the pathways are strictly regulated. In cancer cells, many of the genes that control these functions are mutated or aberrantly expressed, leading to uncontrolled cell proliferation.
What is the G0 phase and how do cancer cells affect this phase?
Cells that stop proliferating enter the G0 phase, in which they do not grow or divide but are metabolically active. Cancer cells are unable to enter G0 and cycle continuously.
What are the checkpoints of the cell cycle?
G1/S, G2/M, and M checkpoints.
G1/S Checkpoint
Cell monitors size and DNA integrity
G2/M Checkpoint
Cell monitors DNA synthesis and damage
M Checkpoint
Cell monitors spindle formation and attachment to kinetochores.
What mediates the regulation of the cell cycle?
Cyclins and Cyclin-Dependent Kinases (CDKs). The complexes then phosphorylate other proteins that advance the cell through the cell cycle.
What causes the cell to halt progress through the cell cycle?
When the cell encounters DNA replication, repair, or chromosome assembly that is aberrant.
Apoptosis
Programmed cell death that occurs if DNA damage is so severe that repair is impossible.
What is responsible for apoptosis?
A series of proteases called capases. They are also responsible for digesting intracellular components.
What are proto-oncogenes?
Genes whose products promote cell growth and division.
What do proto-oncogenes encode?
1. Transcription factors that stimulate expression of other genes.
2. Signal transduction molecules that stimulate cell division.
3. Cell-cycle regulators that move through the cell cycle.
What is an oncogene?
A proto-oncogene that is mutated or aberrantly expressed (gain of function alteration) and contributes to the development of cancer.
What do the products of tumor suppressor genes normally do?
Regulate cell cycle checkpoints and initiate the process of apoptosis.
What happens when tumor suppressor genes are mutated or inactivated?
Cell are unable to respond normally to cell cycle checkpoints or are unable to undergo apoptosis if DNA damage is extensive.
What protein is mutated in nearly all human cancers?
p53 or components of the p53 pathway.
What does p53 do?
Acts by holding cells in G1 arrest to allow for DNA repair. Mutations in p53 allow mutant cells to continue through the cell cycle and allow escape from apoptosis.
What is the "breakage-fusion-bridge cycle"?
1. Cell enters S phase and replicates its DNA despite unrepaired strand break.
2. One daughter cell inherits a chromosome lacking a telomere.
3. Cell enters S phase and replicates itself
4. Sister chromatid ends that lack telomere fuse
5. Fused sister chromatids are pulled apart at mitosis, creating breakage at a new site.
6. One daughter cell inherits a chromosome with duplicated genes but again lacking a telomere.
What can the "breakage-fusion-bridge cycle lead to?
1. Selection that favors the cell with increased numbers of copies of a gene in the affected chromosomal region will lead to mutants in which the gene is amplified to a high copy number.
2. The chromosomal disorder can also lead to loss of genes, with selection in favor of cells that have lost tumor suppressors.
What is replicative cell senescence?
The phenomenon in which somatic cell types can undergo a limited number of divisions in cell culture.
What causes replicative cell senescence?
Changes in telomeres.
How are telomeric sequences synthesized?
By the enzyme telomerase and not replicated like the rest of DNA.
Why do telomeres become shorter in each cell division in most cells?
Because most cells do not express telomerase.
What eventually occurs to cells that do not express telomerase?
DNA damage occurs that the chromosome ends which result in p53 activation of cell cycle arrest.
How might the lack of telomerase activity in cells be beneficial to somatic cells?
It protects against unrestricted proliferation.
How do cancer cells affect telomerase activity?
Most types of cancer cells regain ability to produce telomerase.
What does the loss or mutation of the RB1 tumor suppressor gene cause?
It contributes to the development of many types of cancers due to unregulated progression through the cell cycle.
What happens in familial retinoblastoma?
A mutated RB1 allele is inherited.
What is sporadic retinoblastoma?
The acquisition of two independent mutational events of RB1 within the same cell.
What is pRB?
The retinoblastoma protein is a tumor suppressor protein that controls the G1/S cell cycle checkpoint.
What must the primary tumor do initially before metastasizing?
Cancer cells must digest components of the extracellular matrix and basal lamina that normally inhibit migration of cells.
What is metastasis thought to be controlled by?
A large number of genes, including those that encode cell adhesion molecules and proteolytic enzymes.
How do malignant tumors affect proteolytic enzymes?
