Biology Exam #3

how do we turn of kinase signaling?

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what happens during transcription regulation of eukaryotic gene expression?

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describe g protein coupled receptor signaling

0) before the signal arrives, the g-protein is off. this means that it is GDP bound 1) signal arrives and binds to the receptor 2) the arrival of the signal causes the receptor to change its conformation. - g protein binds GTP and splits in two - In response to hormone binding, the receptor changes the conformation of the G-protein so that it releases the GDP and binds to the GTP molecule. When GTP is attached, the G protein changes shape radically; it splits into two parts 3) The activated G-protein then binds to a specific enzyme, resulting in the enzyme catalyzing a reaction that produces second messengers -- G-protein-activated enzymes catalyze the production of many small nonprotein signaling molecules called second messengers that diffuse rapidly and spread the signal message throughout the cell = amplification. --The same second messenger can trigger different events (e.g. open ion channels, activate protein kinases, etc.) in different cell types 4) G protein hydrolyzes GTP to GDP 5) signal is deactivated

Describe the steps that are taken in PCR to produce many identical copies of a specific gene

1) start with a solution containing template DNA, synthesized primers, an abundant supply to the four dNTPs, and Taq Polymerase 2) DENATURING - heating leads to denaturation of the double stranded DNA, making two single strands (we heat because there is no helicase in the initial solution) 3) Primer ANNEALING - at cooler temperatures, the primers bind to the template DNA by complementary base pairing - remember that the primers bind in the 5' to 3' direction (which means the 3' to 5' direction for the DNA strand) 4) EXTENSION - during incubation, Taq polymerase uses dNTPs to synthesize complementary DNA strands, starting at the primer 5) Repeat the cycle - perform denaturing, annealing and extending over and over again to produce millions of copies of DNA

describe the two general categories of signaling molecules

1. Lipid soluble Hydrophobic molecules that can cross plasma membrane to enter cell. 2. Lipid-insoluble Large hydrophilic molecules that can't cross the plasma membrane Depending on the kind of receptor will determine whether or not the signal is received inside the cell (for lipid soluble signals) vs. on the plasma membrane (for lipid-insoluble signals)

What type of mutation in tumor suppressor genes and oncogenes promote cancerous growth? A) Inactivating mutations in tumor suppressor genes and activating mutations in oncogenes. B) Activating mutations in tumor suppressor genes and inactivating mutations in oncogenes. C) Activating mutations in both. D) Inactivating mutations in both.

A) Inactivating mutations in tumor suppressor genes and activating mutations in oncogenes.

Complete the following sentence. Receptors for lipid-soluble signaling molecules can be _________. Receptors for lipid-insoluble signaling molecules are _________. A) intracellular proteins; transmembrane proteins B) transmembrane proteins; intracellular proteins C) intracellular proteins, intracellular proteins D) transmembrance proteins; transmembrane proteins

A) intracellular proteins; transmembrane proteins

How do we create a complementary DNA (cDNA)?

After the mRNA strand has been produced from the GH gene through transcription and RNA processing, the mRNA gets converted into complementary DNA (cDNA) using an enzyme called reverse transcriptase reverse transcriptase goes against the central dogma formula

During which stage(s) of mitosis can microtubules access the chromosomes? A) Prophase through telophase (all stages). B) Metaphase and anaphase C) Anaphase only D) Prophase only

B) Metaphase and anaphase

What is the major difference between controlling translation by a regulatory protein or miRNA bound to a specific RNA, versus phosphorylation of a ribosomal protein? A) miRNAs and regulatory proteins inhibit translation while phosphorylation of a ribosomal protein enhances translation. B) miRNAs and regulatory proteins enhance translation while phosphorylation of a ribosomal protein inhibits translation. C) A miRNA or a regulatory protein will control translation of one (or a few) proteins, phosphorylation of ribosomal proteins will control translation of all proteins.

C) A miRNA or a regulatory protein will control translation of one (or a few) proteins, phosphorylation of ribosomal proteins will control translation of all proteins.

What is the consequence for a cell in which Rb protein is mutated and cannot bind E2F? A) No cell division will occur B) Cell division will only take place if sufficient nutrients and social signals are present C) Cell division will take place even if there are not enough nutrients and social signals.

C) Cell division will take place even if there are not enough nutrients and social signals.

what is cancer? what are the two different ways in which people can get cancer?

Cancer = uncontrolled cell division, as a consequence of improper control of the cell cycle There are many different types of cancer - depends on the cell type and on the way in which the cell cycle control is disrupted MUTATIONS THAT CAUSE CANCER: Mutation in tumor suppressor genes - an inactivating mutation in a tumor suppressor gene can cause cancer because the tumor suppressor gene wont be able to encode for proteins that are required to enforce the cell cycle checkpoints. therefore, there would be no checkpoints and the cell would continue to form and divide, even with errors - Rb is a tumor suppressor gene because it stops cell division if there are errors and the cell shouldn't divide; eliminating the border patrol would cause cancer Mutation in proto-oncogenes (creating oncogenes) - activating mutations in proto-oncogenes, producing oncogenes, will result in an increased activation of proteins that are required for cell growth and division. this increased level of activation will lead to uncontrolled cell growth / division

describe the activation of MPF

Cdk is only active when bound to M-cyclin. Therefore, MPF activity is low throughout the cell cycle and high at the end of the G2 phase, where there is enough M-cyclin so that most of the Cdk is cyclin-bound. However, this initial cyclin-Cdk complex is inactive because Cdk is phosphorylated (by Cdk-inhibitory kinase) on a specific site, thus inactivating it. At the end of the G2 phase, Cdk is dephosphorylated at one of the two phosphorylation sites - this actives MPF and the M-phase is initiated. MPF phosphorylates other proteins (proteins that are necessary for mitosis) in order to activate them MPF also phosphorylates M-cyclin protease and activates it. This protease degrades M-cyclin. This means that whenever a wave of cyclin-Cdk activation occurs, it promotes its own destruction by activating M-cyclin protease. This is an example of negative feedback inhibition

what are the stages of the cell cycle?

