GENETICS FINAL EXAM!

creating knockout mice

-construct a targeting vector; this creates segment of DNA for introduction into cell -targeting vector then undergoes homologous recombination with gene of interest and renders it nonfunctional -target vector has mutated a copy of gene of interest -foreign DNA disrupts the reading frame and produces nonfunctional protein

Familial Adenomatous Polyposis (FAP)

-genetic disposition to cancer -one mutant copy inherited of APC gene on long arm of chromosome 5 -mutations include deletions, frameshift, and point mutations

eukaryotic gene regulation is more complex than in prokaryotes

-greater amount of DNA that is associated w/ histones and other proteins -mRNAs must be spliced, capped, and polyadenylated prior to transport from nucleus -genes on numerous chromosomes are enclosed in a double membrane nucleus mRNAs have a wide range of half-lives (they don't live forever) modulation of mRNA translation, protein processing, degradation

mRNA localization and localized translational control

-mRNA 3' UTR sequence termed a zip code- cis-regulatory element that serves as a binding site for RBP called zip code binding protein 1 (ZBP1) -blocks translation until mRNA is localized to site of cell where it is needed -when mRNA has reached destination, Src phosphorylates ZBP1 - allows translation

regulation of mRNA stability and degradation

1) adenosine-uridine rich element (ARE)- cis acting sequence regulates mRNA stability- stretch of A and U nucleotides found in 3' UTRs can bind RNA binding proteins which recruit decay machinery - many ARE containing mRNAs encode cell proliferation proteins 2) mRNA surveillance- regulatory sequence that attracts proteins for mRNA surveillance = don't want cancer to form --nonsense-mediated decay (NMD)- mRNA surveillance pathway that efficiently eliminates mRNAs w/ premature stop codons (from nonsense mutation or RNA pol error) -> caught then tagged so cell can get rid of

alternative splicing types

1) cassette exons ("exon skipping") - exons may be excluded from the mature mRNA by joining the 3' end of the upstream exon to the 5' end of the downstream exon, skipping of cassette exons is the most common type in animals (accounts for 40%) 2) alternative splice site - within an exon that may be upstream or downstream of the normally used splice site 3) intron retention - introns are included in the mature mRNAs and are translated, producing novel isoforms, can negatively regulate gene expression at the posttranscriptional level (such mRNAs are degraded or are retained in the nucleus) 4) mutually exclusive exons - allows for swapping of protein domains encoded by different exons, splicing is co-regulated for a cluster of two or more adjacent exons such that inclusion of one exon leads to the exclusion of the others in the same cluster (GET ONE, can't have both) 5) alternative promoters - have more than one site where transcription may be initiated, produces pre-mRNAs w/ different 5' exons, which may be alternatively spliced to downstream exons 6) alternative polyadenylation - spliceforms w/ different 3' ends are produced, when an exon containing a polyadenylation signal is skipped, downstream exons are included and a downstream polyadenylation signal will be used

ras proto-oncogene pathway

1) growth factor stimulates receptor 2) Ras is activated and then activates Raf 3) Raf activates Mek 4) Mek activates map kinase, which goes into nucleus and activates transcription factors

gene expression regulation

1) mRNA stability 2) mRNA degradation

cytoplasmic polyadenylation pathway

1) mRNAs regulated through cytoplasmic adenylation often have cytoplasmic polyadenylation element (CPE) in 3' UTR - UUUUAU 2) sequence recognized by cytoplasmic polyadenylation element binding protein (CPEB) 3) CPEB recruits poly-A specific ribonuclease (PARN) 4) CPEB, PARN, Maskin, PABP, and elF4G form complex that blocks the interaction of elF4G and prevents translation OR... 1) cell received signal to reactivate CPE containing mRNAs = CPEB is phosphorylated by kinases 2) conformational change allows elF4E and elF4G to interact = translation

retroviruses cause cancer by

1) proviral DNA may integrate near proto-oncogenes, which stimulates high levels of transcription 2) acute transforming retroviruses: normal retrovirus w/ normal viral gene products can stimulate inappropriate cell growth (more likely to do it this way) 3) retroviruses associated w/ human cancers: human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV-1)

3 steps of PCR

1. denaturation 2. primer annealing (hybridization/annealing) 3. extension repeated over and over using thermocycler to amplify DNA exponentially DNA strand is doubled in each cycle new strands along w/ old strand serve as templates in next cycle taq polymerase is used to keep the cycle repeating

evidence for role of epigenetic alterations in cancer

1. global hypomethylation: may cause genomic instability and the large-scale chromosomal changes that are a characteristic feature of cancer 2. epigenetic mechanisms: can replace mutations as a way of silencing individual tumor-suppressor genes or activating oncogenes 3. epigenetic modifications: can silence multiple genes, making them more effective in transforming normal cells into malignant cells than sequential mutations of single genes

epigenetic change mechanisms

1. methylation: reversible; addition or removal of methyl groups 2. histone modification and chromatin remodeling: alter the accessibility of the genes -> aka how tightly the DNA is wrapped 3. noncoding RNA

1) long noncoding RNAs binding to chromatin-modifying enzymes 2) chromatin remodeling 3) DNA methylation 4) histone modification involving acetyl, methyl, and phosphate groups 5) binding of RISC to target mRNA molecules

5 major mechanisms of epigenetic genome modification

D) silencer

A regulatory sequence of DNA that is 10,000 base pairs away from the gene it regulates is mutated. The result is that the gene being regulated is now expressed at a higher rate compared to when this regulatory sequence was not mutated. What would this sequence of DNA best be called? A) insulator B) activator protein C) enhancer D) silencer E) zinc finger motif

APC gene

APC (adenomatous polyposis) gene product: tumor suppressor that controls cell-cell contact and growth inhibition FAP is due to one mutant copy of APC gene heterozygous APC mutation: causes formation of many rectal polyps or adenomas in early life -second APC mutation leads to later stage of cancer

genetic testing w/ DNA microarrays

ASO and RFLP analysis are efficient methods for mutation screening but can only detect a few specific mutations have been used to produce DNA microarrays - expression of thousands of genes examined on a single gene -gives you an idea of how the cancer is multiplying

reciprocal chromosomal translocations

Are characteristic of many cancers Include white blood cell cancers such as leukemias and lymphomas Example: Burkitt lymphoma: Reciprocal translocation between chromosome 8 and chromosomes 2, 14, and 22 myc is a proto-oncogene that promotes cell proliferation

CRISPR-Cas9

CRISPR (clustered regularly interspaced palindromic repeats)-Cas system gene editing method that involves the use of specifically engineered DNA-modifying enzymes (nucleases) that create changes in a specific sequence to remove, correct, or replace a defective gene or parts of a gene gene editing is based on using different nucleases to create breaks in the genome in a sequence-specific manner *bacteria have a mechanism against bacteriophages, use CRISPR system = can be used to create knock-out organisms*

synpolydactyly (SPD)

Caused by mutations in HOXD13 Characterized by: Extra fingers and toes Bone abnormalities in hands and feet

transgenic animals

Created for research purposes to study gene function examples: 1) oversized mice containing human growth hormone transgene 2) transgenic atlantic salmon 3) transgenic cows for battling mastitis (infection of mammary glands that reduces output and contaminates milk) --incorporated gene whose protein product destroys S. aureus bacterium --had lysostaphin gene from Staphylococcus simulana; lysostaphin cleaves S. aureus cell wall ex ex) mad cow disease (cows fed cows)-bovine spongiform encephalitis: cattle were produced lacking prion protein PrP gene, early results indicate animals are resistant to the disease enviropig: successful transgenic farm animal, pig w/ salivary glands that express gene encoding enzyme phytase, allows pig to break down dietary phosphorus (major pollutant), reducing its excretion GloFish: transgenic strain of zebrafish, contains red fluorescent protein from sea anemones, useful for assaying heavy metal contamination in water genetically modified A. aegypti male mosquitos: do not bite or spread disease, mate w/ females and pass along lethal gene = die before reaching biting adulthood -originally created by transgenic approaches but now by CRISPR-Cas9

DNA-based vaccines

DNA encoding proteins from pathogen inserted into vector Injected into individual Triggers immune response if individual becomes exposed to pathogen in future Trials are underway using plasmid DNA encoding protein antigens from HIV and ZIKA

alteration of DNA-histone contacts

DNA exposed (by sliding) for transcription

DNA ligase

DNA fragments will seal phosphodiester backbone joins restriction fragments covalently to produce intact DNA molecules

fetal haplotypes

DNA in blood is sequenced to analyze haplotypes Haplotypes: contiguous segments of DNA that do not undergo recombination -Distinguish which DNA segments are maternal and which are fetal

transcription activators and repressors bring changes to RNA pol II transcription

