What are the general features indicating complexity of eukaryotic genomes? Larger size; many introns; tandomly-repeated sequences; repetitious sequences. What are pseudogenes and what is their role in cellÕs life? Non-functional gene copies; they are not transcribed because they are missing promoter sequences. How are processed pseudogenes generated? Reverse transcription of an mRNA which is re-integrated into the DNA; Duplication of a segment of DNA. What are the two types of repetitious DNA? Simple repeats (not expressed); Transposons (some expressed into reverse transcriptase). What is DNA fingerprinting? Analysis of VNTR (variable number of tandem repeats) DNA which is unique to the individual. What percentage of the genome is repetitious DNA? More than 50%. What percentage of the genome is mobile DNA? About 30%. What are the two types of mobile DNA elements? Transposons, retrotransposons. What is evolutionary importance of mobile DNA? Transposable elements have played valuable roles by stimulating gene rearrangements and contributing to the genetic diversity. What are SINEs and LINEs? Short interspersed elements; long interspersed elements. What is the main difference in the two? Their length! SINEs: 100-300 bp; LINEs: 6000-8000 bp. What is a nucleosome? The first level packing of DNA, with histones, into a 10-nm fiber. What are the 10 nm and 30 nm fibers? The first and second levels of DNA packing, respectively. What is genomic imprinting? When genes are only expressed from one inherited parental chromosome, apparently controlled by DNA methylation. How does methylation of cytosine residues correlate with gene expression? It inhibits transcription, likely by disrupting CpG targeted factors. What are telomeres? Special repeated DNA sequences at the ends of linear chromosomes. What is their function? They protect the euchromatin portions of chromosomes from degradation. How do cells maintain telomeres? Telomerase adds special repeated sequences (e.g. AGGGTT in humans) to the 5Õ end of DNA. By lengthening the strand before replication, cells can compensate for telomere shortening during DNA replication. Explain the role of telomeres in development and aging. The amount of telomerase decreases as age increases. This leads to poorly maintained telomeres, which leads to chromosome degradation, which leads to apoptosis. What special advantages does the presence of a nucleus render to eukaryotic gene regulation? Differential processing; regulated import of transcription factors. What is the nuclear lamina and what role does it play in cell cycle? A meshwork of intermediate filaments; serves as site of chromatin attachment. What are nuclear pore complexes? Large spoke-ring complexes of internal and external filaments. Serve as gatekeepers and regulators of active transport in/out of the nucleus. What are NLS and how do they signal specific localization of proteins? Nuclear Localization Signals are specific sequences of amino acids in proteins that are recognized by receptors that direct transport through the nuclear pore complex. Describe in detail how the process of nuclear import of specific proteins brought about by importins. A proteinÕs nuclear localization signal is recognized and bound to an importin. The importin is then responsible for escorting the protein through the nuclear pore complex. The importin is actually a complex of importin + Ran + GDP. The importin complex + the protein is able to bind to nuclear pore complex. After transport through the pore, Ran GEF exchanges the GDP for GTP, which causes the complex to release the protein. How does nuclear import play a part in regulation of gene expression? Transport to/from the nucleus can be regulated, and since many proteins need to return to the nucleus, by regulating their import, you are having the same effect as you would regulation of gene expression at initiation of transcription. How are the RNA molecules that are made in the nucleus but required for translation in the cytoplasm transferred back and forth? Molecules such as rRNAs must be complexed with proteins (hnRNPs), which have import/export signals as needed. Where and how does the assembly of snRNPs take place? snRNAs are transcribed in the nucleus, exported outside the nucleus, assembled into snRNPs, and then are re-imported to the nucleus. How does the nucleus delegate tasks, in regional distribution within itself? The nucleus has a separate region for ribosome production (nucleolus) as well distinct territories for chromosomes. There are also discrete structures (nuclear speckles) which are storage sites for splicing components, Cajal (coiled) bodies which are believe to function as sites of RNP assembly, and PML bodies, whose function is unknown. Why are there special mechanisms for protein trafficking developed for eukaryotes as compared to prokaryotes? Eukaryotes have a nucleus and organelles, which means that they need to provide a way for proteins need to be able to target and enter them. Prokaryotes donÕt have many choices Š just the cytosol, cell membrane, or secretion out of the cell, so they havenÕt needed to evolve more advanced mechanisms. What is the original sorting step in eukaryotes? Translation. How are proteins sorted out in different compartments following this initial selection? The finished protein contains a localization signal which directs the protein to its target destination. What is a signal sequence? If present in a protein being translated, it causes the ribosome to bind to the ER and become a membrane-bound ribosome. How does it help targeting of protein in the cell? It helps by getting the ribosome