Genetics Exam 4

epigenetics

study of phenomena and mechanisms causing chromosome associated heritable changes to gene expression, not dependent on DNA sequence changes

epigenome

specific patterns of epigenetic modifications in the cell at a given period of time

methylome

set of methylated nucleotides in an organism’s genome at a given time, it is tissue and cell specific but not fixed

methylation

addition of methyl groups to cytosine to silence genes by blocking protein binding

DNA methyltransferase

catalyzes methylation after DNA replication and during cell differentiation

CpG islands

where methylation takes place on the cytosine bases adjacent to the guanine bases to create CpG dinucleotides clustered in these island regions in the promoter and upstream sequences; if they are next to essential genes and cell specific genes, they are unmethylated so the genes can be transcribed

heterochromatic methylation

silences transcription, maintains chromatin stability by preventing translocation and abnormalities

royal jelly methylation

suppresses Dnmt3 (dna methyltransferase 3) for the expressions of characteristics of the queen bee

chromatin remodeling

ATP powered protein complexes that move, remove, or alter nucleosomes

histone modifications

covalent post translation modifications of amino acids near the n terminus of histone proteins that extends beyond the nucleosome forming a tail, makes genes accessible or inaccessible to transcription and is passed to daughter cells

writers

proteins that add chemical groups to histones

readers

proteins that interpret modifications

erasers

proteins that remove chemical groups

phosphate chemical modifications

open chromatin structures

methylation

closes chromatin structure

acetylation

opens chromatin structure

histone acetylation

relaxes grip of histone on DNA to make genes more available for transcription, can be reversible, opens DNA configuration

histone code

sum of complex modification patterns and interactions within and between histones, it is reversible as writers and erasers recruit readers to modify structure or regulate transcription

noncoding RNA

transcribed from DNA; impact formation of heterochromatin, histone modifications, site specific dna methylation, gene splicing, and epigenetic regulatory networks

short noncoding RNAs

miRNAs, siRNAs, and piRNAs that repress gene expression

piRNAs (piwi-interacting RNAs)

form RNA protein complexes that epigenetically silence genes in germ and somatic cells

long noncoding RNAs

lack extended open reading frame to prevent insertion of amino acid into polypeptide, have relations to nearby protein coding genes, form RNA-protein complexes with regulators, deliver complexes to specific locations in the genome, participate in remodeling, interact with transcriptions factors, carry out gene regulations

anti-sense lncRNA genes

overlap protein coding genes, transcribed in opposite directions, use their own promoter

intronic lncRNA genes

located within introns and their transcription doesn’t overlap with adjacent exons of protein coding genes

bidirectional lncRNA genes

use the promoter of a protein coding gene but are transcribed in the opposite direction

intergenic lncRNA genes

discrete transcription units outside protein coding genes, independent loci, use their own promoters

decoys

bind to transcriptions factors and prevent them from interacting with target genes

adapters

bring 2 or more proteins together to form a functional complex

guides

target chromatin remodeling enzyme complexes involved in gene silencing to guide the complex to the target gene to silence a specific allele

enhancers

bringing proteins to the upstream regulatory sequence of a gene via looping of the chromosomal region

monoallelic gene expression

1 allele is transcribed and the other transcriptionally silent

genome imprinting

genes are expressed in a parent of origin pattern, certain genes show expression of only the maternal or paternal allele, parentally imprinted genes are marked in male and female germ line cells during gamete formation

random monoallelic expression

random X inactivation in mammalian female cells and the random allele inactivation of autosomal genes

marking

sperm and egg DNA is highly methylated, after fertilization most of the germ line marks are erased to give the embryonic cells a clean epigenetic state to allow for new epigenetic modifications for new cell types, some genomic regions escape demethylation and remethylation

epimutations

resultant dysfunctional epigenetic changes causing mutations in imprinted genes that can impact adjacent imprinted genes

paramutation

an interaction between two alleles leading to a heritable change in expression in one of the alleles

epialleles

genetically identical alleles that produce heritable differences in phenotype through epigenetic processes

MOP1 gene

encodes RNA dependent RNA polymerase that produces siRNA

B-I and B’ structure

B-I has more open conformation, B’ has more closed conformation

random x inactivation in mammalian female cells

1 X chromosome in each cell is randomly inactivated to provide equal expression of X linked genes in males and females

Xist (x inactive specific transcript) gene

encodes long noncoding RNA that coats the X chromosome from which it was transcribed resulting in chromatin structure changes that silence transcription