Cell cycle = alternation of interphase (non-dividing) and mitosis (dividing) stages The lengths of each stage of the cell cycle can vary in different tissues or organisms. STAGES OF THE CELL CYCLE Mitosis (M phase) - When DNA and cell is in the act of dividing G1 Phase (First Gap) - After mitosis and before replication begins DNA Synthesis (S phase) - When DNA replication occurs G2 phase (Second Gap) - The interlude before mitosis. Cells have twice the normal amount of DNA at this time

what are the basics of cell-cell communcation?

Cells release signaling molecules. Receptors inside or on the surface of another cell bind to the signaling molecule to receive it. A Conformational change occurs in the receptor, changing its activity. In the case of cell surface receptors, the "signal" is transduced from one protein to another Response: the activity of the target cell changes as a consequence of receiving and transducing the signal Signal is eventually deactivated.

how can we regulate gene expression through chemical modification?

Chemically modifiying a protein to regulate it - such as adding a phosphate through phosphorylation thanks to the enzyme kinase Tagging a protein with a phopshate group can either activate it or deactivate it, depending on the protein or where the phosphate is added - this tagging is done by kinase Opposite is done by the enzyme phosphatase It is reversible by can be added or removed by the respective enzymes

what happens during pre-transcription regulation in eukaryotic gene expression

Chromatin = a complex of DNA and DNA-associated proteins (histones) that tightly package DNA into nucleosomes Histone = positively charged proteins that have DNA wrapped around them two times Nucleosome = a group of 8 histones length of DNA wrapped around a histone twice Nucleosomes are then organized into 30nm fibers, which are then attached to protein scaffolds (remember from Chapman?) The wrapping of DNA around histones to form nucleosomes helps shorten the length of DNA Tight packing blocks RNA polymerase access to DNA - therefore, chromatin packaging is repressive to transcription Same goes for DNA replication DNase I is an enzyme that can cut DNA, but only DNA that is open and not tightly packaged (condensed chromatin) The portions of the chromatin that are more loosely packaged are more susceptible to DNase digestion compared with condensed chromatin because they are "out in the open: Condensed chromatin is correlated with low gene expression Looser chromatin is correlated with active gene expression - the genes are being expressed Whether or not genes are loosely packaged or tightly packaged is one of the ways that cells regulate which genes get expressed Note that the genes that are loosely packaged have the ability to interact with the environment. This means that RNA Polymerase can react with the DNA, transcribe DNA and begin the process of making proteins (something that can't happen when the DNA is tightly packaged since the RNA Polymerase can't penetrate it

what happens during telophase?

Chromosomes decondense Nuclear envelope reforms Chromosome separation is complete. The nuclear envelopes reform and the spindle fibers disassemble.

How do we determine which DNA sequences produce genes?

Computer programs are used to search a genome sequence to identify open reading frames (ORFs) A reading frame is a sequence of amino acids based on the cDNA - There are a total of 6 different reading frame, three for the top strand of DNA and three for the bottom In order for a reading frame to code for an actual protein, it needs to have a start codon and a stop codon What is an open reading frame (ORF)? - ORF = AUG-(codon-codon-codon)n-STOP

Practice exam question - Exposure of zebrafish nuclei to mitotic cytosol resulted in phosphorylation of L68 protein by MPF. The L68 is a protein of the nuclear lamina attached to the nuclear envelope. What is the most likely role of phosphorylated L68 in mitosis? A) It enables the attachment of the spindle microtubules to kinetochore regions of the centromere. B) It is involved in the disassembly and dispersal of the nucleolus. C) It assists in the movement of the centrosomes to opposite sides of the nucleus. D) It is involved in the disassembly of the nuclear envelope

D) It is involved in the disassembly of the nuclear envelope

What is the key property of DNase that makes it useful for assessing whether chromatin is in a closed (tightly condensed) or open (loosely packed) configuration?

DNase preferentially digests DNA not associated tightly with protein.

describe enzyme linked receptor signaling

Enzyme-linked receptors have an extracellular signal binding domain, a membrane spanning domain (which is hydrophobic), and an intracellular enzymatic domain. 1. Signal arrives and binds to Receptor 2. Signal-receptor complex changes conformation (forms a dimer) and is phosphorylated. 3. Proteins form a bridge between the activated kinase to Ras. - Many of receptors are signaling through Ras = a small G-Protein, which can be GTP or GDP bound. When GDP bound, inactive. GTP bound is active. The bridging proteins form a bridge for Ras, Ras becomes GTP bound and activated 4. Now that Ras is activated, Ras catalyzes the phosphorylation of an intracellular protein, activating it. In other words, the activated Ras protein can bind to an inactive protein kinase 1 and activate it 5. The phosphorylated protein the phosphorylation of another protein, producing a phosphorylation cascade where each protein phosphorylates another until a response is triggered in the cell.

compare the composition of genes vs. genomes in the DNA of prokaryotes and eukaryotes

Exons constitute less than 2% of the human genome, and repeated sequences make up more than 50%. - Less than 2% of a DNA sequence is coding for proteins In prokaryotic cells, usually 90% of the DNA sequences code for a product used by the cell.

what happens during post-translational regulation of gene expression in eukaryotes?

Folding by chaperones can be regulated Degradation by the protease is regulated Chemical modification regulation Transport regulation

where are the three checkpoints during the cell cycle?

G1 checkpoint G2 checkpoint Metaphase checkpoint At each checkpoint, interactions between regulatory molecules determine whether a cell proceeds with division.

how is mitosis different from meiosis?

Higher eukaryotes are diploid = two copies of each chromosome Meiosis creates specialized cells called gametes (ex = eggs or sperm) that contain only one copy of each chromosome (haploid) Mitosis occurs in somatic cells (which form the body and do not directly contribute to the next generation)

what happens during the metaphase checkpoint?

