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BC463 Exam 1 Questions with Accurate
Answers
"Junk" DNA correct answer Non-genic DNA, that is proving to to be functional but not fully understood. 10 nm Fiber correct answer Arises from nucleosome association with DNA. "Beads on a String." 2D Electrophoresis correct answer Is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW). In this way, complex mixtures consisted of thousands of different proteins can be resolved and the relative amount of each protein can be determined. 3' RACE correct answer Rapid Amplification of cDNA Ends. Allows the ends of RNA molecules to be determined. 3' race takes advantage of the poly-A tail and creates a primer that anneals to it. Steps:
- Generate cDNA from mRNA mixture. -Reverse Transcriptase -anchor primer: poly(dt) + additional sequence
- Synthesize 2nd Strand of DNA: -DNA polymerase (Taq) -gene-specific internal sense primer
- Start Standard PCR cycle with: -DNA polymerase (Taq) -anchor primer -gene specific internal sense primer 30nm Fiber correct answer The further compaction of the 10nm fiber turns into this, whose formation relies on H1. This fiber is then further compacted into chromosomes in which large loops of chromatin are anchored to a central scaffold.
5' RACE correct answer Rapid Amplification of cDNA Ends. In this version of RACE, the sequence of the 5' end is unknown. To overcome this problem, an artificial 5' end is generated. A poly(A) tail is generated using a terminal transferase. Steps:
- Internal artisans primer is extended by reverse transcriptase
- Terminal transferase is then used to add a poly(A) tail to the 3' end.
- The RNA is then removed and an anchor primer is added
- Generate cDNA from mRNA mixture. -Reverse Transcriptase -anchor primer: poly(dt) + additional sequence
- Synthesize 2nd Strand of DNA: -DNA polymerase (Taq) -gene-specific internal sense primer
- Start Standard PCR cycle with: -DNA polymerase (Taq) -anchor primer -gene specific internal sense primer A-DNA correct answer Preferred configuration of RNA Right Handed Helix 11 bp's in every turn making major and minor groove very similar Present in RNA-DNA Hybrids Absorption Spectrum correct answer The amount of light absorbed by a sample as a function of wavelength. DNA=260nm Protein=280nm Acetylation correct answer Attachment of acetyl group to a lysine. Regulation of interactions.
cDNA library correct answer This library contains a set of clones representing the mRNA's in a given cell type at a given time. -cDNA libraries from the same organism can be different depending on the tissue or cell type -Libraries made from different cell types or tissues make it possible to identify and study genes from specific biological contexts. Cell Cycle correct answer G1: cells grow to a minimum size required for the nest stage. S: DNA is copied, duplicating the chromosomes. The two copies (sister chromatids) remain associated with one another. G2: The cell prepares for mitosis M: sister chromatids are separated and a full set of chromosomes is moved to each pole of the cell (Chromosome segregation). Centromere correct answer The point on a chromosome by which it is attached to a spindle fiber during cell division. ChIp-CHIP correct answer In this instead of RNA samples, Input DNA and immunoprecipitated DNA are labeled and mixed during hybridization. Each spot on the array represents 1 pair of primers in traditional PCR analysis. Used to investigate interactions between proteins and DNA in vivo. Specifically, it allows the identification of the cistrome, sum of binding sites, for DNA-binding proteins on a genome-wide basis. ChIp-seq correct answer a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. Chromatin correct answer The non-condensed DNA protein complex Chromatin immunoprecipitation (ChIp) correct answer A procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo. Procedure:
- Treat cells with formaldehyde to crosslink the proteins to the DNA
- Break the DNA into short fragments by sanitation or MNase digestion
- Use antibodies against the specific protein to immunoprecipitate the protein
- Reverse crosslink to separate the protein from the DNA
- Identify DNA sequence by PCR hybridization to a microarray (ChIp-chip), or by sequencing (ChIp-seq). Chromatin Remodeler correct answer A large family of protein complexes carry chromatin remodeling out. This activates many eukaryotic genes that require chromatin remodeling: mobilize nucleosome. Also requires ATP hydrolysis. Chromosome correct answer Defined as a structure composed of DNA complexed with specific proteins, which is the form that genetic information is encoded, maintained, and transmitted. Contains one dsDNA molecule==> Eukaryotes have linear chromosomes and most bacteria and archaea have circular chromosomes Eukaryotic cells have additional chromosomal DNA which is from the mitochondria/chloroplast Bacterial cells have additional DNA within their cells called plasmids which are not needed for survival Cloning by Ligation correct answer Steps:
- RE generate compatible ends: -Sticky ends form base pairs -Blunt ends are also compatible.
