BIOL 3090: Exam 2
Describe the structure of RNA polymerase.
Bacterial RNA polymerases are composed of two related large subunits (β and β'), two copies of a smaller subunit (α), and one copy of a fifth subunit (ω). Diagrams of the transcription process generally show RNA polymerase bound to an unbent DNA molecule. However, x-ray crystallography and other studies of an elongating RNA polymerase indicate that the DNA bends at the transcription bubble.
Complementary DNA (cDNA)
DNA copies of cellular mRNAs that lack the noncoding regions present in genomic DNA
Why are heavy metals used for staining in transmission electron microscopy?
Electrons are deflected by the heavy metal stains.
True or False: Only agarose gels are used to separate DNA fragments.
False! Agarose can be used to separate DNA. However, agarose can reflect 0.5-20 kbp. Polyacrylamide is also used to separate DNA. In contrast, polyacrylamide only reflects 20-1500 bp.
True or False: Ethidium bromide is safe to handle with your bare hands.
False! It is mutagenic since it can slide between base pairs, which results in the wrong nucleotide may be included.
T/F: RNA is always linear.
False. RNA can be linear or circular; single- or double-stranded.
What is the function of DNA ligase?
DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligase. For the purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends. They are covalently ligated through the standard phosphodiester bond of DNA. ATP hydrolysis is required for this reaction.
What enzyme introduces supercoils in bacteria?
DNA gyrases introduce supercoils into bacteria.
DNA has what kind of charge?
DNA has a negative charge.
To which electric pole does DNA migrate in gel electrophoresis?
DNA migrates toward the anode during gel electrophoresis.
Describe the DNA proofreading mechanism of DNA polymerase.
DNA polymerase sometimes makes mistakes. However, DNA polymerase is incredibly accurate, and this is largely due to proofreading by DNA polymerase. Eukaryotic polymerases δ and ε employ proofreading mechanisms. Proofreading depends on the 3' to 5' exonuclease activity of some DNA polymerases. When an incorrect base is incorporated during DNA synthesis, base pairing between the 3' nucleotide of the nascent strand and the template strand does not occur. As a result, the polymerase pauses, then transfers the 3' end of the growing chain to its exonuclease site, where the incorrect mispaired base is removed. Then the 3' end is transferred back to the polymerase site, where this region is copied correctly.
Where does DNA synthesis begin?
DNA synthesis starts with a primer.
What kind of helix does this DNA-RNA hybrid have during transcription?
DNA-RNA hybrids are A-form helices.
DNA Vector
DNAs that replicate autonomously
Describe deconvolution microscopy, and how does it battle the inherent problems seen in conventional fluorescence microscopy?
Deconvolution microscopy uses computational methods to remove fluorescence contributed by out-of-focus parts of the sample. Computer algorithms are used to subtract unwanted focal planes.
True or False: Circular DNA molecules are found in prokaryotes, many viruses, mitochondria, and chloroplasts.
True
True or False: In elongation, the amino group of incoming amino acid attacks bond of the peptidyl-tRNA in the P site.
True
True or False: In translation elongation, the peptide chain is briefly attached to the tRNA in the A site.
True
True or False: In vitro organic synthesis of an oligonucleotide is backwards, using a 5' OH rather than a 3' OH.
True
True or False: Nucleotides are read as triplets.
True
True or False: Restriction enzymes can be used to establish a physical map of the DNA.
True
True or False: Ribosomal RNAs have conserved secondary and tertiary structures.
True
True or False: Since cDNAs are made from mature, cytoplasmic mRNAs, cDNAs have no introns, no promoters, and no intergenic DNA.
True
Do rRNAs have a 5' cap?
Nope! RNA polymerase II is the only polymerase affiliated with the 5' cap.
Which eukaryotic polymerase is always associated with the 5' cap?
RNA polymerase II
How should RNA be handled in a lab?
RNA should be handled with care. It is exceedingly sensitive to ribonuclease activity and base-catalyzed hydrolysis. Gloves should always be worn, as ribonucleases could destroy the RNA. The pH of solutions should be less than 8.0 as well.
In DNA replication, RNA primers are removed by ___________________________.
Rnase H and FEN I
Supercoiling
a process by which a DNA molecule folds back on itself
Haploid
a single set of chromosomes
Describe prokaryotic genome organization compared to eukaryotic genome organization.
The most common arrangement of protein-coding genes in bacteria includes gene encoding proteins that function together. Such an arrangement of genes in a functional group is called an operon because it operates as a unit from a single promoter. Transcription of an operon produces a continuous strand of mRNA that carries the message for a related series of proteins. This arrangement results in the coordinate expression of all the genes in the operon. This type of organization produces polycistronic mRNAs, where an mRNA encodes several different proteins. Repressors and co-repressors help regulate the expression of operons in bacteria.
Describe non-homologous end joining.
The predominant mechanism for repairing double-stranded breaks in multicellular organisms involves rejoining of the non-homologous ends of two DNA molecules. Even if the joined DNA fragments come from the same chromosome, the repair process results in the loss of several base pairs at the joining point. The formation of such a possibly mutagenic deletion is one example of how repair of DNA damage can introduce mutations. Occasionally, when broken ends from different chromosomes are joined together, this leads to translocation of pieces of DNA from one chromosome to another. Such translocations may generate chimeric genes that can have drastic effects on normal cell function.
What is the purpose of a Southern blot?
Southern blots allow you to characterize the gene in the genome: such as how many copies of the gene are there in the genome? and determine the restriction or physical map of the gene.
Where do cDNAs come from?
cDNAs are made from tissue-specific or cell-specific mRNAs (liver vs. brain) or perhaps from a particular stage of embryogenesis.
Genetic distance is expressed in ______________________.
cM
Plasmid
circular, double-stranded DNA molecules that replicate separately from a cell's chromosomal DNA
Primary cells have a ___________________ life span in culture.
finite
Transfection
genes are cloned into specialized eukaryotic expression vectors and are introduced into cultured animal cells
If two genes are far apart, the frequency of recombination is ____________________.
high
The amino acid of a tRNA attaches to the 3' hydroxyl group of the tRNA through a _______________________ bond.
high-energy ester
In transcription, the incoming _________________ base pairs with the base in the DNA template strand.
rNTP
Restriction Site
short, palindromic sequences; that is, the restriction site sequence is the same on each DNA strand when read in the 5' --> 3' direction
Non-homologous end joining has implications in:
telomeres and CRISPR
Allele
the different forms or variants of a gene
If two genes are close together, the frequency of crossing-over is _____________.
low
All DNA in living systems is ________________________________ supercoiled. It is supercoiled in __________________________ direction.
negatively; one
Genotype
the particular set of alleles for all the genes carried by an individual
Reading Frame
the sequence of codons that runs from a specific start codon to a stop codon
In transcription, the 3' hydroxyl group of an rNTP attacks the ______________________ phosphate.
α
In transcription, mRNA is synthesized in the _________________________ direction.
5' --> 3'
How is a polynucleotide chain often written as?
A polynucleotide is often written 5' to 3' displaying nitrogenous bases.
Describe the two definitions of synthetic lethality.
Another phenomenon, called synthetic lethality, produces phenotypic effect opposite to that of suppression. In this case, the deleterious effect of one mutation is greatly exacerbated by a second mutation in a related gene. 1. In one situation, a heterodimeric protein is partially, but not completely, inactivated by mutations in either one of its nonidentical subunits. However, in double mutants carrying specific mutations in the genes encoding both subunits, little interaction between subunits occurs, resulting in severe phenotypic effects. 2. In another situation, two pathways produce the same product. If either pathway alone is inactivated by a mutation, the other pathway will be able to supply the need product. However, it both pathways are inactivated at the same time, the essential product cannot be synthesized, and the double mutants will be nonviable.
True of False: The codon and anti-codon is the only parallel interaction in the cell.
False
True or False: The cDNA sequence directly provides the protein sequence.
False
True or False: There are two aminoacyl tRNA synthetases for methionine.
False
What is the effective resolution for TEM?
0.10 nm, which is one Angstrom.
One intact ribosome contains:
1. four ribosomal RNAs 2. ~80 ribosomal proteins in eukaryotes 3. 80S in eukaryotes
In autoradiography, what radioactive isotopes are used to label the following: 1. DNA 2. RNA 3. Protein
1. 3H-Thymidine to label DNA. 2. 3H-Uridine to label RNA. 3. 3H-Amino acid to label protein.
What is the origin of replication in a shuttle vector for eukaryotes? Prokaryotes?
1. ARS for eukaryotes 2. ORI for prokaryotes
Describe the lytic pathway of viruses.
1. Adsorption: Virion interacts with a host cell by binding of multiple copies of capsid protein to specific receptors on the cell surface. 2. Penetration: Viral genome crosses the host plasma membrane. For some viruses, viral proteins packaged inside the capsid also enter the host cell. 3. Replication: Viral mRNAs are produced with the air of the host-cell transcription machinery or viral enzymes. Viral mRNAs are translated by the host=cell translation machinery. Production of multiple copies of the viral genome is carried out either by viral proteins alone or with the help of host-cell proteins. 4. Assembly: Viral proteins and replicated genomes associated to form progeny virions. 5. Release: The host cell either ruptures suddenly (lysis), releasing all the newly formed virions at once, or disintegrates gradually, releasing the virions slowly. Both types of release lead to the death of the infected cell.
What are some commonly used antibiotics used in the selection of recombinant DNA?
1. Ampicillin 2. Tetracycline 3. Chloramphenicol 4. Kanamycin
What are the three forms of DNA?
1. B DNA 2. A DNA 3. Z DNA
What are four ways of introducing DNA into mammalian cells?
1. Ca++-DNA precipitate: You can take recombinant DNA and mix it with CaCl, forming a precipitate. The precipitate is engulfed by pinocytosis. 2. Liposome Fusion: Another way is to take DNA and mix with lisosomes. The phospholipid surrounds DNA, and the liposome binds to cell membrane. 3. Retrovirus Delivery 4. Electroporation: Another is electroporation. You take cells and shock them, and this punches holes in the plasma membrane, allowing DNA to enter the cell.
Give two examples of a DNA vector.
1. Circular plasmid DNAs in bacteria 2. Bacteriophage lambda vectors
DNA strands must separate during these processes:
1. DNA Replication 2. Transcription 3. Recombination 4. DNA Repair
Describe the two basic ways of arranging capsid protein subunits and the viral genome into a virion.
1. In some viruses, multiple copies of a single capsid protein form a helical structure that encloses and protects the viral RNA or DNA, which runs in a helical structure. 2. The other major structural type is based on the icosahedron, a solid, approximately spherical object build of 20 identical faces, each of which is an equilateral triangle
Compare and contrast human primary cells and mouse primary cells grown in culture.
1. Human Primary Cells: When cells are removed from an embryo, most of the adherent cells will divide a finite number of times (about 50) and then cease growing, enter senescence, or G0 of the cell cycle in other words. 2. Mouse Primary Cells: Primary cultures of normal rodent cells commonly undergo spontaneous transformation into a cell line. After the rodent cells are grown in culture for several generations, the culture goes into senescence. During this period, most of the cells stop growing, but often a rapidly dividing transformed cell that has attracted oncogenic mutations arises spontaneously and takes over, or overgrows a culture. A cell line derived from such a transformed variant will grow indefinitely if provided with the necessary nutrients.
Describe the main differences between RNA and DNA.