If all of chromosomes are aligned in the middle of the cell and attached to spindle apparatus, the metaphase checkpoint is passed. The metaphase checkpoint is activated if the sister chromatids are not properly aligned along the metaphase plate and attached to the microtubules.

explain how a microarray experiment works by using cells that grow in normal temperatures versus cells that grow in high temperatures

Isolate mRNAs and use reverse transcriptase to prepare single-stranded cDNA from mRNA 2. Make cDNA probes Add fluorescent green label to control cDNA and fluorescent red label to treatment cDNA (colors are used to tag, mark, each cDNA). 3. Probe a microarray with the labeled cDNAs. cDNA probes will bind to and label spots containing complementary sequences. 4. Shine laser light to induce fluorescence. Analyze the pattern of hybridization between the two cDNAs and the DNA on the microarray. - Spots containing a gene that is expressed under both conditions are yellow, because green and red hybridize - Spots containing a gene that is not expressed under either condition are dark - Spots of genes that are activated by heat are red - Spots of genes that are only expressed at normal temperature are green The cells grown under normal temperatures produce many mRNAs that will code for the proteins that allow it to survive. Similarly, the cells grown under high temperatures will produce many mRNAs that will transcribe the proteins that allow it to survive at these temperatures. Some of the mRNAs will code for the same protein, such as RNA Polymerase since both cells need those, while the mRNA grown translated from the cell grown under the high temperature could code for a protein that creates an outer layer that protects the cell from the heat. Cells produce multiple mRNAs since they need multiple proteins. In order to figure out these proteins, we use reverse transcriptase to create a cDNA that represents the gene that the protein transcribed came from. There will be multiple cDNAs produced because there were multiple mRNAs produced, per cell. These cDNAs tagged with a fluorescent color, in this example either green or red, depending on which cell they came come. Each of the many cDNAs produced are tagged with their own cDNA probe (the probe is the fluorescent color). Each of the many, now tagged, cDNAs are put into a microarray. When you shine a laser light to induce fluorescence, the colors show. If a circle, meaning a cDNA, produces a pure green light, then we know that the genes that would be produced by that cell would have been produced by the cells grown under the normal temperatures. If the light was pure red, then we know that the cDNA would code for proteins that would allow the cells to survive under the high temperature. If the color was yellow, then we know that the cDNA would code for proteins that would allow the cells under both the high temperature and the normal temperature to survive (such as RNA Polymerase). The many spots represent the different cDNAs that that were produced during reverse transcriptase from the many many many mRNAs that the cell produced while it was growing in its respective culture.

Under which condition(s) does it "make sense" for E. coli to express b-galactosidase? Explain why.

Lactose levels are high and glucose levels are low When only glucose is present, beta-galactosidase is not expressed when both glucose and lactose are present, then beta-galactosidase is not expressed when only lactose is present, beta-galactosidase is expressed this means that lactose induces the expression of beta-galactosidase and glucose inhibits the production of beta-galactosidase

explain the steps taken to clone the human growth hormone gene (GH1) to make GH1 in bacteria for treatment of pituitary dwarfism

Low GH1 in people results in pituitary dwarfism Can be treated with growth hormones Researchers wanted to engineer E. Coli to produce large amounts of growth hormone They isolated mRNA from human pituitary gland tissue and converted it to DNA using an enzyme called reverse transcriptase (RT). The DNA made from mRNA by RT is called complementary DNA (cDNA). Short DNA primers are then attached to both ends of each cDNA using DNA ligase. These primers contain a specific sequence that can be recognized and cleaved by a specific restriction enzyme. The cDNA was ligated into a linearized plasmids, and transformed into E. coli. Once the GH gene was cloned, the E. coli produced human GH. GH can be used to treat pituitary drawfism. Also used by athletes to enhance performance!!

describe how MPF is deactivated.

M-Cyclin is degraded by an enzyme that is activated during anaphase of mitosis. The enzyme targets cyclin to the proteosome where it is destroyed. This is an example of negative feedback regulation: a process is slowed down by one of its products.

in regards to gene families, what are the two major surprises?

MAJOR SURPRISE #1 = there is not a clear increase in gene number as cellular complexity increases - There is no strict correlation between the number of genes in an organisms and our perception of which organism is more complex -- For example, mice and rice have more genes than we do - this is because of alternative splicing = different proteins can be made from the same gene depending upon how the mRNA transcript is spliced together MAJOR SURPRISE #2 = the base sequence of human beings and chimp are 98.8% identical - The leading hypothesis focuses on the importance of regulation of gene expression. - Regulatory sequence = a section of DNA involved in controlling the activity of other genes; it may be a promoter, an enhancer, or a silencer - Structural genes = a sequence that codes for a tRNA, rRNA, protein, or other type of product - Regulatory genes = code for regulatory transcription factors that alter the expression of specific genes To resolve the sequence-similarity hypothesis, biologists propose that even though many structural genes in closely related species, such as humans and chimps, are identical or nearly identical, regulatory sequences and regulatory genes in the two species might contain important differences. The difference between humans and our nearest relatives (chimpanzees) is likely not in the coding sequences, but rather in the regulatory information

what is MPF and how does it work?

MPF = mitosis promoting factor MPF is composed of two protein subunits: a protein kinase (Cdk) and a cyclin (M-cyclin) - Cdk is an enzyme that catalyzes the transfer of a phosphate group from ATP to a target protein (it catalyzes the phosphorylation of a protein which triggers the onset of mitosis) How does it work? - Cdk is only active when bound by M-cyclin = when Cdk is bound by M-cyclin, you get MPF - MPF phosphorylates other proteins - MPF is regulated by phosphorylation

what happens at the G2 checkpoint?

MPF functions at entry to mitosis at G2 - the way to pass this checkpoint is that MPF needs to be activated. Without activation, you wont pass checkpoint. In order for MPF to be activated, chromosomes must be fully replicated and there isn't any damage in the DNA If there are issues, then the cell cycle would halt so that the damaged to DNA are corrected

what is the purpose of mitosis and the cell cycle in eukaryotes?

