BIOL 302 Exam IIII

What is constitutive expression?

When proteins are expressed all the time.

What is regulated expression?

When proteins are expressed at very low levels, except in certain circumstances when they are highly expressed.

What are the characteristics of eukaryotes?

• Membrane bound organelles

• Reproduces by sexual reproduction

• True chromosomes

• Multicellular

What are the differences between eukaryotes and prokaryotes?

• Protein-coding sequences are a minority of the DNA

• Genes are larger and interrupted

• Transcripts are modified before translation

• Transcription takes place in the nucleus and the processed mRNA is exported to the cytoplasm for translation

• 3 RNA polymerases instead of one, whereas RNA polymerase II transcribes most genes that encode proteins

• Genes functioning in a common process are not physically linked

• The DNA is wrapped around nucleosomes to form chromatin

What are the examples for protein-coding sequences are a minority of the DNA?

• E. coli has 1 gene per 1000 bp

• Humans have 1 gene per 132,000 bp

• Only 1-2% of the DNA in the human genome codes for protein

What are the examples that genes are larger/interrupted?

• Average human protein is 300 amino acid, which is 1,000 bp of coding DNA

• Human gene is 10,000 bp

• The other 9 kb includes introns and much larger upstream promoter regions than found in bacteria

What are the examples that transcripts are modified before translation?

• Eukaryotic primary transcripts are modified by…

  • Capping

  • PolyA tail addition

  • Splicing out of introns

What are the examples that transcription takes place in the nucleus and the processed mRNA is exported to the cytoplasm for translation?

• Eukaryotes have compartmentalization of transcription and translation

What are the examples that 3 RNA polymerases instead of one are used, and RNA polymerase II transcribes most genes that encode proteins?

• Sigma factor guides RNA polymerase to promoters because RNA polymerase core enzyme cannot distinguish gene start sites on its own.

• The eukaryotic preinitiation complex

What happens in the formation of the eukaryotic preinitiation complex?

• The general transcription factor (TFIID) which contains the TATA-binding protein TBP binds DNA first, at the TATA box.

• TFIID recruits TFIIB

• The TFIIB complex recruits a complex of TFIIF with DNA polymerase II

• TFIIE, then TFIIH join the complex

• DNA strands are melted apart

• Transcription can proceed

What are the examples that the DNA is wrapped around nucleosomes to form chromatin?

• 200 bp of DNA are wrapped around a histone octamer.

  • Made of two each of H2A, H2B, H3, and H4 to make a nucleosome

• Nucleosomes are packaged into higher order structures

What is the general repressor of transcription initiation?

• Chromatin is generally repressive for transcription initiation.

  • Active genes are found in euchromatin.

  • Inactive genes are found in heterochromatin.

What is a difference between E.coli and human genomes cells?

• E.coli cells respond to environmental conditions.

• Human cells respond to environmental conditions and development.

What are some examples of regulated gene expression?

• They are expressed only…

  • In response to specific internal or external conditions

  • At certain times of development

  • In a subset of cells

What are cell-type specific gene products and give some examples?

• Genes that are only in specific cells.

  • Liver-specific genes

  • Neural-specific genes

    • Neurons

  • House-keeping genes

    • Skin cells

What are some of the many mechanisms to regulate gene expression?

• Transcriptional regulation

• mRNA processing

• Regulation of mature mRNA

• Translation

• Post-translation

Explain transcriptional regulation.

• Regulatory proteins and transcription factors bind to consensus DNA sequences.

• Regulatory DNA sequences bind regulatory proteins to facilitate transcription of specific genes in each cell type.

• Open chromatin structure is favorable for transcription.

• Promoters utilized in different cell types to produce different pre-mRNA molecules.

• Methylation of DNA inhibits transcription.

Explain mRNA processing.

• Capping of the 5’ end polyadenylation of the 3’ end, and intron splicing modify pre-mRNA.

• Splicing can also produce different mature mRNA molecules from some cell types.

• RNA editing modifies the base sequences of mRNA.