Tsix gene

transcribed on the active X chromosome, antisense to Xist, produces the Tsix lncRNA that is complementary to Xist lncRNA, Tsix represses Xist expression on active X chromosome

Jpx gene

encodes lncRNA that stimulates the transcription of Xist on the inactive chromosome to transcribed Xist on the inactive chromosome

Xite gene

sustains Tsix expression of the active X chromosome to inhibit Xist and maintain transcription of genes on the active X chromosome

Xist RNA

recruits PCR2 whose proteins produce epigenetic marks that repress transcription

behavioral epigenetics

life experiences especially early in life

epigenetic changes

induced by maternal behavior, epigenetic effects of early stress in humans, in cognition

molecular cloning

large quantities of identical copies of a specific DNA molecule can be produced for research on DNA structure and organization, study of gene expression and protein products, and producing commercial proteins

recombinant DNA

genetic material from multiple sources are joined together to create unique sequences not found in the host genome

Bt gene

bacillus thuringiensis bacterial species that produces the cry protein that is a safe microbial insecticide

restriction enzymes

produced by bacteria as a defense mechanism against bacteriophage infection by restricting or preventing viral infections by degrading virus DNA after binding to specific palindromic restriction sites

digestion

the restriction enzyme cuts both strands of the DNA in the sequence by cleaving the phosphodiester backbone

blunt end

cleavage with no complementary ends, less effective, about 50% are oriented the wrong way, can ligate fragments digested by different sequences or restriction enzymes

sticky ends

complementary ends on a 5’ or 3’ overhang, more effective, able to orientate

complementary DNA (cDNA)

contain DNA copies made from mRNA molecules from cells of tissue samples, represents genes being expressed in cells at the time the library was made, without noncoding regions

genomic DNA (gDNA)

contains all of the DNA, coding and noncoding, in a genome

reverse transcriptase

an enzyme that uses mRNA as a template to make cDNA by forming an mRNA/cDNA duplex; the mRNA is digested with RNAse to produce gaps, the 3’ ends of the mRNA serve as primers for DNA pol I to synthesize a second DNA strand to form ds cDNA that can be closed into vectors

polymerase chain reaction

amplifying a specific gene using a template, primers, polymerase, nucleotides, and a thermal cycler

cloning vector

DNA molecules that accept DNA fragments and replicate them when they are introduced into host cells, they differ in what host they can enter and the size of the DNA fragment inserts; have several restriction sites to allow insertion of DNA fragments, able to replicate in host, contain selectable marker or reporter genes, incorporate specific sequences for sequencing inserted DNA

DNA ligase

used to create phosphodiester bonds to seal nicks in the DNA backbone

transformation

uptake of exogenous DNA by bacterial cell

transfection

forced introduced into the eukaryote chemically, lipid based, or through electroporation

transduction

introduction of exogenous genetic material by a virus

lac Z gene

screens for bacteria containing the recombinant plasmid, if lac Z gene has foreign DNA instead the bacteria are transformed, encodes b-galactosidase

bacteria with the non recombinant plasmid does not disrupt lacZ and produces b-galactosidase that cleaves X-gal and makes the colonies blue

bacteria with a recombinant plasmid disrupts lac Z and does not synthesize b-galactosidase and the colonies remain white

bacteria without a plasmid do not grow because the plate has antibiotic, without the plasmid having an antibiotic resistance gene, the cells can’t grow

gel electrophoresis

load samples into the well, apply electrical current, separate DNA/RNA by size (smaller travels faster), stain gel to visualize bands

probe

DNA or RNA with a base sequence complementary to a sequence within the gene of interest used to locate individual genes/sequences among DNA fragments after electrophoresis

visualizing targets through sandwich

gel placed with specialized paper to bind molecule and a current moves molecules from gel to paper surface, southern blot: DNA, northern blot: RNA, western blot: protein

fluorescence in situ hybridization (FISH)