DNA looping (aka folding to interact w/ enhancer sequence) delivers activators, repressors, and general transcription factors to promoter vicinity recruitment model: enhancers and silencer elements act as donors; affect regulatory proteins at gene promoters enhanceosome: activator and coactivator proteins

alteration of the DNA path

DNA pulled off nucleosome = exposed for transcription

transgenic animals: knock-in animals

Express or overexpress particular gene of interest (transgene) Vector with transgene undergoes homologous recombination into host genome Vector with transgene can also be put into ES cells and injected into embryos Allows for study of effects on appearance and function in mice

southern blot

FOR DNA; used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest methodology: hybridization between complementary nucleic acid (DNA or RNA) molecules determines whether clone contains all or part of gene ascertains size and sequence organization of gene or DNA sequence of interest 2 components: separation of DNA fragments by gel electrophoresis hybridization by using labeled probes

J. Craig Venter Institute

Filed two patent applications for "the world's first-ever human-made life form" Patent will cover minimal genome of M. genitalium (believed to contain genes necessary for self-replication) Synthetic biology poses ethical questions

mutations in the regulatory gene GAL4 prevent activation

GAL4 (activator protein) encodes the Gal4 activator protein (Gal4p) required for transcription of the GAL structural genes -Gal4p consists of 881 amino acids and functions as a homodimer --a DNA-binding domain (DBD) that *recognizes and binds specific DNA sequences* --an activation domain (AD) that activates transcription

GAL operon: galactose absent

GAL4 = activator UASG(subG) = upstream activation sequences of GAL genes Gal80 = repressor Gal4p and Gal80 bind together = they don't fit on the activation sequence UASG so NO TRANSCRIPTION

p53 tumor suppressor gene

GUARDIAN OF THE GENOME Most frequently mutated gene (50% of all cancers) Encodes transcription factor that represses or stimulates transcription different genes is continuously synthesized but is rapidly degraded, so is present at low levels increased levels due to increases in protein phosphorylation, acetylation, and other posttranslational modifications

GAL operon: galactose present

Gal3 binds to Gal80 = Gal80 can't bind to Gal4p so Gal4p binds to activation sequence UASG(subG) = TRANSCRIPTION OCCURS

genomic instability

In cancer cells manifests in gross defects: -translocations -Aneuploidy -Chromosome loss -DNA amplification -Chromosomal deletions MDM2 encodes a ubiquitin ligase MYCN - member of MYC family double-minute chromosomes (as in small) - small fragments of DNA stay separate from chromosomal DNA

agricultural biotechnology

Introduces insect resistance, herbicide resistance, or nutritional characteristics into farm plants and animals

prenatal genetic testing

Many genetic disorders are diagnosed prenatally using cytogenetic, biochemical, and recombinant DNA testing Samples are obtained from amniocentesis (amniotic fluid) and chorionic villus sampling (CVS) (tissue from placenta)

hereditary cancer

Most cancers result from somatic cell mutations However, 50 forms of hereditary cancer (1-2%) are known

heterogenous staining region

Myc (red) and MDM2 (green) mutations in neuroblastoma cells green - heterogenous staining region - multiple copies of MYCN

D) denaturation, annealing primers, elongation

Order of the 3 main steps in a PCR A) denaturation, elongation, annealing primers B) elongation, annealing primers, denaturation C) annealing primers, denaturation, elongation D) denaturation, annealing primers, elongation

pathways of degradation

PURPOSE: to stop translation!!!!! 1) deadenylation-dependent decay: exoribonuclease enzymes shorten the length of the poly-A tail - binding of poly-A binding protein to tail stabilizes mRNA 2) decapping enzymes removes 7-methylguanine cap = mRNA unstable now 3) endonuclease cleaves mRNA internally UTR mRNAs sometimes are localized in P (processing) bodies sometimes they are not destroyed but marked for possible use later

loss of heterozygosity

Phenomenon where second wild-type allele is mutated in tumor Essential first step in expression of inherited cancers Further mutations in proto-oncogenes and tumor- suppresser genes are necessary for full malignancy -inherited one mutated allele and then other mutates

genome-wide association studies (GWAC)

Quest to identify genes that influence disease Result: 700 publications linking 3000 genetic variations to about 150 traits carried out by comparing thousands of unrelated individuals with particular disease to and those w/o disease identifies variations that confer risk of developing disease -helps narrow things down = focus on genes above line

retroviruses

RNA viruses animal viruses that cause cancer reverse transcriptase enzyme copies RNA to DNA DNA copy enters nucleus of infected cell retroviruses integrate into host genome as provirus, which is replicated w/ host's DNA during normal cell cycle acute transforming retroviruses: carry cell-derived oncogenes, infect and transform cells into cancer cells C-onc = host cell oncogene V-onc = when c-onc is transferred into viral genome

tumor suppressor genes

Regulate cell-cycle checkpoints and initiate process of apoptosis mutated tumor-suppressor genes: are unable to respond to cell-cycle checkpoints or undergo apoptosis won't stop the cell cycle leads to more mutations and development of cancer

epigenome projects

Several projects are underway to map the human epigenome, which may help explain how environmental settings in early life can affect predisposition to adulthood diseases The NIH Roadmap Epigenomics Project is based on the idea that many aspects of health and susceptibility to disease are related to epigenetic regulation or misregulation of gene activity

limitation of PCR

Some information about nucleotide sequence of target DNA is required to synthesize primer Minor contamination from other sources can cause problems (e.g., skin cells from researcher) PCR cannot amplify long segments of DNA *contamination and length of sequence are limitations*

GTFs that assist RNA pol II at a core promoter

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFFIIH, and a large multi-subunit complex called mediator

A) bacterial defense against viral infection

The CRISPR-Cas system of gene editing is based on what naturally occurring biological process? A) bacterial defense against viral infection B) eukaryotic defense against viral infection C) viral defense against bacterial defenses D) viral defense against eukaryotic defenses

C) transcription would not be initiated properly

The pre-initiation complex (PIC) contains several proteins. What would be the direct consequence be if PIC failed to form? A) replication would not be initiated B) translation would not be initiated C) transcription would not be initiated D) mRNA splicing would not be initiated E) protein would not fold properly

insulin production in bacteria

Therapeutic proteins can be produced by introducing human genes into bacteria humulin- synthetic human insulin was originally produced in bacteria; insulin regulates glucose metabolism bioengineering process of insulin synthesis: 2 insulin subunits are produced as a fusion protein (hybrid protein) -fusion protein purified -> cleaved to release insulin polypeptides -2 subunits spontaneously unite and form active insulin

G1/S, G2/M, and M checkpoints

Three distinct checkpoints where the cell monitors external signals and internal equilibrium Cells decide whether to proceed to the next stage of the cell cycle

APC gene: adenomatous polyposis coli

Tumor-suppressor gene Encodes protein involved in normal differentiation of intestinal cells in colorectal cancers, adenoma or poly formation requires inactivating mutations in APC gene

western blot

Used for analyzing proteins

applications of PCR

Useful tool; the most widely used technique in genetics and molecular biology Allows for screening of mutations involved in genetic disorders Location and nature of mutation can be determined quickly Allele-specific probes for genetic testing can be synthesized, making PCR important for diagnosing genetic disorders

single-cell sequencing

We can now sequence the genome from a single cell Involves isolating DNA from single cell and executing whole genome amplification Produces sufficient DNA to be sequenced -can look at variation between cells = assuming 1 cell represents all cells (its downfall) -picture = see clusters of cell expression

lysine

______ is a positively charged amino acid

B) detect mutations that may indicate a risk of disease

a DNA microarray can be used for A) generate mutations in gene of interest B) detect mutations that may indicate a risk of disease C) detect RFLPs D) determine protein translation levels

yeast

a eukarote, fungus

what is homeobox

a gene that produces a 60 amino acid binding protein homeobox gene regulates development

variable gene activity

accounts for the variety of cellular structures and functions in eukaryotic cells

histone modification - acetylation

acetylation by histone acetyltransferase (HAT) opens up the chromatin structure - makes genes available for transcription removal of the acetyl groups by histone deacetylase (HDAC) closes the configuration - silences genes by making them unavailable histones acetylated by histone acetyltransferase deacetylated by histone deacetylase picture: spaces are for transcription factors or RNA pol II to come in

