Exam 4 Content

key steps of gene expression

transcription: synthesis of complementary RNA strand, processing: modifications made to RNA in nucleus of eukaryotic cells, translation: creating polypeptide chains based on the processed RNA molecule

gene regulation in the promoter

mutations to the promoter, enhancer, or other proteins that bind to the promoter/enhancer could affect the ability of RNA polymerase to bind to the DNA, altering transcription levels

gene regulation in RNA processing

alternative splicing, mutations to splice donor/acceptor sites, etc. could affect translation of the RNA

gene regulation in enhancers

mutations in the enhancer regions could affect binding affinity with transcription factors which could cause more or less transcription

epigenetics

an area of study focusing on changes in gene expression due to factors that are mitotically/meiotically heritable, NOT focusing on changes to the DNA sequence

epigenetic phenomena

heritable, self-perpetuating changes in gene expression that are not a result of changes to the DNA sequence, typically due to modified cytosine residues, modified histone tails in chromatin, and sRNAs

histone molecules

play an important role in the formation of nucleosome structures in eukaryotic chromatin, the tails that extend from the nucleosome can be modified to change gene expression

acetylation

makes chromatin more accessible to promote gene expression, histone acetyltransferase adds positively charged acetyl groups to histone tails to prevent close packing of nucleosomes and loosen the chromatin, the process is reversed with histone deacetylase

methylation

makes chromatin more inaccessible, histone methyltransferase adds partially positive methyl groups to histone tails, effects can vary depending on the amino acid being modified, adding a methyl group to H3 lysine 9 favors the formation of heterochromatin (packs nucleosomes closer together), the process is reversed by histone demethylase

increased methylation of histone H3

PRE (polycomb response element) is a cis-acting DNA sequence that can bind to repressors to recruit the PRC2 co-repressor complex, PRC2 has a histone methyltransferase that methylates lysine 27 in the histone tail of H3, the H3 lysine 27 marks propagates to adjacent nucleosomes to repress the Hox gene promoter

partial inheritance of histone marks

with PRE the methylated H3 lysine 27 and the closed chromatin get recapitulated at the Hox gene in all descendent cells, without PRE the marks are still self-perpetuating but do not maintain repression indefinitely

DNA methylation

the process of a methyl group being added to the cytosine base in a 5’ CpG 3’ dinucleotide by DNA methyl transferase (DNMTs)

5’ CpG 3’ dinucleotide

a molecule made of a cytosine nucleotide and a guanine nucleotide that are linked together with a phosphodiester bond

CpG island

a cis-acting transcriptional regulatory element that is rich in CpG dinucleotides found upstream in some eukaryotic genes, its activity depends on the amount of methylation done to the cytosine

nonmethylated CpG islands

can induce gene expression by binding to transcriptional activators and recruiting RNA polymerase

methylated CpG islands

decreases transcription by binding to methyl CpG binding proteins (MeCP repressors) and closing the chromatin structure

constant methylation states

DNA methylation is maintained throughout cell division, special DNMTs at the replication forks methylates the newly synthesized DNA strands in DNA replication, some genes pass on the methylation states to the next generation

genomic imprinting

a phenomenon where an allele’s expression depends on which parent transmits it, imprinting means an allele is silenced, paternal imprinting causes the paternal allele to not be transcribed and maternal imprinting causes the maternal allele to not be transcribed

insulators

regulatory elements that isolate the right and left sides of DNA, create independent domains of transcriptional activity in eukaryotic genomes, have anti-enhancer activity that blocks communication between the enhancer and promoter, also have anti-silencer activity that prevents the spread of repressive chromatin

insulation of Igfr2 gene

the imprinting control region (ICR) acts as an insulator, the nonmethylated maternal insulator is functional and can bind to CTCF to silence transcription, the nonmethylated paternal insulator is nonfunctional so transcription is active