1. In RNA, the sugar is a ribose. In DNA, deoxyribose is the sugar used. 2. Three of these bases - A, G, and C - are found in both DNA and RNA; however, T is only found in DNA and U only in RNA.
What are the three types of super-resolution microscopy? Describe each.
1. In structured illumination microscopy (SIM), the specimen is illuminated with a pattern of light and dark stripes, and several images are taken as the illuminating pattern is rotated to generate Moiré fringes. Analysis of the images gives a resolution of about 100 nm. SIM is good at live cell imaging. 2. In stimulated emission depletion (STED) microscopy, the sample is scanned just as in point-scanning microscopy, but the focused excitation laser point is surrounded by a donut-shaped depletion beam, which effective makes the area excited much smaller. Since the spot is precise, it can build up an impressive image. 3. In photoactivated localization microscopy (PALM), a variant of GFP is photoactivated; that is, it can become fluorescent only after being activated by a specific wavelength of light, different from its excitation wavelength. When we then excite the sample, the one activated GFP emits many photons, giving rise to a Gaussian distribution. The center of the distribution can tell us where the GFP is located with nanometer accuracy. When we activated another GFP, we can localize it individually with the same precision. This is done over and over. Thousands of images are generated through a computer.
Compare and contrast classic versus reverse genetics.
1. In the classical genetic approach, we begin with the isolation of a mutant that appears to be defective in some process or interest. Genetic methods are then used to identify and isolate the affected gene. The isolated gene then can be manipulated to produce large quantities of the protein it encodes for experiments and to design probes for studies of where and when the protein is expressed in an organism. 2. In the reverse genetic approach, we begin with the isolation of an interesting protein or its identification based on analysis of an organism's genomic sequence. Once the corresponding gene has been isolated, that gene can be altered and then reinserted into the organism.
Describe the process of transcription in eukaryotes.
1. Initiation: During transcription initiation, RNA polymerase, with the help of initiation factors, recognizes and binds to a specific sequence of double-stranded DNA called a promoter. After binding, RNA polymerase and the initiation factors separate the DNA strands to make the bases in the template strand available for base pairing with the bases of the rNTPs that it will polymerize. This allows the template strand to enter the active site of the enzyme. The active site was where catalysis of phosphodiester bond formation between rNTPs that are complementary to the template strand takes place. The segment of melted DNA in the polymerase is known as the transcription bubble. Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond. 2. Elongation: After several ribonucleotides have been polymerized, RNA polymerase dissociates from the promoter DNA and initiation factors (called σ factors). During the strand elongation stage, RNA polymerase moves along the template DNA, opening the double-stranded DNA in front of its direction of movement and guiding the strands back together so that they reassociate at the upstream end of the transcription bubble. One ribonucleotide at a time is added by the polymerase to the 3' end of the growing RNA chain. 3. Termination: During transcription termination, the final stage in RNA synthesis, the complete RNA molecule is released from the RNA polymerase and the polymerase dissociates from the template DNA. Once it is released, an RNA polymerase is free to transcribe the same gene again or another gene.
What are three methods used to sequence DNA?
1. Maxam and Gilbert Technique 2. Sanger Sequencing 3. Next Generation DNA Sequencing
What are the three functions that the nano-machines, also known as ribosomes, carry out?
1. The ribosome reads mRNA from the 5' --> 3' direction. 2. The ribosome binds activated tRNAs and growing peptide chains. 3. The ribosome catalyzes peptide bond formation, incorporating 3-5 amino acids per minute.
What are the three primary types of RNA? Describe each.
1. Messenger RNA (mRNA) carries the genetic information transcribed from DNA in a linear form. The mRNA reads in sets of three-nucleotide sequences, called codons, each of which specifies a particular amino acid. 2. Transfer RNA (tRNA) is the key to deciphering the codons in mRNA. Each type of amino acid has its own subset of tRNAs, which are covalently bound to that amino acid and carry it to the growing end of a polypeptide chain when the next codon in the mRNA calls for it. The correct tRNA with its attached amino acid is selected at each step because each specific tRNA molecule contains a three-nucleotide sequence, an anticodon, that can base pair with its complementary codon in the mRNA. 3. Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These structures, which move along the mRNA molecule, catalyze the assembly of amino acids into polypeptides. Ribosomes are composed of a large and a small subunit, each of which contains its own rRNA molecules.
A nucleoside consists of:
1. Nitrogenous Base 2. Ribose (or Deoxyribose)
A nucleotide consists of:
1. Nitrogenous Base 2. Ribose (or Deoxyribose) 3. 1-3 Phosphates
What can happen to DNA introduced into eukaryotic cells by transfection?
1. Randomly integrate into chromosomes (transformed). 2. Undergo double homologous recombination with normal endogenous gene (gene knockout technology) 3. Remain as an extra-chromosomal plasmid (episome)
What two things are vital to DNA cloning?
1. Restriction Enzymes 2. DNA Ligase
List some common retroviruses.
1. Some retroviruses contain oncogenes (mice and birds) 2. Human T-cell Lymphotrophic Virus (HTLV): a leukemia 3. Human Immunodeficiency Virus (HIV): AIDS
What are the two types of tRNAs for methionine? What is the difference between the two?
1. tRNA for initiation 2. tRNA for elongation The initiation tRNA can bind to the P site during initiation. The elongation tRNA cannot bind to the P site during initiation, only during elongation.
Peptidyl transferase activity is a function of the __________________.
28S rRNA
RNA polymerase reads the DNA template strand in the _________________________ direction.
3' --> 5'
DNA polymerase reads the template strand __________________ to synthesize the daughter strand ___________________.
3' to 5'; 5' to 3'
What functional group is required on the terminal nucleotide of DNA for replication to continue?
A 3' OH on the last nucleotide is needed for condensation synthesis, which is a nucleophilic attack on alpha phosphate.
Describe genetic complementation analysis.
A common test for determining whether mutations are in the same gene or in different genes exploits the phenomenon of genetic complementation; that is, restoration of the wild type phenotype by mating two different mutants. For example, the screen for cdc mutations in Saccharomyces described previously yielded many recessive temperature-sensitive mutants that appeared to be arrested at the same cell cycle stage. To determine how many genes were affected by these mutations, Hartwell and colleagues performed complementation tests on all of the pair-wise combination of their cdc mutations. Haploids of opposite mating types and carrying different recessive temperature-sensitive cdc mutations were mated. For example, cdcX (mutant type a) and cdcY (mutant type α) were crossed. These were plated and incubated at the permissive temperature. They were replica-plated and incubated at the non-permissive temperature. These mutants saw growth, and growth indicates that mutations cdcX and cdcY are in different genes, providing normal function. This means that the cdcX and cdcY mutants complemented each other. In another example, cdcX (mutant type a) and cdcZ (mutant type α) were crossed. These were plated and incubated at the permissive temperature. They were replica-plated and incubated at the non-permissive temperature. These mutants did not see growth, and no growth indicates that mutations cdcX and cdcZ are on the same gene, making both alleles nonfunctional.
What is a dideoxyribonucleoside triphosphate?
A ddNTP is a dNTP without a 3' OH group.
Describe fluorescence-activated cell sorter (FACS).
A fluorescence-activated cell sorter (FACS), which is based on flow cytometry, can both analyze the cells and select the few fluorescent cells from thousands of others and sort them into a separate culture dish. To achieve this, the cells are mixed with a buffer and forced through a vibrating nozzle to generate tiny droplets. Just before the nozzle, the stream of cells passes through a laser beam so that the presence and size of a cell can be recorded from the scattered light using one detector, and the amount of fluorescent light emitted can be quantified using a second, fluorescent light detector. If a cell is present in a droplet, the droplet is given a negative charge as it emerges from the nozzle. The stream of droplets then passes though two plates that generate an electric field proportional to the fluorescence detected from the cell in the droplet. The field generates a force that moves charged droplets out of the stream and into the collection tube. Since the amount of force is proportional to the fluorescence emitted by the cell in the droplet, cells with different levels of fluorescence can be collected.
Describe the function of the lac operon?
A gene within the bacterial genome encodes an enzyme for regulating lactose. If E. coli is starved for glucose, it will use lactose. The operon synthesizes lactose. Lactose broken down is galactose and glucose, where the glucose can be used for cellular functions.
Describe a plaque assay.
A number of infectious viral particles in a sample can be quantified by a plaque assay. This assay is performed by culturing a dilute sample of viral particles on a plate covered with host cells and then counting the number of local lesions, called plaques, that develop. A plaque develops on the plate wherever a single virion initially infects a single cell. The virus replicates in this initial host cell and then lyses the cell, releasing many progeny that infect the neighboring cells on the plate. After a few such cycles of infection, enough cells are lysed to produce a clear area, or plaque, in the layer of remaining infected cells.
Describe the structure of a polynucleotide.
A single nucleic acid strand has a backbone composed of repeating pentose-phosphate units from which the purine and pyrimidine bases extend as side groups. Like a polypeptide, a nucleic acid strand has an end-to-end chemical orientation: the 5' end has a hydroxyl or phosphate group on the 5' carbon of its terminal sugar; the 3' end usually has a hydroxyl group on the 3' carbon of its terminal sugar. This directionality, plus the fact that synthesis proceeds in the 5' --> 3' direction, has given rise to the convention that polynucleotide sequences are written and read in the 5' --> 3' direction. The chemical linkage between adjacent nucleotide is commonly called a phosphodiester bond. The linear sequence of nucleotides linked by phosphodiester bonds constitutes the primary structure of the nucleic acid molecule.
Describe the process of PCR.
A typical PCR procedure begins with heat-denaturation of a DNA sample into single strands at 95 ºC. Next, two synthetic oligonucleotides complementary to the 3' ends of the DNA segment of interest (the target sequence) are added in great excess to the denatured DNA, and the temperature is lowered to 50-60 ºC. These specific oligonucleotides hybridize to their complementary sequences in the DNA sample, whereas the long strands of the sample DNA remain apart because of their relatively low concentration. The hybridized oligonucleotides serve as primers for DNA chain synthesis in the presence of deoxyribonucleotides (dNTPs) and a temperature-resistant DNA polymerase. When synthesis is complete, the whole mixture is reheated to 95 ºC to denature the newly formed DNA complexes. After the temperature is lowered again, another cycle of synthesis takes place because excess primer is still present.
Describe functional complementation screening.
A yeast genome can be used to isolate the wild type gene corresponding to one of the temperature-sensitive cdc mutations. The starting yeast strain is a double-mutant that requires uracil for growth due to a URA3 mutation and is temperature-sensitive due to cdc28 mutation. Recombination plasmids isolated from the yeast genomic library are mixed with yeast cells under conditions that promote transformation of the cells with foreign DNA. Transformation is done by a treatment of lithium acetate, PEG, and heat shock. Since transformed yeast cells carry a plasmid-borne copy of the wild type URA3 gene, they can be selected by their ability to grow in the absence of uracil. This collection of yeast transformants can be maintained at 23 ºC, the permissible temperature. The entire collection is then transferred to replica plates, which are maintain at 36 ºC, the nonpermissive temperature. Yeast colonies that carry recombinant plasmids expressing a wild type copy of cdc will be able to grow. Once temperature-resistant yeast colonies have been identified, plasmid DNA can be extracted from the cultured yeast cells and analyzed by DNA sequencing.
How are amino acids coupled to the correct tRNAs?