A) embryonic stem cells

after fertilization of an egg the totipotent cell divides and the cells begin to specialize. these 30-40 cells are called ______ and are pluripotent. A) embryonic stem cells B) somatic cells C) germ line cells D) T celsl

Cancer: Multistep process requiring multiple mutations

age-related incidence of cancer indicates that cancer develops from accumulation of several mutagenic events in single cell incidence of most cancers rise exponentially w/ age independent and random mutations are necessary for cell to become malignant carcinogens: cancer-causing agents, delay between exposure to carcinogen and appearance of cancer is an indication of the multistep process ex) leukemia from radiation exposure (Hiroshima) had an incubation period of 5-8 years

evidence that epigenetic changes are involved in cancer

all cancers this far have exhibited a pattern of global hypomethylation, although specific regions are hypermethylated. the genes that are silenced include tumor suppressor genes

genetic tests

allele-specific oligonucleotides (ASOs): short, single-stranded fragments of DNA, used as probes to identify alleles that differ by a single oligonucleotide (single-nucleotide polymorphisms) or SNPs PCR method using ASOs: now available to screen for many disorders, including sickle-cell anemia and cystic fibrosis

genetic engineering

alteration of organism's genome using recombinant DNA technologies to add or remove gene from genome -produces genetically modified organisms (GMOs)

intron retention

alternative splicing type introns included in mature mRNA -> novel isoforms -> negatively regulate gene expression

alternative promoters

alternative splicing type more than 1 site where transcription may be initiated = produces pre-mRNAs w/ different 5' exons, which may be alternatively spliced to downstream exons

alternative polyadenylation

alternative splicing type spliceforms w/ different 3' ends are produced -when an exon containing a polyadenylation signal is skipped, downstream exons are included and a downstream polyadenylation signal will be used

mutually exclusive exons

alternative splicing type swapping of protein domains encoded by different exons splicing is co-regulated for a cluster of 2 or more adjacent exons i.e. inclusion of 1 leads to exclusion of others in the same cluster

molecular clock

amino acid or nucleic acid sequences in which evolutionary changes accumulate over time ideally at a constant rate so that the degree of differentiation is correlated to the length of time of divergence

synthetic genome

an artificially constructed genome for artificial cells or designer organisms bacterium mycoplasma genitalium = simple self-replicating bacteria, serves as model for understanding minimal elements of genome necessary for self-replicating cell

PIC

an assembly of proteins that provides a platform for RNAII polymerase to begin transcription

epigenetic trait

an epigenetic trait is stable, mitotically and meiotically heritable phenotype that results from the changes in the gene expression w/o alterations in the DNA sequence

gene-expression microarrays

analyze gene-expression patterns in genetic diseases contain probes for specific genes expressed differently in cell types mRNA expression detected through gene expression microarrays - powerful genetic disorder diagnostic tool gene expression microarrays: have revealed that certain cancers have patterns of gene expression; patterns correlate w/ factors - stage of cancer - clinical course - response to treatment - example: diffuse large B-cell lymphoma

carcinogens

any substance or event that damages DNA and causes mutations to occur in proto-oncogenes or tumor-suppressor genes carcinogens include chemicals, radiation, some viruses, and chronic infections can be natural or human-made -our environment contains abundant carcinogens

control of apoptosis

apoptosis: programmed cell death, occurs when DNA or chromosomal damage is too severe to repair, cell halt progress through cell cycle, prevents cancer, eliminates cells not contributing to final adult organism steps in apoptosis: 1) fragmentation of nuclear envelope 2) disruption of internal cellular structures 3) dissolution of cell into small, spherical apoptotic bodies 4) engulfing of apoptotic bodies by phagocytic cells caspases: series of proteases responsible for initiating apoptosis and digesting intracellular components

inherited cancers

are caused by defects in DNA repair genes xeroderma pigmentosum (XP) - defective nucleotide excision repair leading to skin cancer hereditary nonpolyposis colorectal cancer (HNPCC) - autosomal dominant syndrome (200/1000 affected), increased risk of colon, ovary, uterine, and kidney cancers 8 genes implicated; 4 involved in DNA mismatch repair

Metastasis suppressor genes

are mutated or disrupted in metastatic tumors affect growth and development of metastatic tumors, not primary tumor ex) C D82 protein: expression of it is lost in metastatic tumors

what is methylation (cue jeopardy theme)

associated w/ decreased gene expression, mostly attached to cytosines

DNA methylation

associated with decreased gene expression methylation occurs most often on cytosine of CG doublets in DNA aka CpG islands methylation can repress transcription by binding to transcription factors of DNA

Cleidocranial Dysplasia (CCD)

autosomal dominant trait, caused by mutation in human runt gene, RUNX2 affected heterozygotes have a number of skeletal defects, including a hole in top of skull where infant fontanel fails to close; collar bones don't develop or only form small stumps = individuals w/ CCD can fold their shoulders across their chests

disadvantages to using bacteria

bacteria are unable to process and correctly modify many eukaryotic proteins = cannot add carbohydrates or phosphorylate like eukaryotic organisms solution: many biopharmaceuticals are now produced in eukaryotic hosts -bioreactors/biofactories: living factories, such as a herd of goats or cows -baculovirus: gene delivery system in which a virus is used to infect insect cells

Benign vs. Malignant

benign tumors: result from unregulated cell growth that forms a multicellular mass, removed by surgery, causing no serious harm malignant tumors: result from metastasized cells invading other tissue and causing life-threatening problems

genes act as switches

binary switch genes: 2 alternative developmental fates for cell, initiate complete development or organ or tissue type gene-regulatory networks: binary switch genes in conjunction w/ signaling pathways

Gal80

binds Gal4 when galactose is absent = no transcription

Gal 3

binds to Gal80 when galactose is present TO ALLOW TRANSCRIPTION

TFIID

binds to the TATA box to begin formation of PIC

blue-white screening

blue-white selection: used to identify cells containing recombinant and nonrecombinant DNA, plasmid contains lacZ gene, which encodes beta-galactosidase agar plates contain X-gal = analog of lactose, substrate for beta-galactosidase; when X-gal is cleaved by enzyme it turns blue bacterial cells w/ functional lacZ gene carrying a nonrecombinant plasmid = blue bacterial cells w/ recombinant plasmid = white

UV light and ionizing radiation

both UV light and ionizing radiation (X rays and gamma rays) induce DNA damage -UV sunlight can cause skin cancer -radon gas: responsible for up to 50% of ionizing radiation exposure of US population and contributes to lung cancer

methylation CpG and heterochromatic methylation

bulk of methylated CpG dinucleotides are found in repetitive DNA sequences located in heterochromatic regions of the genome including the centromere euchromatin - expressed heterochromatic - not expressed = most methylation Barr body = heterochromatic region heterochromatic methylation- maintains chromosome stability by preventing translocations and other chromosomal abnormalities X chromosomes in mammalian females are inactivated by converting them into heterochromatin (dosage compensation) CpG methylation in euchromatic regions causes a parent-specific pattern of gene transcription -> where find differences between males and females

oncogene

cancer causing gene mutated proto-oncogene has a *gain-of-function* alteration contributes to development of cancer only 1 allele needs to be mutated or misexpressed to contribute to cancer -*confers a dominant cancer phenotype*

chromatin remodeling and histone modification in cancer

cancer cells also show disrupted histone modification profiles mutations in genes encoding members of the histone-modifying proteins histone acetyltransferase (HAT) and histone deacetylase (HDAC) are linked to the development of cancer ex) Rubinstein-Taybi syndrome

mutator phenotype

cancer cells show higher than normal rates of mutation, chromosomal abnormalities, genomic instability mutator phenotype: high level of genomic instability in cancer cells

vectors

carrier DNA molecules can replicate cloned DNA fragments in host cell must be able to replicate independently have several restriction enzyme sites to allow insertion of DNA fragment carry selectable gene marker to distinguish host cells that have taken them up from those that have not

signal transduction

cells in G0 are stimulated to reenter cell cycle by external growth signals signal transduction initiates gene expression that propels cell out of G0 and back into cell cycle cancer cells often have defects in signal transduction pathways

cell cycle

cellular events in sequence from one division to another phases: interphase, G1, S phase, G2, M phase

leucine zipper, helix turn helix, zinc finger

characteristic domains of DNA binding proteins

enhancers regulate transcription of eukaryotes

cis-acting transcription regulatory elements enhancers: located on either side of gene, some distance from gene, or even within gene (are important in reaching maximum level of transcription) insulators: found *between an enhancer and a promoter* for a nontarget gene, allow some enhancer-promoter interactions and block others

silencers regulate transcription of eukaryotic genes

cis-acting transcription regulatory elements silencers: repress the level of transcription initiation

homeotic genes

class of genes that control developmental identity of segments along the anterior-posterior axis

clonal origin of cancer cells

clonal origin: all cancer cells in primary and secondary tumors are clonal clonal: originated from a common ancestral cell that accumulated numerous specific mutations reciprocal translocations and x-inactivation demonstrate that cancer cells are clonal