- Joined by DNA ligase: form phosphodiester bonds
- transform DNA Cloning by Recombination correct answer Cloning by recombination involves adding recombination sites to the PCR product and then inserting the PCR product into your plasmid via recombinases. These enzymes recognize sequences of homology. Cloning correct answer Commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. Cloning of any DNA fragment essentially involves four steps:
- Fragmentation - breaking apart a strand of DNA
DNA Methylation correct answer Is an epigenetic mechanism used by cells to control gene expression. A number of mechanisms exist to control gene expression in eukaryotes, but DNA methylation is a commonly used epigenetic signaling tool that can fix genes in the "off" position. Methyl groups can be added to cytosine residues to make 5-methyl cytosine by DNA methyltransferases. DNase Footprinting correct answer Detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease (DNase, for short), to cut the radioactively end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern. The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the "footprint". Drosophila melanogaster correct answer Key model for multicellular development. Takes about a week to develop from a egg to an adult fly. Genetic studies exploit phenotypic differences between wild type and mutant flies. Also used to examine chromosome structure and gene expression-polytene chromosomes in the salivary glands facilitate this. E. Coli correct answer A bacterial model organism that is widely used for cloning and genetic engineering processes because of the ease of its manipulation. EMSA or Gel shift correct answer A common affinity electrophoresis technique used to study protein- DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Requires non-denaturing conditions (native). Depends on charge, size, and shape. Epigenetics correct answer The study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself. Eraser correct answer A histone modifier that removes groups from the histone tail. Ex. Deacetylases, demethylases, and phosphatases. Ethidium Bromide correct answer A fluorescent dye that intercalates between base pair stacks.
Euchromatin correct answer Relatives extended and open chromatin that is potentially transcriptionally active Eukaryote correct answer organism having a nuclear compartment that contains its genetic information. Forward Genetics correct answer Where a mutant phenotype is obsessed and the gene that causes the phenotype is then discovered. Gel Electrophoresis correct answer Laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. Gels: Agarose- Good for larger molecules, due to the larger pores Polyacrylamide- Great for smaller molecules due to the smaller pores Gene correct answer Region of DNA that controls a discrete hereditary characteristic. Genome correct answer The complete DNA sequence of an organism, which serves as its instructional blueprint. I should be noted that the genome of some viruses consist of RNA. Genomic Library correct answer Contains clones of all the DNA from an organisms genome. Genotype correct answer The collective DNA sequence of an organism Germaine Cells correct answer Cells in multicellular eukaryotes that are involved in the production of the progeny. Gibson Cloning correct answer This type of cloning allows several fragments to be cloned simultaneously. Requirements are that the end sequences are 20 to 40 bp long that match the end sequences of the other fragments and the vector. Three steps in one pot:
Insulators/Boundary element correct answer These are elements that separate euchromatin from heterochromatin. These elements also prevent the spreading of heterochromatin. Isoelectric Focusing correct answer A technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings. pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode (negatively charged electrode). "positive" anode and "negative" cathode end. Karyotype correct answer A display of the chromosomes from an individual. Derived from microscopy images prior to separation of sister chromatids. Labeled Tracers correct answer Allows you to detect extremely small amounts of a particular substance Can be radioactive or non-radioactive Linker Histone correct answer H1 is the linker histone, binds to the linker DNA in between successive nucleosomes, helping compaction. Living organisms are constructed from what 4 basic molecules? correct answer a. DNA b. Protein c. Lipids d. Carbohydrates LUCA correct answer Last Universal Common Ancestor. This represents the first branch point on a phylogenetic tree. Lytic Viruses correct answer Cause host cell to burst, releasing progeny into medium Burst cells appear as clear areas on plates which are called plaques Major Groove correct answer Occurs where the backbones are far apart, and varies more in available chemical groups than in the minor groove, because the T-A and A-T are the same, and G-C and C-G are
the same, so these can't be distinguished by binding proteins. So DNA binding proteins most frequently bind in this groove. Methylation correct answer Attachment of methyl group to lysine or arginine. Regulation of interactions. Microarray correct answer A grid of DNA segments of known sequence that is used to test and map DNA fragments, antibodies, or proteins. Micrococcal Nuclease (MNase) correct answer enzyme (nuclease) that digests DNA Used in experiment to test the model of how DNA is packaged into chromatin Not able to digest DNA that is bound to histones!!! Minor Groove correct answer Occurs where the backbones are closer together, and binding of proteins in this groove causes distortion in the DNA that can benefit certain reactions. Model Organism correct answer Are organisms that have unique features that can be used to answer biological questions. They are usually small, easy and fast to grow. One to know: 1.E. Coli: Commonly studied bacteria used mainly for recombinant DNA research.
