Genetics Exam 3

What is histone methylation, and how does it affect gene expression?

Activating or repressing; can increase or decrease gene expression

What are common transcription factor structural motifs?

Alpha helix

Tumor suppressor

Block tumor progression; loss of function results from mutations in both copies -Recessive -Both copies are mutated Examples: TP53, BRCA1, & BRCA2 -normal function: control cell division, slow down

RNA interference

Blocking gene expression by means of a miRNA (micro-RNA) silencing complex. -bind to mRNA and prevent it from becoming a protein

5. What is cDNA? How is it generated in the laboratory? What advantages does the study of cDNA have over genomic DNA for understanding the genetics of a disease or other trait? What disadvantages does it have?

Coding DNA (DNA derived from mRNA); no introns. Use reverse transcriptase to use RNA as a template to make a complementary DNA strand. Pros: Researchers can focus on coding sequence, allows expression of encoded protein Cons: Regulatory sequences not represented

postive eugenics

Encouraging the reproduction of those with beneficial traits

All cells in the human body are derived through mitotic division of a single cell -- the fertilized egg, or zygote. The cells are therefore genetically identical (i.e., contain the same DNA). So how do cells in the body look and function so differently from one another?

Gene expression

RNA-seq

Sequencing technique used to determine directly the nucleotide sequence of a collection of RNAs. --high-throughput sequencing of cDNA - whole transcriptome can be sequenced; measures number of RNA strands

penetrance

The percentage of individuals with a particular genotype that actually displays the phenotype associated with the genotype.

What is the "two-hit" hypothesis?

Those without a germline mutation will take longer to develop cancer than those who are born with a mutation

How did researchers find the location of the gene associated with PAP? How did they determine the specific mutation responsible?

Used polymorphic markers until they found a gene that showed no recombination with the phenotype. -They saw what chromosome the marker was on; this narrows down what the specific mutation is.

Repressors

downregulation

Repressible negative regulation

usually on, but repressed when a repressor binds - TRP operon

Operator

(site) is a sequence of bases that provides a binding site for a repressor protein called the lac repressor. The "switch" that controls transcription.

What is the major difference between 454 Pyrosequencing and Ion Torrent sequencing?

-454 pyrosequencing: the wells flood instead of dNTPS being flooded across the plate. -Ion Torrent measures pH instead of taking a picture of colors

Describe how PCR can be used in infectious disease diagnostics, and how it might have an advantage over traditional (culture-based) approaches.

-Amplify the gene sequence for the suspected disease. If it amplifies, it is present; if the sample stays the same, it is not. -This takes a few hours rather than a few days.

Alternative Splicing of mRNA

-One gene can code for more than one protein -Exons can be spliced together into a variety of different mRNAs

gel retardation assay

-The binding of a protein to a fragment of DNA retards its rate of movement through a gel -Must be performed under a nondenaturing conditions -Proteins must be properly folded and DNA must be a double helix

Ion Torrent Sequencing

-based on the release of an H ion when nucleotide is added. -- resulting change in pH read on a semiconductor chip

What are some applications of next generation sequencing?

-isolate mutant genes from forward genetic screens -reconstruction of human population history -bacterial genome sequencing in the clinic: bioinformatic challenges and solutions -cancer genome-sequencing study design -de novo mutations -the human microbiome --sequence comparisons

differentiation

A cell that has become a specialized type of cell within a multicellular organism

What is the definition of a suppressor?

A mutation in a second gene that eliminates the mutant phenotype of a mutation in the first gene.

Pleiotropy

A single gene having multiple effects on an individuals phenotype

Southern blotting

A technique that enables specific nucleotide sequences to be detected in a sample of DNA. It involves gel electrophoresis of DNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe.

Northern blotting

A technique that enables specific nucleotide sequences to be detected in samples of mRNA. It involves gel electrophoresis of RNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe.

What is the purpose of backcrossing?

Backcrossing: cross of F1 hybrids to individuals that have genotypes of parental generation -Purpose: remove the other mutations that were induced by the mutagen from the mutation that you are interested in

What are induced pluripotent stem cells (iPS cells)? Why might the use of iPS cells be important from a scientific standpoint? From an ethical standpoint?

Cells reprogrammed back into embryonic-like pluripotent state -Come from the patient - no immune response; avoid "killing a baby" ---immature cell that is generated from an adult (mature) cell and that has regained the capacity to differentiate into any type of cell in the body. -Induced pluripotent stem cells differ from embryonic stem cells, which form the inner cell mass of an embryo but also are pluripotent, eventually giving rise to all the cell types that make up the body.