Mitosis is used for wound repair and growth in multicellular eukaryotes and for asexual reproduction in single celled organism

how does regulation of folding proteins affect gene expression?

Molecular chaperones assist proteins to fold properly. A translated polypeptide needs to be folding in order to be useful for the organism. Chaperones help proteins fold Therefore, you can regulate proteins (and therefore gene expression) by regulating the ability of protein (molecular) chaperones

What is the key to PCR?

Need to have sequence information to design primers One of the catches with PCR - sequence information is required because to do a polymerase chain reaction, you have to start by synthesizing short lengths of of single-stranded DNA that match sequences on either side of the gene of interest. These short segments act as primers for the synthesis reactions. To design the appropriate primer, the base sequences at the "primer-annealing sites" (where the primer binds to DNA) must be known - And the primers are extremely important because without them, DNA Polymerase wont work. Taq Polymerase is a DNA Polymerase taken from an extremophile, because we need this extremophile DNA polymerase to work under the high temperatures found following denaturing

what is the difference between negative regulation and positive regulation?

Negative regulation = when a regulatory protein binds to DNA and shuts down transcription (turning things off); the repressor is the "off switch" to a specific DNA sequence and turns the gene off Positive regulation = when a regulatory protein binds to DNA and triggers transcription (turning on); is done by an activator; binds to a different DNA sequence than the repressor

What is Polymerase Chain Reaction?

PCR = Polymerase Chain Reaction PCR is an alternative to DNA cloning in E. Coli PCR rapidly makes millions of copies of a specific DNA sequence in a few hours by a chain reaction amplification process PCR is an in vitro DNA synthesis reaction in which a specific section of DNA is replicated over and over, by DNA Polymerase, to amplify the number of copies of that sequence. It is a technique for generating many identical copies of a particular section of DNA Inserting a gene into a bacterial plasmid in one method for cloning DNA. PCR is another

What are the different levels of eukaryotic gene expression regulation?

Pre-transcription >> chromatin structure Transcription >> transcription factors Post-transcriptional >> alternative mRNA splicing and differential mRNA degradation Translation >> RNA bound proteins, ribosomes Post-translational >> folding, chemical modification, transport, activation, degradation

describe the different in identifying genes between prokaryotes and eukaryotes.

Prokaryotes - looking at DNA allows you to determine the genes of the organism because prokaryotes don't contain introns; during DNA replication, all of the nucleotides are converted to mRNA and used in translation of an amino acid Eukaryotes - it is difficult to identify genes by looking at DNA because DNA contains both introns and exons. Since only exons produce genes, you don't know if you are reading an amino acid sequence that was produced from only exons, introns, or both. Therefore, the production of cDNA from mRNA (which only contains exons) allows us to produce a DNA sequence that contains the information used to make a gene

how can we regulate gene expression through degradation?

Protease is used if a protein has been around too long and the cell wants to get rid of it or if a protein has been folded incorrectly (and it therefore dysfunctional) There is a molecular machine (protease) that can take in a protein and chop it into many little pieces This protein must be tagged by ubiquitin for it to be degraded. You add ubiquitin to the protein, creating an ubiquitin-tagged protein If the protein gets added to the machine, then it will be degraded

What are proto-oncogenes? What does it do? Explain how a mutation in a proto-oncogene could lead to cancer.

Proto-oncogenes are involved in the cell growth control at the G1 checkpoint - they are the ones that will promote the cell cycle activating mutations in proto-oncogenes cause the protein to become hyperactive. this hyperactivity means that DNA synthesis of not necessarily good DNA will occur no matter what

what are the four categories of cell to cell signaling?

SHORT RANGE SIGNALING CONTACT-DEPENDENT SIGNALING Molecule on surface of one cell is a signal, and a receptor on the surface of another cell "talk" to one another - only when the cells are touching one another PARACRINE SIGNALING LONG RANGE SIGNALING - don by specialized cells SYNAPTIC SIGNALING - electrically excited cells communicaate at synapses - found primarily in the nervous system ENDOCRINE SIGNALING - occurs when signals travel throughout the body via the bloodstream - primarily done by hormones

go into detail about how cloning works using plasmids

STEP 1 = CUTTING AND PASTING DNA - Plasmids contain a recognition site for a restriction enzyme. This site is a palindromic sequence, meaning that the sequence in the 5' to 3' direction of one strand is the same as the sequence in the 5' to 3' in the antiparallel strand - Researchers attach the same palindromic sequence to the ends of each cDNA in their sample - Restrictions enzymes cut the recognition sites in each plasmid and at the end of each DNA (such as EcoR1) - After making their cuts, most restriction enzymes create staggered cuts, sticky ends, that are capable of hydrogen bonding with a complementary sequence, because there are ends 'hanging off'. The other possible cut is made in the form of a blunt end - Finally, researchers use DNA ligase to seal the recombinant pieces of DNA together STEP TWO = TRANSFORM (INSERT) PLASMID INTO BACTERIA - If a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides. In this way, researchers can obtain millions or billions of copies of specific gene sequences STEP THREE = "GROW" MORE PLASMID - aka creating a new cDNA library - Only a very small fraction of the treated bacterial cells will pick up a plasmid (become transformed) - Need for selection: only cells that have picked up a plasmid can grow in the presence of antibiotics. - This is where the antibiotic characteristic comes in handy - Our plasmid carries a gene that allows it to survive in presence of antibiotic (ampicillin) - by placing bacteria cells on an antibiotic-containing growth plate, the ampicillin kills bacteria cells that doesn't have the plasmid - Therefore, plasmid will be able to keep growing in the presence of ampicillin without the presence of other bacterial cells

