Genetic 3
What are 2 main techniques of transformation used to introduce plasmids into bacteria?
using calcium ions and brief heat shock to pulse DNA into cells -electroporation: a brief but high-intensity pulse of electricity to move DNA into bacterial cells
What are the 3 most common forms of histone modification?
The 3 most common forms of histone modification are methylation, acetylation, and phosphorylation.
What are the two functional domains of transcription factors?
The DNA-binding domain binds to specific DNA sequences in the cis-acting regulatory site and the trans-activating domain activates or represses transcription by binding to other transcription factors or RNA polymerase.
Explain what posttranscriptional regulation is?
The control of gene expression at the RNA level. It occurs once the RNA polymerase has been attached to the gene's promoter and is synthesizing the nucleotide sequence.
What are positive and negative control mechanisms?
A negative control requires the process to be actively shut off when a regulator molecule bonds while a positive control will only start transcription when a regulator molecule bonds.
What are mutations classified by?
Molecular change
What is mono allelic expression (MAE and its three major classes?
Mono allelic expression is when only one allele is transcribed, while the other allele is transcriptionally silent. The three major classes are 1) Parent-of-Origin Monoallelic Expression, 2) Random Monoallelic Expression, and 3) Random Monoallelic Expression of Autosomal Genes.
Compare and contrast activators and repressors.
Activators and repressors are both transcription factors that regulate transcription initiation by binding to either enhancer/promoter elements, other transcription factors, or RNA polymerase. Activators increase transcription initiation, while repressors decrease transcription initiation.
How do bacteria discriminate between which strand of DNA is older and which is newer?
Adenine methylase adds methyl groups to DNA and the older DNA will have methyl groups while the new strand will not.
What are the mechanisms of posttranscriptional gene regulation?
Alternative Splicing mRNA stability Translation RNA silencing
what are two methods of post transcriptional gene regulation?
Alternative splicing and RNA silencing.
Which of the following is a mechanism of posttranslational control?
Alternative splicing/RNA processing Control of mRNA stability Translation RNA silencing
What is the result of a gene being imprinted?
An imprinted gene will not be expressed.
T or F: In the interphase nucleus, chromosomes uncoil and intertwine with other chromosomes.
F: Each chromosome occupies a discrete domain and stays separate from the other chromosomes.
What is not something a riboswitch does?
Breaks down antiterminator
Which of the following is not an example of a post-transcriptional gene regulation mechanism ?
D. Chromatin remodeling is not an example of a mechanism for post-transcriptional gene regulation. This is an example of something that occurs prior to transcription.
What are restriction enzymes? What are the two kinds of fragment ends they produce?
DNA cutting enzymes that bind at specific, palindromic (same forwards and backward) restriction sites and cleave both strands of DNA to produce fragments. Sticky/cohesive ends are fragments with overhands while blunt ends are fragments with double-stranded ends with no overhangs
True or False: Coactivators interact with transcription complex.
False, coactivators interact with proteins and form enhanceosomes, which those interact with transcription complex.
What are the four functions of lncRNA
Decoy, adapter, guide, enhancer
DNA methylation is associated with __________ transcription and DNA acetylation is associated with ________ transcription.
Decreased, increased
Explain the difference between depurination and deamination.
Depurination is an apurinic site due to the loss of a nitrogenous base Deamination is a change of base pairing due to an amino group being converted such as adenine converting to hypoxanthine.
What is an isoform?
Different forms of a protein that are made from the same gene.
T or F: DNA mutations resulting from exposure to UV light are classified as spontaneous mutations because UV light is natural.
False: They are considered induced mutations.
What is the difference between focused and dispersed promoters?
Focused promoters are binary in functionality, with a single promoter producing a single, predictable transcript. Dispersed promoters offer various "start sites" that produce transcripts of variable composition relative to their proximity to the gene being transcribed.
What are sticky ends
Fragments produced with overhangs
What is gene knockout?
Gene knockout is the disruption or elimination of a specific gene or genes to see what happens, it can be used to understand the purpose of a gene as well as genetically modify organisms (such as knockout mice).
What is monoallelic expression, and what is one example of it?
Monoallelic expression is when only one allele is transcribed, while the other allele is transcriptionally silent. One example of this is parent-of-origin monoallelic expression, such as imprinting. Imprinting is when a gene that is imprinted only shows the expression of either the maternal allele or paternal allele. For example, if a gene is maternally imprinted, the gene would only show the male expression.