GAL gene system in yeast

composed of four structural genes and 3 regulatory genes products of structural genes transport galactose into cell for metabolism positive control: activator protein must be present to turn on gene transcription GAL genes are inducible: 1) transcription is regulated by presence or absence of galactose, 2) absence of galactose = GAL structural genes not transcribed, 3) galactose present = transcription begins

computer-automated DNA sequencing

computer-automated high-throughput DNA sequencing: since early 1990s, DNA sequencing has been done through computer-automated Sanger reaction-based technology -generates large amounts of sequence DNA -enabled rapid progress of Human Genome Project large-scale genome sequencing: automated; uses fluorescent dye-labeled dideoxynucleotides (ddNTPs)

A) important function for that sequence

conservation of amino acid sequence among distantly related groups of organisms is suggestive of a(n) A) important function for that sequence B) lack of selective pressure on that sequence C) lack of common ancestor D) intrinsic resistance to mutations in that sequence

genomic library

contains at least one copy of all sequences in genome of interest constructed by cutting genomic DNA w/ restriction enzyme and ligating fragments into vectors

complementary DNA (cDNA) library

contains complementary DNA copies made from mRNAs present in cell population represents genes active transcriptionally at the time cells were collected for mRNA isolation cDNA library is constructed 1) isolating mRNA from cells 2) synthesizing complementary DNA using reverse transcriptase -reverse transcriptase is used to generate cDNA from mRNA, it extends oligo(dT) primer and synthesizes complementary DNA copy of mRNA, produces mRNA-DN = double-stranded hybrid molecule 3) cloning cDNA molecules into vector

histone modification

covalent bonding of functional groups onto N-terminal tails of histone proteins; most common: acetyl, methyl, phosphate (methylation = shutting down, acetylation = activation) -acetylation decreases positive charge, which reduces affinity of histone to DNA = loosens DNA -histone acetylation of nucleosome is catalyzed by histone acetyltransferase enzymes (HATs) - associated w/ increased transcription and acetylation of nucleosome

processing bodies

cytoplasmic structures that will aggregate mRNA molecules for either degradation or later translation

lncRNA mechanisms of action

decoy - binds to proteins so other things can't adapter - helps protein assemble guide - pulls molecular machine together and connects stuff enhancer - connects a sequence far away from the promoter

determination vs differentiation

determination- early commitment of a cell to an eventual developmental fate differentiation- process of becoming fate

tumorigenesis

development of malignant tumor; result of two or more genetic alternations -progressively release cells from normal controls on cell proliferation and malignancy

Dideoxynucleotides (ddNTPs)

dideoxynucleotide chain-termination sequencing (Sanger) most common method of DNA sequencing -dideoxynucleotide: deoxynucleotide w/ a hydrogen at the 3' end instead of an OH -dideoxynucleotide causes DNA synthesis to terminate Sanger sequence = using labeled dNTPs

A) toward an extreme of the phenotype

directional selection generates a phenotypic shift A) toward an extreme of the phenotype B) away from either extreme of the phenotype C) toward both extremes of the phenotype D) toward the mean of the extremes of the phenotype

single-cell RNA sequencing

enables a comparison of the DNA sequence (genes) present in a cell and the relative levels of expression for each transcript by RNA sequencing in that cell

Gal4 protein

encoded by GAL4 gene negatively regulated by Gal80 protein has DNA-binding domain (DBD) that recognizes and binds to sequences in UASG(subscript G, called upstream activation sequences of GAL genes) binding of phosphorylated galactose to Gal80p and/or Gal4p exposes activation domain

cis-acting regulatory elements

enhancers, promoters, insulators, silencers

environmental induction of epigenetic change

environmental agents: nutrition, chemicals, physical factors i.e. temperature can alter gene expression by affecting the epigenetic state of the genome women pregnant during 1944-45 famine in the Netherlands had children w/ increased risk of obesity, diabetes, and coronary heart disease F2 generation also had abnormal patterns of weight gain and growth RATS: reduced protein diet fed to rats during pregnancy results in permanent changes in the expression of several genes in the F1 and F2 offspring - variable expression of yellow phenotype in mice caused by diet-related epigenetic changes in the genome -to evaluate it, the diet of pregnant mice was supplemented w/ methylation precursors (folic acid, vitamin B12, choline) = variation in coat color in the offspring was reduced and shifted toward the pseudoagouti phenotype

histone modification - epigenome alteration

epigenetic mechanism of gene regulation chromatin is composed of DNA wound around an octamer (8 histone) of histone proteins to form nucleosomes amino acids in the N-terminal region of the histones can be covalently modified by acetylation, methylation, and phosphorylation modifications occurs at conserved amino acid sequences in the N-terminal histone tails, which protrude from the nucleosome chemical modification of histones alters the structure of chromatin, making genes accessible or inaccessible for transcription

genetic and epigenetic regulation of development

epigenetic process begins early in embryogenesis w/ global DNA demethylation that erases parental epigenetic marks contributes to establishment of pluripotency by blastula stage, methylation gradually resumes and is associated w/ initial steps in specification and determination of embryonic stem cells (ESCs) and embryonic germ cells (EGCs) ex) SRY gene

chromatin modifications and cancer epigenetics

epigenetics: study of factors that affect gene expression but do not alter nucleotide sequence of DNA epigenetic effects may be present in somatic or germ-line cells examples of epigenetic modifications: DNA methylation, histone acetylation and phosphorylation

restriction map

establishes the number of, order of, and distances between restriction enzyme cleavage sites on cloned segment of DNA provides information on length of cloned insert and location of restriction sites within clone restriction maps: created by cutting DNA w/ different restriction enzymes and separating DNA fragments by gel electrophoresis, which separates fragments by size smallest fragments move farthest down the gel fragments visualized on gel by staining with ethidium bromide and illuminated by UV light

dideoxynucleotides

every time fluorescent dNTPs are added to the end of a sequence, the computer picks it all up and sequences it based on the fluorescent

remodeling of nucleosome core particle

exchange of histone variants change histone = change affinity of histone to DNA = loosens DNA strand from nucleosome to expose it for transcription

metalloproteinase gene MMP1

expressed in breast cancer cells that metastasize to bone MMP1 and MMP2 genes that expressed in cancer spread to the lungs level of aggressiveness correlates positively with levels of proteolytic enzyme activity expressed by tumor

plasmid

extrachromosomal double-stranded DNA molecule replicates independently from chromosomes within bacterial cells plasmids used in DNA cloning: genetically modified bacterial plasmids--first vectors developed engineered to contain a number of convenient restriction sites and a marker gene to select for presence in host cell

imprinting

father and mother have 1 gene, one has it turned off and the other has it turned on SO it depends on the location of the gene as to whether it is expressed or not AND how it is expressed

epigenetic cancer therapy

focus of epigenetic therapy is the reactivation of the genes that have been silenced by methylation or histone modification FDA approved drug (decitabine, marketed as Vidaza) for treatment of acute myeloid leukemia and myelodysplastic syndrome (precursor to leukemia)

great diversity exists in eukaryotic promoters, in structure and function

focused promoters: specific transcription initiation at start site, major type of initiation for lower eukaryotes dispersed promoters: direct initiation from several weak transcriptional start sties; can get many transcripts = many different gene products

northern blot

for RNA used to determine whether a gene is actively being expressed in given cell or tissue used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders

insulator

found between the enhancer and promoter of the target gene preventing their interaction

promoter structure

found in focused promoters made up of DNA sequence elements including: initiator (Inr), TATA box, TFIIB recognition element (BRE), downstream promoter element (DPE), motif ten element (MTE) ^these serve as a platform for assembly of RNA pol II and transcription factors

