Uworld Bio - DNA and Gene Expression

gel electrophoresis - with PAGE or agarose gel

gel electrophoresis with PAGE - Separates DNA molecules based on size - using a polyacrylamide gel (PAGE) or an agarose gel. The polyacrylamide gel is generally used to analyze proteins or shorter fragments of DNA because it has smaller pores through which the molecules can traverse without falling through.

native PAGE gel

A native PAGE gel contains - no denaturants or - reducing agents and - allows DNA molecules to maintain their double-helical structure and traverse the gel in their native (unaltered) state. Individuals with the mtDNA mutation would be expected to show - one band, representing the undigested product - - and two bands, representing digested products Samples without the mutation would show only the - undigested band

DNA ligase

DNA ligase - joins Okazaki fragments.

DNA polymerase

DNA polymerase - is involved in replication, not transcription.

DNA polymerase

DNA polymerase - synthesizes daughter strands in a 5′ → 3′ direction only. One daughter strand is synthesized continuously toward the replication fork (leading strand); the other strand is synthesized discontinuously in a direction away from the replication fork (lagging strand), with more and more segments added as the replication fork progresses. This process results in the formation of Okazaki fragments, short stretches of newly synthesized DNA separated by RNA primers.

SDS PAGE under nonreducing conditions

DNA samples are analyzed in nonreducing conditions as DNA has no disulfide bonds.

DNA sequencing

DNA sequencing - is a molecular technique used to determine the nucleotide order of DNA molecules. By analyzing the nucleotide sequences, DNA sequencing of whole genomes may be used to precisely quantify the copies of the gene present within a given tissue

RNA Polymerase

RNA polymerases - are responsible for carrying out transcription and must bind DNA at a promoter region for transcription to be initiated.

SDS PAGE under nonreducing conditions

Reducing substances are used to break disulfide bonds in protein,

constitutively active gene model

constitutively active gene model - the gene of interest is transcribed at a relatively constant rate regardless of current cell conditions. - This results in increased gene mRNA and protein activity.

Conjugation

Conjugation - is the exchange of genetic information between prokaryotes, typically in the form of plasmid DNA. - It does not occur in eukaryotes.

DNA helicase

DNA helicase - unwinds the DNA double helix and separates the parent strands at the origin of replication.

Transformation

Transformation - occurs when prokaryotes pick up foreign genetic material from their surroundings. - Eukaryotes do not participate in transformation.

Northern blotting = RNA

1) Load RNA samples unto gel and apply electric field 2) Transfer samples from gel to blotting membrane 3) Hybridize with probes and visualize results - Probe hybridize with target RNA

dimers

A dimer - is a protein that is composed of two noncovalently bound polypeptide chains (also called subunits, or monomers). A homodimer - is a protein in which the polypeptide chains of the two monomers have the same sequence (number, order, and type) of amino acids (AA).

Oncogene

According to the passage, c-Myc overexpression leads to early cancer development (ie, B-cell lymphoma) in Eμ-Myc mice. - Therefore, c-Myc is most likely an *oncogene* Oncogene - is a mutated or overexpressed gene that induces uncontrolled cell growth by promoting cell cycle progression or inhibition of apoptosis. As a result, c-Myc levels would be higher in Eμ-Myc (Eμ) mice than in wild-type mice to promote cancerous cell growth.

Alternate splicing

Alternate splicing - produces multiple protein products from the same gene, not multiple similar genes. It's a process that allows various proteins to be generated from a single pre-mRNA transcript based on differential inclusion and exclusion of exons during splicing.

Apoptosis, or the programmed death of preexisting cells

Apoptosis, or the programmed death of preexisting cells, - limits the rate of cell proliferation. However, cancerous tumors develop as cells grow/divide uncontrollably and fail to follow predetermined apoptotic pathways. As a result, the fraction of apoptotic cells within a group of cancer cells is markedly decreased (inhibited). Educational objective: Apoptosis is the programmed and controlled death of aged, unnecessary, or damaged cells. Cancerous cells exhibit rapid and uncontrolled proliferation, which is caused by cell growth that outpaces normal apoptosis or by dysfunction of the apoptotic process itself.