- Saccharomyces cerevisiae: Single celled eukaryote, and is a budding yeast. Exist as both haploid and diploid, so useful in studying dominant and recessive mutants.
- Schizosaccharomyces Pombe: Single celled eukaryote and is a fission yeast. Used primarily to study spatial cue such as cells middle, is formed.
- Dictyostelium discoidum: Singe celled eukaryote slime mold, that is used to study intercellular communication and signaling.
- Drosophila Melanogaster: Fly, multicellular eukaryote that is use to study developments and modern recombinant DNA.
- Caenorhabditis elegans: nematode worm, and multicellular eukaryote. Used because is has very few cells (909) and is used to study cell differentiation.
- Arabidopsis thaliana: Plant, and multicellular eukaryote, used because it is diploid and has smaller genome then other plants.
- Fragments are then attached to a surface that has primers complementary to the ligated adapters- the adapters and primers bas pair. DNA is then amplified to form a bridge of double stranded DNA. DNA is then denatured to give clusters of identical amplified molecules.
- The molecule in each cluster are then sequenced simultaneously. A specific primer and flourescently labeled nucleotides are used for DNA synthesis. After each addition, the added nucleotide is determined by its fluorescence Sequencing reads are typically short and mapped to known reference genome Non-Coding RNA correct answer Often fold into complex, molecule specific, three dimensional structures. Primary Structure: RNA sequence, in 5' to 3' direction Secondary Structure: short doblie helical regions Tertiary Structure: arrangement of double helices and single stranded regions in the final configuration of the RNA Non-Covalent Interactions correct answer Interactions between atoms or molecules that do not depend on formation of covalent bonds. Such as... Charge-Charge, Charge-dipole, Dipole-dipole, Charge-induce dipole, Dipole-induced dipole, Van der Waals, and Hydrogen bonding. Non-Template Strand of DNA correct answer This is the Coding strand, sense strand, or mRNA like strand. 5' to 3' directionality. For convenience, the ORFs and regulatory signals are drawn on the mRNA- like strand. Nucleoid correct answer An irregularly shaped region within the cell of a prokaryote that contains all or most of the genetic material, called genophore. In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear membrane. Nucleosome Core Particle correct answer 146 bp DNA wrapped around a histone octane in 1.67 turns. The DNA is wound around the octamer in a left-handed direction-when the octamer is removed, this leaves negatively supercoiled DNA. Negative supercoiling makes strand separation easier, which is required for replication and transcription.
Origin of Replication correct answer A particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. PCR correct answer Polymerase Chain Reaction, this is a technique used to amplify a DNA fragment of interest. It is able to amplify 1 billion copies of DNA from one template molecule. Advantages: Sensitive, fast, safe (non-radioactive) Disadvantages: contamination. can be finicky to work. Three main steps:
- Denaturation of DNA. Temp around 95C
- Annealing of primers. Temp around 50-70C
- Synthesis of new DNA. Temp around 70C Components: -dsDNA template -Two primers (ss olives) -Thermostable DNA polymerase (eg. Taq) -dNTPS -Mg^2+, required for catalysis and buffer components. Peptide Bond correct answer Another name for an amide bond, a chemical bond formed when a carboxylic acid condenses with an amino group with the loss of a water molecule. The term peptide bond is used only when the reactive groups come from amino acids. This is also a covalent bond with double bond character. Phenotype correct answer Visual features and properties of an organism Phosphodiester Bond correct answer Is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA. Strong covalent bonds form between the phosphate group and two 5-carbon ring carbohydrates (pentoses) over two ester bonds. Phosphorylation correct answer Attachment of phosphate group to a Serine, Threonine, or a Tyrosine. regulation of interactions or activity.