Aminoacyl tRNA synthetases link specific amino acids to its cognate tRNA. There are 20 different aminoacyl tRNA synthetases, and each enzyme is specific for only one of the 20 amino acids. These coupling enzymes link an amino acid to the free 2' or 3' hydroxyl group of the adenosine at the 3' terminus of the tRNA molecule. In this reaction, the amino acid is linked to the tRNA by a high-energy ester bond and is thus said to be activated, or charged.
Describe the aminoacyl tRNA synthetase "proofreading" mechanism.
Aminoacyl-tRNA synthetases recognize their cognate tRNAs primarily by interacting with the anticodon loop and the acceptor stem. Furthermore, specific bases in incorrect tRNAs that are structurally similar to a cognate tRNA will inhibit charging of the incorrect tRNA. However, sometimes, mistakes are still made. These mistakes are corrected by the enzymes themselves, which has a proofreading activity that checks the fit in their amino acid-biding pocket. If the wrong amino acid becomes attached to a tRNA, the bound synthetase catalyzes removal of the amino acid from the tRNA.
Describe mismatch excision repair.
Another process principally eliminates base-pair mismatches and insertions or deletions of one or a few nucleotides that are accidentally introduced by DNA polymerases during replication. The cell recognizes mistakes in the new daughter strand, and MSH2 and MSH6 remove the wrong base. The gap is much larger than it is in base excision repair. The gap is sealed via DNA polymerase and DNA ligase.
Are the helical regions of secondary RNA structure parallel or antiparallel?
Antiparallel
What form of DNA is typically found in cells?
B-form is standard.
When does base excision repair occur: before or after DNA replication?
Base excision repair must occur before DNA replication.
Describe the base pairing rules seen in DNA.
Base pairing rules are based on the size, shape and composition of the bases. G pairs with C using 3 hydrogen bonds. A pairs with T using 2 hydrogen bonds. The presence of these hydrogen bonds contribute to the stability of the double helix. Hydrophobic and van der Waals interactions between the stacked adjacent base pairs further stabilize the double-helical structure.
Describe the mechanism of DNA replication in SV40 cells.
Because simple viruses such as SV40 depend largely on the DNA replication machinery of their host cells, they offer an opportunity to study the replication of multiple identical small DNA molecules by cellular proteins. Multicomponent complexes permit the cell to carry out an ordered sequence of events that accomplishes their essential cell functions. The molecular machine that replicates SV40 DNA contains only one viral protein; all other proteins involved in SV40 DNA replication are provided by the host cell. This vial protein, large T-antigen, forms a hexameric replicative helicase, a protein that uses energy from ATP hydrolysis to unwind the parent strands at the replication fork. Primers for leading and lagging daughter strands are synthesized by a complex of primase and DNA polymerase α. The primer is extended into daughter-strand DNA by DNA polymerase δ, which is less likely to make errors. During the replication of cellular DNA, polymerase δ synthesizes the lagging strand while DNA polymerase ε synthesizes the leading strand. Each polymerase forms a complex with PCNA (proliferating cell nuclear antigen), which displaces the complex following primer synthesis. PCNA is a homotrimeric protein that has a central hole through which the daughter DNA complex passes, thereby preventing dissociation from the template. A pentameric protein called RFC (replication factor C) functions to open the PCNA ring so that is can encircle the short region of double-stranded DNA synthesized. RFC is the clamp loader. The single-stranded template for lagging strand synthesis is bound by multiple copies of RPA (replication protein A), a heterotrimeric protein. Binding of RPA maintains the template in a uniform conformation that is optimal for copying.
Describe the origin of replication in SV40 cells.
Both parent DNA strands that are exposed by local unwinding at a replication fork are copied into daughter stands. Two replication forks might assemble at a single origin and then move in opposite directions, leading to bidirectional growth of both daughter strands. In SV40 DNA, replication is initiated by the binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins form two replication forks. These forks move away from the SV40 origin in opposite directions, and leading and lagging strand synthesis occurs at both forks. Eukaryotic chromosomal DNA molecules contain multiple replication origins separated by kilobases. A six-subunit protein called ORC, for origin recognition complex, binds to each origin and associates with other proteins required to load cellular helicases composed of six homologous MCM proteins. Two MCM helicases, oriented in opposite directions, separate parent strands at an origin, and RPA proteins bind to the resulting single-stranded DNA. Synthesis of primers and subsequent steps occur.
On a tRNA, the amino acid covalently attaches to a 3' _______________.
CCA
Upon rare occasion, _____ and ______ encode the initial Met.
CUG; GUG
Describe nucleotide excision repair.
Cells use nucleotide excision repair to fix DNA regions containing chemically modified bases, often called chemical adducts, that distort the normal shape of DNA locally. Certain proteins slide along the surface of a double-stranded DNA molecule looking for bulges or other irregularities in the shape of the double-helix. For example this mechanism repairs thymine-thymine dimers, a common type of chemical adduct caused by UV light. These dimers interfere with both replication and transcription of DNA. Five polypeptide subunits of TFIIH, a general transcription factor required for transcription of all genes, are also required for nucleotide excision repair. Two of these subunits have homology to helicases. First, a DNA lesion that causes distortion of the double helix is initially recognized by a complex of the XP-C and 23B proteins. This complex then recruits TFIIH, whose helicase subunits, powered by ATP hydrolysis, partially unwind the double helix. XP-G and RPA proteins then bind to the complex and further unwind and stabilize the helix until a bubble is formed. Then, XP-G and XP-F, a second endonuclease, cut the damaged strand on each side of the lesion. This releases the DNA fragment with the damaged bases, which is degraded. Finally, the gap is filled by DNA polymerase exactly as in DNA replication , and the remaining nick is sealed by DNA ligase.
Describe confocal microscopy, and how does it battle the inherent problems seen in conventional fluorescence microscopy?
Confocal microscopy uses optical methods to obtain images from a specific focal plane and exclude light from other planes. Confocal microscopes collect a series of images focused through the vertical depth of the sample, from which an accurate three-dimensional representation can be computationally generated.
What inherent problem exists in the conventional fluorescence microscope?
Conventional fluorescence microscopy has two major limitations: 1. The fluorescent light emitted by a sample comes not only from the plane of focus, but also from the molecules above and below it, inducing a blurry image. 2. To visualize thick specimens, consecutive images at various depths throughout the sample must be collected and then aligned to reconstruct structures in the original thick tissue.
Describe gain of function genes and give an example.
Dominant alleles are often the consequence of a mutation that causes some kind of gain of function. Such dominant mutations may increase the activity of the encoded protein, confer a new function on it, or lead to a new spatial or temporal pattern of expression. A good example of a gain of function gene is an oncogene.
Describe translation initiation in eukaryotes.
During the first stage of translation, the small and large ribosomal subunits assemble around an mRNA that has a Met-tRNA correctly positioned at the start codon in the ribosomal P site. In eukaryotes, the assembly of this complex is mediated by a special set of proteins known as eukaryotic translation initiation factors (eIFs). As each individual component joins the complex, it is guided by the eIFs. Several IFs bind GTP, and the hydrolysis of GTP to GDP functions as a proofreading switch that allows subsequent steps to happen. The large and small ribosomal subunits are kept apart by the binding of eIFs to the small 40S subunit. This first step of translation initiation is the formation of a 43S preinitiation complex. This preinitiation complex is formed when the 40S subunit with eIFs associates with eIF5 and a ternary complex consisting the Met-tRNA and eIF2 bound to GTP. The mRNA to be translated is bound by the multisubunit eIF4 complex, which interacts with both the 5' cap and the poly(A)-binding protein (PABPC) bound in multiple copies to the mRNA poly(A) tail. The binding results in the formation of a circular complex. The eIF4E subunit binds the 5' cap on mRNAs. The large eIF-4G subunit binds to several PACPC proteins bound to the poly(A)-tail, and also forms a scaffold to which the other eIF4 subunits bind. The eIF4-mRNA complex associates with the preinitiation complex through an interaction between eIF4G and eIF3. The initiation complex then slides along, or scans, the associated mRNA as the helicase activity of eIF4A, stimulated by eIF4B, uses energy from ATP hydrolysis. eIF4A unwinds any stem-loop structures in the 5' UTR. Scanning stops when the Met-tRNA anticodon recognizes the start codon, also known as Kozak's sequence, which is the first AUG downstream from the 5' cap. Recognition of the start codon leads to the hydrolysis of GTP associated with eIF2, resulting in eIF5 in the GAP and the formation of the 48S initiation complex. Association of the large 60S subunit with the small subunit, which is mediated by eIF5B bound to GTP, results in the displacement of many of the IFs. Correct association between the ribosomal subunits results in hydrolysis of the eIF5B-bound GTP to GDP and the release of eIF5B-GDP and eIF1A, completing the formation of an 80S initiation complex. The ribosome is now ready for elongation.
Describe the reaction that adds an incoming rNTP to a polynucleotide chain.
During transcription, one DNA strand acts as the template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are linked together to form a complementary RNA chain. Bases in the template DNA strand base-pair with complementary incoming rNTPs, which are then joined in a polymerization reaction catalyzed by an RNA polymerase. The polymerase reaction involves a nucleophilic attack by the 3' oxygen in the growing RNA chain on the α phosphate of the next nucleotide precursor, which results in a phosphodiester bond and the release of pyrophosphate. RNA molecules are always synthesized in the 5' --> 3' direction.
Electrons have a wavelength of 0.005 nm. How does light microscopy compare to transmission electron microscopy (TEM)?
Electron microscopic imaging of biological samples offers a much higher resolution of ultrastructure than can be obtained by light microscopy. The effective resolution of the transmission electron microscope in the study of biological systems is considerably less than ideal. Under optimal conditions, a resolution of 0.10 nm can be obtained, about 2000 times better than with conventional light microscopes.
Why do you think eukaryotic cells use alternative splicing of their pre-mRNAs? How many genes are there in humans? What if one gene alternatively splices its pre-mRNA? So could humans produce more different kinds of proteins than the gene number indicates?
Eukaryotic cells use alternative splicing to produce multiple protein products as a powerful source of gene regulation. There are approximately 19,000-20,000 genes in the human genome. If alternative splicing is carried out, this means that humans can produce more protein products that the gene number indicates.
Describe how fluorescence-activated cell sorter (FACS) separates cells based on cell markers.
FACS can separate out individual cell types provided they have specific cell surface markers that identify them. Monoclonal antibodies are used to bind these cell surface markers. The antibodies are tagged with green or red fluorescent molecules, such as fluorescein or rhodamine. FACS then selects green vs red or both green and red.
Describe Forster resonance energy transfer (FRET) microscopy.
Fluorescence microscopy can be used to determine if two proteins interact in vivo by taking advantage of Forster resonance energy transfer (FRET). This technique uses a pair of fluorescent proteins in which the emission wavelength of the first is close to the excitation wavelength of the second. For example, when CFP is excited with 433 nm light, it fluoresces and emits light at 475 nm. If YFP is close by, however, instead of emitting 475 nm light, CFP transfers energy to YFP by FRET, and YFP emits light at 530 nm. Another application of FRET, called FRET biosensor, can be used to sense local biochemical environments in live cells. The idea is to express a single polypeptide containing both CFP and YFP separated by a region that undergoes a conformational change when it senses a biochemical signal. In the absence of the signal, CFP and YFP are too far apart to undergo significant FRET. However, when the signal is detected, the conformational change brings CFP and YFP close enough together to generate FRET. A version of this technique can be used to measure the local activity of a specific protein kinase. In this case, between the CFP and YFP lies a region of polypeptide containing the substrate for the protein kinase - the sensor domain - and a domain that binds specifically to the phosphorylated substrate - the ligand domain. When the sensor domain is phosphorylated by the kinase, the ligand domain binds to it and brings the CFP and YFP sufficiently close to undergo FRET. Dephosphorylation will deactivated the FRET biosensor.