Explain regulation of mature mRNA.

• Translational regulatory proteins bind mature mRNA to delay translation initiation.

• RNA silencing by RNA interference blocks mature mRNA translation.

• Transport of mature mRNA to cytoplasm is regulated.

• Regulation of mRNA stability.

Explain translation.

• Masking of mRNA delays or precents translation.

Explain post-translation.

• Polypeptides are processed and modified in the Golgi body before transportation out of cell.

• Regulatory molecules bind to a polypeptide to alter its function.

• Regulation of protein stability.

What is the examples that most gene regulation occurs at the level of transcription initiation?

• Activation of gene expression by transcription activatory proteins is the most common mechanism in eukaryotes

What are transcription activators?

• They determine when, where and how much a gene is expressed.

  • For example, the basal transcription apparatus determines the start point for transcription.

What is a domain?

A portion of a protein that performs one specific function, often folds into a separate shape.

What is the DNA-binding domain?

Binds a short specific DNA sequence at the gene to be regulated.

What is the activation domain?

Physically interacts with other proteins to recruit them to the promoter of the gene and activate transcription.

What is a heterodimer?

• Transcription factors that bind DNA in a complex with different type of protein.

  • Helix-loop-helix motif.

What is a homodimer?

• Transcription factors that bind DNA in a complex of two of the same protein.

  • Leucine zipper motif.

What is an enhancer?

• It contains the binding sites for one or more transcription factors which acts to enhance expression of a nearby gene.

• They have flexible distance and orientation.

  • Could be upstream downstream, or in intron of the promoter.

Explain how regulation of eukaryotic genes is more complex.

• Eukaryotic genes are regulated by multiple transcription factors.

  • Different transcription factors bind different DNA sites.

• All the genes with a binding site for a factor could be turned on by binding of that factor.

• Genes expressed in a similar way are scattered across the chromosomes, but all have binding sites for the same transcription factor.

What are the eukaryotic transcription factor activation mechanisms?

• They recruit the preinitiation complex and RNA pol II to the promoter of a gene.

• They recruit enzymes that alter chromatin structure, making transcription initiation by RNA pol II easier.

How can some transcription activators help to form the preinitiation complex?

• Transcription activators function by helping to assemble a functional preinitiation complex at the transcription start site.

• They interact with a mediator, which serves as a bridge between transcription activator proteins and RNA pol II.

What are the mechanisms of eukaryotic transcription repressors?

• Transcription repressor binding sites can overlap transcription activator binding sites and block the activator from binding.

• Transcription repressors can bind to the activation domain of a transcription activator and keep it from functioning.

• Transcription repressors can recruit enzymes that modify chromatin and make it less accessible to PIC formation.

• Transcription repressors can insulate downstream genes from the effects of enhancers in their vicinity.

What are insulators?

Binding sites for proteins that function to keep an enhancer for one gene from working on another nearby gene.

What are some examples of eukaryotic transcription regulators?

• GAL genes in yeast are regulated by GAL4 and GAL80.

• Activation of gene expression by steroid hormones.

What are saccharomyces cerevisiae?

Single-celled eukaryote.

How does the induction of the GAL gene in yeast work?

• In the presence of galactose, four genes are induces that encode the enzymes needed to metabolize the sugar, GAL1, GAL2, GAL7, and GAL10.

  • They bring the galactose into the cell and convert it into a compound that can feed directly into glycolysis to make ATP.

• 3 genes are close together, but not in an operon, they all have an enhancer called the upstream activation sequence (UAS.)

  • The binding site for transcription factor GAL4.

• Without galactose, GAL4 is bound to UAS and GAL80.

  • GAL80 blocks transcription activation domain of GAL4 by covering it.

• With galactose, the inducer galactose binds the GAL3 protein, which binds to GAL80 and makes it fall off. GAL4 can now function.

What are hormones?

Factors secreted by cells to signal to other cells and cause a change in their behavior. Often cause changes in gene expression.

What are the two types of hormones?

• Peptide

• Steroid

What do steroid hormones do, and give some examples.