DNA methylation is responsible for

gene silencing associated w/ parental imprinting, heterochromatin gene expression, X chromosome inactivation histone modifications: disrupted in cancer cells, genes that encode histone acetylases, deacetylases, methyltransferases, and demethylases are often mutated or aberrantly expressed in cancer cells

recombiant DNA technology

gene testing: one of first successful applications of recombinant DNA technology; currently >900 gene tests in use, detect DNA mutations associated w/ SINGLE GENE diseases i.e. sickle-cell anemia... use of gene tests: perform prenatal diagnosis of genetic diseases, identify carriers, predict future development of diseases in adults, confirm diagnosis of disease detected by other methods, identify genetic diseases in embryos created by in vitro fertilization

cytoplasmic polyadenylation (translational regulation)

general info: mRNAs are not always translation immediately but stored for translation at a later time or in response to certain cues i.e. mRNAs in oocyte held until fertilization mechanisms to regulate poly-A tail: - cis-regulatory element in 3' UTR = cytoplasmic polyadenylation element (CPE) - recognized and bound by the cytoplasmic polyadenylation element binding protein (CPEB) - CPEB recruits poly-A specific ribonuclease (PARN) and Maskin

core promoter TFIID

general transcription factor that assists RNA pol II at core promoter TFIID *first step in forming PIC--binding of TFIID to TATA box via TATA binding protein (TBP) fully formed PIC* (whole clump of proteins) mediates unwinding of promoter DNA at start site and transition of RNA pol II from transcription to initiation to elongation

formation of RNA pol II initiation complex

general transcription factors (GTF): proteins, required at promoter to initiate basal or enhanced levels of transcription, assembly of proteins in specific order forms pre-initiation complex (PIC), PIC provides platform for RNA pol II to recognize transcription sites

proto-oncogenes

genes whose products promote cell growth and division -encode transcription factors that stimulate expression of other genes, signal transduction molecules that stimulate cell division, cell-cycle regulators that move cell through cell cycle PROTO-ONCOGENES TO KNOW: c-myc: transcription factor, regulates cell cycle, differentiation, apoptosis -alteration in cancer: translocation, amplification, point mutations -associated cancers: lymphomas, leukemias, lung cancer, many types cyclins: bind to CDKs, regulate cell cycle -gene amplification, overexpression -associated cancers: lung, esophagus, many types

preimplantation genetic diagnosis

genetic analysis of single cells from embryos created by in vitro (in glass) fertilization ASO used for PGD

GMOs

genetically modified organisms; includes transgenic crop plants (combine different species together) ex) alfalfa, corn rice, potatoes... reasons for generating transgenic crops: -improving growth characteristics and yield of culturally valuable crops -increasing nutritional value of crops -providing crop resistance against insect and viral pests, drought, and herbicides

cancer: genetic disease

genomic alterations associated w/ cancer include single-nucleotide substitutions, chromosomal rearrangements, amplifications, and deletions cancer caused by mutations in somatic cells -only 5% of cancers are associated w/ germ-line mutations cancer: genetic disease at somatic level results from mutated gene products or abnormally expressed genes, mutations affect multiple cellular functions, cancer cells share 2 fundamental properties: abnormal cell growth and division (unregulated cell proliferation) and metastatic spread

newborn genetic testing

genomic sequencing and newborn sequencing disorders program: underway to sequence exomes of more than 1500 babies, early screening of PKU and may other crucial genetic disorders may be required by law in the future

driver mutations

give growth advantage to tumor cells the total number that occur in any particular cancer is small--between 2 and 8

hox genes

group of related genes that specify regions of the body plan of an embryo along the head-tail axis of animals

functional domains of transcription factors

have 2 functional domains (clusters of amino acids w/ a specific function) DNA-binding domain: binds to specific DNA sequences in the cis-acting regulatory site trans-activating 1) helix-turn-helix (HTH): present in both eukaryotic and prokaryotic transcription factors 2) zinc-finger: found in wide range of transcription factors that regulate gene expression 3) basic leucine zipper (bZIP): allows for protein--protein dimerization (sometimes need proteins to form dimers) domain: activates or represses transcription by binding to other transcription factors or RNA pol

passenger mutations

have no direct contribution to the cancer phenotype acquired over time possibly from the high levels of DNA damage that occurs in cancer cells

homeobox

hox genes contain a 180-bp domain encodes DNA-binding sequence of 60 amino acids (homeodomain) expression of hox genes is collinear w/ the anterior-to-posterior organization in embryo homeotic genes: regulate development of segments and specific body structures order of genes correlates w/ sequential anterior borders of their expression domains

abnormal regulation of the epigenome leads to...

human genetic disorders (cause by ________________________________) prader-willi syndrome angleman syndrome beckwith-weidemann syndrome *the loss or alteration of other epigenetic states can result in cancer*

clusters of hox genes

humans and most vertebrates have 4 clusters of hox genes: HOXA, HOXB, HOXC, HOXD - containing 39 genes cluster of 2 to 4 hox genes is involved in forming specific structures

epigenetics and cancer

hypomethylation is in all cancers for complex diseases, there are strong links to environmental factors i.e. smoking and lung cancer 1980s- Feinberg and Vogelstein observed colon cancer cells had much lower levels of methylation than normal cells derived from the same tissue other half of picture: promoter and CpG island hypermethylation -> tumor-suppressor genes (turn off) genetic mutations -> tumor-suppressor genes (turn off) OR oncogenes (turn on)

Jim Lewis and Jim Springer

identical twins separated at birth until age 39 but are very similar both suffered from tension headaches, were prone to nail biting, smoked Salem cigarettes, drove same type of car, vacationed at the same beach in Florida

cancer stem cells

identified in leukemia, brain, breast, colon, ovarian, pancreatic, and prostate cancer

e) microRNA (miRNA) and small interfering RNA (siRNA)

if the protein Dicer is not functioning, then which kind of posttranscriptional gene regulation is most likely affected? A) alternative splicing B) alternative adenylation C) only microRNA (miRNA), but not small interfering RNA (siRNA) D) small interfering RNA (siRNA) E) microRNA (miRNA) and small interfering (siRNA)

C) the temperature is raised so that taq polymerase can extend the primers

immediately after the primers have annealed to the target sequence ______ A) the temperature is lowered so that taq polymerase can extend the primers B) the temperature is raised to cause denaturation C) the temperature is raised so that taq polymerase can extend the primers D) the annealing temperature is maintained until polymerase has finished extension of the new strands

epigenetics - imprinting

imprinted genes show expression of only the maternal or paternal allele this parent-specific pattern of allele expression occurs during gamete formation differential methylation of CpG-rich regions produces allele-specific imprinting and subsequent gene silencing one gene methylated and imprinted, it remains transcriptionally silence during embryogenesis and development most imprinted genes direct aspects of growth during prenatal development - in mice, genes on X chromosome are expressed in the placenta, but genes on the paternal X chromosome are silenced

imprinting - mammals

imprinting in mammals is reprogrammed every generation when gamete formation begins in female/male germ cells, both chromosome sets have their imprints erased and are each reprogrammed by changing the pattern of methylation to carry female/male imprint that is transmitted to the next generation through the egg/sperm reprogramming occurs in the parental germ line and in the developing embryo just before implantation after implantation, differential genomic remethylation recalibrates which maternal and paternal alleles will be inactivated

condition knockout

in certain place (not knocked out everywhere) and at a certain time

methylation

in mammals, *methylation of DNA takes place after replication and during differentiation of adult cells* -> enzymes methylate certain things it involves the addition of a methyl group catalyzed by methyltransferase enzymes addition of a methyl group (-CH3) to cytosine on the 5-carbon of the cytosine nitrogenous base results in 5-methylcytosine (5mC) reaction catalyzed by a family of enzymes called DNA methyltransferases (DNMTs) occurs on cytosine bases adjacent to guanine called CpG dinucleotides, clustered = CpG islands -> are located and near promoter sequences adjacent to genes = essential genes (housekeeping genes) and cell-specific genes are unmethylated and available for transcription other genes are methylated and transcriptionally silenced