Reader correct answer These molecules read histone modifications. Ex. Bromodomain, Chromodomain, PHD finger, WD40 repeat. Real time PCR correct answer Real-time polymerase chain reaction is a laboratory technique, based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, in real-time, and not at its end, as in conventional PCR. Real-Time PCR correct answer Detects the accumulation of amplicon (DNA or RNA) during the reaction (can track the progression of a PCR reaction) SYBR Green is a highly sensitive dye used in this method Used in gene expression analysis, and others Restriction Enzyme correct answer Are bacterial proteins that often cut specific short palindromic DNA sequences. Restriction Modification System correct answer Bacteria prevent their DNA from being spliced by methylating their DNA which prevents restriction enzymes from acting on it Reverse Genetics correct answer Where a gene of interest is disrupted, and the phenotype observed. RNA correct answer ribonucleic acid. It is an important molecule with long chains of nucleotides. A nucleotide contains a nitrogenous base, a ribose sugar, and a phosphate. Just like DNA, RNA is vital for living beings. RNA has a extra 2'OH that facilitates a reaction that can break phosphodiester bonds. This also means that it favors A-type helix rather than the B-type helix. Saccharomyces cerevisiae correct answer A key eukaryotic model organism used to better understand transcription, recombination, cell division and other processes. Even though it has some fundamental differences from other eukaryotes. It also divides by budding. Exists has either Haploid (a or alpha cells) or diploid (a/alpha cells) Salt Bridge correct answer Non-Covalent interaction: Ionic interaction between two charged atoms
Sanger Sequencing correct answer A method of DNA sequencing, based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. The extension of the polynucleotide will stop if a dideoxy nucleotide is included in the chain as no 3' OH group will be available on the sugar. The reaction generates a set of molecules of different sizes, each terminating at a dideoxynucleotide. Schizosaccharomyces pombe correct answer Yeast that is extremely distantly related to S. Cerevisiae. The centromeres and DNA structure of this organism are more similar to those in more complex eukaryotes than those of S. Cerevisiae. Known as a fission yeast. SDS-PAGE correct answer A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. SDS binds to the hydrophobic regions of proteins, denaturing them and giving the proteins a uniform negative charge. Complete denaturation requires the addition of reducing agent that will break the disulfide bonds. Senescence correct answer When a cell loses its ability to divide Shotgun Sequencing correct answer The sequence of a whole genome can be obtained by assembling sequence fragments 1)Genomic DNA is broken into many fragments and linkers are attached
- Fragments are amplified based on linker sequence
- Fragments are then sequenced
- Computationally, linker sequences are removed from the sequence data and the overlapping regions used to determine the order of the fragment sequences Site Directed Mutagenesis correct answer By using PCR reactions in which the primers are designed so they have a complimentary sequence to the flanking regions but have different bases at the mutation site. Because the parent strand is from Ecoli it will be methylated and you can digest the plasmids with Dpn1 to remove the non mutants and leaves you with the mutant plasmid. Sodium Dodecyl Sulfate (SDS) correct answer A surfactant that binds to the hydrophobic regions of proteins, denaturing them and giving them a uniform negative charge.
Template Strand of DNA correct answer The DNA strand that is used as a template to synthesize an RNA copy of the coding strand. 3' to 5' directionality. Also know as the antisense strand. Tetahymena thermophila correct answer Chosen for it unique biology this ciliate posses's two nuclei: a diploid micronucleus that acts as the gremlin vessel and a polyploid micronucleus in which gene expression occurs. The macronucleus DNA undergoes gene amplification and rearrangement. Study of this system lead to identification of self-splicing rRNA and telomeres. Transfer RNA (tRNA) correct answer Involved in Translation of protein by bringing in correct amino acid that matches its codon Ubiquitination correct answer Is a 76 amino acid protein, that is added by a chain of chemical reactions (E1, E2, E3) to Lysine and removed by deubiquitinating enzyme (DUB) Van Der Waal's correct answer Non-Covalent Interaction: Weak interaction between atoms at a certain distance Vector correct answer DNA molecule that is used in cloning. Often contains an origin of replication, a multiple cloning site and a selectable marker. A DNA fragment of interest is clone into the vector, which can then be introduced into cells and propagated. Viruses correct answer Not considered to be a living organism. Needs host cell in order to replicate its genome Contain small nucleic acid genomes (DNA or RNA) surrounded by a protein coat Why Culture Cells correct answer Single celled organisms can be grown in culture or on agar plates (single colony = identical cells) Multi-cellular organisms = different cell types Proteins and nucleic acids are usually extracted for the single cell type for homogeneity Wobble Base Pair correct answer G-U base pair, which has two hydrogen bonds
Writer correct answer A histone modifier that adds groups to a histone tail. Ex. Acetylases, methylases, and kinases. Xenopus correct answer The frog has large oocytes and eggs which allow easy manipulation and extracts are highly concentrated, making it feasible to perform many biochemical reactions. Not used commonly for genetic studies because it is a partial tetraploid. Z-DNA correct answer Left Handed Helix Can result from Methylation of Cytosine, torsional stress, and high salt concentrations