Why does EcoRI not chop up the E. coli genome?
For each restriction enzyme, bacteria also produce a modification enzyme, which protects a host bacterium's own DNA from cleavage by modifying the host DNA at or near each potential cleavage site. The modification enzyme adds a methyl group to one or two bases, usually within the restriction site. When a methyl group is present there, the restriction endonuclease is prevented from cutting the DNA. Together with the restriction endonuclease, the modification enzyme forms a restriction-modification system that protects the host DNA while it destroys foreign DNA.
How would you ligate a PCR-amplified DNA product into a plasmid?
For organisms in which all or most of the genome has been sequenced, PCR amplification starting with the total genomic DNA is often the easiest way to obtain a specific DNA region of interest for cloning. In this application of PCR, the two oligonucleotide primers are designed to hybridize to sequences flanking the genomic region of interest and to include sequences that are recognized by specific restriction enzymes. After amplification of a target sequence, cleavage with the appropriate restriction enzyme produces sticky ends that allow efficient ligation of the fragment to a plasmid vector whose polylinker has been cleaved by the same restriction enzyme. The resulting recombinant plasmids can then be cloned in E. coli cells.
What is formylmethionine?
Formylmethionine is a modified methionine amino acid. It has a formyl group attached. This is the first amino acid in a bacterial polypeptide.
What does G-418 allow you to do?
G-418, or neomycin, allows researchers to select for recombinant cDNAs in mammalian cells that have the neoR gene.
Describe gel electrophoresis.
Gel electrophoresis is used as a sieving activity, separating large and small molecules of DNA. First, the DNA fragments are resolved on an agarose gel. An electrical field runs through the gel. The DNA fragments are separated by charge as well as size. The larger molecules will move slower than smaller molecules. DNA will move towards the anode of the electrical field since DNA is negative. After running the gel, you dye with ethidium bromide, and the DNA fluoresces orange-red in UV light.
Describe Gibson Assembly.
Gibson Assembly uses PCR products to produce a recombinant plasmid. Fragments are taken, mixed together with the vector, and the components are assembled correctly. This is due to long overhangs on the DNA fragments. The 5' to 3' exonuclease chew back on these long overhangs. Compatible stick ends are annealed, and 3' extension with a polymerase fills in gaps. Ligase is then used to seal the phosphodiester bond.
Describe cryoelectron microscopy.
Hydrated, unfixed, and unstained biological specimens can be viewed directly in a transmission electron microscope if the samples are frozen. In cryoelectron microscopy, an aqueous suspension of a sample is applied to a grid in an extremely thin film, frozen in liquid nitrogen, and maintained in this state by the means of a special mount. The frozen sample is placed in the electron microscope. The sample can be observed in detail in its native, hydrated state without fixing or heavy metal staining. Cryoelectron tomography allows researchers to determine the three-dimensional architecture of organelles or even whole cells embedded in ice. A single picture is a two-dimensional representation of a structure that lacks depth. However, looking at the same structure from different angles gives a three-dimensional perspective. The specimen holder is tiled in small increments around the axis perpendicular to the electron beam. The images are merged computationally.
18S, 5.8S, and 28S pre-rRNAs are transcribed by RNA polymerase ______.
I
True or False: Biosafety committees have no problem with researchers using Lentivirus.
I think false?
5S rRNA is transcribed by RNA polymerase _____.
III
Describe homologous recombination.
If a break in the phosphodiester backbone of one DNA strand is not repaired before the replication fork passes, the replicated portions of the daughter chromosomes become separated when the replication helicase reaches the nick in the parent strand because there are no covalent bonds between the two fragments of the parent strand on either side of the nick. This is known as replication fork collapse. The first step in the repair of the double-stranded break is the exonucleolytic digestion of the strand with its 5' end at the broken end of the DNA, leaving the strand with the 3' end at the break single-stranded. The lagging nascent strand base paired to the unbroken parent strand is ligated to the unreplicated position of the parent chromosome. A protein required for the next step is RecA/Rad51. Multiple RecA/Rad51 molecules bind to the single-stranded DNA and catalyze its hybridization to a perfectly or nearly perfectly complementary sequence in another, homologous, double-stranded DNA molecule. The complementary strand of this target double-stranded DNA is displaced as a single-stranded loop of DNA over the region of hybridization to the invading strand. This RecA/Rad51-catalyzed invasion of a duplex DNA by a single-stranded complement of one of the strands is key to the recombination process. This process is called strand-invasion. Next, the hybrid region between target DNA and the invading strand is extended in the direction away from the break by proteins that use energy from ATP. This process is called branch migration because the position where the target DNA strand crosses from one complementary strand to its complement in the broken DNA molecule. The resulting structure is called a Holliday structure. Cleavage of the phosphodiester bonds that cross over form one parent strand to the other and ligation of the 5' and 3' ends base paired to the same parent strands result in the generation of a structure similar to a replication fork. Rebinding of replication fork proteins results in extension of the leading strand past the point of the original strand break and re-initiation of lagging strand synthesis, thus regenerating a replication fork.
Describe how supercoiled DNA can produce a relaxed circle of DNA.
If a molecule is supercoiled, DNase is used to introduce a nick into the helix by cutting one strand of the DNA. The supercoils run out, producing a relaxed circular DNA molecule. All DNA in living systems is negatively supercoiled. It is supercoiled in one direction.
Describe stable transfection.
If an introduced vector integrates into the genome of the host cell, that genome is permanently altered, and the cell is said to be transformed. Integration is most likely accomplished by endogenous enzymes that normally function in DNA repair and recombination. A commonly used selectable marker is the gene for neomycin phosphotransferase (neoR), which confers resistance to a toxic compound chemically related to neomycin, known as G-418. The basic procedure for expressing a cloned cDNA by stable transfection starts with relatively few transfected cells that integrate into the exogenous DNA are selected on medium containing G-418. Because the vector is integrated into the genome, these stably transfected cells will continue to produce the cDNA-encoded protein as long as the culture in maintained. Because integration occurs at random sites in the genome, individual transformed G-418 resistant clones will differ in their rates of transcription, and those are usually screened to identify those that produce the protein of interest at the highest levels.
There are 61 codons, but only 20 amino acids. What does this say about the genetic code? What are the two exceptions to the degeneracy of the genetic code?
If there are 61 codons and only 20 amino acids, obviously most amino acids are encoded by two or more codons. This fact is why the genetic code is called "redundant" or "degenerate." Methionine and tryptophan are exceptions to the degeneracy to the genetic code.
What technology can be used to locate a particular mRNA inside the cell or tissue?
In Situ Hybridization
What is autoradiography?
In autoradiography, tritium is used due to its very short emission path. The emission in this instance is a β particle, or a high-speed electron. A photographic film emulsion coating is used. The β particle is emitted from the slide. The radioisotopes of the tritium are interacting with the film's AgBr particles. On the x-ray film, as a result, dark spots appear.
In bacteria, there are 30-40 different tRNAs. However, there are 61 functional codons. Explain this phenomenon, using inosine as an example.
In bacteria, a single tRNA may recognize more than one codon in the mRNA. It does so by the wobble base position, which is the third (3') base in an mRNA codon and the corresponding first (5') base in its tRNA anticodon. The first and second bases of a codon almost always form standard Watson-Crick base pairs with the third and second bases of the corresponding anticodon, but four nonstandard interactions can occur between bases in the wobble position. For example, inosine, a deaminated product of adenine, can form nonstandard base pairs with A, C, and U. A tRNA with inosine in the wobble position thus can recognize the corresponding mRNA codons with A, C, or U in the third wobble position. For this reason, inosine-containing tRNAs are heavily employed in translation.
In eukaryotes, there are 50-100 different tRNAs. However, there are only 20 amino acids and 61 functional codons. Explain this phenomenon.
In eukaryotes, more than one tRNA can attach to a certain amino acid to one codon. Or in other words, there are multiple tRNAs for a single amino acid. These tRNAs are known as cognate tRNAs.
Describe fluorescence recovery after photobleaching (FRAP) microscopy.
In fluorescence recovery after photobleaching (FRAP), a high-intensity light, permanently bleached fluorochrome is used. In the location of the fluorochrome, there is initially no fluorescence coming from it, and it will look dark in the fluorescence microscope. However, if the components of the patch are in dynamic equilibrium with unbleached molecules elsewhere in the cell, the bleached molecules will be replaced by unbleached ones, and the fluorescence will begin to come back. The rate of fluorescence recovery is a measure of the dynamics of molecules, allowing us to measure in vivo diffusion rates of intracellular components.
How does one create and screen for a conditional mutation?
In haploid yeast cells, essential genes can be studied through the use of conditional mutations. Among the most common conditional mutations are temperature-sensitive mutations, which are useful in organisms, such as bacteria and lower eukaryotes, that can grow at a range of temperatures. Hartwell and colleagues set out to identify genes important in regulation of the cell cycle. Exponential growth of a single yeast cell for 20-30 divisions forms a visible yeast colony on solid agar medium. Because mutants with a complete block in the cell cycle would not be able to form colonies, conditional mutants were required to study mutations that affect this basic cellular process. To screen for such mutants, the researchers first exposed yeast cells to mutagens and then identified yeast cells that could grow normally at 23 ºC, but could not form a colony when placed at 36 ºC. Once temperature-sensitive mutants were isolated, further analysis revealed that some indeed were defective in cell division. These mutants were therefore designated cdc (cell division cycle) mutants. Most cdc mutations in yeast are recessive; that is, when haploid cdc strains are mated to wild-type haploids, the resulting heterozygous diploids are neither temperature-sensitive nor defective in cell division.
Describe light-sheet microscopy.
In light-sheet microscopy, the sample is illuminated from the side and then viewed in an orthogonal direction. A focused laser beam (the illumination objective) sweeps back and forth across the sample, illuminating a single plane to generate a light sheet. A detection objective, also known as the orthogonal lens, at right angles to the illuminated plane, then images the sample by detecting fluorescence. To generate a three-dimensional image, all the planes in the sample have to be imaged. This is achieved by coordinately stepping the illuminating sheet and detection objective through the sample to generate a stack of images, which can then be assembled computationally into a three-dimensional rendering.
Describe Mendel's Law of Segregation.
In meiosis, one round of DNA replication is followed by two separate division, yielding haploid cells known as gametes that contain only one chromosome of each homologous pair. The apportionment, or segregation, of the replicated chromosomes to daughter cells during the first meiotic division is random and different chromosomes segregate independently of one another, yielding gametes with different mixes of paternal and maternal chromosomes.
Describe pulse-field electrophoresis.
In pulse-field electrophoresis, the top of the gel has a positive charge and the bottom a negative charge. Similar to gel electrophoresis, agarose gel is used; however, the field is constantly flipped from left to right, top to bottom. They have huge relays. They are good for isolating and resolving huge fragments of DNA. You can use this to resolve yeast chromosomes.
Describe dominant-negative genes.
In rare instances, a dominant mutation in one allele may lead to a structural change in the protein that interferes with the function of the wild type protein encoded by the other allele. This type of mutation is referred to as a dominant-negative.
Describe the process of in situ hybridization.