Autoradiography

Autoradiography - only detects molecules that contain radioactive atoms; - lanes containing molecules with no radiolabel will appear empty. However, it is also possible that those lanes appear empty because they contain no molecule at all, with or without radiolabels.

ITC experiment = (an in vitro technique) - performed outside of living organisms Western Blot = (an in vivo technique) - performed in living organisms

In the ITC experiment (an in vitro technique that uses purified proteins), - the dissociation constant Kd is used to determine the binding affinity of proteins. - The low Kd shows a higher binding affinity . In the in vivo experiment, (in which proteins were extracted directly from cells), - the results are the opposite. - the thicker band of the western blot indicates increased binding. Smallest Kd = Highest affinity Thickest band = Highest affinity

Isoelectric focusing

Isoelectric focusing - uses a stable pH gradient to separate proteins based on their isoelectric point.

Loading controls = Housekeeping genes expressed ubiquitously

Loading controls - are used to normalize protein detection and ensure that protein loading and mobility are standardized across the gel. - Proteins used as loading controls tend to be ubiquitously expressed as they have consistent concentration levels across all cell/tissue types regardless of varied experimental conditions. The most common loading controls are proteins necessary for baseline cellular function that are transcribed from housekeeping genes (eg, α- and β-tubulin proteins are structural/mobile cytoskeleton components). Educational objective: Loading controls normalize protein detection and ensure that protein loading is standardized across the gel. Proteins used as loading controls tend to be ubiquitously expressed and have consistent concentrations across all cell/tissue types. Housekeeping genes are the most common loading controls.

Primase

Primase - synthesizes RNA primers and positions them at the beginning of each DNA strand. Only one primer is needed for leading strand synthesis, but lagging strand synthesis requires many RNA primers.

Single-strand DNA-binding protein

Single-strand DNA-binding protein - binds to each strand to prevent spontaneous reannealing of unwound single-stranded DNA.

Telomeres and Centromeres = Heterochromatin regions

Telomeres are regions at chromosomal ends that are repeatedly truncated with each round of cell division. Centromeres join two sister chromatids and are required for proper chromosome division during mitosis. Despite their different chromosomal locations, both telomeres and centromeres are composed of heterochromatin, a tightly condensed complex of DNA wrapped around histones. Because of its structurally restrictive form, heterochromatin is transcriptionally inactive, meaning that proteins responsible for regulating gene expression cannot access the tightly packed DNA. As a result, hetechromatic regions are often gene-poor and contain repetitive DNA.

p-value

The p-value is the probability of observing a result due to chance alone, assuming that the null hypothesis is true The p-value ranges from 0 to 1 and is generally interpreted as follows: p > 0.05 signifies that - There is a greater than 5% probability that the observed result is due to chance alone, not due to an actual association between the variables under study. - Therefore, they are not statistically significant. p ≤ 0.05 signifies that - There is a 5% or lower probability that the observed result is due to chance alone. - Therefore, they are statistically significant. The plasma triglyceride level in mutation carriers is significantly lower than the plasma triglyceride level in mutation noncarriers (p = 0.023)

The rough endoplasmic reticulum (RER)

The rough endoplasmic reticulum (RER) - has long, folded membranes coated with attached ribosomes that translate proteins destined for secretion into the rough ER lumen.

The smooth endoplasmic reticulum (SER)

The smooth endoplasmic reticulum (SER) - has varying metabolic functions depending on the cell type; examples include lipid synthesis (testes/ovaries), drug/poison detoxification (liver), and calcium ion storage (muscle).