They are present at higher than normal levels and are not susceptible to the normal controls conferred by regulatory molecules such as tissue inhibitors of metalloproteinases (TIMPs).
What do most cancers result from?
Somatic cell mutations, but 50 forms of herediatry cancers are known.
Are most inherited cancer-susceptiblity genes sufficient to trigger cancer development?
No, at least one other somatic mutation in the other copy of the gene must occur to drive a cell toward tumorigenesis.
What is usually necessary for cancer phenotypes to be expresssed?
Mutations in other genes. Familial Adenomatous Polyposis (FAP) in an example.
What activity poses the biggest risk factor for developing cancer?
Smoking.
What is the second biggest risk factor for developing cancer?
15% of cancers are caused by viruses.
What are Acute Transforming Retroviruses?
Viruses that cause cancer in animals.
How can a retrovirus cause cancer?
By integrating near a proto-oncogene or by integrating a copy of a host proto-oncogene into its genome.
In what two ways can a proto-oncogene be converted to an oncogene upon incorporation into retrovirus?
1. The gene sequence may be altered or truncated such that it has abnormal activity.
2. The gene may be brought under the control of powerful and enhancers such that the product is made in excess, or in inappropriate circumstances.
What is a insertional mutation?
Genetic disruption that occurs via insertion of retroviruses into host genomes at sites close or within proto-oncogenes.
What drug has been developed to fight the human papillomavirus (HPV)?
Gardasil
What is a carcinogen?
A carcinogen is any substance, radionuclide or radiation, that is an agent directly involved in the exacerbation of cancer or in the increase of its propagation. This may occur by mutations in proto-oncongenes or tumor supressor genes.
What are two ways that chemical carcinogens affect DNA?
1. Some act directly on DNA.
2. Others become damaging only after they have been changed to a more reactive form by metabolic processes.
What is an example of a chemical carcinogens?
Intracellular enzymes, cytochrome P-450 oxidases, normally help convert ingested toxins into harmless secreted products. Their activity on certain compounds (such as benzopyrene, present in coal tar and tobacco smoke) generates products that are highly mutagenic.
What is an example of metabolic activation of a carcinogen?
The metabolism of aflatoxin B1, is a toxin from a mold. It may contribute to liver cancer in the tropics and its associated with characteristic mutations of the p53 gene.
How does the AMES test work?
1. Salmonell bacteria culture requiring histidine to grow is divided into to two samples.
2. One sample is added to an agar plate with nutrients but with no histidnes or additions to the medium.
3. The other sample is aded to an agar plate with nutrients and no histidine but the suspected mutagen is added.
4. The plates are incubated for 12 hours.
5. The first plate with not additions to the medium should have no colonies since there was not histidine present.
6. If the second plate has no colonies, then it means that the suspected mutagen did not cause an increases of mutations. If there are colonies, it may mean that the suspected mutagen caused an increase in mutation which allowed the cells to grow in the abscence of histidine.
Which is generally agreed to be more effective in treating cancers: sequential or multidrug treatments?
Multidrug treatments because the drugs act together to inhibit cells that may be resistant to one of the other drugs in the mixture.
Mice and Humans % genetically similar, but how does aging different in the two species?
Humans live much longer (48 years) which means the key to aging must lie within a few select genes.
What are some factors that cause aging?
1. Nuclear DNA damage
2. Telomere maintenance (senescence)
3. The mitochondrial theory of aging
4. Endocrine regulation
5. Altered metabolism
Transgenic mice with lacZ transgenes are rescued in E. coli and the beta-galctosidase activity was measured, what was concluded from the experiment?
1. Point mutations accumulate with age and genomic rearrangements appear.
2. It was suggested that DNA repair machinery becomes less efficient and more error prone with age.
What suggests that nuclear DNA and not proteins are good targets for aging?
Nuclear DNA must last a lifetime unlike protein which can be replaced. This means that nuclear DNA may become damaged over many years.
What are some sources of nuclear DNA damage?
1. Extrinsic sources such as ionizing radiation and genotoxic drugs.
2. Replication errors (for example, MMR enzymes don't catch everything)
3. Spontaneous chemical changes (deanimation)
4. Reactive Oxygen Species (ROS), which may come from the mitochondria or antimicrobial burst of phagocytes.
What do patients with mutations in the ATM gene suffer from?
Ataxia-telangiectasia, a neurodegenrative disease characterized by a prematurely aged appearance among other things.
What is the ATM gene responsible for?