What is chromatin remodeling, and what types of proteins are involved?

Chromatin remodeling causes dynamic changes in the structure of chromatin during the life of the cell. Contain multiple ATP-dependent chromatin remodeling complexes. -The remodeling process is carried out by a protein complex that recognizes nucleosomes and uses ATP to alter their configuration. 3 ways chromatin remodeling complexes change chromatin structure: -Change in nucleosome position -Eviction of histone octamers -Replacement with histone variants

Enhancers

DNA sequence that functions as a regulatory element, when bound to activator TF increases level of transcription

Silencers

DNA sequence that functions as a regulatory element, when bound to repressor TF decreases level of transcription

What is germline gene therapy?

DNA transferred into the cells that produce reproductive cells, eggs, or sperm in the body. --correct the genetic variants of the reproductive cells of an individual, and this would be passed down to future generations. This therapy removes a hereditary disorder from a family line forever.

negative eugenics

Discouraging the reproduction of those with undesirable traits

Describe how regulatory transcription factors function.

Enhance or inhibit binding of general TFs, effects on mediator protein complex (replaces enhancer) -Binding of a small effector molecule (hormones) -Protein-protein interactions -Covalent modifications

Where and when does gene regulation take place - where in the cell, when in the cell cycle, where in the stages of processing of genetic material from DNA to RNA to proteins?

Gene regulation can take place during any stage of processing, anywhere, anytime, although during transcription in the nucleus is most common.

Describe the difference between general and regulatory transcription factors.

General are typical transcription factors while regulatory transcription factors regulate gene expression. -Enhance or inhibit binding of general TFs

How do housekeeping genes and tissue-specific genes differ in regard to their methylation patterns?

Housekeeping genes are unmethylated, so they are always expressed, while tissue-specific genes are methylated since they are often silenced.

zinc-finger domains

Is coordinated by Zn; DNA is recognized by an a helix; Multiple fingers may exist in a protein and each recognizes a different sequence; Strong binding affinity

Understand the process of gel electrophoresis.

Negative charge on DNA backbone causes migration in electric current; larger fragment = slower migration

How does an organism benefit from the ability to regulate the expression of a gene?

Not wasting metabolic energy on expressing a gene they don't need

Forward genetic approach

Observe a phenotype, screen a collection of mutants, identify gene(s) causing the phenotype. -an approach of determining the genetic basis responsible for a phenotype.

What is DNA methylation, and how does it affect gene expression?

Occurs in CpG island, usually silences DNA expression - occurs on cytosine. It occurs on the cytosine base and usually inhibits gene transcription. It occurs via an enzyme called DNA methyltransferase.

What makes C. elegans a good model organism for genetic screens?

Quick generation time, mostly selfing hermaphrodites (self-cross), can also mate them together.

How are regulatory transcription factors regulated?

Regulated by mediator protein complex

Describe how recombinant DNA is produced.

Restriction enzymes used to cut two strands of DNA at palindromic sequences, then are fused together via DNA ligase

Understand how restriction enzymes are used.

Restriction enzymes: bind to specific DNA sequences and then cleave the DNA at 2 defined locations, one on each strand

What is meant by a reversion mutation? Be able to list two possible types of revertants.

Reversion mutation: a mutation that returns a mutant allele back to the wild-type allele. b. True revertants --ade2 mutant allele to ADE2 wildtype c. Suppressors --a mutation in a different gene eliminates the ade2 mutant phenotype

Reverse genetic approach

Select a gene of interest, generate a null mutant of the gene, observe a phenotype. -Identify interesting genes: RNA sequencing (highly expressed in one population but not another)

What is meant by a suppressor mutation? Be able to distinguish from a revertant.

Suppressor mutation: a mutation at a second site that suppresses the phenotypic effects of another mutation -Doesn't remove mutant phenotype like reversion, just suppresses mutant phenotype

Describe the process of Somatic Cell Nuclear Transfer.

The nucleus from an adult/differentiated cell is implanted into a denucleated (nucleus removed) egg and then either reimplanted into surrogate or grown in a dish -dolly!

Totipotency

a cell that possesses the genetic potential to produce an entire individual

Microarray

a grid of DNA segments of known sequence that is used to test and map DNA fragments, antibodies, or proteins.