In situ hybridization, researchers are looking for a particular RNA inside a tissue. They want to know its location and what cell is expressing the RNA. They know a little about the sequence, so they can devise a probe complementary to the RNA. They can make this probe radioactive. Back in the day, tritium was used. RNA probes can be used as well. The probe is added to cells, and the probe will find the RNA and bind to it. It is radioactive, so you can find the RNA. You can buy a photographic emulsion. You dip microscope slide into the photographic emulsion, let it dry, and allow the tritium to expose the AgBr particles. The mRNA appears as silver grains in the light microscope.
Describe Sanger sequencing.
In the 1970s, Sanger and colleagues developed the chain-termination procedure, which served as the basis for most DNA sequencing methods for the next 30 years. The idea behind Sanger sequencing is to synthesize from the DNA fragment to be sequenced a set of daughter strands that are labeled at one end and terminate at one of the four nucleotides. Separation of the truncated daughter strands by gel electrophoresis, which can resolve strands in length by a single nucleotide, can then reveal the lengths of all strands ending in G, A, T, and C. From these collections of strands of different lengths, the nucleotide sequence of the original DNA fragment can be established. The method today is fully automated.
Describe the behavior of the lac operon with the following characteristics: - lactose + glucose
In the presence of glucose and no lactose, the operon is not turned on. It's repressed. Repressor proteins bind to the operator and prevent RNA polymerase from binding to the operator. This is negative control.
Describe the behavior of the lac operon with the following characteristics: + lactose + glucose
In the presence of high lactose and high glucose levels, the cell has a choice of which substance to use; however, the cell prefers the use of glucose. The lactose binds to the repressor proteins, allosterically changing its configuration and popping off the regulatory proteins. The lacZ gene is transcribed at a low rate.
Describe the behavior of the lac operon with the following characteristics: + lactose - glucose
In the presence of no glucose and high lactose levels, lactose binds to repressors, allosterically changing its configuration and popping off the repressor proteins. This allows RNA polymerase to attach to promoter. cAMP is produced. It binds to CAP, and CAP recruits RNA polymerase. Transcription rates are really high. The enzymes convert lactose into galactose and glucose, which keeps the cell alive.
Describe the Meselson and Stahl experiment. Why is the Meselson and Stahl experiment considered a Pulse-Chase experiment?
In this experiment, Meselson and Stahl grew bacteria in the presence of N-15, which is a non-radioactive, but heavier isotope of regular N-14, for several generations. If DNA contains N-15, it will be heavier and denser than normal DNA. You can use a CsCl gradient to isolate the different DNAs. All of the DNA is heavy at the beginning of the experiment. The cells divide in the presence of N-15. Chase with N-14 for two generations. There were two models: conservative, where the parent strand was completely conserved, and semiconservative, where the DNA is partially conserved. The results revealed that the semi-conservative model was the correct model of DNA replication, as intermediate density DNA was found in all replicates after several generations.
What's inosine doing in a tRNA? Why is inosine "promiscuous?"
Inosine, a deaminated product of adenine, can form nonstandard base pairs with A, C, and U. A tRNA with inosine in the wobble position thus can recognize the corresponding mRNA codons with A, C, or U in the third wobble position, which is why it is known as "promiscuous." For this reason, inosine-containing tRNAs are heavily employed in translation.
Describe immuno-electron microscopy.
Instead of fluorescent dyes, an antibody is tagged with gold, which can deflect the electron beam. To make the antibody visible in the electron microscope, it must be attached to an electron-dense marker. One way to do this is to use electron-dense gold particles coated with protein A, a bacterial protein that binds the Fc segment of all antibody molecules. Because the gold particles diffract incident electrons, they appear as dark spots.
A tRNA has a 3D _________-shaped structure.
L
Peptidyl-transferase activity is a function of the _______ ( ____ rRNA in E. coli; _____ in eukaryotes).
LSU; 23S; 28S
Mutations in genes encoding _______ and _______ are linked to colorectal cancers.
MSH2; MLH1
What is the function of a restriction enzyme?
Many restriction enzymes make staggered cuts in the two DNA strands at the corresponding restriction site, generating fragments that have a single-stranded "tail" at both ends. These tails are complementary to those on all other fragments generated by the same enzyme, allowing ligation of foreign DNA in those sites.
DAPI is a fluorescent dye that binds DNA. Could you use DAPI and FACS to separate cells that are in G1, S phase, and G2?
Meh?
When does mismatch excision repair occur: before or after DNA replication?
Mismatch excision repair occurs just after DNA replication.
Describe some of the uses of monoclonal antibodies today.
Monoclonal antibodies are very useful. They are commonly deployed in affinity chromatography to isolate and purify proteins from complex mixtures, immunofluorescence microscopy to bind to and so locate a particular protein within cells, immunoblotting, and as a therapeutic tool in medicine. For example, monoclonal antibodies against the mutant HER2 cell surface receptor on breast cancer cells can select for infected cells.
Describe the structure of standard B-form DNA.
Most DNA in cells takes the form of a right-handed helix. The x-ray diffraction pattern of DNA indicates that the stacked bases are regularly spaced 0.34 nm apart along the helix axis. The helix makes a complete turn every 3.4 to 3.6 nm; thus, there are about 10-10.5 base pairs per turn. The diameter is about 2 nm, or 20 Angstroms. This is known as the B-form of DNA. On the outside of the helix, the spaces between the intertwined strands form two helical grooves of different widths, described as the major grooves and the minor groove
What is negative control of the lac operon?
Negative control of the lac operon is the action of the operon in the absence of lactose.
What would happen if DNA polymerase made mistakes?
Mutation!
Describe the 5' cap found on mRNAs. Are 5' caps found in prokaryotes?
No, 5' caps are not found in prokaryotes. At the 5' end of eukaryotic mRNAs, as the RNA chain emerges from the surface of RNA polymerase, it is immediately acted on by several enzymes that together synthesize the 5' cap, a 7-methylguanylate that is connected to the terminal nucleotide of the RNA by an unusual 5',5' triphosphate linkage. The 5' cap is synthesized by RNA polymerase II.
Describe the 3' poly(A) tail found on mRNAs. Are poly(A) tails found in prokaryotes?
No, poly(A) tails are not found in prokaryotes. Processing at the 3' end of a pre-mRNA involves cleavage by an endonuclease to yield a free 3' hydroxyl group, to which a string of adenylic acid residues is added one at a time by an enzyme known as poly(A) polymerase. The resulting poly(A) tail contains 100-250 bases and is part of a complex of proteins that can locate and cleave a transcript at a specific site and then add the correct number of A residues.
What is the purpose of a Northern blot?
Northern blots allow researchers to detect a particular mRNA within a cell type. Its relative abundance, tissue type expression, and the induction profile of the gene encoding the mRNA can be figured out by a Northern blot.
Why is the melting temperature of DNA important to PCR?
Oligonucleotides (primers) anneal to DNA, and they have their own unique melting temperature.
How many times will an origin start DNA replication in a typical cell cycle of a diploid cell?
Once!
How does one preserve a stably transfected cell line?
One can preserve a stably transfected cell line by freezing mammalian cell lines. The cell line must be frozen gradually (slowly) in 10% dimethyl-sulfoxide (DMSO) and 90% fetal calf serum, which is protein-rich. These are cryo-preservatives that prevent the formation of ice-crystals within the cells. The cells are stored in liquid nitrogen (-340 F) from years to decades. When ready to be used, the cells must be thawed quickly, but very gently at 37 C. One can resume growth in normal culture medium.
Describe some of the most frequent causes of point mutations in DNA.
One of the most frequent causes of point mutations is deamination of cytosine, which converts it into a uracil. Another is deamination of the common modified base 5-methyl cytosine, which forms thymine when it is deaminated.
How do you assemble short DNA sequences into a set of chromosomes (a genomic sequence)?
Overlapping DNA sequences link one fragment to the next, so the chromosome walking method can be used.
What is the proliferating cell nuclear antigen (PCNA)? What is its function?
PCNA is a homotrimeric protein that forms a clamp around the new helix to ensure the processivity of DNA polymerases δ and ε.
Why is PCR considered the most powerful molecular biology technique ever devised?
PCR is so powerful because it amplifies your sequence of DNA of choice.
What did Paul Nurse do with the complemented (rescued) genomic cdc mutation?
Paul Nurse re-isolated the "red" plasmid from the rescued yeast cells, sequenced the "red" gene that was ligated into the plasmid, and deduced the amino acid sequence to identify the protein product. These yeast proteins were determined to control the cell cycle; his and other labs determined how these conserved regulatory proteins work. And from there, cancer research takes a surging leap forward.
Describe how restriction enzymes provide a physical map of DNA.
Physical maps are associated with DNA sequences. In order to produce a physical map, take one restriction enzyme and cut bp fragment. Take another restriction enzyme to cut the DNA again. Take both restriction enzymes and cut the same fragment. You could generate several possibilities. What is the right orientation? That is determined by taking the digestion and running on agarose. If you get certain sizes, you know the orientation. You have mapped the sites.
What three regions of a plasmid are essential to DNA cloning?
Plasmids contain three regions essential for DNA cloning: a replication origin, a marker that permits selection (usually antibiotic resistance gene), and a region in which exogenous DNA fragments are inserted.
What is positive control of the lac operon?
Positive control of the lac operon is the action of the operon in the presence of lactose.
Describe primary cell cultures.
Primary cells are cells isolated directly from tissues. Normal animal tissues or whole embryos are commonly used to establish primary cell cultures. To prepare a primary cell culture, the cell-cell and cell-matrix interactions must be broken. To do so, tissue fragments are treated with a combination of a protease (trypsin, collagenase, or both) and a divalent cation chelator (EDTA) that depletes the medium of free calcium. Many CAMs require calcium and are thus inactivated when calcium is removed. The released cells are placed in a nutrient-rich, serum-supplemented medium in dishes, where they adhere to the surface and one another. However, primary cells have a finite life span in culture.
How do most proteins bind DNA? What groove is usually involved? What is the one exception?
Protein bind DNA through the major groove. Proteins bind to DNA through hydrogen bonds and van der Waals interactions. The TATA-binding protein (TBP) is the one exception. TBP binds the minor groove to bend the DNA thus separating the two strands and allowing RNA polymerase to read the strand more easily during transcription.
What interactions/bonds occur between DNA and protein?
Proteins make contact with bases via H-bonds and van der Waals, usually within the major groove.
Describe the mRNA processing of introns found in eukaryotes. Are introns spliced in prokaryotes?
RNA splicing is the internal cleavage of a transcript to excise introns and stitch together the coding exons. The functional eukaryotic mRNAs produced by RNA processing retain noncoding regions, referred to as untranslated regions (UTRs) at each end. The 5' UTR and 3' UTR sequences participate in regulation of mRNA translation and stability, and the 3' UTRs also function in the localization of many mRNAs to specific regions of the cytoplasm. Splicing is very rare in prokaryotes.
What is replication protein A (RPA)? What is its function?
RPA is a heterotrimeric protein that maintains the single-stranded DNA template in a uniform conformation to ensure rapid replication. RPA displaced by DNA polymerase α.
Describe loss of function genes and give an example.
Recessive alleles usually result from a mutation that inactivates the affected gene, leading to a partial or complete loss of function. Such recessive mutations may remove part of the gene or the entire gene from the chromosome, disrupt expression for the gene, or alter the structure of the encoded protein, altering its function. A good example of a loss of function gene are tumor suppressor genes.
What is the most abundant RNA-protein complex in a cell?