The ATM gene produces a protein in the nucleus that is used to regulate the cell cycle and assists in recognizing damage in DNA and prompting the repair of the DNA by activating other enzymes.
What do ATM mutants also endure besides premature aging?
1. They grow poorly.
2. Genomic Instability.
3. They senesce prematurely.
What is the purpose of KU80 and what happens when it is knocked out of the mouse genome?
It activates other proteins and enzymes to repair non-homologous recombinatorial double-stranded breaks. If it is knocked out, mice prematurely age.
What is the purpose of BRCA1 and RAD50 and what happens when it is hypomorphic?
BRCA1 and RAD50 are responsible for recruiting proteins to aid in homologous double-stranded breaks. If it is hypomorphic, mice prematurely age.
What happens when genes are lost that are associated with global genome and transcription coupled nucleotide excision repair?
Loss of these genes is associated with accelerated aging.
What does MMR do?
Removes mispaired bases from replication errors, such as recombination between imperfectly matched sequences and oxidative damage.
What is the importance of MMR for microsatellites?
MMR is important in stabilizing microsatellites.
If instability of microsatellites and the precision of MMR decreases with age, why not overexpress them?
Overexpression of these genes may have unforeseen effects because of the complexity of interactions in the cell and nucleus.
Why have perfect DNA repair systems not evolved in life on earth?
In the wild, organisms do not live forever and often succumb to predation or accidents long before they accumulate enough mutations to show manifestations of aging. Therefore, there was no advantage in investing into maintenance of perfect DNA repair systems.
What are age related diseases and premature aging characterized by?
Short telomeres.
What happens to telomeres with subsequent cell divisions?
Telomeres continuously lose TTAGGG repeats.
What has the progressive telomere shortening been proposed to act as?
A 'molecular clock' that underlies organismal aging.
How do germ cells and some cancer cells maintain cell viability?
By expressing high levels of telomerase.
What is TRF1 and TRF2?
Telomere Repeat binding Factors that directly binds the TTAGGG repeats and interact with many other factors forming large protein complexes.
What is telomerase?
A reverse transcriptase that recognizes the 3' OH at the overhang and elongates telomeres by extending from an RNA primer.
How do telomeres protect chromosomes?
The G strand overhang can fold back and anneal with the double stranded region of the TTAGGG repeat to form a large telomeric loop known as the T loop. This helps to protect the ends from exonucleases and prevents the free ends from fusing with one another.
What is Bloom Syndrome?
Characterized by short telomeres. It is caused by a faulty BLM helicase. It causes short stature, facial rash from sun exposure, diabetes, immune deficiency, and average age of cancer onset is 25 years of age.
What is Werner's Syndrome?
Characterized by telomeric instability. It is caused by a faulty WRN helicase. The introduction of telomerase into Werner's Syndrome cells can rescue their premature senescence.
What is WRN's roles in the cell?
It probably plays a role in providing telomeric access to other factors in telomere maintenance. Also plays a role in overall genome stability and is involved in multiple DNA repair pathways.
What happens when RB1, RBL1, and RBL2 are inactivated and what might this cause?
When RetinoBlastoma1, RetinoBlastomaLike1, and RetinoBlastomaLike2 are inactivated, there is a loss in telomeric heterochromatin. This could theoretically cause telomeres to become much longer causing immortality, but cancer would likely develop.
What is DC?
Dyskeratosis Congenita which is a disorder in which mutations in TERC cause a decrease in telomerase activity or mutations in the dyskeratosis congenita 1 (DKC1) gene which encodes a protein that promotes TERC stability.
What does DC cause?
Short stature, infertility, defects of the skin, bone marrow failure, and premature death.
If shorten telomeres is so deterimental, why don't eukaryotes have circular chromosomes?
The most likely answer is that linear chromosome allow for genetic recombination which allows for more complex organisms.
What is the mitochondrial theory of aging?
The theory that most aging can be attributed to the progressive accumulation of somatic mutations in mtDNA that leads to an inevitable decline in mitochondrial funciton. As the mitochondria 'burn out', the cells undergo apoptosis and the organism slowly develops diseases and dies.
When do clinical aging symptoms appear?
When the number of cells in a tissue declines below the miniumum necessary to maintain function. This relates to the rate at which mtDNA damage accumulates within cells leading to activation of the mitochondrial permeabiltiy transition pore (mtPTP) and apoptosis.