HATS

increase transcription rate, responsible for acetylation of nucleosome

coactivators

interact w/ proteins and enable activators to make contact with promoter-bound factors coactivators form complex "enhanceosome" -proteins that need to be around to activate something else

enhanceosome

interacts with transcription complex repressor proteins at silencer elements decrease rate of PIC assembly and RNA pol II release -referring to body/machine that interacts w/ promoter sequences (generic term)

interphase

interval between mitotic divisions cell grows and replicates its DNA (G1, S, G2) cells that stop proliferating enter G0: do not grow or divide but are metabolically active (neurons), cancer cells are unable to enter G0 and cycle continuously

fluorescent in situ hybridization (FISH)

involves hybridizing probe directly to chromosome or RNA w/o blotting carried out w/ isolated chromosomes on slide or in situ in tissue sections or entire organisms helpful when embryos are used for various studies in developmental genetics *tag specific fragments of the genome*

chromatin remodeling

involves repositioning or removal of nucleosomes on DNA repositioned nucleosomes make chromosome regions accessible to: transcription regulatory proteins, transcription activators, RNA pol II

chronic myelogenous leukemia (CML)

involves translocation of C-ABL gene on chromosome 9 into BCR gene on chromosome 22 structure = philadelphia chromosome

epigenetics

is the study of the ways in which these changes alter cell- and tissue-specific patterns of gene expression implicated in: progressive restriction of gene expression during development, allele-specific expression in gene imprinting, environment genome interactions during prenatal development that affect adult phenotypes

lncRNA linc-MD1 pathway

lncRNA regulates muscle differentiation by sequestering miRNAs - in DMD, linc-MD1 expression is low --miR-133 and miR-135 downregulate mRNAs for TFs and MEF2C and MAML1 when linc-MD1 is introduced via viral vector, miRNAs are bound, MEF2c and MAML1 RNAs are upregulated = increased muscle differentiation --*increase linc-MD1 = transcription factors unregulated = transcription*

cis-acting sequence

located on same chromosome as gene that it regulates required for accurate regulated transcription of genes: promoters, enhancers, silencers

promoters contain proximal-promoter elements

located upstream of TATA and BRE motis enhance levels of basal transcription ex: CAAT and GC boxes shows transcription levels when proximal promoter elements are mutated

lncRNA

long noncoding RNA (lncRNA) like mRNAs w/ 5' caps, 3' poly-A tails, splicing of introns 4 classes of lncRNA unlike mRNA because lack frame that codes for insertion of amino acids into a polypeptide lncRNA binds to chromatin-modifying enzymes - affects chromatin modification and gene expression loci: 1) antisense - go backwards 2) intronic - affect introns 3) bidirectional - affect 2 genes in 2 different directions 4) intergenic - can function between genes

D) to eliminate mRNAs with a premature stop codon

mRNA surveillance mechanisms are necessary A) in determining which alternative splice site should be used B) to measure the concentration of mRNAs in the cytoplasm C) for polyadenylation at the 3' end of mRNA D) to eliminate mRNAs with a premature stop codon E) to recruit general transcription factors to the promoter

cherry blossom mice

male mice conditioned to fear the smell of cherry blossoms (got electric shock) had pups... when smelled cherry blossoms, they became more jumpy and nervous than pups whose fathers hadn't been conditioned to fear them grand-pups also showed heightened sensitivity to cherry blossoms --linked back to epigenetic modifications in their sperm DNA, chemical markers on their DNA were found on a gene encoding a smell receptor

gene targeting

manipulates specific gene in genome to learn its function

cancer stem cell hypothesis

many scientists believe most cells within tumors do not proliferate cancer stem cell hypothesis: tumor cells that do proliferate give rise to cancer stem cells that have capacity for self-renewal stem cells: undifferentiated cells w/ capacity for self-renewal *hypothesis predicts that every cell in tumor has potential to form new tumor*

in drosophila, hox genes regulated by

maternal effect genes: from egg, set up head-tail polarity gap genes: define large, multi-segment regions of the fly pair-rule genes: turned on by interactions between gap genes

posttranscriptional regulation

more significant than transcriptional control mechanisms: 1) control of alternative splicing 2) mRNA stability 3) translation 4) RNA silencing

galactose

must be present for transcription of GAL genes to occur

spliceopathies

mutations that affect regulation of splicing and contribute to several genetic disorders - myotonic dystrophy - spinomuscular atrophy (SMA)

beckwith wiedmann syndrome imprinting

normal human chromosome 11 = imprinting control region (ICR) on paternal chrom is methylated, IGF2 is actice, H19 is silent maternal chrom ICR is not methylated = IGF2 silent, H19 open in Beckwith Wiedmann Syndrome = H19 silenced in both and IGF2 active in both (maternal and paternal) symptoms: prenatal overgrowth, abdominal wall defects, enlarged organs, high birth weight, predisposition to cancer

PCR picture

not shown: the amplification cycles

promoter

nucleotide sequence that serves as recognition sites for transcription machinery

promoters (core and proximal)

nucleotide sequences that serve as recognition sites for transcription machinery located immediately adjacent to regulatory genes critical for transcription initiation *core promoter: determines accurate initiation of transcription* *proximal-promoter elements: modulate efficiency of basal levels of transcription* ^are sequences, recognition sites that attract molecular machinery to them

isoforms

number of proteins made from 1 gene

methyl groups

occupy the major groove of DNA and silence genes by blocking the binding of transcription factors and other proteins necessary to form transcription complexes

x chromosome inactivation

occurs in early development at random all cancer cells within a tumor, both primary and metastatic, within one female individual contain the same inactivated X chromosome

metastasis

once cancer cells have disengaged, they enter the blood or lymphatic system 0.01% of metastatic cells become metastatic tumors metastasis is controlled by a large number of gene products proteolytic enzymes: ex) metalloproteinases present at higher than normal levels in highly malignant tumors not susceptible to normal controls conferred by regulatory molecules such as tissue inhibitors of metalloproteinases (TIMPs)

monoallelic expression (MAE)

only one allele is transcribed while the other is transcriptionally silent 3 classes of MAE: 1) parent-of-origin monoallelic expression: imprinting 2) random monoallelic expression: inactivation of the X chromosome 3) random monoallelic expression of autosomal genes

direct-to-consumer (DTC) genetic tests

over 1900 diseases can be tested for saliva sample or cheek swab is mailed to company for testing tests are not FDA regulated; questions about accuracy arise *intellectual property (IP)* rights: debated as aspect of ethical implications of genetic engineering, patents on intellectual property (isolated genes, new gene constructs, recombinant cell types, GMOs) are lucrative for patent holders, but may also pose ethical and scientific problems

p53 and cell cycle

p53 can stop cell cycle at several phases -cells lacking p53 unable to stop at cell-cycle checkpoints or enter apoptosis -cellular stress events increase p53 levels: DNA damage, double-stranded breaks in DNA, presence of DNA-repair intermediates due to UV light

translational and posttranslational regulation

p53 protein - p53 levels increase if cell suffers DNA damage or metabolic stress - p53 is a transcription factor that induces transcription of Mdm2 gene (ubiquitin ligase, blocks transcription) -ubiquitin tags proteins for degradation by enzymes

palindrome

palindrome - symmetry exhibited by recognition sequences, nucleotide sequence reads same on both strands restriction enzymes cut DNA in characteristic cleavage pattern sticky ends (cohesive ends): fragments produced with overhangs blunt ends: fragments produced with double-stranded ends

posttranslational modification- phosphorylation

phosphorylation = most common type of posttranslational modification -kinases catalyze the addition of a phosphate group to a serine, tyrosine, or threonine amino acid side chains -phosphates are enzymes that remove phosphates phosphorylation usually induces conformational changes and therefore functional changes

imprinted genes

play major roles in controlling growth during embryonic and prenatal development external or internal factors that disturb the epigenetic pattern of imprinting or the expression of imprinted genes can have serious phenotypic consequences -in vitro fertilization (IVF) in humans can cause problems with imprinted genes --children born after IVF and other ART (assisted reproductive technologies) are at risk of very low birth weight, 4-9x risk beckwith-wiedemann syndrome, increased risk for prader-willi and angelman syndrome

alternative splicing

posttranscriptional regulation: how exons are spliced determines what protein is made - generates different forms of mRNA from identical pre-mRNA -> increases # of proteins - expression of one gene gives rise to many proteins w/ similar and different functions increases # isoforms- # of proteins made from 1 gene types: 1) cassette exons 2) alternative splice site 3) intron retention 4) mutually exclusive exons 5) alternative promoters 6) alternative polyadenylation number of proteins cell can make, or proteome, is not directly related to the # of genes in genome at least 2/3 of protein-coding genes in humans undergo alternative splicing