Oncogene

a mutant gene that promotes cancer; only 1 copy needs to be mutated -rapid cell growth and divsion -gas pedal

Negative transcriptional regulation

a repressor binds to the operator, preventing transcription

20. What is histone acetylation, and how does it affect gene expression?

a. "dissolving" of histones; increases gene expression through transcription activation

23. What is a CpG island, and what is its importance in terms of gene regulation?

a. Activator protein binding site

18. How is the structure of chromatin related to gene transcription?

a. Allows DNA accessibility to transcriptional machinery and TFs.

Which structural properties of DNA are important in manipulating DNA?

a. Complementary binding of double-stranded DNA b. Negative charge of phosphate group c. Increased strength of G-C bonding

What kinds of biological questions can be answered by examining DNA? RNA? Proteins?

a. DNA: How is the genome organized? How do individuals differ in their genomes? What genome features are associated with a given disease? b. RNA: Which genes are expressed during certain times/conditions? Which genes are associated with a given disease? How do regulatory RNAs influence biology and disease? c. Proteins: How do proteins function and interact?

17. What types of factors influence the binding of regulatory transcription factors?

a. Effector molecules, protein-protein interactions, or covalent modifications

Describe how Sanger (dideoxy) sequencing works. How is it like a PCR? How is it different?

a. Four dideoxyribonucleotides are added to a tube and after incubation and polymerization, a sample is loaded into the tube (capillary). They are separated according to length and visualized, with fluorescent markers distinguishing between nucleotide bases. b. Similar to PCR: amplification, running on gel, separating by size; Different: marks nucleotides individually by fluorescent rather than weight

What is gene therapy? How might "good" genes be introduced into a patient's cells, and what are some of the potential pitfalls?

a. Gene therapy: introduction of cloned genes into living cells in an attempt to cure or alleviate disease b. Use of a virus that alters genes - triggers immune response

What is a germline mutation, and what influence does a germline mutation in BRCA1 have on the probability of developing a cancer? Understand what it means to be predisposed to cancer and be able to determine inheritances from a cancer pedigree.

a. Germline mutations are inheritable; first mutation occurs in a parental gamete b. Cancer risk associated with BRCA1 follows autosomal dominant inheritance pattern i. Inherited 1 mutated copy, chance of cancer 80%

16. What is a mediator protein complex, and how (in general) does it function? How is it regulated by transcription activators and repressors?

a. Mediates interaction, Activator activates the mediator and repressors suppress the mediator -It mediates the interaction between RNA polymerase II and regulatory transcription factors.

Understand how to conduct a PCR reaction - the reagents needed, the steps included in each cycle (including temperatures, what happens at each), and approximately how many cycles are conducted. Be able to design PCR primers.

a. Primers bind to either side of the gene of interest, this segment is copied over several PC cycles until it is amplified enough to be visualized. b. Denaturation: Separate DNA strands at 94 degrees Celsius to break apart H bonds c. Primer annealing: Lower temp, allows primers to bind to template DNA d. Primer extension: Incubate at slightly higher temp, allows DNA synthesis to occur e. Starting materials: Template DNA, Oligonucleotide primers, dNTPs, Taq polymerase f. 20-30 cycles

For each of the points along the "central dogma" pathway (DNA --> mRNA --> protein) where gene expression can be regulated, describe at least one mechanism for gene regulation.

a. Replication: N/A b. Transcription: Regulatory Transcription Factors c. Translation: Alternative Splicing

What are the six hallmarks of cancer?

a. Self-sufficiency in proliferation signals b. Insensitivity to antigrowth signals c. Tissue invasion and metastasis d. Limitless replicative potential e. Sustained angiogenesis - grow new blood vessels f. Evading apoptosis

Be able to describe the steps involved in a forward genetic screen in C. elegans. Be able to draw the genotypes of worms at each step in the screen. Be able to draw Punnett squares and describe inheritance patterns.

a. Treat with EMS b. Remove parental generation after ~100 eggs laid c. Each F1 carries 2 mutagenized chromosomes (m/+ heterozygotes for each mutation) d. Remove F1 worms after ~2000 eggs for 20 hours e. Worms with m/m genotype for each mutation mixed with m/+ and +/+ animals f. Mutant worms (m/m) individually picked onto fresh plate

Be able to describe the steps involved in a forward genetic screen in yeast.

a. Treat wt yeast with mutagen b. Colonize a "complete plate" c. Place -adenine plate on top d. Any colonies on the complete plate not on the -adenine plate are mutants e. "genetic screen"

What are embryonic stem cells? During which stage of embryonic development are they obtained? Why are they considered superior to adult stem cells in terms of their therapeutic potential?