What is said to account for the exponential increase in oxidative damage during aging?
Some pathogenic mutations, such as 8993 T>G which leads to increased ROS (Reactive Oxidation Species) production.
How do many mutations that extend life affect endocrine signaling?
They interrupt the processes.
What happens when the insulin/IGF-1 pathway in worms, flies, and mammals is mutated?
The insulin/IGF-1 pathway activates daf-2, a transcripiton factor, when it's mutated, it extends life span by 80% in drosophila.
What did the mutation in insulin/IGF-1 seem to cause?
Downstream genes were analyzed with RNAi. Ones that affected aging were antioxidants, chaperones, and heat shock proteins. (Some of these were overexpressed in the mutant). This demonstrated the hormonal regulation of aging.
What is the INDY gene?
(AKA I'm not dead yet gene) A gene that is a dicarboxylate transporter found in the plasma membrane of organs and is important in the intermediary metabolism of the fly.
What type of mutation occurs in the INDY gene?
It is a hypomorphic mutation that has been postulated to reduce the uptake, utilization, or storage of important nutrients, somehow altering the metabolism in a way that increases life span.
What was the important effects of the INDY gene mutation?
There was no detectable change in resting metabolism or flight activity but they were still able to live twice as long.
If the INDY gene mutation is beneficial, why hasn't it dominated the wild type gene?
It was observed that under poor food conditions that flies with this mutation lay fewer egg.
Why haven't mutations that increase the life span been able to dominate the wild type genes in organisms?
Because many of these mutations have unacceptable trade-offs in metabolic rate, growth, physical activity, or early-life fecundity.
How do yeast divide?
Asymmetrically by micromanipulation, in which the older yeast take on a larger volume.
How was gene screening used to find a relationship between starvation resistance and life span?
Yeast were mutagenized. Then those with the ability to survive starvation conditions were observed.
What was found from the gene screening?
A family of proteins deacetylases, SIRtuins, were identified, specifcally SIR2, 3, and 4 genes.
What do Sirtuins do?
Deacetylate lysine residues on histones, closing chromatin and discouraging transcription.
What are some Salmonella characteristics?
1. Gram negative enteric (intestinal) pathogen
2. 1 billion new human infections per year, 3 million deaths
3. Multi-drug resistant strains
What two main characteristics make Salmonellla a good tool?
1. Genetically tractable (easily managed)
2. Amenable to molecular biology manipulation
What are the two main types of models of S. typhimurium in animals?
1. Acute typhoid fever model (In BALB/c mice)
2. Chronic carrier model (In wild type mice)
What are some barriers that Salmonella must overcome to spread within the host?
1. Must travel considerable distances.
2. GI (gastrointestinal) epithelium provides a physcial barrier.
3. Brush border, thick mucus coat, acidic environment, perstalis, anti-microbial peptides, cell turnover and endogenous flora (competition with indigenous microbes of the intestines)
What makes phagocytes good vehicles for Salmonella?
1. Readily engulf microbes.
2. Can traverse epithelial barriers.
3. Can shield pathogens from other components of the immune system.
What is a possible drawback to Salmonella using phagocytes as vehicles?
Phagocytes release cytokines that strongly inhibit their motility when infected.
What are the two main pathways that Salmonella uses to enter the bloodstream?
From the GI tract:
1. CD18 pathway
2. Lymphatic System
What normally happens in the GI tract?
Microbes are taken up by dendritic cells and transported through the lymphatic system.
What can happen in the GI tract with Salmonella?
Salmonella is taken up by dendritic cells and transported to the bloodstream rather than the lymphatic system.
What two major organs do Salmonella occupy after they have entered the bloodstream?
1. Liver
2. Gall Bladder
What is a Type III secretion system?
One cell uses a complex "arm" that runs from the cytosol of the bacterium to the host's cytosol, alllowing for direct injection.
What are Type III secretion systems encoded with?
Pathogenicity Islands which are large blocks of virulence genes absent from the nearest non-pathogenic relative.
How were the pathogenicity islands acquired?
Horizontally from another organism.
What are the two primary Salmonellla pathogenicity islands?
1. SPI-1 (Salmonella Pathogenicity Island 1)
2. SPI-2 (Salmonella Pathogenicity Island 2)
What does SPI-1 allow Salmonella to do?
Gain entry into the macrophage.
What does SPI-2 allow Salmonella to do?
Promote growth inside the phagolysosome.
What is ssrAB?
