Module 3 Flashcards

Question

Describe the key steps in transgenic technology

Answer 1

Pronuclear injection to insert a transgene into a fertilized egg. Integration into the fertilized egg's genome is random.

Answer 2

Homologous recombination of targeting vector in embryo-derived stem cells is selected for, and then injected into a blastocyst-stage embryo. Disrupts or mutates the targeted gene, which can be used to knockin or knockout a gene.

Answer 3

Precise editing of the targeted genome using CRISPR-Cas9 CRISPR-Cas system is used to precisely edit any cell type.

Answer 4

A genetically-identical organism created by nuclear transfer from the adult somatic cells to an egg Method is to transfer the entire nucleus from the donor into an eunucleated, unfertilized egg. This creates (mostly) genetically identical individuals.

Answer 5

Yes. All nuclear DNA in the cell comes from the donor, and all the egg’s chromosomes are removed from the egg during metaphase.

Answer 6

No. Half of the mitochondrial DNA would come from the original Smarty, and the other half from the egg. This is because we leave the mitochondrial DNA in the egg.

Answer 7

Analyze polymorphic markers, which are unique biological identifiers that follow simple Mendelian patterns of inheritance, to match an individual with their DNA. Everyone inherits two of each STR one from the egg and the other from the sperm. The basis of DNA typing is analysis of polymorphisms. The goal is to calculate a probability that only one person could have the same profile of markers. A variety of analytic techniques are used for this, including minisatellite analysis, PCR-based analysis, STR analysis, mtDNA analysis, Y chromosome analysis, and random amplification of polymorphic DNA (RAPD) analysis. Only about 0.1% of the human genome differs from one person to another. The majority of the human genome represents intergenic DNA, regions that aren’t controlled by selection pressures, where mutations are usually maintained and transmitted from generation to generation. Short tandem repeats (STRs) are variable regions that forensic geneticists like to use because they can generate the profile of the individual without giving information about the individual’s phenotypic traits. STR analysis provides higher discriminating power than minisatellite analysis while requiring a smaller DNA sample size. STRs are widely distributed throughout the human genome. STR analysis is usually combined with a multiplex PCR reaction (multiplex PCR enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers). The preferred technique for separating the STR loci is multichannel capillary electrophoresis, however regular gel electrophoresis can be used as well. For multiplex PCR analysis of STRs, one primer in each pair is labeled with a different-colored fluorescent tag. The PCR amplification products are then mixed with labeled internal lane standards and detected using an automated sequencer. Different STR loci and alleles are separate by size based on their migration rate in the sequencer gel matrix and detected by color after laser-induced excitation of the fluorescent dyes

Answer 8

The accumulation of genetic mutations (8-15) that lead to unregulated cell growth and division.

Answer 9

The mutation that leads to tumor growth. There can be other mutations, but the driver is the one that sets off the cancer.

Answer 10

Genes whose products have the ability to induce malignant transformation of eukaryotic cells

Answer 11

Genes that will become oncogenes if they are mutated

Answer 12

Genes that inhibit cell growth, and cause cancer if they’re not expressed

Answer 13

It's the master switch of the cell cycle

Answer 14

Speculates that two independent mutations (“hits”) cause the loss of function of both alleles leading to cancer. If the loss of a single allele is inherited, the individual is said to have a genetic predisposition for cancer.

Answer 15

The guardian of the genome. In a healthy cell, relatively little is expressed, but expression can be triggered by stress (eg sun exposure) and suppress tumors through apoptosis. If p53 is a homotetramer, it is expressed correctly, but if it is a heterotetramer, it leads to unrestrained cell growth and cancer.

Answer 16

Benzo(a)pyrene isn’t a carcinogen itself, but when it is consumed and metabolized, it becomes a bulky DNA adduct that is resistant to NER, and then undergoes translesion synthesis and G →T transversion that damages the genome. These mutations can occur in p53, and then it’s game over.

Answer 17

You have a greater chance of developing Alzheimer's. This isn’t a guarantee that you will get it; it's just an increased risk. Live a healthy life, take care of yourself, and eat lots of blueberries.

Answer 18

This study was done on a relatively small and specific group of people. Before bragging about your findings, more research on a wider range of demographics is required. All you can conclude is that among college students, this allele was found to be linked to reckless behavior.

Answer 19

Intergenic DNA

Answer 20

STR analysis

Answer 21

Unique biological identifiers for each person that have simple Mendelian patterns of inheritance

Answer 22

To establish parentage, to exonerate those wrongly accused of crimes, and to determine if a clone is genetically identical to the donor nucleus

Answer 23

only one person in a quintillion could have the same profile of markers.