DNA methylation in non-coding RNA regions

ICR in the Igfr2 gene includes a noncoding RNA (ncRNA) whose transcription is regulated by CpG islands, nonmethylated paternal CpG islands allow for Air ncRNA which silences Igfr2 transcription, methylated maternal CpG islands prevents Air production which allows Igfr2 transcription to be active

transgenerational inheritance of agouti alleles

mutations in the agouti genes of mice can cause an Avy allele that can be methylated differently to create different fur phenotypes of various shades of yellow, also causes excessive hunger from the agouti protein in mice brains

retrotransposon

a transposable element that undergoes transposition from one location to another in the genome by forming an intermediate RNA transcript that is used to create of a copy of the DNA of the transposable element through reverse transcriptase, the newly synthesized DNA is then inserted to the new genome location

retrotransposons in the agouti gene

Avy alleles occur due to a retrotransposon with a promoter region and CpG island being jumped upstream the agouti gene, an unmethylated CpG island causes high transcription from the retrotransposon promoter which causes a yellow coat, a methylated CpG island causes regular transcription from the normal agouti promoter which causes a gray coat

inheritance status of the Avy allele

color mostly represented by the methylation state of the mother, variable fur color indicates instability of methylation in that area

ATAC-seq (transposase-accessible chromatin sequencing)

method to assess open or closed DNA, chromatin isolated from a specific tissue, DNA is fragment with Tn5 transposase that simutaneously adds adapters, sequencing by synthesis and bioinformatics are used to identify the closed and open DNA

effect of cellular metabolism on epigenetics

metabolites taken up by cells can passively/actively diffused through the plasma/nuclear membrane to modify chromatin, metabolic enzymes can process metabolites and converted to substrates or cofactors for chromatin-remodelling enzymes

genetic screens

the process of manipulating genomes to determine the function of a specific gene

forward genetic screen

starting with a well established phenotype to determine the unknown genotype, after phenotype is established, DNA is randomly mutagenized, hundreds to thousands of individuals are screened for phenotypic differences, complementation analysis, gene mapping and cloning to study the mutated gene

reverse genetic screen

start with a gene of interest to determine the phenotype, manipulate the gene in various ways to test for the phenotype

transgene

an experimentally constructed piece of DNA integrated into the genome of a recipient organism

transgenic organism

an organism that contains the transgene in their genome, the transgene can come from an individual in the same or different species or can be newly created using recombinant DNA technology

cloned DNA in mouse zygotes

harvest fertilized eggs from female mouse, inject a transgene into one of the pronuclei of the fertilized egg, transgene is randomly integrated in the genome 25-50% of the time, injected mice embryos are born, if the transgene was integrated after cell division began it will only be in some cells, mice are inbred to create stable transgenic lines

mosaic

an organism where a transgene was integrated after cell division such that some cells have the transgene and others don’t

P element

a type of transposable element in drosophila

transformation plasmid

a constructed plasmid that contains the P element and visible marker gene that are surrounded by inverted repeats

helper plasmid

a plasmid that contains the gene for transposase, is injected with the transformation plasmid to transpose the new DNA

transgene integration in drosophila

a transformation plasmid with the P element and another visible marker (with inverted repeats) is injected into fly embryos with a helper plasmid, results in mosaic adult flies with the transgene having been integrated at a random location, the visible markers identify the transgenic flies in later genetic crosses

reporter gene

a gene that can be fused with a specific gene in a transgene such that it is expressed wherever the promoter is activated, can identify where genes are expressed

GFP in the arc-2 gene

the arc-2 transgene has both the A-beta and GFP gene under the arc-2 promoter, wherever arc-2 is expressed, the gene is also labeled with GFP

overexpression studies

a method where genetic constructs that express too much of a gene within an organism or express a gene in a tissue where it is normally not expressed, allows for screening of gene function and gene modifiers

rescue experiments

a method to determine if a mutation is the true cause of a certain phenotype, a cloned wildtype form of the putative gene (transgene) causing the phenotype is combined with the mutant phenotype and injected into an animal, if the combination results in the wildtype phenotype then it can be concluded that the mutation did cause the phenotype change

embryonic stem cells

undifferentiated cells from embryo blastocysts, can grow and divide in culture (in a petri dish), are totipotent (can become ANY type of cell), can be mutagenized in a petri dish for more targeted gene manipulations

homologous recombination with neomycin

new and modified DNA molecule can be cloned to insert at a specific location, homology arms (100-1000 bp with the same sequences as insertion sites) added on both sides for homologous recombination, low efficiency rate so neomycin resistance gene included to screen for the ES cells that integrated the new DNA, usually neomycin resistance gene in an exon which causes a non-functional protein

chimera

a new organism made of two or more different individuals, ~10 mutated ES cells injected into another blastocyst, mutated cells integrate (are totipotent) and contribute to the normal development of the new organism, modified DNA must be in meiocyte-producing cells to pass the mutation to the next generation