It is a two part SPI-2 regulator.
What is srfH?
A gene that is involved in the mobile elements in macrophages.
Does SrfH promote intracellular growth?
No.
How is a hybrid screen used to show protein-protein relationships?
1. Transcription factor is broken apart into its two components (the binding domain and activation domain).
2. A protein is bound to both of these components.
3. A reporter gene is placed downstream.
4. If the two proteins attached to the components of the transcription factor interact, then the transcription factor will become whole again and the reporter gene will expressed.
Out of the 1 million cDNA clones screened, how many of these proteins were isolated?
2 unique isolates of TRIP 6 were recovered. They are found in the Zyxin famiy of proteins which regulate adhesion and motility. (Subfamily of adaptor protiens and enriched focal adhesions).
What were the exerimental results of SrfH?
SrfH promotes the motility of infected macrophages in vitro. Also, SrfH alone is sufficient for stimulating motility.
Does SrfH affect motility through TRIP 6?
Yes. A mouse was infected through oral inoculation and 30 mins later blood was withdrawn and the host cells lysed. It was found that SrfH promotes the systemic spread of Salmonella in mice and accelerates the colonization of internal organs.
What is the summary for the activities of Salmonella?
1. Salmonella invades the host cells.
2. It survives within phagocytes.
3. Exploits them as vehicles for intra-host dissemination.
4. Induces apoptosis.
What is the relationship between SrfH, TRIP 6, CAS, and CRK?
SrfH binds to TRIP 6 and allows it to recognize CAS and CRK which then allows for migration of the host.
What is the relationship between SrfH, TRIP6, CAS & CRK inside a focal adhesion?
SrfH acts as glue that holds the CAS, CRK, TRIP 6, and SrfH complex to a filamin attached to the interior of a focal adhesion.
What is a summary for the activity of SrfH?
1. SrfH is activated greater than 100-fold inside of macrophages.
2. SrfH is secreted into macrophages by SPI-2.
3. SrfH does not promote intracellular growth.
4. SrfH binds the host protein TRIP 6 which regulates motility.
5. SrfH also binds filamin
6. SrfH forces macrophage to move faster in vitro through an as of yet uncharacterized interaction with the host protein TRIP 6.
7. SrfH accelerates the systemic spread of infection.
What did the paper that was released opposite Prof. Worley conclude about SrfH?
That SrfH did not bind TRIP 6 with its amino terminus, instead it binds IQGAP-1 with its carboxyl terminus-cysteine 178 which inhibited productive macrophage motility in vitro and vivo.
What are the two major strains of Salmonella typhimurium used in experimentation and what is the difference between them?
1. 14028s
2. SL1344
The difference is a single nucleotide polymorphism (SNP).
What was the conclusion when the experiment was performed again?
SrfH has antagonistic domains. The amine end binds uses Residue 103 to bind TRIP 6 and promote motility. The carboxylic end uses Residue 178 to bind IQGAP-1 to prevent motility.
What is the coding (nontemplate) strand?
The DNA strand that has the same sequence as the mRNA that is related by the genetic code to the protein sequence that it represents.
What is RNA polymerase?
An enzyme that synthesizes RNA using DNA template (formally described as DNA-dependent RNA polymerase).
What is a promoter?
A region of DNA where RNA polymerase binds to initiate transcription.
What is a startpoint?
The position on DNA corresponding to the first base incorporated into RNA.
What is a terminator?
A sequence of DNA that causes RNA polymerase to terminate transcription.
What is a transcription unit?
The sequence between sites of initiation and termination by RNA polymerase; it may include more than one gene.
What does upstream mean?
Sequences in the opposite direction from expression.
What does downstream mean?
Sequences proceeding farther in the direction of expression within the transcription unit.
What is the primary transcript?
The original unmodified RNA product corresponding to a transcription unit.
What is the RNA transcription bubble?
1. RNA polymerase separates the two strands of DNA in a transient "bubble".
2. RNA polymerase uses one strand as a template to direct synthesis of a complementary sequence of RNA.
How long is the RNA transcription bubble?
The bubble is 12 to 14 bp and the RNA-DNA hybrid within the bubble is 8 to 9 bp.
What are the stages of RNA transcription?
1. Initiation
2. Elongation
3. Termination
What occurs during initiation?
1. Template recognition; RNA binds to duplex DNA.
2. DNA is unwound at promoter.
3. Very short chains are synthesized and released.
What occurs during elongation?