Answer 24

Secondary transfer DNA

Answer 25

The simultaneous amplification of many STR loci in one reaction tube by using more than one pair of primers.

Answer 26

The individual inherited a different marker from each parent at this locus

Answer 27

The molecular cradle. The RNA tertiary structure folds into a molecule cradle which holds everything together, in addition to being the catalyst, for group 1 and 2 intron splicing

Answer 28

Archaeal splicing pathway is completely different from spliceosomal introns Spliced by an endonuclease protein Archaeal introns are enzymatically removed by a cut-and-rejoin mechanism. This mechanism requires ATP, an endoribonuclease, and a ligase Archaeal intron transcripts generate a bulge-helix-bulge motif at the exon-intron junction, which is recognized by the splicing endoribonuclease, which cuts within the three-nucleotide bulges at symmetric positions Exons are ligated by a specific ligase. Sometimes structural rearrangement occurs post-ligation

Answer 29

After cleavage and release of mRNA, the 3’ end is polyadenylated by the enzyme poly(A) polymerase, which adds adenosine 5’-monophosphates to the 3’ end The poly(A) tail enhances stability and translation efficiency poly(A) tails are coated with sequence-specific poly(A)-binding proteins. In the nucleus the tail is coated with PABPN1 and in the cytoplasm the tail is coated with PABPC. PABPN1 increases the processivity of poly(A) polymerase and defines the length of the newly synthesized tail PABPC initiates translation and regulates mRNA decay Histone mRNAs have a conserved stem-loop structure instead of a poly(A) tail

Answer 30

Adding Us can correct internal frameshifts, create start and stop codons, and create open reading frames. U insertions and deletions are mediated by guide RNAs (gRNA), which are transcribed from the gRNA gene in the DNA. Many gRNAs are required to edit each pre-mRNA transcript, and the gRNAs confer specificity by complementary base pairing with the mature mRNA. The editing is catalyzed by the editosome.

Answer 31

No. The finding that nearly half of the sequence information in an mRNA was not present in the gene encoding it was difficult to rationalize at first, but once the mechanism for RNA editing was discovered in trypanosome mitochondrial DNA, the central dogma of biology was no longer in question. RNA editing is the post-transcriptional modification of an mRNA transcript’s base sequence. It changes gene-specific codons, thus changing protein structure and function. The U insertions and deletions are mediated by gRNAs, which are transcribed gRNA genes in the kinetoplast “minicircle” DNA (note that this DNA is not found in the nucleus!) The RNA editing mechanism found in the trypanosome mtDNA → guide RNA is used to alter the nucleotide sequence of the pre-mRNA

Answer 32

Apolipoprotein B (ApoB) is a plasma protein that plays a key role in the assembly, transport,, and metabolism of plasma lipoproteins. There are two forms of ApoB that circulate in the plasma, long form ApoB-100, which is liver specific, and short form ApoB-48, which is small intestine specific. These two proteins are produced from the same gene, but generated by RNA editing. The ApoB pre-mRNA transcript undergo splicing, polyadenylation, and editing in the nucleus. ApoB-100 is produced from unedited ApoB pre-mRNA transcripts. ApoB-48 is produced from a C→U transition that creates a premature stop codon, truncating the protein.

Answer 33

The editing complex confers specificity using the APOBEC1 complementation factor (ACF) subunit of the RNA-specific cytidine deaminase that carries out the recognition and catalysis of ApoB RNA editing. The ACF subunit is an RNA-binding protein and recognizes an 11-nucleotide “mooring sequence” just downstream of the edited C in the ApoB mRNA.

Answer 34

RNA turnover is a critical part of maintaining steady-state RNA levels. It’s a quality control mechanism. Some mRNAs are stored in the cytoplasm, but most are immediately translated and later degraded. Once in the cytoplasm, the mRNA might be held in a translationally silent state, it might be translated and then degraded, or it might be rapidly degraded via nonsense-mediated mRNA decay if it contains a premature termination codon.

Answer 35

The P site had peptidyl transferase activity, catalyzing the formation of peptide bonds.

Answer 36

Transgenic technology is the insertion of transferred genetic material (the transgene) into an organism at a random site, using pronuclear injection. mRNA levels (i.e. transcription) can be analyzed using reverse-transcription PCR Protein levels (i.e. translation) can be analyzed using western blots Changes in morphology can be observed directly Expression of reporter genes, like a fluorescent one, can be observed directly

Answer 37

Both the non-homologous end joining (NHEJ) and homology directed repair (HDR) pathways can be used, as Cas9 generates a double stranded break, which can activate either repair pathway. NHEJ is much more error prone than HDR, as it has a tendency to induce insertion or deletion mutations. Instead researchers are focused on promoting HDR to prevent these mutations.