• Regulate development and physiology in multicellular organisms.

  • Hydrocortisone

  • Aldosterone

  • Testosterone

  • Progesterone

What are steroid hormone receptors?

• Transcription factors that regulate gene expression in response to specific steroid hormones.

• If a cell expresses the steroid hormone receptor for a specific hormone, then the cell will respond to that hormone.

What are the steroid hormone receptor domains?

• DNA binding domain that binds a steroid response element on DNA

• Transcription activation domain

• Domain for interaction with HSP90 protein

• Ligan that binds hormone

What is used in the absence of steroid hormones?

HSP90

How do steroid receptors work?

• In the presence of a steroid hormone, hormone binds the steroid hormone receptor, causing a conformational change which causes it to release from HSP90.

• The steroid hormone receptor is now free to enter the nucleus.

• It binds a specific DNA sequence upstream of some genes and activates their expression.

  • ERE, the estrogen response element

  • GRE, the glucocorticoid response element

• All the genes in the cell with the same steroid response element will be activated when the cell is exposed to that steroid hormone.

What are the mechanisms of gene regulation in eukaryotes?

• Regulation of gene expression by interactions between proteins and DNA sequences.

  • Transcription factors

• Regulation of gene expression by chemical modifications to histone proteins and DNA.

  • Modifications of chromatin structure

    • Histone proteins (acetylation, methylation)

    • DNA bases (methylation)

• Regulation of gene expression by small RNA molecules.

  • mRNA degradation by miRNAs and siRNAs

What can increase the rate of transcription initiation?

• An open chromatin conformation.

  • Nucleosomes are displaced, activator binds.

  • RNA pol II and transcription factors bind promoter.

  • Transcription is initiated.

What is a transcription activator protein?

Determine when, where, and how often a gene is expressed.

What are the eukaryotic transcription factor activation mechanisms?

• They recruit the preinitiation complex and RNA pol II directly to the promoter.

• They recruit enzymes that alter chromatin structure, making transcription initiation by RNA poly II easier.

  • Chromatin remodeling complexes

  • Enzymes that chemically modify histone proteins

What complex can help open chromatin and increase gene expression?

The SWI/SNF complex, as it is one of several chromatin remodeling complexes.

How do pioneer transcription factors bind their sites even when covered by a nucleosome?

• Most transcription factors cannot access their binding sites in heterochromatin.

  • Pioneer transcription factors can access their binding sites in heterochromatin.

• Pioneer transcription factors bind first, recruit enzymes that open up the chromatin, then other factors can bind and the gene can become fully open or accessible, so the PIC can form.

What histone modifications are found in open versus closed chromatin?

• Open chromatin have acetylation and demethylation.

• Closed chromatin have deacetylation and methylation.

Where do the chromatin modifications come from?

• Histone acetyltransferases (HATs) acetylate N-terminal amino acids and help open the chromatin structure to activate transcription.

  • Transcription activators recruit the HAT complex to a gene.

• Histone deacetylases (HDACs) deacetylate amino acids in N-terminal histone protein tails and close the chromatin structure.

  • Transcription repressors recruit the HDAC complex to a gene.

What do histone modifications make it easier to do?

Keep a gene on or off in specific cell types or conditions.

What is the transition from DNA modification to a change in gene expression?

• Methylated CpGs serve as binding sites for proteins that recruit histone deacetylases to the chromatin, putting it into a repressive state.

  • Modification to DNA (CH3)

  • Modification to histones (deAc)

  • Change in gene expression (repression)

What can chromatin modifications do?

Keep certain genes off in cells that will never need those genes, and keep other genes on.

What is epigenetic mean?

Not due to a permanent alteration in the DNA.

Are all the DNA and histone modifications reversible.

Yes, they are all epigenetic gene regulation.

What is the relationship between parental conditions and offspring?

• Conditions affecting a parent can lead to epigenetic marks on the DNA of developing offspring, causing phenotypes in the offspring.