conditional knockouts

almost identical process to ES cell path with added loxP sites in parallel orientation, Cre recombinase can recognize and bind to adjacent loxP sites to delete the DNA in between, loxP sites only 34 bp long and are typically in introns so do not have an effect on gene expression

tissue specific expression of Cre recombinase

mouse line with loxP-flanked gene of interest and coding sequence of Cre protein in tissue specific promoter, Cre recombinase brings loxP sites together to delete the sequence inbetween resulting in a mutated gene ONLY in the tissue it is expressed in, mutations are not present in the rest of the animal

meganucleases

similar to restriction enzymes but use longer (18+ bp) sequences to induce double strand breaks and mutate individual genes

zinc finger nucleases and TALEN

cloning of individually linked proteins, new cloning is required for each cut position, develops mutated individual genes

CRISPR/Cas9

uses nuclease with different guide RNAs to induce double strand breaks and develop individual mutated genes

CRISPR repeat sequences

encode CRISPR RNA (crRNA) to prevent self-targeting and to target and destroy foreign DNA

guide RNA (gRNA)

homologous RNA to the target DNA sequence to base pair with it

tracrRNA

can base pair with a segment of gRNA and recruit Cas9 nuclease

Cas9 nuclease

cleaves both strands of the target DNA, induces the double strand break

non-homologous enjoining in CRISPR/Cas9

sgRNA targeting the gene of interest and Cas9 are introduced in a cell, sgRNA invades the target DNA, Cas9 cleaves the target DNA allowing for exonuclease degradation, ends are rejoined which usually created targeted deletions, is an error prone process

homology directed repair (HDR) in CRISPR/Cas9

similar to creating a targeted deletion but also provides an edited DNA template, the DNA template has ~100 bp homologies on both sides to the endogenous sequences for base-pairing to occur, Cas9 and exonuclease digestion create a gap where the edited DNA template is inserted

repression with inactive Cas9

mutations to the Cas9 coding sequence can remove ability to induce double strand breaks (no genome editing), mutated Cas9 can be placed in the promoter region of a gene to block binding of transcription activators or RNA polymerase II, fusing repressor proteins with Cas9 creates stronger blocking of the recruitment of the transcription complex causing increased repression

targeted gene therapy methods

different methods depending on the disease, if caused by loss of function: a wild type copy of the gene is added, if caused by gain of function: a therapeutic gene is added to inactivate the disease gene or the protein product, if is has a complex origin: a gene in the genetic pathway is targeted

viral particle packaging

recombinant/therapeutic DNA has sequences needed for genome modifications and sequences needed for packaging, recombinant DNA transferred to a packaging cell with helper DNA (has capsid protein, reverse transcriptase, and RNA polymerase genes), recombinant DNA transcribed to RNA, RNA packaged into a viral particle, the viral particle has reverse transcriptase to turn the RNA to DNA after infection

pattern formation

a process in development, where spatially organized and specialized cells in the embryo from cell division and differentiation of the fertilized egg

C. elegans model

a nematode that has virtually identical patterns of cell division and differentiation from one individual to the next, hermaphrodites have 959 somatic cells and males have 1031 somatic cells, the developmental history of each somatic cell can be tracked

cell lineages

ancestor-descendant relationships within a group of cells, comparable to a cell pedigree showing each cell division, indicating terminal differentiated states of each cell

autonomous development

from genetically programmed changes within the cells, one of the ways cell fate is determined

positional information

restrictions due to the positions of cells in the embryo

intercellular signaling

interactions between neighboring cells or gradients in concentration of certain molecules, one of the ways cell fate is determined

loss of function

no mRNA is produced or the protein is inactive, are recessive mutation

gain of function

genes are overexpressed or expressed at the wrong time, mutations are dominant

lin-12 gene

a gene in C. elegans that codes for a transmembrane receptor protein important for vulva development

recessive lin-12 mutation

a loss of function mutation that fails to produce the transmembrane signal, causing both cells to differentiate into anchor cells (AC), evidence that lin-12 is necessary for vulva differentiation

dominant lin-12 mutation

a gain of function mutation that causes both cells to produce the transmembrane signal so both will differentiate into vulva, evidence that lin-12 is sufficient to differentiate into vulva