describe the steps taken during DNA sequencing

STEP 1 = INCUBATE REACTION MIX create a reaction mix that includes: - template DNA - primer - DNA polymerase - deoxynucleotides (dNTPs) - dideoxynucleotides (ddNTPs) STEP 2 = DNA SYNTHESIS OCCURS - the primer anneals to sequence it is complementary to - primer allows DNA polymerase to add a nucleotide - DNA polymerase can add either a dNTP or a ddNTP - there are more dNTPs than there are ddNTPs in the reaction mix and the choice that DNA Polymerase makes as to whether or not to add a dNTP or ddNTP is completely random - if a dNTP is added, DNA polymerase can add another nucleotide because it can use the 3' hydroxyl group - however, it a ddNTP is added, then the synthesized DNA strand would stop being synthesized because DNA polymerase doesnt have a 3' hydroxyl group to react with (since ddNTPs are missing the 3' hydroxyl) STEP 3 = COLLECT DNA STRANDS THAT ARE PRODUCED - fragments of the newly synthesized DNA have distinctive labels - the labels come from the fluorescent tags that were given to the ddTNPs STEP 4 = SEPARATE FRAGMENTS BASED ON SIZE - the short DNA can go through the special machine faster than longer DNA - this allows the different synthesized DNA sequences to be separated based on size - Since we are looking to sequence an entire DNA strand, there will be fragments of every length up to the length of the original DNA strand STEP 5 = READ SEQUENCE - the fluorescent tags from the ddNTPs allow scientists to read the sequence of DNA because each nucleotide is given a different tag - depending on the color that is presented by the tag will determine which nucleotide is present

describe what happens during shotgun sequencing

Shotgun Sequencing = an approach used when researchers set out to sequence the genome of a certain species for the first time. In shotgun sequencing, a genome is broken up into a set of overlapping fragments that are small enough to be sequenced. The regions of overlap are then used as guides for putting the sequenced fragments back into the correct order. STEP 1 = CUT GENOME (DNA) INTO LARGE FRAGMENTS STEP 2 - CLONE USING BACs - insert the DNA fragments into bacterial artificial chromosomes (BACs), which are bigger versions of plasmids - grow in bacterial cells to obtain large numbers of each fragment STEP 3 = CUT DNA AGAIN, EACH BAC, INTO SMALLER PIECES and grow in plasmid - we needed to cut the BACs into the size of a normal plasmid - the plasmids are copying many times as the bacterial cells grow into a large population STEP 4 = SEQUENCE EACH FRAGMENT - find region where different fragments overlap STEP 5 - ASSEMBLE SEQUENCE - assemble all of the fragments based on their overlapping regions to create a draft sequence

what is the difference between DNA sequencing and shotgun sequencing?

Shotgun sequence gives you the entire DNA genome DNA Sequencing finds out only an unknown stretch of DNA

what happens during anaphase?

Sister chromatids are pulled to opposite poles of the cells Sister chromatids separate and the chromosomes are pulled to opposite ends of the cell by spindle fiber shortening

give an example of intracellular receptors using steroid hormone receptors

Steroid hormones are lipids (hydrophobic) Can cross plasma membrane Steps of steroid signaling pathway: - Binding proteins transport the hormone through the blood stream - Steroid hormone diffuses across the plasma membrane and binds to an intracellular receptor - The receptor-hormone complex enters the nucleus and alters gene transcription

in prokaryotes, what are the three levels that gene expression can be regulated?

TRANSCRIPTION - efficient, but it can be slow - transcriptional regulation = controlling the amount of mRNA that is made because regulatory proteins affect RNA Polymerase's ability to bind to a promoter and initiate transcription - regulation at the transcriptional level is efficient because the cell does not invest any energy in making a gene product that may not end up being used - however, it can be slow TRANSLATION - translational regulation = controlling the amount of mRNA being translated into a protein - speed is inbetween POST-TRANSLATIONAL - controlling the activity of a protein after it is synthesized - can be very fast. this wastes energy because transcribing and translating a protein that may not be needed

describe the concentration levels of M-cyclin and Cdk over the course of the cell cycle

The active protein kinase from the phosphorylation cascade is the kinase enzyme that phosphorylates ATP to AD For example, active protein kinase 1 is the phosphoprotein of one of the reactions of the phosphorylation cascade, and in another reaction, t acts as the kinase enzyme The phosphate group on the active protein kinase is not going to lose its phosphate until phosphatase removes it through hydrolysis

Why do we know the genes that we know (their functions)?

The function of many genes can be inferred from similarity to known genes. - The reason why we know the genes that we know is because once an ORF is found, its sequence is compared with the sequences of known genes from well-studied species But the function of many genes is still unknown. - If the ORF is unlike any gene that has so far been described in any species, the gene is considered "unknown" because more research is required before it can actually be considered a gene

how does the cell regulate the tightness of chromatin packaging?

There are three mechanisms eukaryotic cells use to open or close chromatin (to regulate tightness of packaging): 1) DNA methylation -In most plants and some animals, cytosines in DNA can be methylated -DNA methylation generally results in tighter chromatin packaging -DNA methyltransferase (DNMT) adds methyl groups to cytosines -When DNA is in an open conformation, a methyl group can be added to tighten the packaging 2) modification of histones (e.g. acetylation) - Histone acetylation opens up chromatin Acetylation has the opposite effect of methylation -Histone acetyl transferases (HATs) add acetyl group to positively charged lysine residues -Histone deacetylases (HDACs) remove acetyl groups, converting loose chromatin into condensed chromatin Both methylation and acetylation can work together to package chromatin 3) chromatin remodelling (uses ATP to slide nucleosomes along DNA)

what are repetitive sequences in eukaryotes?

There are two kinds of repetitive sequences in eukaryotes: 1) transposable elements (~45% of the genome) - Transposable elements are parasitic pieces of DNA that got into our genome and have been spreading from time to time - Essentially, they are pieces of DNA that can move 2) short tandem repeats (~3% of the genome) - There are two major classes of STRs: -- Microsatellites are repeats of 1-5 bases -- Minisatellites are 6-500 bases that are repeated - These repeats make up 3% of out genome - The number of these repeats varies from person to person Minisatellites and microsatellites are very useful in DNA fingerprinting because they vary so much from person to person

If reverse transcriptase only creates a single stranded cDNA, how does it become double stranded cDNA?