Answer 38

Mostly. All of the clone’s nuclear DNA would come from the donor, as the egg’s chromosomes are removed during metaphase. However, only half of the clone’s mtDNA would come from the donor. The other half would be from the egg. This happens because we leave the mtDNA inside the egg when we transfer the nucleus into the egg, we don’t remove it.

Answer 39

Constitutive However, the splicing of some exons is regulated by splicing regulator proteins, where the exons are either included or excluded from the mature mRNA. This is a mechanism for generating protein diversity from a relatively small set of genes

Answer 40

Codon-anticodon recognition and aminoacyl-tRNA synthesis

Answer 41

A specialized cytoplasmic ribonuclease. It processes longer dsRNAs into short ds siRNAs. It associates with a partner protein, binds to 3’ overhang present at the base of the pre-mRNA hairpin, and liberates a 22 bp RNA duplex with 3’ overhangs of two nucleotides. A single duplex is generated from each hairpin precursor.

Answer 42

Part of the RISC complex, cleaves the mRNA at the site where the guide strand siRNA is bound.

Answer 43

RNA induced silencing complex. The argonaut unit has slicer activity. Thought to be an ancient, highly conserved viral defense mechanism (because some viruses use dsRNA genomes)

Answer 44

siRNA and miRNA interacting with RISC in RNAi The catalytic core of the RISC complex is an Argonaute protein. When associated with a siRNA or miRNA that is fully complementary to the target RNA, RISC cleaves the RNA at that discrete position.

Answer 45

I would use gene editing since CRISPR is precise, but gene targeting would also work to knockout a gene. RNAi knockdowns expression, but it doesn’t knockout expression. Since CRISPR knocks out expression, it’s more efficient and is more likely to be successful long-term.

Answer 46

This is how restriction endonuclease moves along the DNA. Linear diffusion, motion is stochastic, random. Equal probabilities for forward and reverse steps. Binding is nonspecific.

Answer 47

sliding over short distances of 30-50 bp between nonspecific binding sites and recognition sites. Involves helical movement from tracking along a groove of the DNA over short distances. This is the main mode of translocation over long distances. The restriction endonuclease moves between binding sites by dissociating from its initial site before reassociating elsewhere in the same DNA chain.

Answer 48

coupling and catalysis in order to cut DNA. This is the recognition process that triggers large conformational changes of the enzyme and DNA, leading to the activation of the catalytic center

Answer 49

ID SNPs by their length after digestion by a restriction endonuclease. RFLPs are visualized by digesting DNA from different individuals with restriction endonucleases, followed by gel electrophoresis to separate the fragments by size, then using southern blotting and hybridization to a labeled probe to identify the target locus. RFLP+PCR analysis is used as a diagnostic test for maple syrup urine disease (mutation of the protein-metabolism pathway, causes a buildup of keto acids in urine which makes it smell like syrup, also interferes w/ brain function. This kills infants within 2-3 weeks after birth if untreated??!?). A mismatch PCR-RFLP assay identifies the Y393N allele, which is the allele with a single nucleotide change resulting in the disease. This test is vital since it provides rapid turnaround times (8 hours max) In the analysis, DNA samples are amplified by PCR using primers specific for the diseased allele site, After digestion with restriction endonucleases, the samples are visualized and stained in a gel electrophoresis.

Answer 50

ADA cDNA is introduced into the target T lymphocytes by retroviral-mediated gene transfer. Psi is required for the inclusion of RNA in a viral particle.

Answer 51

CRISPR is being used to treat sickle cell anemia. ADA-SCID (ADA is an essential enzyme for adenine and guanine metabolism. Also T lymphocytes are destroyed in the absence of ADA, predisposing patients to recurring and persistent infections. ADA-SCID is a severe x-chromosome linked form of of the ADA disease) Traditional treatment for ADA-SCID is bone marrow transplants and weekly ADA injections An ex vivo gene therapy has been used in which ADA cDNA is introduced into T lymphocytes by retroviral-mediated gene transfer. These gene-corrected T lymphocytes are then infused back into the patients. For the majority of patients, this gene therapy treatment, when combined with the weekly ADA injections, is enough to live a long and healthy life.

Answer 52

CDKs regulate the replication licensing system and progression through each phase of the cell cycle. G1 → low S → high G2 → low M → high

Answer 53

One of pBR’s normal functions is preventing the cell from entering S phase (the phase in which the cell is replicated) If pRB is absent, the E2F complex will continue to stimulate S phase specific genes, resulting in unrestrained cell growth

Module 3 Flashcards

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