Ribosomes!
Describe the main structural difference between ribose and deoxyribose.
Ribose contains a 2' hydroxyl group while deoxyribose does not contain a 2' hydroxyl group. The 2' hydroxyl group on ribose makes RNA unstable and highly reactive, which is why it does not serve as the basis of genetic information.
Describe scanning electron microscopy (SEM).
Scanning electron microscopy (SEM), a type of microscopy with 10 nm resolution, allows researchers to view the surfaces of unsectioned metal-coated specimens. The tissue is fixed, critical-point dried, and coated with a thin layer of gold or platinum. An intense electron beam inside the microscope scans rapidly over the sample. Molecules in the coating are excited and release secondary electrons that are focused onto a scintillation detector; the resulting signal is displayed on a cathode-ray tub, which forms three-dimensional images of the sample.
Describe polysomes. What are they?
Simultaneous translation of an mRNA by multiple ribosomes is readily observable in electron micrographs and by sedimentary analysis, revealing mRNA molecules attached to multiple ribosomes bearing nascent growing polypeptides. These structures, referred to a polyribosomes or polysomes, were seen to be circular in electron micrographs of some tissues.
How are thick tissues prepared to be viewed under transmission electron microscopy (TEM)?
Single cells and pieces of tissue are too thick to be viewed directly in the standard transmission electron microscope. To overcome this problem, methods were developed to prepare and cut thin sections of cells and tissues. To prepare thin sections, it is necessary to chemically fix the sample, dehydrate it, impregnate it with a liquid plastic that hardens, and then cut sections of about 5 to 100 nm in thickness using an ultra-microtome. The sample has to be stained with heavy metals such as uranium and lead salts.
Describe the enzymatic functions carried out by RNA.
Some RNAs fold to achieve enzymatic functions. These RNAs are called ribozymes. Some ribozymes display nucleotidyl transferase activity, in which the RNA adds a free nucleotide to itself, and site specific nuclease activity. Some RNAs self-splice, or remove their own introns. Some RNAs catalyze pre-mRNA splicing. These RNAs are known as small nuclear RNAs (snRNAs). Ribosomal RNA displays catalytic activity in protein synthesis, specifically, peptidyl-transferase activity.
Describe haploinsufficient genes and give an example.
Some gene are haploinsufficient, in that removing or inactivating one of the two alleles of such a gene leads to a mutant phenotype because not enough gene product is made. A good example of haploinsufficiency is Treacher-Collins syndrome.
Describe the Southern blotting technique
Southern blotting is used to detect DNA fragments of a specific sequence. This technique is capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme. When the DNA form a whole genome is fragmented by digestion with a restriction enzyme, the mixture of DNA fragments that is produced is so complex that even after gel electrophoresis, so many different fragments of nearly the same length are present. The restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter by blotting. This procedure preserves the distribution of fragments in the gel, creating a replica of the gel on the filter. The filter is then incubated under hybridization conditions with a specific labeled DNA probe. The DNA restriction fragment that is complementary to the probe hybridizes to it, and its location on the filter can be revealed by autoradiography or by fluorescent imaging.
Localized unwinding or over winding of a DNA molecule, which occurs during DNA replication and transcription, induces torsional stress into the remaining portion of the molecule because the ends of the strands are not free to rotate. How is this torsional stress relieved?
Stress is relieved by supercoiling. All cells contain topoisomerase I, which can relieve the torsional stress that develops in circular and viral DNA during replication and transcription. This enzyme binds to DNA at random sites and breaks a phosphodiester bond in one strand. Such a one-strand break in DNA is called a nick. The broken end then winds around the uncut strand, leading to loss of supercoils. Finally, the same enzyme joins the two ends of the broken strand. Topoisomerase II makes breaks in both strands of a double-stranded DNA and then relegates them.
How are samples prepared for transmission electron microscopy?
Structures can easily be viewed in the transmission electron microscope, provided that they are stained with a heavy metal that scatters the incident electrons. To prepare a sample, it is first absorbed to a 3 mm electron microscope grid, which is coated with a thin field of plastic or carbon. The sample is bathed in a solution of a heavy metal. As a result, the metal coats the grid, but is excluded from the regions where the sample has adhered. When we view the sample in the TEM, we see where the stain has been excluded, so the sample is said to be negatively stained.
Describe how ribosome recycling works.
Studies revealed that multiple copies of PABPI interact with both an mRNA poly(A) tail and the eIF4G subunit of eIF4. Since the eIF4E subunit of eIF4 binds to the cap structure on the 5' end of an mRNA, the two ends of an mRNA molecule a bridged by the intervening proteins, forming a circular mRNA.
Where in the cell does large and small ribosomal subunit assembly take place?
Subunit assembly takes place in the nucleolus.
Describe gene suppression.
Suppose that point mutations lead to structural changes in one protein (A) that disrupt its ability to associate with another protein (B) involved in the same cellular process. Similarly, mutations in protein B lead to small structural changes that inhibit its ability to interact with protein A. The normal functioning of proteins A and B depends on their interacting. In theory, a specific structural change in protein A might be suppressed by compensatory changed in protein B, allowing the mutant proteins to interact.
Why is the synthesis of polynucleotide chains always in the 5' --> 3' direction?
Synthesis is always in the 5' --> 3' direction because of the free 3' hydroxyl group. We need that hydroxyl group for hydration synthesis.
What is a purine? What are the purines?
The bases adenine and guanine are purines, which contain a pair of fused rings.
What is a pyrimidine? What are the pyrimidines?
The bases cytosine, thymine, and uracil are pyrimidines, which contain a single ring.
What is the URA3 gene?
The URA3 gene is a selectable marker gene in yeast. URA3- yeast cells require uracil, so they must be cultured on a plate that contains uracil in order to grow.
Describe translation elongation in eukaryotes.
The correctly positioned ribosome-Met-tRNA complex is now ready to begin the task of stepwise addition of amino acids by in-frame translation of the mRNA. A set of specialized proteins, termed elongation factors (EFs), is required to carry out this process. At the completion of translation initiation, Met-tRNA is bound to the P site on the assembled 80S ribosome. The second aminoacyl-tRNA is brought into the ribosome as a ternary complex in association with EF1α-GTP and becomes bound to the A site. The tRNA anticodon base pairs with the second codon in the coding region. When that occurs properly, the GTP in the associated EF1α-GTP is hydrolyzed. The hydrolysis of GTP promotes a conformational change in EF1α that leads to the release of the resulting EF1α-GDP complex and tight binding of the aminoacyl-tRNA in the A site. This conformation change also positions the aminoacylated 3' end of the tRNA in the A site close to the 3' end of the Met-tRNA in the P site. The α-amino group of the second amino acid reacts with the "activated" methionine on the initiator tRNA, forming a peptide bond. The peptidyl transferase reaction is catalyzed by the large 28S rRNA, which precisely orients the interacting atoms. Following peptide bond synthesis, the ribosome translocates a distance equal to one codon along the mRNA. This translocation step is monitored by the hydrolysis of GTP in EF2-GTP. As a result of conformational changes in the ribosome that accompany proper translocation and the resulting GTP hydrolysis of EF2, Met-tRNA is moved to the E site on the ribosome concurrently, the second tRNA, now covalently bound to a dipeptide, is moved to the P site. Translocation thus returns the ribosome conformation to a state in which the A site is open and able to accept another aminoacyl-tRNA complexed with EF1α-GTP, beginning another cycle.
Describe the experiment that led to the deciphering of the genetic code.
The deciphering of the genetic code requires the use of synthetic RNAs. A bacterial extract composed of a cell lysate that contains ribosomes, tRNAs, and ATP among other components was used to input these synthetic DNAs. The cell was used to translate those codons, and the amino acid sequence was recorded. Continue with the same process to decipher the code. Synthetic transcripts now have two different nucleotides with three distinct reading frames. More experiments like this cracked the genetic code.
Describe eukaryotic genome organization compared to prokaryotic genome organization.
The economical clustering of gene seen in prokaryotes devoted to a single metabolic function is rarely found in eukaryotes. Rather, eukaryotic genes encoding proteins that function together are most often physically separated in the DNA; such genes are usually located on different chromosomes. Each gene is transcribed from its own promoter, producing one mRNA, which is generally translated into a single polypeptide. This means that eukaryotes do not produce polycistronic mRNAs; however, these affiliated genes are coordinately regulated using their own promoters.
Why is a partial digestion done to produce a genomic library?
The enzyme, Sau3A, is at low concentrations so the fragments will not cut at every Sau3A site. That produces overlap in the fragments. The overlapping fragments allow you to walk down the chromosome. In addition, chromosomal overlap is also required for sequencing and linkage studies.
Describe the Northern blotting technique
The expression of a particular gene can be followed by assaying for the corresponding mRNA by Northern blotting. An RNA sample, often called the total cellular RNA, is denatured by treatment with an agent such as formaldehyde that disrupts the hydrogen bonds between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation. The individual RNAs are separated according to size by gel electrophoresis and transferred to a nitrocellulose filter to which the extended denatured RNAs adhere. The filter is then exposed to a labeled DNA probe that is complementary to the gene of interest; finally, the labeled filter is subjected to radiography.
Describe translation termination in eukaryotes.
The final stages of translation require specific molecular signals that decide the fate of the mRNA-ribosome-peptidyl-tRNA complex. Two types of specific protein release factors (RFs) have been discovered. eRF1 acts by binding to the ribosomal A site and recognizing stop codons directly. eRF3 is a GTP binding protein that acts in concert with eRF1 to promote cleavage of the peptidyl-tRNA bond, thus releasing the completed protein chain and terminating translation. Release of the completed protein leaves a free tRNA in the P site and the mRNA still associated with the 80S ribosome, to which eRF1 and eRF3-GDP are still bound in the A site. Ribosome recycling occurs when this post-termination complex is bound by a protein called ABCE1, which uses energy from ATP hydrolysis to separate the subunits and release the mRNA and tRNA in the P site. Initiation factors eIF1, eIF1A, and eIF3, which are also required for the separation of the subunits, load onto the 40S subunit, making it ready for another round of initiation.
How do you make cDNAs?
The first step in preparing a cDNA library is to isolate the total mRNA from the cell type of tissue of interest. Because of their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs present by the use of a matrix to which oligo-dTs are linked. This is done by an oligo-dT affinity columns. These RNAs anneal to the oligo-dT on the beads. The tRNAs and rRNAs and washed away while the mRNAs are eluted off the column. The enzyme, reverse transcriptase, which is found in retrovirus, is used to synthesize a strand of DNA complementary to each mRNA molecule, starting from an oligo-dT primer. The resulting cDNA-mRNA hybrid molecules are converted in several steps into double-stranded cDNA corresponding to all the mRNA molecules in the original preparation. This is done by the hybrid being introduced to an alkali solution, denaturing the hybrid. Terminal transferase adds Gs to the 3' end of the single-stranded DNA. An oligo-dC primer is used to construct the complementary strand of cDNA. Methylation of the cDNA protects it from subsequent restriction enzyme cleavage. Short double-stranded DNA molecules containing the recognition site for a particular restriction enzyme are ligated to both ends of cDNAs using DNA ligase. The resulting molecules are then treated with the restriction enzymes specific for the attached linker, generating cDNA molecules with stick ends. The plasmid is treated by the same procedure. The plasmid vector and the collection of cDNAs are then mixed and joined covalently by DNA ligase. The resulting DNA molecules are introduced into E. coil cells to generate individual clones; each clone carries a cDNA derived from a single mRNA.