RNA sequencing

powerful tool for transcriptome-wide analysis of genes expressed by cells within a population allows researchers to differentiate genetic variations between cells

common cancer treatments

precision medicine: new future - customized therapy based on YOUR factors chemotherapy: chemo delivered through IV to wipe out cancer cells... healthy cells affected too surgery: cut cancer out immunotherapy: activates your immune system to help recognize and kill cancer cells... only effective for certain types of cancer

miRNA pathway

primary miRNAs (pri-mRNAs) transcribed and processed have hairpin structures 1) drosha removes noncomplementary 5' and 3' ends to make pre-miRNA 2) hairpins to cytoplasm cleaved by Dicer - processed by RISC 3) target complementary sequences on mRNAs (serve as binding sites for miRNA response elements (MREs)) - if perfect match = degraded - if partial match - translation inhibited

bipharming

production of proteins in genetically modified plants

product of hMTIIA (human metallothionein 2A (MT2A))

protein that binds heavy metals and protects cells from toxic effects protects cells from oxidative stress expressed in low levels in all cells transcribed at high levels when exposed to heavy metals under stress, the body releases glucocorticoids = activate and interact with the gene that can take care of the problem

cyclin genes

proto-oncogene function: binds to CDKs, regulate cell cycle alteration in cancer: gene amplification, overexpression associated cancers: lung, esophagus, many types

c-myc gene

proto-oncogene normal function: transcription factor, regulates cell cycle, differentiation, apoptosis alteration in cancer: translocation, amplification, point mutation associated cancers: lymphomas, leukemias, lung, many types

polymerase chain reaction (PCR)

rapid method of DNA cloning that eliminates the need to use host cells for cloning copies specific DNA sequence via in vitro reactions; can amplify target DNA sequences present in very small quantities PCR requires 2 primers: primers- short, single-stranded sequences, one complementary to 5' end and another complementary to 3' end dsDNA to be cloned is put in tube w/ DNA pol, Mg2+, and dNTPs primers anneal to denature DNA complementary strands are synthesized by heat-stable DNA pol

ras proto-oncogene

ras genes = proto-oncogne some of most frequently mutated genes in human tumors encode signal transduction molecules associated w/ cell membrane regulate cell growth and division

recombinant DNA

recombinant DNA - joining of DNA molecules, produced by artificially joining DNA from different biological sources not found together in nature clones: recovered copies of recombinant DNA molecule - used to study structure and orientation of DNA recombinant DNA technology is used to isolate, replicate, and analyze genes

ethics of recombinant DNA and genomic technologies

recombinant DNA and genomic technologies: identify genes, diagnose and treat genetic disorders, produce commercial and pharmaceutical products, solve crimes applications of knowledge raise ethical, social, and legal issues: 2007 = Genetic Information Nondiscrimination Act - designed to inhibit improper use of genetic information in health insurance and employment

biopharmaceuticals

recombinant proteins; therapeutic proteins that treat disease

epigenome

refers to the epigenetic state of a cell

signaling pathways in development

regulate development act both independently and in coordinated networks send and receive developmental signals and elicit specific transcriptional responses ex) notch signaling pathway

CDKs: cyclin-dependent kinases

regulation of cell-cycle progress is mediated by cyclins and cyclin-dependent kinases (CDKs) regulate synthesis and destruction of cyclin proteins

SWI/SNF

remodels histones

SWI/SNF remodeling complexes remodel histones

remodels histones; loosens attachment between histone and DNA loosens DNA strand from nucleosome core causes reorganization of internal nucleosome component

DNA libraries

represent a collection of cloned DNA two main types: genomic and complementary (cDNA)

silencer

represses the level of transcription

restriction enzymes

restriction enzymes produced by bacteria as a defense mechanism against bacteriophage DNA-cutting enzymes bind to DNA at specific recognition sequence (restriction site) and cleaves DNA to produce restriction fragments enzyme cleaves both strands of DNA (digestion)

RT-PCR and qPCR

reverse transcriptase PCR (RT-PCR): methodology for studying gene expression (mRNA production by cells or tissues) reverse transcriptase is used to generate ds-cDNA quantitative real-time PCR (qPCR): real-time PCR allows researchers to quantify amplification reactions as they occur in real time mRNA to DNA to amplification big computer systems, fluorescence used

southern blot picture

run gel put together system - gel binding membrane, wet and dry things add probe to layers to hybridize layers that are complementary to it = they will GLOWWW

runt homolog gene (in mice)

runt gene = Cbfa1 1 mutant copy = skeletal abnormalities as seen w/ humans 2 mutant copies = complete absence of bone formation, skeletons containing only cartilage runt gene important in controlling initiation of bone formation

organization and patterns of expression in Hox genes

see the arrangement and how it fits spatially to the organism

selective breeding

selection and breeding of naturally occurring or mutagen-induced variants technique utilized by farmers for generations manipulates genetic makeup of plan and animal to enhance food production ex) selective breeding in corn

RNA interference

sequence specific posttranscriptional regulation short RNA molecules regulate gene expression in cytoplasm of plants, animals, and fungi; repress translation and trigger mRNA degradation known as RNA-induced gene silencing

enhancer

sequence that allows for maximum transcription

siRNA pathway

siRNAs are derived from dsRNA 1) cleaved into 22 nucleotide long ds siRNAs by Dicer 2) siRNAs associate w/ RISC (contains argonaute) which binds RNA and has silencer activity 3) RISC "cleaves and evicts" 1 of 2 strands = keeps others as a guide to recruit RISC to a complementary RNA 4) RISC cleaves mRNA in the middle of region of siRNA-mRNA complementarity 5) leftover "parts" degraded by exoribonucleases

miRNA

small, noncoding RNA molecules -> epigenetic regulation - miRNAs, siRNAs, piRNAs after transcription, these miRNA (microRNA) molecules associate w/ protein complexes to form RNA-induced silencing complexes (RISCs)

long noncoding RNAs posttranscriptional regulation

some lncRNAs function as competing endogenous RNAs (ceRNAs)- sponges that soak up miRNAs = mitigate the repression of miRNAs by sequestering them a ceRNA is important for the differentiation of muscle cells in mice and humans -undifferentiated muscle cells (myoblasts) express a lncRNA called long intergenic noncoding RNA muscle differentiation 1 (linc-MD1) -linc-MD1 was introduced into cultured myoblasts from DMD (duchenne muscular dystrophy) patients, muscle cell differentiation was partially restored

nucleic acid blotting

southern blot: used to identify which clones in library contain given DNA sequence -> characterize size of fragments from restriction digest northern blot: used to determine whether gene is actively expressed western blot: used for analyzing proteins

regulation of alternative splicing

splicing enhancers and splicing silencers- cis-acting sequences that regulate alternative splicing 2) SR (serine arginine rich) proteins- bind to splicing enhancers and *activate splicing* by recruiting spliceosome components 3) heterogenous nuclear ribonucleoproteins (hnRNPs)- class of proteins that bind splicing silencers and *inhibit splicing*

control of mRNA stability

steady-state level of mRNA, amount of mRNA in cell available for translation, determined by combination of transcription and mRNA degradation rates half-life- mRNA is degraded at some point after synthesis, lifetime varies... regulated by cell need

vaccines

stimulate immune system to produce antibodies against disease-causing organisms 2 TYPES inactivated vaccines: prepared from killed samples of infectious virus or bacteria attenuated vaccines (MOST VACCINES): live viruses or bacteria that can no longer reproduce but can cause mild form of disease subunit vaccines: surface proteins from virus or bacterium developed by genetic engineering hepatitis B virus: causes liver damage and cancer, surface protein cloned into yeast expression vector = purified from yeast = packaged for use as vaccine gardasil: vaccine that provides immunity against human papillomavirus (HPV)

stress-induced behavior is heritable

stress-induced epigenetic changes that occur prenatally or early in life can influence behavior ex) newborn rats raised w/ low levels of maternal nurturing (low-MN) had low glucocorticoid receptor (GR) expression and did nit adapt to stress well as adults BUTTTT newborn rats exposed to high levels of maternal nurturing care early in life (high-MN) had increased GR expression and adapted to stress well as adults the GR expression levels were associated w/ differences in histone acetylation and DNA methylation levels --GR gene promoter: -low-MN rats w/ low expression of GR had significantly higher levels of promoter methylation -high-MN rats w/ high expression of GR had low levels of promoter methylation

ubiquitin-mediated protein degradation

targets a protein for degradation by covalently modifying it w/ ubiquitin (TAG) ubiquitinated proteins are recognized by the proteasome (multi-subunit protein complex) w/ protease (protein cleaving) activity the proteasome unwinds target proteins, removes their ubiquitin tags and breaks the protein into small peptides about 7-8 amino acids long

population

term for the given group of individuals belonging to the same species that live in a defined geographic area that actually or potentially interbreed