  • For example, 60 years after the Dutch Hunger Winter, those conceived during this time had less methylation, impaired glucose tolerance, raised blood pressure, and higher rates of obesity.

  • For example, BPA in maternal mice caused a higher proportion of yellow coated, obese offspring. Dietary supplements that were high in methyl-donors negated the BPA effects.

What is RNA interference?

• Post-transcriptional gene silencing mechanism that uses dsRNA.

• Small RNA molecules regulate gene expression by affecting the stability or the translation of a specific mRNAs.

How does RNA interference by miRNA work?

• A miRNA gene is transcribed, the RNA folds and is cut into a short dsRNA molecule of 21-25 nucleotides by an enzyme called dicer.

• Fragments from one of the dsRNA strands are bound by the RNA-induced silencing complex or RISC and become the guide RNA.

• The guide RNA allows the RISC complex to bind any mRNAs that are complementary to the 21 nucleotide guide RNA and either.

  • Destroys the mRNA by digesting it or…

  • Blocks translation of the mRNA.

How can investigators use this system to decrease expression of any gene and see what phenotype results?

• Introduce a dsRNA fragment for the gene of interest into cells or organism and let the RNAi machinery do its work to reduce expression of the normal gene mRNA.

  • The dsRNA can be synthesized and injected directly or…

  • A gene can be designed that transcribes an RNA that fold up to make an RNA stem/loop structure.

    • siRNAs, short interfering RNA.

Who discovered RNAi?

Andrew Z. Fire, and Craig C. Mello as they were studying gene regulation in the nematode work, C. elegans, in Baltimore, MD.

What is a neoplasm?

A disorder of cell growth that is triggered by a series of acquired mutations affecting a single cell and its clonal progeny. The causative mutations give the neoplastic cells a survival and growth advantage, resulting in excessive proliferation that is independent of physiologic growth signals.

What are benign tumors?

• Not cancerous

• Often can be removed, and, in most cases, do not come back.

What are malignant tumors?

• Cancerous

• Cells in these tumors can invade nearby tissues and spread to other parts of the body.

What are liquid tumors?

• Cancers that do not form solid tumors.

  • Leukemia

What is metastasis?

• Process whereby cells leave a primary tumor and travel through the bloodstream or lymphatic system to a new location.

  • Most cancer deaths are due to metastatic disease

  • Can remain dormant, then re-emerge later

  • Cancers are most likely metastasized in the liver, lung, and bone

What is cancer?

• Not one disease, but several diseases that result in uncontrolled cell proliferation

• Driven by genetic mutations, epigenetic changes, and changes in gene dosage

• These changes disrupt a multitude of signaling pathways that change phenotypes such as cell proliferation, survival death, repair, movement and invasion

How does pathological analysis work?

• Biopsy

• Tissue is processed to remove water, paraffin is infused, slices are cut

• Hematoxylin and eosin dyes are used to visualize cellular architecture

What do pathologists look for when analyzing biopsies?

• A loss of differentiation

• Differences in arrangement of cells

• Cell shape changes

• Nuclear to cytoplasm ratio changes

• Morphology of he nucleus and/or chromatin changes

• Increase in frequency of mitotic cells

What does a cancer grading depend on?

The extent of differences from normal.

What are the differences in structure between a normal cell and a cancerous cell?

• A normal cell has a large cytoplasm, whereas cancer has a small cytoplasm.

• A normal cell has a single nucleus, whereas cancer has multiple nuclei.

• A normal cell has a single nucleolus, whereas cancer has multiple and large nucleoli.

• A normal cell has fine chromatin, whereas cancer has coarse chromatin.

What is a carcinoma?

Cancer that begins in epithelial cells.

What is adenocarcinoma?

Carcinoma of glandular origin in epithelial tissue, includes most breast, prostate, and colon cancers.

What is a sarcoma?

A cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.

What is leukemia?

Cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.

What is lymphoma and myeloma?

Cancers that begin in the cells of the immune system.

What are central nervous system cancers?

Cancers that begin in the tissues of the brain and spinal cord.