There are two options in creating double stranded cDNA 1) reverse transcriptase also has the ability to synthesize the cDNA to produce a double stranded DNA 2) researchers add a primer to the single stranded cDAN so that DNA Polymerase can synthesize the double stranded cDNA

describe the similarities between enzyme-linked kinase signaling and g protein signaling

They both can activate proteins, either by adding a phosphate to a protein or by converting a G-protein from the GDP to GTP bound form Both activation events are reversible (phosphatase can remove the phosphate or the G-protein, hydrolyzing the GTP to GDP)

what happens at the G1 checkpoint?

This is where the cell decides if it wants to commit to the whole thing. You wouldn't start the process if you don't have the resources. If the cell hasn't grown sufficiently during G1, it wont go into S phase Sufficient nutrients are required to pass this checkpoint because you need the nutrients to make the stuff that you still don't have and organelles If social signals aren't present (signals from other cells or the environment saying that you can divide (such as growth factors - something that can tell the cell it can divide), then the cell wont pass this G1 checkpoint Damaged DNA can cause a block in G1

describe how transposable elements move from one part of the DNA to another

Transposable Elements spread within a Genome to produce most of the extra DNA nucleotides that don't make protein and we don't know what they are there for (the extra "junk" in DNA) Long Interspersed Nucleus Elements (LINEs) are a few 1000 base pairs long. Active LINEs have a functional promoter and two genes, a reverse transcriptase and a DNA integrase. Most LINE sequences in the genome are no longer able to transpose (once they move, they can't move again; they get old) - The reason why LINEs only have two genes, one for reverse transcriptase and one for integrase, is because the reverse transcriptase allows it to "enter" the cDNA form and a ligase so that it can enter itself Into the strand 1) parent LINE = a LINE exists in DNA 2) transcription = RNA polymerase transcribes LINE, producing LINE mRNA 3) translation - LINE mRNS exits nucleus and is translated - ribosome makes two proteins (reverse transcriptase and integrase) 4) into nucleus - LINE mRNA and proteins enter nucleus - proteins that enter are reverse transcriptase and integrase 5) cDNA copy - reverse transcriptase makes LINE cDNA from mRNA, then makes cDNA double stranded 6) insertion - integrase cuts chromosomal DNA and inserts LINE cDNA 7) daughter LINE = new copy of LINE is integrated into genome

A specific miRNA controls the translation of the LIN-41 protein. What do you expect to see in cells that lack the gene encoding this microRNA (i.e. the miRNA gene is deleted). A) Increased levels of LIN-41 RNA and increased levels of LIN -41 protein. B) Decreased levels of LIN-41 RNA and decreased levels of LIN -41 protein. C) Unchanged levels of LIN-41 RNA and increased levels of LIN-41 proteins. D) Unchanged levels of LIN-41 RNA and decreased levels of LIN-41 proteins

Unchanged levels of LIN-41 RNA and increased levels of LIN-41 proteins.

describe the experiment that proves how the chromosomes move to the opposite side of the cell during anaphase

Use fluorescent labels to make the chromosomes fluoresce blue and the microtubules fluoresce yellow Photobleaching = use a laser to bleach (destroy) the fluorescent label - photobleach a segment of the microtubule (turning it dark) result - since the distance between the chromosomes and the darkened section lesson, then this proved that microtubules shortened at the kinectore in order to move the chromosomes closer to the ends of the cell

explain the regulation of lacZ and lacY based on the levels of lactose

When lactose isn't present, the repressor protein (coded for by lacI) can bind to the operator (a sequence of DNA), preventing transcription of the lac operon (lacZ and lacY...) - an example of negative regulation by the repressor protein When lactose is present, lactose binds to the repressor, changing its conformation such that the repressor can no longer bind to the DNA. When the repressor is not bound, there is no more "negative regulation" and transcription of the lac operon can occur. This is an example of positive regulation (where lactose is the inducer, the activator, since it is allowing transcription to occur, thus activating the lacZ and lacY genes). Lastly, when the normal lacI is mutated (now there isn't a repressor present), it doesn't matter whether lactose levels are high or low because if lactose levels were low, there wouldn't be a repressor to stop the transcription of lacZ and lacY, and if lactose was present then transcription would occur no matter what. Therefore, a mutation in the lacI gene causes constitutive expression. Also, a mutant form of lacI could produce a nonfunctional repressor protein that wouldn't function, allowing transcription to still occur no matter the levels of lactose.

describe the regulation of the G1 checkpont

When the cell receives signals to divide, growth factors (social signals) promote the synthesis of S-cyclin (which is different from M-cyclin) and E2F, leading to a rise in S-cyclin and E2F levels S-cyclin works like M-cyclin E2F is a transcription factor that activates many genes required for the S-phase When S-cyclin binds to Cdk, Cdk is phosphorylated and becomes inactive (just like when M-cyclin binds to Cdk) When Rb binds to E2F, it inactivates it When the S-cyclin-Cdk complex is activated (through dephosphorylation), it is able to use that phosphate group to phosphorylate Rb The phosphorylation changes the conformation of Rb, causing it to no longer be able to bind to E2F E2F is now able to initiate the S-phase In essence, E2F is the S-phase promoting factor (its activation will trigger everything needed for S-phase) Rb is the gatekeeper border patrol, regulating whether or not the cell will enter the S-phase

what are the differences between enzyme linked kinase signaling and g protein signaling

When using ATP, you form a covalent bond between phosphate and protein - with G-protein, you bond GDP in a non-covalent matter (non-covalent interaction). This difference has a huge impact For protein kinases, the hydrolysis of ATP is required for the activity of these proteins. For G-proteins, hydrolysis of GTP is required for the inactivation of the G-proteins A non-hydrolyzable form of ATP inhibits protein kinases while a non-hydrolyzable form of GTP results in constitutive activation (always on) of G-proteins

what happens during post-transcriptional regulation in eukaryotes?