Topoisomerase

Topoisomerase - introduces negative supercoiling in the DNA double helix ahead of the replication fork to reduce the strain produced by unwinding, which causes positive supercoiling.

knockout gene model

knockout gene model - the gene of interest is removed, disrupted or inactivated. - This results in absence of protein activity.

The sequence below is a portion of exon 7 of the SMN1 mRNA transcript. 5′ - UCAAGUGAUUCUCCU - 3′ Which of the following represents the corresponding DNA coding strand sequence for this particular transcript?

5′ - TCAAGTGATTCTCCT - 3′ Sense (coding) DNA strand - 5′-TCAAGTGAT-3′ mRNA Strand - 5′-UCAAGUGAU-3′ AntiSense (non-coding) DNA- 3'-AGTTCACGA-5'

Reverse transcription polymerase chain reaction (RT-PCR)

During reverse transcription polymerase chain reaction (RT-PCR), - the enzyme reverse transcriptase converts mRNA into double-stranded cDNA, which is then amplified in cycles to yield thousands of copies. The cDNA is initially denatured into single strands using heat, and forward primers and reverse primers anneal to the denatured cDNA strands so that polymerase can elongate the DNA sequence.

Chromatin compaction

Educational objective: Chromatin compaction can influence accessibility of regulatory factors to DNA. The known H1 function is to stabilize chromatin compaction by securing nucleosome packaging. However, competing proteins would have the opposite effect, which would be to destabilize chromatin compactness and enhance accessibility to DNA.

Polymerase Chain Reaction (PCR)

1. Denaturation of DNA Template - separate double to single stranded DNA 2. Annealing - Add primer to single strand 3. Elongation by DNA Polymerase Step is 1-3 repeated for multiple cycles Measure DNA amplification

Tumor suppressor genes

Based on the passage, regulation of p21 expression by c-Myc also contributes to B-cell lymphoma development in Eμ-Myc mice. In addition, the passage states that p21 inhibits cell cycle progression, - a key feature of a tumor suppressor gene. Tumor suppressor genes - regulate DNA repair by repressing or pausing the cell cycle to ensure that only normal cells proceed to the division stage and induce programmed cell death if repair fails. As a result, p21 levels would be lower in Eμ-Myc mice compared to wild-type mice because p21 was likely inactivated by loss of function mutations and lost the ability to prevent abnormal growth/division of cancerous cells. Compared to wild-type mice, c-Myc should be expressed more abundantly and p21 should be expressed less abundantly in Eμ-Myc mice.

DNA sequencing and Southern blotting

Educational objective: DNA sequencing and Southern blotting are DNA assays that may be used to assess the relative quantity of genes between tissue types. Northern blotting is an RNA assay used to assess gene expression in different tissues.

Green fluorescent protein (GFP)

Educational objective: Green fluorescent protein (GFP) - is frequently used to track the mobility of proteins in a cell. The protein of interest is tagged with GFP, and its movement, localization, and expression can be studied in cells based on the fluorescence emitted.

cDNA cloning

Educational objective: In cDNA cloning, 1) reverse transcriptase generates a single strand of cDNA from a target mRNA sequence. 2) DNA polymerase synthesizes the second complementary DNA strand and amplifies the target cDNA sequence. 3) The target cDNA can then be inserted into a plasmid vector via the actions of a restriction enzyme (cuts both plasmid and vector) 5)The foreign cDNA and vector are cut by a restriction enzyme generating complementary sticky ends that anneal when both molecules are mixed together. 4) DNA ligase (joins the cDNA to the vector). *Therefore, Reverse transcriptase, DNA polymerase and restriction enzymes are required in cDNA cloning*

Western Blot = Protein immunoblotting

Educational objective: Western blot (protein immunoblotting) - uses antibodies to detect specific proteins. Greater quantities of the protein of interest will manifest as larger bands on the resultant blot. In general, proteins with the shortest amino acid sequences have lower molecular weights and migrate quickly through the gel. Band intensity (thickness) demonstrates the relative levels of expression of the target proteins. Shortest AA sequence = Bottom Highest Concentration/Highest MW = Top