A) gene pool

term for the total genetic information carried by all members of a population A) gene pool B) genome C) chromosome complement D) breeding unit E) race

NIH Roadmap Project

the human epigenome project: collects and catalogs data on a set of human epigenomes to serve as reference standards; this atlas allows researchers to perform integrative and comparative analysis of epigenome data across genomic regions or entire genomes a multinational, public/private consortium - identify, map, and establish the functional significance of all DNA methylation patterns in the human genome analysis may show that genetic responses to environmental cues mediated by epigenetic changes are pathways to disease

C) to define the target region and provide a 3' end that can be extended by taq polymerase

the role of primers in PCR is ________ A) the annealing temperature is maintained until polymerase has finished extension of the new strands B) to denature the template DNA and define the target region C) to define the target region and provide a 3' end that can be extended by taq polymerase D) solely to define the target region

histone code

the sum of the complex patterns and interactions of histone modifications that change chromatin organization and gene expression can guess what is methylated

B) transgenic animal

the use of biotechnologies to introduce the antibiotic gene lysostaphin from ~fancy bacteria name~ is an example of generating what type of animal? A) knock-out animal B) transgenic animal C) conditional knock-out D) synthetic animal

gene knockout

to disrupt or eliminate specific gene/genes and see "what happens"

selective breeding use

to enhance growth and yield from domesticated plants and animals

cancer cells metastasize and invade other tissues

to metastasize from primary tumor, cancer cells must digest components of extracellular matrix (EM) and basal lamina (BL) EM and BL normally separate the body's tissues and inhibit migration of cells -cancer in 1 place -cancer moves towards blood vessels and eats into it (NOM) -once get to blood vessel, cells can float away in blood to spread some cancers are more likely to spread than others

environmental carcinogens

tobacco smoke: most significant environmental carcinogen, contains at least 60 mutagenic chemicals, giving smokers a 20-fold increased risk of developing lung cancer red meat and animal fat: associated with colon, prostate, and breast cancer alcohol may cause inflammation and lead to liver cancer natural substances and natural processes are potentially carcinogenic aflatoxin: mold on bread and corn; one of most carcinogenic chemicals known nitrosamines: meat preservative; known to cause cancer naturally occurring and synthetic pesticides and antibiotics can be carcinogenic

totipotent v pluripotent

totipotent- capable of becoming any cell in mature organism pluripotent- capable of becoming any cell EXCEPT placental and embryonic cell types

GAL1 and GAL10 genes

transcription of both genes is positively regulated controlled by central control region, UASG(subG) contains four binding sites for Gal4p UAS is functionally similar to enhancers in eukaryotes -chromatin structure of UAS is constitutively open, or DNase hypersensitive (it's loosely associated w/ nucleosomes) DNase breaks things apart yo

transcription factors

transcription regulatory proteins (physical connectors) target cis-acting sites of genes regulating expression activators increase transcription initiation repressors decrease transcription initiation multiple transcription factors bind to several different enhancer and promoter elements and fine-tune the level of transcription initiation -human metallothionein IIA gene (hMTIIA): example of how a gene can be transcriptionally regulated due to promoters, enhancer elements, & the transcription factors that bind to them

whole-genome transcriptome analysis of pathogens

transcriptome - mRNA, all transcripts coming out -- can take tissue and look at cells to see what transcripts are being made = determines what genes are expressed Informs researchers about genes that are important for pathogen infection and replication Pathogens are used to infect host in vitro Expression microarrays are used to analyze gene expression and host responses to pathogens

DNA cloning with plasmids

transformation treat cells with Ca2+ ions, heat shock causes plasmid DNA to go into cell OR electroporation - apply brief, high-intensity pulse of electricity to move plasmid DNA into cell cut bacterial and plasmid DNA with same restriction enzyme, fragments w/ complementary stick ends are added, annealing, DNA ligase seals phosphodiester bonds

plant produced vaccine proteins

transgenic plants are easily grown and are a source of recombinant proteins - recombinant plant proteins from transgenic plants not yet approved by FDA edible vaccines ~ currently in clinical trials, produced in food plants, decrease production cost, vaccine against cholera produced in genetically engineered potatoes

RB1 gene

tumor-suppressor gene function: cell-cycle checkpoints, binds E2F alteration in cancer: mutation, deletion, inactivation by viral oncogene products associated cancers: retinoblastoma, osteosarcoma, many types

TP53 gene

tumor-suppressor gene function: cell-cycle checkpoints, binds E2F alteration in cancer: mutation, deletion, viruses associated cancers: many types

BRCA1 and BRCA2 genes

tumor-suppressor genes function: DNA repair alteration in cancer: point mutations associated cancers: breast, ovarian, prostate cancers

DNA hypomethylation and hypermethylation - cancer

turns genes on, leading to high transcription of many gene sets including oncogenes hypermethylation at CpG islands and inactivation of certain genes are also found in many cancers - includes tumor-suppressor genes BRCA1 (repair gene)- hypermethylated and inactivated in breast cancer and ovarian cancer -> combination of mutation and hypermethylation occurs in familial forms of cancer ex) CDKN2A mutation in bladder cancer hypermethylated genes include those involved in DNA repair, differentiation, apoptosis, and drug resistance

cassette exons

type of alternative splicing exon may be skipped from mature mRNA joining the 3' end of the upstream exon to the 5' end of the downstream exon

alternative splice site

type of alternative splicing within exon that may be upstream/downstream of normally used splice site (BASICALLY CUTTING IT IN A DIFF PLACE)

RNA-induced gene silencing mechanisms

types of small noncoding RNAs (sncRNAs) = siRNA (small interfering) and microRNAs; are short, double-stranded ribonucleotides *siRNAs*- arise in cell due to viral infection or transposons (jumping genes), produce double-stranded RNA which is recognized and cleaved by Dicer *microRNAs*- noncoding RNAs that negatively regulate gene expression PURPOSE: regulation, rapid change in gene expression

C) inactivated or attenuated viruses

typical composition of a vaccine? A) naked DNA B) RNA from bacteria C) inactivated or attenuated viruses D) genes encoding for virulence factors

cre-lox system

used for knocking out a gene conditionally (in a certain place and at a certain time) - scientists can control when target gene is disrupted cre-lox system promoter for cre designed as tissue-specific (Cre will only inactivate gene in desired tissues) conditional KOs made by inserting sequencing called lox P sites sequences are introduced into germ line

embryonic (ES) stem cells

using ES cells, scientists introduce targeting vectors into cells via electroporation ES cell takes in targeting vector and endogenous enzyme recombinase catalyzes homologous recombination recombinant ED cells are injected into mouse embryo results: chimeras

biotechnology

utilizing living organisms to create a product or process that helps improve life for humans or other organisms

what GWAS tells you

what genes are upregulated or downregulated in a series of samples

D. phosphorylation

what posttranslational modification occurs on the protein CPEB to initiate mRNA translation? A) acetylation B) ubiquitination C) methylation D) phosphorylation E) glycosylation

cell signaling pathways determine cell differentiation

which genes are activated cell lineage and cell to cell interactions define differentiation

D) transformation

which of the following is not a common system for generating knock-out organisms? A) cre-lox B) CRISPR-CAS C) homologous recombination D) transformation

binary switch genes

wild-type allele of binary switch gene programs eye formation instead of antenna mutant allele eyeless: eyes are reduced in size and have irregular facets eyeless gene is part of a seven-gene network - master regulator of eye formation are at the top of the hierarchy of gene action in eye development, other genes are second-level genes that are regulated by the master control genes -complex program w/ genes interconnected via feedback loops = NOT A LINEAR SYSTEM

notch signaling pathway

works via direct cell-cell contact & controls developmental fate of interacting cells notch gene encodes signal receptor (transmembrane protein) embedded in plasma membrane notch signal only works between adjacent cells

Nucleosomes modified via change in composition

*wrap DNA around nucleosomes = get chromosomes* Important step in gene regulation; involves changes to either nucleosome or DNA Histones contain normal histone H2A Variant histones (H2A.Z) affect nucleosome mobility and positioning on DNA Nucleosome position may repress or activate transcription via gene promoter