Polymerase synthesizes RNA.
What occurs during termination?
RNA polymerase and RNA are released.
In what direction does RNA transcription occur?
The transcription bubble moves along DNA and the RNA chain is extended in the 5' ---> 3' direction by adding nucleotides to the 3' end.
What is the holoenzyme of bacterial RNA polymerase?
The RNA polymerase form that is competent to initiate transcription. It consists of the five subunits of the core enzyme and a sigma factor.
Where does the catalysis of the holoenzyme come from?
the beta and beta' (prime) subunits.
What is the CTD (C-terminal domain)?
The domain of RNA polymerase that is involved in stimulating transcription by contact with regulatory proteins.
What two parts can the holoenzyme of the bacterial RNA polymerase be divided into?
1. The alpha-2-beta-beta'-omega core enzyme that catalyzes transcription
2. The sigma subunit that is required for initiation only.
What role does the sigma factor specifically play in transcription?
It changes the DNA-binding properties of RNA polymerase so that its affinity for general DNA is reduced and its affinity for promoters is increased.
How does RNA polymerase locate promoters on the DNA?
The rate at which RNA polymerase binds to promoters is too fast to be accounted for by simple diffusion. Therefore, RNA polymerase binds to random sites on DNA and exchanges them with other sequences until a promoter is found.
What is the ternary complex?
The complex in initiation of transcription that consists of RNA polymerase and DNA as well as dinucleotide that represents the first two bases in the RNA product.
When is the sigma factor usually released from RNA polymerase?
When the nascent RNA chain reaches ~10 bases in length.
What usually occurs before the enzyme moves to the next phase of transcription?
A cycle of abortive initiations.
What is a conserved sequence?
Sequences in which many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations.
What is a promoter defined by?
The presence of short consensus (conserved) sequences at specific locations.
What is the general structure of a promoter consensus sequence?
It usually consists of a purine at the startpoint, a hexamer with a sequence close to TATAAT centered at ~-10(called -10 element or TATA box) and another hexamer with a sequence similar to TTGACA centered at ~-35 (called -35 element).
What factors can affect the efficiency of a promoter?
1. UP elements
2. Down mutations
3. Mutations in the -35 sequence
4. Mutations in the -10 sequence
What is an UP element?
A sequence in bacteria adjacent to the promoter, upstream of the -35 element, that enhances transcription.
How do down mutations affect promoter efficiency?
They usually decrease conformance of the promoter to the consensus sequences.
How do mutations in the -35 element affect promoter efficiency?
It affects the initial binding of RNA polymerase.
How do mutation in the -10 sequence affect promoter efficiency?
It affects the binding or melting reaction that converts a closed complex to an open one.
What is footprinting?
A high resolution method for characterizing RNA polymerase-promoter and DNA-protein interactions in general. It identifies these by virtue of the protection of bonds in the region against attack by nucleases. The bonds are protected in the region by the bound proteins.
Where on the DNA are the most contact points for RNA polymerase to bind to the promoter?
The consensus sequences at -35 and -10 element.
How does the DNA which strand to bind to?
The points of contact for the RNA polymerase lie primarily on one face of the DNA.
Where is the sigma factor on the holoenzyme located?
It occupies the RNA exit channel and must be displaced to accommodate RNA synthesis.
When do abortive initiations usually occur?
Before the enzyme forms a true elongation complex.
How does an arrested RNA polymerase restart transcription?
By cleaving the RNA transcript to generate a new 3' end.
What is a terminator?
Discrete sequences of DNA that are used to terminate transcription.
What are the two classes of RNA transcription terminators?
1. Intrinsic terminators
2. RHO-Dependent terminators
What is an intrinsic terminator?
A terminator that is recognized by RNA polymerase itself without the requirement of any cellular factors.
What is a RHO-dependent terminator?
Termination sequences that require that help of the cellular protein called RHO to terminate the transcription of the RNA polymerase.
Where are almost all termination sequences found?
In the transcribed region of DNA. This means that termination relies on scrutiny of the template and/or the RNA product that the polymerase is transcribing.
What does the intrinsic terminator code for?
A hairpin structure in the RNA product.
What is a readthrough?
A event that occurs at transcription or translation when RNA polymerase or the ribosome, respectively ignores a termination signal because of a mutation of the template or the behavior of an accessory factor.
What is antitermination?
A mechanism of transcriptional control in which termination is prevented at a specific terminator site, allowing RNA polymerase to read into the genes beyond it.