after transcription, mRNA processing must occur - this mean splicing ALTERNATIVE SPLICING = splicing the same primary RNA transcript in different ways to produce different mature mRNAs and thus different proteins During splicing, changes in gene expression are possible because selected exons may be removed from the primary transcript along with the introns. As a result, the same primary RNA transcript can yield more than one kind of mature, processed mRNA, consisting of different combinations of transcribed exons. This is important because if these mature mRNAs contain differences in their ribonucleotide sequence, then the polypeptides translated from them will also differ. - different exons can be cut, thus producing a different mRNA and therefore resulting in a different gene being expressed. DIFFERENTIAL MRNA DEGRADATION - Once mRNA is spliced it enters the cytoplasm where additional regulatory mechanisms control mRNA stability (and therefore gene expression) - The stability of mRNAs in the cytoplasm is highly variable. Some are degraded rapidly, allowing for only a short period of translation - Others are quite stable, and could remain in cytoplasm for months - In many cases, the life span of an mRNA (before translation) is controlled by proteins binding to the 3' UTR of mRNAs. Regulation by mRNA interference - microRNAs (miRNAs) and short interfering RNAs (siRNAs): these short RNAs regulate mRNA stability and translation Production and regulation by miRNA: - Transcribe one strand of DNA to produce a single stranded RNA (ssRNA) - The ssRNA folds upon itself to create a hairpin RNA (because pairs of sequences within the RNA transcript are complementary). - The actual hairpin is cut out to make "mature" miRNA, which gets incorporated into the protein complex to produce a miRNA-RISC protein - miRNA-RISC will find the target mRNA, bind to it, and will inhibit translation Production and regulation by siRNA: - Transcribe both strands of DNA to produce a double stranded RNA (dsRNA) dsRNA will be recognized as something that needs to be chopped into pieces. - Just one of these pieces will get incorporated into a protein complex to produce siRNA-RISC - siRNA-RISC will find target mRNA and cutting the mRNA in half will cause it to degrade (aka the "kiss of death")

how is bioinformatics used during shotgun sequencing?

bioinformatics = Development and application of computing, mathematical and statistical methods to analyze biological, biochemical and biophysical data during shotgun sequencing, computing is used to recognize similar stretches and this mix and matching can put a sequence together - done through aligning of overlapping stretches

how can we regulate gene expression through protein transport?

by transporting the protein from the cytoplasm to the nucleus, we can regulate gene expression

explain how glucose can create feedback inhibition on beta-galactosidase? in other words, how does glucose regulate the lac operon?

cAMP allows CAP proteins to bind to the CAP binding site on RNA Polymerase and this triggers transcription of the lac operon when glucose levels are high, cAMP levels are low - this means that CAP can't bind to the CAP binding site on the RNA Polymerase, and therefore transcription of the lac operon doesn't occur - the details: The enzyme Adenylyl cyclase converts ATP into two phosphate groups and cAMP. - high glucose concentration inactivate adenylyl cyclase, which results in low cAMP levels, which means CAP cant bind to operator, which means infrequent transcription of the lac operon when glucose levels are low, cAMP levels are high - therefore, CAP is able to bind to RNA Polymerase and transcription of the lac operon can occur - the details: The enzyme Adenylyl cyclase converts ATP into two phosphate groups and cAMP. - low glucose concentration means adenylyl cyclase is active, converting ATP into two phosphates and cAMP - high levels of cAMP allows CAP to bind to RNA Polymerase, which allows RNA Polymerase to transcribe the lac operon

what happens during interphase?

chromosomes are replicated during the S phase = replication of DNA - replicated chromosomes are joined together at the centrosome organelles are replicated and cell growth occurs during G1 and G2 phases

what happens during prophase?

chromosomes condense spindle apparatus begins to form

what happens during metaphase?

chromosomes line up in the middle of the cell Spindles on each side of the sister chromatids align the chromosomes in the middle of the cell (the metaphase plate)

what is the difference between constitutive expression and regulated expression?

constitutive expression = gene production made continuously regulated expression = gene product made on demand; expression can be induced or repressed

what happens during cytokineses? describe what happens in both plants and animal cells

cytoplasm is divided two daughter cells form Cytokinesis = splitting the cell to form to daughter cells Cytokinesis in animals, fungi, and slime molds occurs when a ring of actin and myosin filaments contracts inside the cell membrane, causing it to pinch inward in a cleavage furrow. Cytokinesis in plant cells: Vesicles are transported from the Golgi apparatus to the middle of the dividing cell along microtubules. These vesicles fuse to form a cell plate. - Since plants have a rigid cell wall, the pinching and splitting can't happen like it does in animal cells. Instead, they build another cell membrane and cell wall in between the two cells. You bring vesicles that contain the material to form a cell plate

Provide examples of real-life applications of PCR

detect mutations find out who the daddy is to solve a crime when you need to recombine (recombinant DNA)

What are dideoxynucleotides and why are they so important?

dideoxynucleotides (ddNTPs) are deoxynucleotides, but without a hydroxyl group on the 3' carbon (hence the 'di') the lacking of the 3' hydroxyl is important because without the 3' hydroxyl, DNA Polymerase doesn't have anything to excite it. Therefore, DNA sequencing will automatically end when a ddNTP is added by the DNA polymerase (compared to the normal dNTP)

define functional genomics

functional genomics = how genes work together to produce a phenotype example = use DNA microarray to study RNA transcripts

what are gene families?

gene families = clusters of closely related genes. - they are thought to have arisen from gene duplications (the creation of an additional copy of a gene) some members of a gene family may be nonfunctional pseudogenes. they are remnants of a functional copy of the gene but, due to mutation, do not produce a working product - If the two genes remain identical functionally, then one or the other might start to accumulate mutations and become non-functional. Degraded genes that are no longer encoding functional proteins are called pseudogenes. Sometimes you will find something that looks like a gene but isn't a gene = pseudogene