a. Type of pluripotent stem cell found in the early blastocyst b. Obtained at early-stage c. Pluripotent rather than multipotent - can make anything (sans placenta)

Know what a vector/plasmid is. What are common components of a vector? Know the difference between a tissue-specific promoter and a constitutive promoter, and why one may be more beneficial over another in a given situation. Know what GFP is and how it can be used.

a. Vector/plasmid: used as carrier for DNA of interest b. Common components: Origin of replication, antibiotic resistant gene, multiple cloning site, selectable marker (eukaryotic cells only), promoter (if gene expression desired) c. Tissue-specific promoter: has activity in only specific types of cells

What are riboswitches/ribozymes? What level(s) of gene regulation do ribozymes represent?

a. When RNA binds a small molecule; can regulate transcription, translation, RNA, stability, and splicing b. Can regulate transcription and translation

Positive transcriptional regulation

an activator binds to the operator, stimulating transcription

Activator

binds to DNA and INCREASES the rate of transcription.

Repressor

binds to operator and prevents transcription

Stem cell

cell that has the capacity to divide and to differentiate into one or more specific cell types

haplotype association studies

conducted to identify disease-causing alleles -need a lot of families with a lot of kids to narrow down the disease causing allele -use huge pedigrees that show affected chromosomes, carriers, and crossing over of chromosomes. Crossing over narrows down where the mutation is on the chromosome.

Promoter

contains a polymerase binding site and an operator; genes of operon follow the promoter -signals beginning of transcription

What is a ddNTP (be able to recognize its chemical structure), and how does it function in a Sanger sequencing reaction? Be able to "read" a gel sequencing reaction.

ddNTP: dideoxynucleosides triphosphate; added to nucleotide base in sequencing. -synthesize nucleotides that are missing the -OH group -Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow --chain termination

Screen

each member of population examined to determine if they fit phenotype criteria

454 pyrosequencing

emulsion PCR: beads encapsulated in oil droplets --beads placed on microtiter plate --dNTPs added sequentially over plate --produces light that are read by camera and computer -Slow, expensive, & not super accurate

What is a housekeeping gene? When are these genes typically expressed? Would you expect that they are typically methylated or unmethylated?

genes that are required for the maintenance of basic cellular function and are expressed in all cells of an organism. They encode proteins that are involved in fundamental cellular processes, such as cell cycle regulation, DNA replication, and metabolism. Housekeeping genes are also known as reference genes, because they can be used as a baseline for comparing the expression of other genes.; unmethylated

Operon

groups of (usually functionally related) genes that are adjacent to one another and expressed as a unit.

Selection

individuals not meeting criteria don't survive or are otherwise eliminated from the population

Regulation of Lac operon

inducible, off when lactose is absent, repressor is active. Inducer is allolactose (different form of lactose), binds to repressor and changes its shape so it cannot bind to operator anymore; also regulated by an activator protein - cAMP and CAP at CAP site - wants to digest present glucose before having to breakdown allolactose

sporadic

late onset -single tumor -not hereditary -inherit two normal genes -get cancer due to a rare event, tumor, lose a gene

Downregulation

less transcription

Gene regulation

level of gene expression can vary under different conditions -gene regulation occurs during any step in the central dogma

Upregulation

more transcription

Gene expression

multistep process that proceeds from transcription to translation and may involve posttranslational effects on protein structure and function. -Gene regulation can occur at any of the steps in the pathway of gene expression. --central dogma of gene expression -the accessing of genetic information, synthesis of RNA and (usually) proteins, and their effects on phenotype

Proto-oncogene

normal cellular gene that does not cause cancer, but which may incur a gain-of-function mutation or become incorporated into a viral genome and thereby lead to cancer -mutated form: oncogene

Linkage

occurs when different traits are inherited together more often than they would have been by chance along; it is assumed that these traits are linked on the same chromosome

Western blotting

procedure that uses labeled antibodies to detect specific antigens in a mixture of proteins separated according to their molecular weight

Regulation of Trp operon

repressible, when no TRP present, repressor is inactive. TRP acts as corepressor, binding to the repressor to activate it when enough is present

Control elements

sequence of DNA that binds a regulatory protein and thereby influences gene expression -enhancers & silencers

expressivity

the degree to which a genotype is expressed in an individual

Pluripotency

type of stem cell that can differentiate into all or nearly all the types of cells of the adult organism

Multipotency stem cells

type of stem cell that can differentiate into several different types of cells

Activators

upregulation

Inducible negative regulation

usually off, but activated when a regulatory molecule binds to the repressor (making the repressor inactive) - lac operon