What is not one of the three major classes of monoallelic expression (MAE)?
Monoallelic expression of oncogenes
In expression regulation what is Negative and Positive Control?
Negative control is when the gene is expression unless shut off my regulator molecule, while positive control the gene is expression only when stimulated by the regulator control.
Compare positive and negative control.
Negative control is when transcription occurs unless shut off by regulator molecule. Positive control is when transcription occurs only when the regulator molecule stimulates RNA production.
What are the two ways of fixing double stranded DNA breaks?
Non-homologous end joining and homologous recombination repair
Why is eukaryotic gene expression more complex than eukaryotic gene expression?
Greater amount of DNA associated with histones, mRNAs have to be spliced, capped and polyadenylated before transport to the nucleus, genes on numerous chromosomes are enclosed in a double membrane nucleus and mRNAs have a wide range of half-lives
What are hnRNPs
Heterogeneous nuclear ribonucleoproteins that bind splicing silencers and inhibit splicing
What are transcription factories?
Nuclear sites that contain active RNA polymerase and transcription regulatory molecules that can form and disassemble rapidly.
Why does methylation decrease gene expression?
Methyl groups occupy the major groove of DNA. Because of the size and location of the methyl groups, they physically block and prevent transcription factors from binding the DNA.
What is RNA interference (RNAi)
Sequence-specific posttranscriptional regulation- Short RNA molecules regulate gene expression in the cytoplasm of plants, animals, and fungi; repress translation and trigger mRNA degradation
: What are the different classifications of mutations?
There are four mutations. Point mutation results in a change from one base pair to another. Missense mutation results in a new triplet code for a different amino acid. Nonsense mutation results in triplet stop codon coding. Silent mutation the new triplet code continues to code for the same amino acid.
Explain what mutagens are and provide an example
They are natural or artificial agents that induce mutations. An example would be medical x-rays.
T/F: Coactivators form complex "enhanceosome" that interacts with transcription complex
True
T/F: There is greater structural and functional diversity in eukaryotic promoters.
True
True or false: Small noncoding RNAs can be negative regulators.
True
What were the three major mechanisms of epigenetic alteration to the genome? Briefly describe each.
Methylation - reversable addition of methyl groups to DNA that silence genes by blocking transcription Histone modification and chromatin remodeling - covalent modifications added to histone tails which alter chromatin structure and the accessibility of genes for transcription Noncoding RNA - RNA molecules that associate with proteins to form silencing complexes after transcription
What are the three major epigenetic mechanisms?
Methylation, Histone modification and Chromatin remodeling
WHat are the three major mechanisms of epigenetics?
Methylatioon 2. histone modification 3. noncoding RNA
By which mechanism do intercalating agents induce DNA damage?
Wedging between nucleotides, distorting base pairings
Describe similarities and differences between nonsense mutations and missense mutations.
Similarities: Both nonsense mutations and missense mutations stem from a change of one nucleotide of a triplet within a protein coding portion of a gene. Differences: Missense mutations result in the creation of a new triplet which codes for a different amino acid in the protein product. Nonsense mutations occur when the triplet changes into a stop codon, resulting in the termination of translation of that protein.
What are the four mutations classified by location and their location?
Somatic mutations - occur in any cell except germ cells Germ-line mutations - occur in gametes Autosomal mutations - occur within genes located on autosomes X- and Y-lined mutations - occur within genes located on (respective) X and Y chromosomes
What are spliceopathies? Provide one example
Spliceopathies are mutations that affect regulation of splicing and contribute to several genetic disorders. One (possible) example is myotonic dystrophy or spinomuscular atrophy (SMA).
What is the fundamental difference between "sticky" and "blunt" ended DNA fragments?
Sticky" fragments have an overhang of nucleotides with which a complementary sequence can interact. Blunt-ended fragments are completely paired.
______ ends produce fragments with overhangs, while _______ ends produce fragments with double-stranded ends and no overhangs.