DNA probes determine chromosomal location to visual a gene’s expression in a cell

quantitative real time PCR

determines amount of DNA amplified as reaction proceeds, housekeeping genes used as baseline internal control for gene expression

taq-probe

probe with complementarity to your specific target with both a fluorophore and quenching molecule

sanger sequencing

uses a special substrate for DNA synthesis; ddNTP (dideoxy) lacks 3’ OH groups that terminates DNA synthesis and polymerase randomly inserts a ddNTP instead of a dNTP; 4 different reactions all with 4 regular dNTPs and a small amount of one ddNTP

modified sanger sequencing

computer automated DNA sequencing where 1 tube has a small amount of all ddNTPS labeled with different dyes that are passed through capillary gel and laser detector

knockout gene

disrupts or deletes part of a gene leading to a loss of function mutation; if changes are observed, then the gene of interest has a role in the phenotype

targeting vector

creates a segment of DNA that can be introduced into embryonic stem cells; can undergo homologous recombination with the target gene using flanking regions to mutate or disrupt the gene of interest

selectable marker gene

put in the insertion sequence and selects cells based off mutations and markers to verify that the target sequence has been disrupted

recombinase enzyme

catalyzes the homologous recombination between the targeting vector and sequence in a small percentage of cells taking up the target vector

chimeras

mice with some cells from the original blastocyst and some from manipulated embryonic stem cells sometimes generated from mice with different coat colors

conditional knockout gene

can induce the knockout in certain circumstances like time in development, specific cell types, and chemical cues

cre-lox system

inserts loxP into targeting vector on either side of gene of interest, little mouse bred with mouse with Cre gene to encode Cre recombinase to cut and recombine DNA at loxP sites and inactivate the gene

transgenic/knock in gene

alters gene sequence to change the gene to express or overexposes a gene of interest by introducing something new

engineered nucleases

enzymes made to cut in unique sequences in the genome leading to dsDNA breaks and repair in hopes of an error prone repair to genera a deletion in gene of interest

transcription activator-like effector nucleases and zinc finger nucleases

cut sequences determined by the amino acid sequence of the nuclease, can be costly

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated genes endonuclease that cuts DNA) genome editing

powerful way to cut and edit the genome by combining a single guide RNA with a nuclease to attach specific DNA sequences to make double stranded cuts at specific locations, non homologous end joining or homology-directed repair repairs the cuts to introduce alterations to the genome

genetic engineering

alteration of an organisms genome using rDNA; manipulating DNA in vitro and then introducing the DNA into living cells to generate new plants, animals, and other organisms (gmos)

biotechnology

using living organisms to creates products or processes to improve life

biopharming

production of valuable proteins in genetically modified plants or animals using recombinant DNA

recombinant protein production in bacteria

a human gene is cloned into a plasmid, a recombinant vector is introduced into the host, large quantities of transformed bacteria are grown, the recombinant protein is recovered and purified

recombinant insulin

first therapeutic proteins produced by recombinant DNA technology to regulate glucose metabolism

fusion protein

hybrid proteins consisting of the amino acid sequence of b-galactosidase attached to the amino acid sequence for A or B insulins subunits, they are extracted from host cells and purified

eukaryotic hosts

overcome improper folding of eukaryotic proteins in bacterial cells to increase yields

biofactories

living factories that could continuously produce desired therapeutic proteins isolated in a noninvasive way

transgenic animals

therapeutic proteins isolated from animal products like milk

human antithrombin

anti-clotting protein produced by farm animals and extracted from the milk; extracted by placing the gene adjacent to a promoter for beta casein targeting antithrombin expression in mammary glands and the protein is abundantly present in animals like goats

inactivated vaccines

prepared from killed samples of an infectious virus or bacteria to produce antibodies against the disease

attenuated vaccine

contains live viruses or bacteria that no longer reproduce but cause mild disease to confer resistance

subunit vaccine

has one or more surface proteins from the virus or bacteria instead of the whole pathogen

DNA vaccines

DNA encoding proteins from a particular pathogen are inserted into plasmid vectors and directly injected into an individual or delivered by a virus; the pathogen proteins encoded by the DNA trigger and immune response to provide protection in case of future exposure

selective breeding

breeding of natural of mutagen induced mutations to produce a desired result

mastitis in cows

infection of mammary glands that blocks milk ducts reducing milk output and contaminating it with pathogenic microbes; transgenic cows produce lysostaphin that cleaves the cell wall of the most common bacterial pathogen allowing the production of a natural antibiotic to fight S. aureus infection, the enzyme is not effective against other pathogens and the pathogen may develop resistance

gene drive

enables a particular gene to be transmitted to a majority of an individual’s offspring to more than just half