in place where it can deposit the finished protein directly into the ER for further processing. How can membrane proteins that still utilize the SRP end up having different orientations through the membrane and some have C or N termini towards the cytoplasm? Internal non-cleavable signal sequences can result in the termini on either side. What processes the proteins that go through in the ER? Chaperones, enzymes. Why are they important to protein function? Chaperones assist in folding the protein to the correct conformation, and other enzymes are needed to splice out the signal sequences, glycosylation, methylation, phosphorylation, lipidation, and to assemble the subunits. WhatÕs dolichol and what role does it play in glycosylation? Dolichol phosphate is a lipid molecule in the ER upon which oligosaccharides are assembled for the glycosylation of proteins. What is the Golgi Apparatus? A cytoplasmic organelle involved for processing and sorting of proteins and lipids. What is the characteristic bulk flow of proteins in the cell? The Secretory Pathway: Rough ER --> Golgi --> secretory vesicles --> cell exterior Explain what you understand by vesicular transport and the role of Golgi in it. Vesicles bud from the ER and fuse with the cis Golgi. The proteins are processed by the Golgi, and then leave the Golgi at the trans side by budding into new vesicles which can proceed to their final location. How do proteins that are meant to function in the ER diverted towards the ER? After arriving at the cis Golgi, proteins can leave after processing in new vesicles which are destined for the ER. This requires the KDEL sequence. Is there a special amino acid sequence for membrane proteins similar to the ER? Yes, the KDEL (lysine, aspartic acid, glutamic acid, leucine) signal sequence. What is the importance of differentiation in multicellular organisms? It allows a eukaryote to produce specialized cells/tissues. What are stem cells and what are the different levels of commitment of a precursor cell? Stem cells are special undifferentiated cells which can divide and form more stem cells, or can differentiate into specific cell types. There are totipotent stem cells, which can form any cell type, including embryonic cells; pluripotent stem cells, which can form most cell types, but not embryonic cells; and multipotent stem cells which can form cells of a certain class (such as the various types of blood cells). What are the control points for gene expression? Transcription; processing; stabilization; translation. What are the three types of signaling systems utilized by cells? Endocrine: distant target cell; Paracrine: adjacent target cell; Autocrine: same cell. What are receptors? Special molecules attached to the cellÕs exterior that can interact with signaling molecules to trigger a response inside the cell. How does possession of receptors allows for refined control of signaling? ? Also mention versatility of receptor systems. Type of receptor determines type and identity of response; no receptor = no response; same receptor on many cell types = coordinated response; different receptors for same ligand = different response to one signal. What are the receptors parameters? Binding specificity (Kd = [Receptor][Hormone]/[Receptor+Hormone]), effector specificity. What are the properties of steroid hormones? Lipophilic; crosses membrane; hormone + receptor becomes transcription factor; binds enhancer element in DNA; changes gene expression; all derived from cholesterol. What types of receptors do they use? Intracellular receptors Š from the nuclear receptor superfamily. What are the functional domains of steroid hormone receptors? An activation domain; a hormone-binding domain determines binding specificity; a zinc finger serves as the DNA binding domain to the enhancer. The activation and DNA binding domains determine the effector specificity. Using a prototype example of a G-protein coupled receptors please explain how the signal received at the surface manages to change cell behavior. At the receptorÕs extracellular domain, the hormone binds, which transmits structural change through the membrane. The receptorÕs intracellular domain responds by binding the G-protein. The G-protein can then activate adenylyl cyclase, which in turn synthesizes cAMP. What is the mechanism of control of G-protein receptor activity? ? (alpha is masked with binding to beta/gamma) What are second messengers? When receipt of a signal produces another signaling molecule. For example, for a G-protein coupled receptor, processing of the signal leads to the production of cAMP which is a secondary messenger. How does cAMP-dependent protein kinase get activated? cAMP activates cAMP-dependent protein kinase by binding to the R subunits, which cause the kinaseÕs R and C subunits to separate. What happens upon activation of cAMP-dependent protein kinase? After splitting the kinase into C and R subunits, the C subunit is free to phosphorylate cellular proteins. How does the binding of cell surface receptor lead to amplification of the signal with a very small dose of the ligand in any receptor-ligand interaction? The receipt of one signal molecule at a single receptor can activate hundreds of internal signaling molecules, each of which can catalyze enzymatic reactions. Once the cascade is activated what needs to happen so that it can get reversed? What are the different steps in this process? The cascade is deactivated in reverse, starting with enzymes that break down the products of the catalyzed reactions, and then breaking down the original catalysts, and even removing the receptor by endocytosis.