Describe the Maxam and Gilbert DNA sequencing technique.
The first step of Maxam and Gilbert DNA sequencing involves the end-labeling of DNA by P-32 with a kinase. The DNA is then cut by restriction enzymes. The DNA is then denatured. Then the DNA is exposed to various chemical techniques. One chemical technique will cleave after G, one after A and G, one after C and T, and one after C. Let reaction go on a while to generate different size fragments. Take fragments and run on gel. Autoradiography is used. To read sequence, you read form the bottom to the top. You must compare to the respective lane. This is cumbersome, difficult to read.
What does the frequency of recombination between two genes provide?
The frequency of recombination between two genes provides an estimate of how far apart the two genes lie on the same chromosome.
Describe the structure of tRNAs.
The function of tRNA depends on their three-dimensional structure. In solution, tRNA folds into a stem-loop structure. The four stems are short double helixes stabilized by Watson-Crick base pairing; three of the four stems have loops containing seven or eight bases while the remaining stem contains the free 3' and 5' ends of the chain. The three nucleotides composing the anticodon are located at the center of the middle loop. In all tRNAs, the 3' end of the acceptor stem, to which a specific amino acid is attached, has the sequence CCA, which in most cases is added after synthesis and process of the tRNA are complete. Several bases in tRNAs are also modified after transcription, creating nonstandard nucleotides such as inosine, dihydrouridine, and pseudouridine.
Why is the genetic code considered "never" ambiguous?
The genetic code is considered to be "never" ambiguous because codons encode a specific amino acid in all organisms. This allows us to clone and express, for example, human genes in E. coli. However, some rare exceptions do exist to this rule.
Describe retroviruses and their method of infection.
The genomes of a number of animal viruses can also integrate into the host-cell genome. Among the most important of these are the retrovirus, which are enveloped viruses with a genome consisting of two identical strands of RNA. These viruses are so named because their RNA genome acts as a template for the formation of a DNA molecule. In the retroviral life cycle, a viral enzyme called reverse transcriptase initially copies the viral RNA genome into single-stranded DNA that is complementary to the viral RNA. The same enzyme then catalyzes the synthesis of a complementary DNA strand. The resulting double-stranded DNA is integrated into the chromosomal DNA of the host by an integrase. Finally, the integrated DNA, called a provirus, is transcribed by the host cell's own RNA polymerase into RNA, which is either translated into viral proteins or packaged within virion capsid proteins to form progeny.
Why is gold used in immuno-electron microscopy?
The gold particles are electron-dense, so the gold deflects electrons, producing a contrasting image.
Why are heavy metal salts like lead citrate and uranium acetate used to stain thin sections of cells for transmission electron microscopy (TEM)?
The heavy metal salts deflect the electron beam, the structures that take up the heavy metal stain appearing as a dark spot to produce a contrasting image.
How does a cell determine the correct reading frame?
The initial AUG start codon sets the reading frame.
Describe transmission electron microscopy. What kind of lenses are used in TEM?
The major difference from light microscopy is that in electron microscopes, electromagnetic lenses focus a high-velocity electron beam instead of the visible light used by optical lenses. In transmission electron microscope (TEM), electrons are emitted from a filament and accelerated in an electric field. A condenser lens focuses the electron beam onto the sample; objective and projector lenses focus the electrons that pass through the specimen and project them onto a viewing screen. The entire tube between the electron source and the detector is maintained under an ultrahigh vacuum, so no living material can be imaged by electron microscopy.
What does the G-C content of DNA do to the melting temperature of DNA?
The melting temperature as which DNA strands separated depends of several factors. Molecules that contain a greater proportion of G-C pairs require higher temperatures to denature because the three hydrogen bonds in G-C pairs make these base pairs more stable than A-T base pairs, which only have two hydrogen bonds. As a result, the number of G-C base pairs can be estimated from its melting temperature.
The genetic code consists of _________________ nucleotides within DNA and RNA.
four
Describe the mechanism of base excision repair.
The most common type of point mutation in humans is a change from C to a T, which is caused by deamination of 5-methyl C. Since a G-T mismatch is almost invariably caused by chemical conversion of C to U or 5-methyl C to T, the repair system evolved to remove the T and replace it with a C. The G-T mismatch is recognized by a DNA glycosylase that flips the thymine base out of the helix and then hydrolyzes the bond that connects it to the sugar-phosphate DNA backbone. Following this initial incision, an endonuclease, APE1, cuts the DNA strand near the now abasic site. The deoxyribose phosphate lacking the base is then removed and replaced with a C by a DNA polymerase β that reads the G in the template strand. This repair must take place prior to DNA replication!
Would you use the lac promoter to express a cDNA in a mammalian cell? If not, what would you use?
The promoter used to express cDNA in a mammalian cell cannot prokaryotic. The promoter must be a eukaryotic promoter.
Describe the rabies virus and its method of infection.
The rabies virus has a nucleocapsid that consists of a single-stranded RNA genome bound by multiple copies of a nucleocapsid protein. The rabies virus is replicated in the cytoplasm and does not require host-cell nuclear enzymes. A rabies virion is adsorbed by endocytosis, and release many progeny virions occurs by budding from the host-cell plasma membrane.
How do you select for recombinant DNA?
The rare transformed cells can be easily selected by the use of a selectable marker. For instance, if the plasmid carries a gene that confers resistance to the antibiotic ampicillin, transformed cells can be selected by growing them in an ampicillin-containing medium. All antibiotic resistant progeny will contain plasmids with the same inserted DNA. Since all the cells in a colony arise from a single transformed parent cell, the constitute a clone of cells of the same recombinant plasmid.
Describe transient transfection.
The simpler of the two transfection methods, called transient transfection, employs a plasmid vector similar to the yeast shuttle vectors described. Plasmid vectors are engineered to carry a replication origin derived form a virus, a strong promoter recognized by mammalian RNA polymerase, and then cloned cDNA encoding the protein to be expressed adjacent to the promoter. Once such a plasmid vector enters the cells, the viral replication origin allows it to replicate efficiently, generating numerous plasmids form which the protein is expressed. However, during cell division, such plasmids are not faithfully segregated into both daughter cells, and in time, a substantial fraction of the cells in a culture will not contain a plasmid, hence the name transient transfection.
Name and describe two examples of RNAs with higher order structures.
The simplest secondary structures in single-stranded RNAs are formed by pairing of complementary bases. "Hairpins" are formed by pairing of bases within about 5-10 nucleotides of each other, and "stem-loops" by pairing of bases that are separated by eleven to several hundred nucleotides. These simple folds can cooperate to form more complicated tertiary structures, one of which is termed a "pseudoknot."
Describe the process of renaturation.
The single-stranded DNA molecules that result form denaturation form random coils without an organized structure. Lowering the temperature, increasing the ion concentration, or neutralizing the pH causes two complementary strands to reassociate into a perfect double helix. The extent of renaturation is dependent on time.
In humans, what constitutes the large subunit (LSU), and what constitutes the small subunit (SSU)?
The small subunit, which is 40S in eukaryotes, contains a single 18S rRNA molecule. The SSU also contains 33 small subunit proteins, called S1, S2, S3, and so on. The large subunit, which is 60S in eukaryotes, contains a molecule of 28S rRNA and one molecule of 5S rRNA, plus an additional molecule of 5.8S rRNA in vertebrates. The LSU also contains 47 large subunit proteins, called L1, L2, L3, and so on. The assembled ribosome is 80S in eukaryotes.
Compare and contrast transmission electron microscopy (TEM) to scanning electron microscopy (SEM).
The specimen is located in two different locations in these two types of microscopy. In TEM, the specimen is above the electromagnetic lens. In SEM, the specimen is located beneath the electromagnetic lens. The electron beam scans the specimen, and the beam is refracted off the specimen and onto a detector to produce a three-dimensional image of the sample.
What is the start codon? What amino acid does the start codon encode?
The start codon is AUG. AUG encodes methionine.
Describe polysome profiles using sucrose gradients. What would these profiles look like if a nucleolar protein required for protein assembly was knocked out? What do you predict the polysome profile would look like for the 40S SSU, the 60S LSU, and the 80S intact ribosome? What about the polysomes?
The thick black line represents normal protein synthesis while the thin black line represents inhibited protein synthesis.
Describe the 5' untranslated region (UTR) of mRNA.
The translation start site (AUG) is many nucleotides downstream from the 5' end. The beginning of the RNA segment that is untranslated due to the start site being downstream is known as the 5' untranslated region (UTR).
Describe the process of denaturation.
The unwinding and separation of DNA strands, referred to as denaturation, or melting, can be induced experimentally by increasing the temperature of a solution of DNA. As the thermal energy increases, the resulting increase in molecular motion eventually breaks down the hydrogen bonds and other forces that stabilize the double helix.
Describe the basic mechanism of DNA replication.
The unwinding of the parent DNA strands is performed by enzymes called helicases. Unwinding begins at segments in a DNA molecules called the replication origins, or simply origins. The nucleotide sequences of origins differ, although they usually contain AT-rich sequences. Once helicases have unwound the parent DNA at an origin, a specialized RNA polymerase called primase forms an RNA primer complementary to the unwound template strands. The primer, still base paired to its complementary DNA strand, is then elongated by DNA polymerase α for another 25 nucleotides or so, forming a primer made of RNA at the 5' end and DNA at the 3' end. This primer is further extended by DNA polymerase δ, thereby forming a new daughter strand. The DNA region at which all these proteins come together to carry out synthesis of daughter stands is called the replication fork. As replication proceeds, the replication fork and the associate proteins move away form the origin. Local unwinding causes torsional stress that is relieved by topoisomerase I. A major complication in the operation of a DNA replication fork arises from two properties of DNA: the two strands of the parent DNA duplex are antiparallel, and DNA polymerases synthesize daughter strands in the 5' to 3' direction. Synthesis of one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5' to 3' direction, the same direction as the movement of the replication fork. The problem comes in synthesis of the other daughter strand, the lagging strand. Because growth of the lagging strand must occur in the 5' to 3' direction, copying of its template strand must somehow proceed in the opposite direction from the movement of the replication fork. A cell accomplishes this feat by synthesizing new primers as more of the strand is exposed from unwinding. Each of these primers is elongated in the 5' to 3' direction, forming discontinuous segments named Okazaki fragments. The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth; finally, DNA ligase joins the adjacent fragments.
Describe the function of alternative splicing in eukaryotes.
The vast majority of genes in multicellular eukaryotes contain multiple introns. Individual repeated protein domains are often encoded by one exon or multiple exons that are repeated in genomic DNA and encode identical or nearly identical amino acid sequences. The presence of multiple introns in many eukaryotic genes permits the expression of multiple, related proteins from a single gene by means of alternative splicing. In higher eukaryotes, alternative splicing is an important mechanism for production of different forms of a protein, called isoforms, by different types of cells.
Describe the polylinker region of a typical cloning vector.
The versatility of an E. coli plasmid vector is increased by the addition of a polylinker, a synthetically generated sequence containing one copy of each of several different restriction sites that are not present elsewhere in the plasmid sequence. The polylinker is cleaved with two different restriction enzymes in preparation to accept a DNA fragment prepared with two different sticky ends corresponding to the same two enzymes.
How many codons are possible in the genetic code? How many of those codons encode amino acids?
There are 64 possible codons, and 61 of those codons encode amino acids. The other three codons are "stop" codons.