Sticky, blunt
As studied in rats, explain the role the Glucocorticoid receptor plays in the heritability of stress-induced behavior
Stress-induced epigenetic changes that occur prenatally or early in life can influence an organism's behavior due to varying levels of expression of the Glucocorticoid receptor (GR). GR expression levels were associated with differences in histone acetylation and DNA methylation levels. Newborn rats raised with low levels of maternal nurturing had low GR expression, did not adapt well to stress as adults, and had significantlyhigher levels of promoter methylation. Newborn rats exposed to high levels of maternal nurturing early in life had increased expression of GR, adapted better to stress as adults, and low levels of promoter methylation.
In prokaryotes, gene activity is ______ when lactose is absent and _______ when available.
c) Repressed, induced
What is a mutation?
change in DNA
Describe lacZ, lacY, and lacA
lacZ- encodes β-galactosidase, an enzyme that converts disaccharide lactose to monosaccharides glucose and galactose lacY- specifies primary structure of permease, an enzyme that facilitates entry of lactose into bacterial cell lacA- encodes enzyme transacetylase, which may be involved in removal of toxic by-products of lactose digestion from the cell
Decoy, adapter, guide, and enhancer are the four class of __
lncRNA
Which of the following is not a major mechanism of epigenetic alterations to genomes?
methylation, noncoding, histone modification and chromtin remodeling, decoding
What are inducible and constitutive enzymes
: Inducible enzymes are only made when certain substrates are present and constitutive enzymes are always being made.
What is a gene mutation?
A gene mutation is an alteration in the DNA sequence of a gene.
What are Riboswitches?
Bind with small ligands (molecules that bind to another); cause conformational change and induce a second RNA domain.
How do siRNA and microRNA differ from one another?
As a result of viral infection, siRNAs produce double-stranded RNA that is identified and cut by a dicer. MicroRNAs are noncoding RNAs that influence gene expression negatively.
______ blotting is a technique used to analyze RNA, while ______ blotting is a technique used to analyze proteins.
B. Northern, Western is the correct answer. Northern blots are used for analyzing RNA and Western blots are used to analyze proteins.
What are some differences between BACs and YACs?
BACs- large, low copy number, plasmid based, circular, 1-2 vectors per cell YACs- have telomeres at each end, ORI and centromere based, linear, 1 vector per cell, high copy number
An exact copy; genes, cells or whole organism that carry identical genetic material because they are derived from the same original DNA
Clone
What is the correct meaning of "clones" when it comes to genetics?
Clones refer to recovered copies of recombinant DNA.
What is the major purpose of the mRNA 3' untranslated region ("UTR")?
Colloquially termed a "zip code," this region of mRNA allows for protein binding (by zip-code binding protein 1 ["ZBP1"] in particular) to prevent translation until the mRNA strand has localized to the region of the cell in which transcription of the product is desired.
What is the difference between inducible and constitutive enzymes?
Constitutive enzyme is an enzyme that is always generated whether or not a suitable substrate is present whereas inducible enzyme is an enzyme that is expressed only when it is clearly of adaptive use.
What is the Cre-Lox system and what is it used for?
Cre is a promoter and LoxP are sequences that cut DNA. Is it used for conditional gene knockouts that allow scientists to control when the target gene is disrupted.
Which blot is used to analyze protein post-translational modifications, including the addition of lipids and phosphates?
Eastern blot
Explain epigenetics?
Epigenetics is the study of changes in organisms caused by the modification of gene expression instead of the change of the genome itself.
What is epigenetics?
Epigenetics is the study of the ways in which these changes alter cell- and tissue-specific patterns of gene expression.
What are two structural features of eukaryote DNA or how it's packaged that make it distinct from prokaryote DNA?
Eukaryote genes are on chromosomes in a distinct location and eukaryote DNA is wrapped around histones and interacts with non-histone proteins to make chromatin.
Name two ways in which eukaryotic gene regulation is more complex than that in prokaryotes.
Eukaryotes have greater amounts of DNA associated with histones and other proteins and (2) eukaryotic mRNA must be processed before transport from the nucleus.
Which of the following is NOT a type of alternative splicing?
Exon and intron swapping
What is the difference between spontaneous mutations and induced mutations?
Induce Mutations to result from the influence of extraneous factors, either natural or artificial. Spontaneous mutation: Changes in the nucleotide sequence that occur naturally.
What are epigenetics?
Is the study of how these changes alter cell- and tissue-specific patterns of gene expression
What is the blue-white selection used for?
It is used to identify cells containing recombinant and nonrecombinant DNA.
Which is not a structural gene of Lac (lactose) operon?