What is the most important cancer risk factor?

Age, because mutations continuously build up.

What are the key attributes of cancer cells?

• Grow where they should not.

• Go through cell division cycle when they should not.

• Escape from their home tissues and move to and proliferate in foreign sites.

• Have abnormal stress responses, enabling them to survive and continue dividing in conditions of stress that would arrest or kill normal cells.

• Less prone to apoptosis.

• Genetically and epigenetically unstable.

• Escape replicative cell senescence, either by producing telomerase or by acquiring another way of stabilizing their telomeres.

What are the theories for how cancers arise?

• Chemical carcinogenesis theory

  • Chemical and environmental factors.

• Viral oncogenesis theory

  • Viruses

• Somatic mutations theory

  • Genes and genetic alterations

What is the evidence that cancer is a genetic disease?

• DNA damaging agents increase risk for cancer

• Recurrent chromosomal abnormalities

What is hereditary cancer?

Cancer risk is transmitted through the germline.

What is sporadic cancer?

Cancer not transmitted to offspring.

What is Knudson’s “two hit” hypothesis?

There must be a recessive gene that, in its wild type form, prevents retinal cancers.

What are oncogenes?

Dominant-acting stimulatory genes that promote cancer, usually by stimulating cell division.

What are proto-oncogenes?

Responsible for basic cellular functions in normal cells, when mutated, become oncogenes.

What are tumor-supressor genes?

Recessive-acting inhibitory genes that generally slow or block cell division.

How was the first oncogene discovered?

• Bishop and Varmus showed that the RSV genome has an extra gene they called v-src that was required for cancer phenotypes in cells.

• They also showed that mammalian cells have their own src gene, c-src that when mutated, caused cancer phenotypes in cells.

Describe the life cycle of an RNA tumor.

• Entry into cell and shedding of envelope.

• RNA

• Reverse transcriptase makes DNA/RNA and then DNA/DNA double helix.

• Integration of DNA copy into host chromosome.

• Transcription.

• Translation

• Assembly of new virus particles, each containing reverse transcriptase into protein coats.

How are non-viral oncogenes discovered?

• Extract DNA from a tumor

• Transfect it into cells in culture

• Identify clumps of cells that can pile up on each other

• Characterize DNA that has been taken up to identify the oncogene

What do proto-oncogenes typically do?

Encode growth factors, growth factor receptors, GTP-binding proteins, transcription factors, and signal transduction pathways.

What is a signal transduction pathway?

• External signal triggers a cascade of intracellular reactions producing a specific response.

• Ras, is a part of the signaling pathway, is frequently mutated, leaving it in an on state, constantly sending pro-growth signals.

• MYC, is turned on by signaling, very rarely mutated, but overexpressed in most cancers.

What are transmembrane receptors?

Mutations that can make tyrosine kinase constitutively active.

What is p53, and where is it found?

Transcription factor that regulates apoptosis, found in the nucleus.

What is RB, and where is it found?

Transcription factor, found in the nucleus.

How does RB control the G1 to S transition?

• RB binds E2F and keeps it inactive.

• This increases the concentrations of cyclin D-CDK and cyclin-E-CDK, which phosphorylate RB.

• RB is activates and releases E2F.

• E2F binds to DNA and stimulates the transcription of genes required for DNA replication.

What are the alterations in cell physiology that underlie malignant growth?

• Self-sufficiency in growth signals.

• Evading apoptosis.

• Sustained angiogenesis.

• Limitless replicative potential.

• Tissue invasion and metastasis.

• Insensitivity to anti-growth signals.

What is BLC2 and its evasion of apoptosis?

• Follicular B-cell lymphoma.

• Translocation between chromosome 18 and 14.

• BCL2 gene comes under the control of an immunoglobulin gene enhancer, gets too overproduction of BCL2 protein.

  • Too many B-cells hang around, some acquire mutations and epigenetic changes and become malignant.

What is VEGF?

• Vascular endothelial cell growth factor

• Therapeutically targeted with an antibody