epistatic gene

a gene that masks the phenotype of another gene

hypostatic gene

a gene whose phenotype gets masked by another gene

principle of epistasis

the products of epistatic genes act downstream relative to the products of hypostatic genes in a switch-regulation pathway

switch regulation pathway

a regulatory pathway where a gene or a gene product can be switched “on” and “off” to control the expression of other genes

drosophila model

development is genetically controlled by maternal effect genes, zygotic segmentation genes and homeotic (Hox) genes

maternal effect genes

from the mother in her eggs, establish differences in the embryo before gene expression starts

zygotic segmentation genes

a zygote’s own gene expression starts to subdivide the body into different segments

homeotic genes

genes expressed later in development, assign specific functions to tissue

early development in drosophila

occurs in the egg case, first nine mitotic divisions are rapid and do not divide the cytoplasm, create a cluster of nuclei in the egg (syncytium), some nuclei move to the edge of the embryo, pole cells (future germ line) cellularized at the posterior end, more mitotic divisions occur in the syncytial blastoderm, membranes form around the nuclei to form the cellular blastoderm

embryo polarity by maternal effect genes

in early development there is limited gene expression from the zygote which makes maternally deposited mRNAs essential, mRNA gets deposited at specific locations, bicoid mRNA is mostly at the anterior part where it gets translated, translation of the bicoid mRNA creates a protein gradient with high levels at the anterior to none in the posterior

bicoid protein

represses translation of caudal mRNA, caudal mRNA causes posterior to anterior gradients of caudal protein

nanos protein

represses hunchback mRNA translation, causes anterior to posterior gradient of hunchback proteins

gap genes

first zygotic genes expressed (Kruppel, hunchback, knirps, giant), binding sites in promoter regions have different affinities for bcd, cad, and hb proteins, can encode transcription factors to control expression of others, products control division of the body axis into generalized regions, establish rough body plans for development

pair rule genes

sharply divide the embryo into seven stripes, expression controlled by transcription factors encoded by maternal genes and zygotic gap genes, upstream has multiple binding sites for transcription activation/repression, each stipe has two segments

segment polarity genes

expressed in 14 different sections, activation after cellularization, intra-segmental patterning mostly determined by diffusion of secreted factors between cells, transcription factors encoded by pair rule genes initiate expression of segment polarity genes in each segment, periodicity maintained by interactions between polarity genes

segmentation

occurs in drosophila embryo and larva, defined by successive indentations from sites of muscle attachment in larval cuticle

segment genes

genes that play an important role in establishing the basic body plan and defining segments in embryonic development, include gap genes, pair-rule genes, segment polarity genes and homeotic genes

segment gene mutations

causes segment loss in drosophila, usually lethal

coordinated gene expression

allows for segmented body plans in insects, from segment gene expression

imaginal discs

larval structures leading to organ formation

properties of homeotic (HOX) genes

mutations transform one body segment into another, usually have 1+ copies of a homeobox (characteristic sequence ~180 nt long) that is highly conserved, are transcriptional regulators, can function at various levels in regulatory hierarchy

pax6 gene

important in eye development, mutant phenotype is lack of eyes in adults, homozygous mutations cause eye defects, introducing WT to homozygous recessive flies causes normal fly eye development, turns on eye development pathway in vertebrates, arthropods, cnidarians, cephalopods, and mollusks

mitosis

process of nuclear division such that each daughter cell receives a copy of the DNA of the parent cell, the daughter cells are genetically identical to the parent cell

cytokinesis

the division of the cytoplasm of the parent cell to produce two daughter cell

cell cycle

the stages a cell goes through, under genetic control, is a true cycle (not reversible)

growth factors

proteins that can bind to transmembrane receptors on the outside of cells, initiating a cell respond to initiate cell division

signal transducers

also called second messengers, first response of the cell when a growth factor binds to a receptor, can lead to the activation of the transcription of certain genes, examples include cAMP or Ca2+

Ras protein

a molecule that can be linked to the growth factor receptors, when there is no growth factor binding it is inactive, when a growth factor binds it is activated and induces a kinase cascade, the kinase cascade can initiation transcription of genes for cell proliferation (cell division)

cyclin

a molecule that binds to cyclin-dependent kinases to regulate transitions between stages of the cell cycle

cyclin-dependent kinases

kinases that regulate transition through the cell cycle, are only active when bound to cyclins