LacC
What are three different types of lac operons and what enzymes do you encode for?
LacZ encodes for beta-galactosidase, LacY encodes for permease, and LaxA encodes for transacetylase.
Name two types of specific gene recovery.
Library screening - Used to sort through the library and isolate specific genes ofinterest Probes- Used to screen library and recover clones of specific gene- A probe is any DNA or RNA sequence complementary tothe target gene of the sequence being identified
Give an example of a vector used in genetic modification
Plasmids
Types of Mutations
Point or Base Mutation MIssense mutation Nonsense mutation Silent Mutation
What are the 4 types of mutations?
Point, Missense, nonsense and silent
Describe one of the benefits of clustering genes involved in the same pathway under a single operon.
Potential answer: Synthesis of various pathway proteins isn't required under every possible context, and so being able to "toggle" these proteins on or off depending on external factors conserves valuable energy and resources.
What is RNAi and include a description of it?
RNA interference, which is a short RNA molecule that regulates gene expression in the cytoplasm of plants, animals, and fungi; represses translation and triggers m RNA degradation.
Why can RNAi be used to diagnose and treat cancer?
RNAi molecules bind to mRNA, preventing the mRNA from coding for a protein. RNAi molecules can function as gene suppressors. Cancer is commonly caused by gene over-expression. Suppressing the gene expression of cancer could be therapeutic
What are the four types of mutations
The first type of mutation is point mutation/base substitution which is a change in one base pair, this can result in a change of amino acid but often does not due to degeneracy. Another type of mutation is a missense mutation which results in a new triplet code for an amino acid, and can changes the protein that DNA codes for. Nonsense mutations change an amino acid triplet into a stop codon, which results in the termination of the protein prematurely, which can heavily alter the protein. The last mutation is a silent mutation where the change in the triplet code does not change the amino acid, and does not have any effect on the protein being created.
We learned about two operons in class, the Lac Operon and the Trp operon, compare and contrast these two operons. Make sure to mention what is being produced, what causes the operon to start/stop, and whether it is inducible/repressible/etc.
They both contain structural genes, however the amount varies with the Lac operon only having three (lacZ, lacY, and lacA) and the trp operon having five (trpE, D, C, B, and A). They also both produce polycistronic mRNA that encodes for enzymes. The lac Operon produces enzymes that metabolize lactose, while the trp Operon produce enzymes that synthesize tryptophan. The lac Operon is an inducible operon that only starts when in the presence of lactose, and stops when there is no lactose present. While the trp Operon is repressible, and is always creating tryptophan, until tryptophan is present at which point it then turns off.
True or false: The phenomenon monoallelic expression (MAE) can be described as only one allele being transcribed while the other allele is transcriptionally silent.
This statement is true
True or false: In prokaryotes, gene activity is repressed when lactose is absent.
This statement is true. When lactose is available, gene activity is induced.
Which of the following is NOT a form of post-transcriptional gene regulation
Transcription
Repositions of the nucleosomes make what access to the chromosomes?
Transcription regulatory proteins - Transcription activators- RNAP II (RNA polymerase II)
What are transposons?
Transposons are "jumping genes" that move within and between chromosomes to insert themselves into various locations within the genome.
Compact chromatin structure inhibits gene transcription, replication, and DNA repair. For anything to be done the DNA must be unwound.
True
Epigenetic marking is responsible for the consistent inactivation of the same X chromosome(s) in future cell generations.
True
Eukaryotic gene regulation is more complex than that in prokaryotes
True
T/F Enhancers regulate transcription of eukaryotic gene
True
What 2 techniques happens with transformation?
heat shocking and use of calcium ions to pulse DNA into cells and Electroporation
What are the four different types of mutations classified by location and their different qualities?
the four different mutations are somatic mutations, germ-line mutations, autosomal mutations, and X- and Y-linked mutations. Somatic mutations occur in any cell except germ cells and are not heritable. germ-line mutations occur in gametes and are heritable, autosomal mutations occur within gerns located on autosomes. X- and Y-linked mutations occur within genes located on X and Y chromosomes.
What is the purpose of alternative promoters?
to offer more than one site where transcription can start and to produce pre-mRNAs with different 5' exons, which may be alternatively spliced to downstream exons
List the mechanisms of posttranscriptional gene regulation
translation, RNA silencing, alternative splicing, and mRNA stability