Heterochromatin & Euchromatin

Gene transcription from DNA to RNA depends partly on the chromatin structure of the DNA that contains the gene. 2 forms of Chromatins - heterochromatin and euchromatin. Heterochromatin - consists of DNA that is tightly coiled around histone proteins, bound by an ionic interaction between the negatively charged phosphates on the DNA backbone and positively charged lysine residues in the histone. - DNA in heterochromatin is not readily accessible to RNA polymerase and so cannot be readily transcribed. Euchromatin - forms when histones are modified, often by acetylation of lysine residues. The added acetyl group neutralizes the positive charge on the histone, reducing interactions between histones and DNA. The reduced interactions yield a more open form that is more accessible to RNA polymerase, allowing euchromatin to be more readily transcribed. - Relatively open, uncoiled DNA is characteristic of euchromatin Because somatic cells transcribe TERT at low levels, the gene that encodes TERT is most likely found in *heterochromatin* within these cells. In somatic cells, the TERT gene is therefore most likely in a portion of the chromosome that is tightly wound around histones.

Positive control (+) and Negative control (-)

POMK + - + + - + FUKU + + - + + - Positive control (+) - help ensure that each step of the experimental procedure performs as expected. - The first lane in each autoradiogram is also a positive control, showing that radiolabels are added when all enzymes are present.(+,+) Negative control (-) - shows what the outcome would look like if the results were negative and helps confirm that positive results come from the expected source. -- In this case, the lane with no POMK in the 32P experiment and the lane with no fukutin in the 3H experiment are negative controls because it is expected that the radiolabels will not be added when the relevant enzymes are absent.

Origin of replication

Prokaryotes (eg bacteria) - have circular DNA with a single origin of replication in the cytoplasm Eukaryotes (eg yeast and humans) - have linear DNA with multiple origins of replication in the nucleus. An origin of replication expands to form a replication bubble, which contains two replication forks that move apart in opposite directions during DNA synthesis.

Western Blot + Autoradiography Western Blot = control used to detect the presence of protein of interest

The western blot was performed with an anti-Dg antibody that detects all Dg molecules and confirms that they are present, even if they are not radiolabeled. - The researchers can then be sure that the absence of bands in the autoradiogram is due to failure of the relevant enzyme to modify Dg and not due to a problem with isolating Dg itself. Note that unmodified Dg runs further on the gel because the absence of carbohydrates yields a smaller molecule. Educational objective: A proper experimental approach requires controls to confirm that the results are related to the conditions being tested rather than experimental error or confounding factors. Controls can be positive or negative, showing what a positive or a negative result should look like, respectively.

Transcription factors

Transcription factors - are proteins that can bind to DNA near gene promoter regions and either increase transcription (activators) or decrease transcription (repressors). Activators facilitate *RNA polymerase* binding to the promoter, and repressors inhibit binding. Educational objective: Transcription factors can upregulate or downregulate transcription by influencing the ability of *RNA polymerase* to bind a promoter. Transcription factors that increase transcription are called activators and facilitate RNA polymerase binding whereas those that decrease transcription are called repressors and inhibit binding.

pre-mRNA

Transcription is the process in which the nucleotide sequence of a DNA region is used as a template to synthesize a new RNA strand. (mRNA) is produced for the transcription of protein-expressing genes. Transcription begins with - the binding of transcription factors and RNA polymerase II to a specific AT-rich sequence (TATA box) in the promoter region of double-stranded DNA. - RNA polymerase II processes the noncoding DNA strand in a 3′ to 5′ direction until a termination sequence is recognized. - As the enzyme travels down the DNA strand, it unravels the DNA double helix and relies on the complementary pairing of bases to catalyze the synthesis of a pre-mRNA strand, which grows in the 5′ to 3′ direction.