What is an RHO factor?
A protein that binds to nascent RNA and tracks along the RNA to interact with RNA polymerase and release it from the elongation complex.
What is RUT?
An acronym for RHO utilization site, the sequence of RNA that is recognized by the RHO termination factor.
What is the process of RHO on nascent RNA to release it?
1. RNA polymerase transcribes DNA.
2. RHO attaches to RUT site on RNA.
3. RHO translocates along RNA
4. RNA polymerase pauses at hairpin and RHO catches up
5. RHO unwinds DNA-RNA hybrid
6. Termination; all components released from DNA
What is polarity?
The effect of a mutation in one gene in influencing the expression (at transcription or translation) of subsequent genes in the same transcription unit.
What is the antitermination complex?
Proteins that allow RNA polymerase to transcribe through certain terminator sites.
How does supercoiling affect transcription?
1. Negative supercoiling increases the efficiency of some promoters by assisting the melting reaction.
2. Transcription generates positive supercoils ahead of the enzyme and negative supercoils behind it. These must be removed by gyrase and topoisomerase.
How can sigma factors regulate initiation factors?
Competition. E. Coli has seven sigma factors, each which causes RAN polymerase to initiate at a set of promoters defined by specific -35 and -10 sequences.
How are activities of different sigma factors regulated?
Through different mechanisms.
What is an anti-sigma factor?
A protein that binds to a sigma factor to inhibit its ability to utilize specific promoters.
What is heatshock response?
A set of loci that is activated in response to an increase in temperature that causes proteins to denature (and other abuses to the cell). All organisms have this response and the gene products usually include chaperones that act on denatured proteins.
How can sigma factors be organized?
By cascades.
What are cascades of sigma factors?
A cascade of a sigma factor is created when one sigma factor is required to transcribe the gene coding for the next sigma factor.
What is an example of a cascade for the phage SPO1?
1. The early genes of SPO1 are transcribed by host RNA polymerase.
2. One of the early genes codes for a sigma factor that causes RNA polymerase to transcribe the middle genes.
3. Two of the middle genes code for subunits of a sigma factor that causes RNA polymerase to transcribe the late genes.
How does transcription and translation occur in bacteria?
Simultaneously (AKA coupled transcription/translation) as ribosomes begin translating an mRNA before its synthesis has been completed.
What is the stability of bacterial mRNA?
It is very unstable and has a half-life of only a few minutes.
What is nascent RNA?
A ribonucleotide chain that is still being synthesized, so that its 3' end is paired with DNA where RNA polymerase is elongating.
What is monocistronic mRNA?
mRNA that encodes only one protein.
What is polycistronic?
mRNA that encodes several proteins and represents different genes.
What is the 5' UTR?
The region in a mRNA between the start of the message and the first codon.
What is the 3' UTR?
The region in a mRNA between the termination codon and the end of the message.
What is the intercistronic region?
The distance between the termination codon of one gene and the initiation codon of the the next gene.
What are basal transcription factors?
Transcription factors required by RNA polymerase II to form the initiation complex at all RNA polymerase II promoters. They are identified as TFIIX (where X = a letter).
What must happen before RNA can bind to the promoter?
The chromatin must be opened.
What is the core promoter?
The shortest sequence at which an RNA polymerase can initiate transcription (typically at a much lower level than that displayed by a promoter containing additional elements)
What is the core promoter for RNA polymerase II?
The minimal sequence at which the basal transcription apparatus can assemble, and it includes three sequence elements; the INR, the TATA box, and the DPE. It's typically ~40bp long.
What is an enhancer?
A cis-acting sequence that increases the utilization of (most) eukaryotic promoters, and can function in either orientation and in any location (down or up stream) relative to the promoter.
What is a silencer?
A short sequence of DNA that can inactivate a gene in its vicinity.
What are housekeeping genes?
Genes that are (theoretically) expressed in all cells because they provide the basic functions for sustenance of all cell types.
What is a coactivator?
Factors that are required for transcription that do not bind DNA, but are required for (DNA-binding) activators to interact with the basal transcription factors.
What does RNA polymerase I synthesize?
rRNA in the nucleolus.
What does RNA polymerase II synthesize?
mRNA in the nucleoplasm.
What is hnRNA?
Heterogenous nuclear RNA. RNA that comprises transcripts of nuclear genes made primarily of RNA polymerase II; it has a wide size distribution and variable stability.