What is genetic engineering?

genetic engineering = taking a piece of DNA and making it different; designing a piece of DNA

describe the experiment that located the beta-galactosidase gene

in order to figure out which gene coded for beta-galactosidase, researchers wanted to find e coli cells that were not capable to metabolizing lactose because whatever is broken is what is used to breakdown lactose Start with a bunch of cells and mutagenize them (induced a mutation). By mutating a bunch of cells, the hope is that one of them is going to have the mutation that prevents it from being able to break down lactose. 1. Plate mutagenized cells: grow a master plate of mutagenized cells on a medium that contains glucose (we want to make sure that these cells can utilize glucose as a source of energy) 2. "Copy" onto second plate: take the mutagenized cells growing in glucose and transfer cells from the growing colonies to a plate that only contains lactose as the energy source 3. Identify mutant colony: Compare the colonies that survive on the plate with glucose as the energy source with the colonies using lactose as an energy source. The colonies that are growing in the presence of glucose but are missing in the presence of lactose are the cells with the mutation causing them to be deficient in lactose metabolism (aka, they are missing beta-galactosidase)

what were the genes found that e coli uses to breakdown glucose?

lacZ = encodes for beta-galactosidase lacY = encodes galactoside permease which allows lactose to enter the cell It was found that lacZ and lacY are located very close to each other We know that lacZ and lacY are expressed on a single mRNA This type of mRNA is called polycistronic - can have 2-10 genes on one mRNA The regulatory region of a polycistronic mRNA and the sequences corresponding to the polycistronic mRNA is called an operon. LacZ and lacY and their control regions are called the lac operon

What is p53? What does it do? Explain how a mutation in p53 would cause cancer.

p53 = tumor suppressor gene If DNA damaged for whatever reason, some molecule will sense the damage and activate p53, causing its levels to rise. p53 will bind to enhancers of genes to repair proteins. If damaged DNA is little enough, then you can activate DNA repair genes (you must stop cell cycle to give cell enough time to repair) If there is no hope to repair this damage, then the cell is killed - apoptosis Cancer causing mutations in p53 prevent the protein from binding to its target sites in DNA. Cells that contain inactive p53 will tolerate DNA damage and accumulate more mutations, including more cancer-causing mutations. Over 50% of human cancer cells have p53 mutations.

what are the effects of activated MPF?

phosphorylate chromosomal proteins and initiate M phase phosphorylate nuclear lamins and initiate nuclear envelope breakdown phosphorylate microtubule-associated proteins and activate the mitotic spindle phosphorylate an enzyme that degrade cyclin causing cyclin concentrations to decline (thus the deactivation of MPF)

describe the composition of a plasmid in the laboratory

plasmid in the laboratory = circular double stranded DNA that contains three essential things: 1) an origin of replication - if they don't have this, then they will never be replicated 2) an antibiotic resistance gene (ex = ampicillin) 3) a cloning site to insert foreign DNA these three characteristics make plasmids useful in genetic engineering

what are plasmids? why are they important to genetic engineering?

plasmids = small, circular molecules of DNA distinct from the chromosome plasmids are important to genetic engineering because researchers can clone a gene by inserting it into a plasmid - plasmids are used for DNA cloning

describe the basics behind using plasmids in the laboratory

plasmids are used for cloning step 1 = create recombinant plasmid step 2 = insert plasmid into bacteria step 3 = bacterium will make copies of plasmid

what are restriction enzymes? why are they important for DNA cloning?

restriction enzymes are endonucleases that cut DNA at specific sites there are hundreds of restriction enzymes that have the ability to make cuts a different recognition sites - the restriction sites are often palindomes, meaning that they read the same in either direction after cutting DNA, restriction enzymes create either a sticky end or a blunt end - sticky ends have overhangs - blunt ends dont have overhangs

What is reverse transcriptase?

reverse transcriptase = an enzyme that converts mRNA into cDNA it will bind to an mRNA and make a cDNA copy reverse transcriptase initially produces a single-stranded cDNA

in the lac operon, what does the lacI gene do?

the lacI gene codes for a repressor protein that exerts negative regulation on lacZ and lacY, therefore turning them off How the repressor works is determined by lactose levels - Lactose binds to the repressor protein (which is produced by lacI), changing its conformation so that it can no longer bind to DNA (an example of allosteric regulation). This allows transcription to occur because the repressor can't do its thing

what happens during prometaphase?

the nuclear envelope breaks down mucrotubules start attaching to chromosomes at kinectocores

what happens during translational regulation of eukaryotic gene expression?

translational control done by miRNAs and siRNAs RNA binding proteins can control whether or not or how efficiently an mRNA is translated Ribosome activation (response to extracellular signals) - phosphorylation of ribosomal proteins can prevent or slow translation - translation of all proteins in the cell can be affected by regulating the activity of the ribosome

What is DNA sequencing?

use to determine an unknown stretch of DNA, nucleotide by nucleotide The most widely used method of DNA cloning relies on dideoxynucleotides (ddNTPS), such as ddATP, ddTTP, ddGTP, and ddCTP (a ddNTP for each DNA nucleotide)

describe the different variations in cell cycle length

varies from tissue to tissue and organism to organism rapidly dividing cells (such as dividing cells of a young embryo) have very brief or no gap phases S-phase and M-phasr are generally a fixed length in a living organism non-dividing cells (also known as terminally differentiated cells, such as mature neurons) are stuck in G1, also called the G0 phase rate of cell division can vary in response to specific conditions - for example, you scrape your knee, the response to loss of skin cells (the stimulus) will increase cell division of skin cells to fill in the hole regulation of the cell cycle is achieved by specific factors promoting or inhibiting specific stages - such as mitosis-promoting factor

if DNA sequencing is used to determine a stretch of DNA, then how to determine the sequence of an entire genome?

we use shotgun sequencing