Where are plasmids found?
These extrachromosomal DNAs occur naturally in bacteria and lower eukaryotic cells and exist in a symbiotic relationship with their host cell.
Who is Thomas Hunt Morgan, and what did he discover?
Thomas Hunt Morgan was a scientist who discovered the role of the chromosome in heredity.
How many possible reading frames can be found in a mRNA?
Three!
What is the Tm?
Tm is the melting temperature of DNA. Tm is dependent on the base composition of DNA.
How do you construct a yeast genomic library that can be assayed by functional complementation?
To construct a plasmid genomic library that is to be screened by functional complementation in yeast cells, the plasmid vector must be capable of replication in both E. coli cells and yeast cells. This type of vector is known as a shuttle vector. To increase the probability that all regions for the yeast genome will successfully be cloned and represented, the genomic DNA is usually only partially digested to yield overlapping restriction fragments. These fragments are then ligated into a shuttle vector in which the polylinker has been cleaved with a restriction enzyme that produces sticky ends complementary to those on the yeast DNA fragments.
How do you make a genomic library?
To make a genomic library, we are taking the entire genome, chopping it up, and taking the fragments and ligating them into a shuttle vector. In addition to the genes, there is DNA between the genes. This is intergenic DNA. In addition, the genomic library contains promoter sequences to turn genes on and off. The shuttle vector will contain introns as well. First, we are open up the plasmid with BamHI. We are cutting open the cell with Sau3A. The enzyme is in low concentrations, so you're only hitting a few of the Sau3A sites. Sau3A and BamHI have compatible stick ends, meaning that these sticky ends can be ligated together. And that is what is exactly done: the sticky ends are ligated together, producing a genomic library.
What is the purpose of culture medium? What does culture medium provide?
To permit the survival and normal function of cultured tissues or cells, the temperature, pH, ionic strength, and access to essential nutrients must simulate as closely as possible the conditions within an intact organism. Isolated animal cells are typically placed in a nutrient-rich liquid, called the culture medium, within specially coated plastic dishes or flasks, atmosphere, and humidity can be controlled. Antibiotics are often added to the culture medium. Media for culturing animal cells must supply the nine amino acids (phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, and histidine) that cannot be synthesized. In addition, most cultured cells require three other amino acids (cysteine, tyrosine, and arginine) that are synthesized only by specialized cells as well as glutamine, which serves as a nitrogen source. The other necessary components are vitamins, various salts, fatty acids, glucose, and serum. The medium must have a proper pH for the culture to thrive as well as growth factors. Growth factors are protein hormones directing cells to divide such as, for example, epidermal growth factor (EGF). These growth factors come from the serum added to the medium.
Describe how to produce monoclonal antibodies.
To produce and then purify monoclonal antibodies, one first needs to be able to grow the appropriate B-lymphocyte clone. However, primary cultures of normal B lymphocytes are of limited usefulness for the production of monoclonal antibodies because they have a limited life span. Thus, the first step in producing a monoclonal antibody is to generate immortal antibody-producing cells. Immortality is achieved by fusing normal B lymphocytes from an immunized animal (typically a rat) with transformed, immortal lymphocytes called myeloma cells that themselves do not synthesize antibodies. The fusion of a myeloma cell with a normal antibody-producing cell from a rat or mouse spleen yields a hybrid that proliferates into a clone called a hybridoma. Hybridoma cells grow rapidly and are immortal. Each hybridoma produces monoclonal antibody encoded by its B-lymphocyte parent. Second, the hybridoma cells must be selected from the unfused parent cells and the cell fused with another of the same type. This selection is usually performed by incubating a mixture of cells in a special culture medium, called a selection medium, that permits the growth of only the hybridoma cells because of their novel characteristics. Finally, each selected hybridoma clone is tested for the production of the desired antibody; any clone producing that antibody is then grown in large cultures, from which a substantial quantity of pure monoclonal antibody can be obtained.
Describe Next-Generation Sequencing.
Today, a single sequencing instrument can carry out billions of sequencing reactions simultaneously by localizing then in tiny clusters of the surface of a substratum. This is known as next-generation sequencing. In one popular method, billions of different DNA fragments to be sequenced are prepared by ligating the double-stranded linkers to their ends. Next, the DNA fragments are amplified by PCR using primers that match the linker sequences. The primers used are covalently attached to a solid substratum, and at the end of the amplification about a thousand identical PCR products are linked to the surface in a tight cluster. These clusters can then be sequenced using a special microscopy to image fluorescently labeled dNTPs as they are incorporated by DNA polymerase one at a time into a growing DNA chain. First, one strand is cut and washed out, leaving a single-stranded DNA template. Then sequencing is carried out on the thousand or so identical templates in each cluster, one nucleotide at a time. All four dNTPs are fluorescently labeled and added to the sequencing reaction. After they are allowed to anneal, the substratum is imaged and the color of each cluster is recorded. Next, the fluorescent tag is removed and a new dNTP is allowed to bind. This cycle is repeated about a hundred times, resulting in billions of hundred-nucleotide long sequences.
What is in situ hybridization used for today?
Today, in situ hybridization is used for: 1. Locating a particular mRNA in both space and time (development). 2. Single-stranded DNA or RNA probes containing modified nucleotides anneal to the target mRNA. 3. Antibodies directed against the modified nucleotides are tagged with a reporter enzyme like peroxidase: yields a purple product.
Who discovered transformation? Who showed that DNA is the transforming material?
Transformation was first discovered by Frederick Griffith in 1928 using Streptococcus pneumoniae. Avery, McCarty and McLeod later showed that DNA is the transforming material in 1944.
Describe translation initiation in bacteria.
Translation begins with the small 30S subunit. The 30S SSU plus Initiation Factors (IFs) 1, 2-GTP (IF2 has GTP bound that provides energy through its hydrolysis to GDP), and 3 form a pre-initiation complex. These IFs keep the small subunit from interacting with the large subunit. This pre-initiation complex binds the charged fMet-tRNAiMet and the mRNA. The small subunit scans the mRNA for the first AUG. The Shine-Dalgarno sequence helps position 30S complex. The 50S LSU joins. IF2-GTP is hydrolyzed into IF2-GDP and is popped off the complex. fMet-tRNAiMet is now in the P site and is ready for elongation.
What are the three stop codons?
UGA, UAA, UAG
Describe how A-form DNA can be produced.
Under laboratory conditions in which most of the water is removed from DNA through 70% EtOH, the crystallographic structure of DNA changes to the A form, which is wider and shorter than B-form DNA, with a wider and deeper major groove and a narrower and shallower minor groove. RNA-DNA and RNA-RNA helices also exist in the A-form in vitro. Base stacking in the A form is more compressed (about 11 bp per turn) as well as the base pairs are severely tilted and off-center.
How do you select for recombinant eukaryotic cells?
We can select for mammalian cells that take up the DNA. We can select for drug resistance (neomycin). This kills eukaryotic cells that do not have the neoR gene.
Describe the process of transformation.
When E. coli cells are mixed with recombinant vector DNA and subjected to a stress such as heat shock, a small fraction of the cell will take up the plasmid DNA, a process known as transformation. Those cells that incorporate the plasmid into the genome are considered "transformed."
If you were to track the movement of rNTPs during transcription, would you use alpha- or gamma- P-32-labeled rNTPs?
When RNA and DNA is synthesized, the alpha is incorporated in the growing chain. So for labeled rNTPs, use alpha.
Describe the segregation of alleles in yeast.
When a/α diploids are placed under starvation conditions, the cells undergo meiosis, each giving rise to a tetrad of four haploid cells, two of type a and two of type α. Sporulation of a heterozygous diploid cell yields two spores carrying the mutant allele and two carrying the wild type allele.
Give a basic synopsis of meiosis.
Whereas the body cells of most multicellular organisms divide by mitosis, the germ cells that give rise to gametes undergo meiosis. Premeiotic germ cells are diploid, containing two homologs of each morphological type of chromosome. During Meiosis I, synapsis, or crossing over occurs. This is the exchange of DNA between homologous chromosomes. Homologous chromosomes then separate. Sister chromatids remain attached to make one homolog. During Meiosis II, sister chromatids that constitute one homolog now separate.
Can you crystallize ribosomes?
Yes!
What is the importance of super-resolution microscopy?
With super-resolution microscopy, a resolution below 0.2 μm can be achieved.
Are the two strands making up the hybrid helix in transcription anti-parallel?
Yes!
How is the melting temperature of DNA measured?
You can measure denaturation by using a spectrophotometer. Because the stacked base pairs in duplex DNA absorb less UV light than the unstacked bases in single-stranded DNA, the change leads to an abrupt increase in absorption of UV light. This phenomenon, known as hyperchromicity, is useful for monitoring DNA denaturation.
How do you pick the colony that has your cDNA of interest?
You pick the colony that has your cDNA of interest through colony lift hybridization. First, you grow out your library on plate. Lay nitrocellulose on top of colonies. Remove. Cells are left on the plates, so they can grow. However, the nitrocellulose is placed in alkaline solutions with SDS, and the cells are broken open. The DNA will adhere to the nitrocellulose. The DNA is denatured by the alkaline solution. You can devise an oligo nucleotide probe that is radioactive (say, labeled with P32). Use the probe, and the probe will specifically anneal to that cDNA. Wash off excess probe. The only probe left is the one attached to your cDNA. You can use X-ray film to reveal dark spot to identify the colony from the plate. This is known as autoradiography.
Describe Z-form DNA. Does it exist in vivo?
Z-form DNA is a type of DNA artificially created in the lab. Z-form demonstrates alternating G and C nucleotides in the two strands. The sugar-phosphate backbone appears to zigzag. It is possible that Z-form DNA exists in vivo, as many gene promoters are G-C rich.
Homozygous
a diploid individual that carries two identical alleles
Heterozygous
a diploid individual with two different alleles
Polylinker
a synthetically generated sequence containing one copy of each of several different restriction sites that are not present elsewhere in the plasmid sequence
Phenotype
a term that refers to all the physical attributes or traits of an individual that are the consequence of a given genotype
Mutation
a term usually reserved for instances in which an allele is known to have been newly formed
Mutagen
an agent that causes a heritable change in the DNA sequence
What is the genetic marker typically inserted in a plasmid?
antibiotic resistance gene
DNA strands are _____________________, meaning that the DNA strands are upside down in respect to one another.
antiparallel
Recombinant DNA
any DNA molecule composed of sequences derived from different sources
Nucleocapsid
capsid coat plus nucleic acid genome
Recessive
defined as one in which both alleles must be mutant in order to the mutant phenotype to be observed; that is, the individual must be homozygous for the mutant allele to show the mutant phenotype
Restriction Enzyme
endonucleases produced by bacteria that typically recognize specific 4-8 bp sequences, called restriction sites, and cleave both DNA strands at these sites
Dominant
observed in a heterozygous individual carrying one mutant and one wild type allele
During DNA replication, ______________________ lays down the RNA primer.
primase
Capsid
protein coat surrounding the nucleic acid genome
What two types of RNA contain higher order structure?
tRNAS and rRNAs contain higher order structures.
Wild Type
the normal, nonmutant allele
Transcription
the process by which RNA is synthesized
Translation
the whole process by which the nucleotide sequence of an mRNA is used as a template to join the amino acids of a polypeptide chain in the correct order
NAD+ (nicotinamide adenine dinucleotide) also has 5' - 5' linkage using _________ phosphates.
two
Diploid
two sets of chromosomes
