genetic engineering

genetic engineering

manipulating genes and genomes w many techniques

plasmids

plasmids

small circular dna molecules found in bacterica that replicate separately from the bacterial chromosome

restriction enzymes

special endonucleases (enzymes) that cut restriction sites and dna to make restriction fragments

restriction sites

strands of dna at specific locations

restriction fragments

single-stranded end w at least 1 sticky end

recombinant dna

artificially made dna using dna from different sources
ex. human gene inserted into e coli

dna ligase

joins dna fragments

cloning vector

carries dna sequence to be cloned into the cell
ex bacterial plasmid

reverse transcriptase

makes complementary dna (cdna) start w small segments of rna and code for dna w dna nucleotides

transgenetic plants and animal for human use

animals produce human protein secreted in milk for medical use

humans can't ____ in plants and plants can't be stopped from being _

digest plats in plants (allergies), plants can't be stopped from being eatened by bugs, they have to be cooked before eating

dna cloning purpose

make mult copies of a dna and protein products

gene cloning process (recombinant dna)
step 1

extract plasmid dna from bacteria cell

step 2

cut the plasmid dna (gene of interest) using restriction enzymes

step 3

use ligase to seal plasmid and gene of interest

step 4

transform bacteria cell w recombined plasmid

step 5

reproduction in the bacterial cell clones gene of interest

gel electrophoresis

separates dna strands by size/charge to make pure sample of specific gene

process of gel electrophoresis

dna cut up w restiction enzyme then put in gel's lttle pits called "wells"

step 1 add _____ to the ______

add buffer to the machine
liquid that covers gel that conducts electricity so it doesn't dry out

step 2 put the _____ in the _____

put the gel in the machine

step 3 apply _________ to separate ________

apply electrical current to separate dna fragments
dna has a negative charge and will move to positive end

step 4 _____ separates ______ by _____

agrase gel separates fragments by size

bands

dna fragments of similar size clump tgt

_______ fragments can move _____ so they travel ______
_______ fragments can't move _____ so they travel ______

smaller fragments can move easier

polymerase chain reaction (pcr)

makes many copies of a specific target segment of dna

3 step cycle

denaturation
annealing
extension

denaturation

heat dna to separate strand

annealing

cools to form primers and hydrogen bonds w target sequence

extension

dna polymerase adds nucleotides to 3' end of each primer

crispr gene editing

cuts out specific genes to study gene function

2 part complex

cas 9 and guide rna

cas 9

dna cutting enzyme

guide rna

genetically engineered rna sequences that comes from pre-existing dna of known gene

process of guiding dna

complex moves along dna for a match (remove/cut

advantages of guiding dna

cure disease and improve medicine

disadvantages of guiding dna

not used it on human genetics which could change lives of humans with defects

differential gene expression for regulation

only 20% of genes are expressed in typical human cell

_____ cell types (w _______ genomes) turn on ______ genes to ______which cause _______

different cell types (w identical genomes) turn on different genes to carry out specific functions which cause different cell types

________ regulated at ________

eukaryotic gene expression regulated at different stages

chromatin structure

tightly bound dna w less genes accessible for transcription

dna methylation

methyl groups added to dna makes tightly packed and decreases transcription

histone acetylation

acetyle groups added to positively charged lysines in the histone tails to make dna more loose and increases transcription

epigenetic inheritance

chromatin modifications can be passed on or be reversed (de-methylation of dna) unlike dna mutations
mexplains difference between identical twins

ex of epigenetic inheritance

genomic imprinting

transcription intitiation control elements

noncoding dna segments regulating transcription and bind transcription factors to start transcription
enhances gene expression

transcription factors

general proteins that start transcription by binding w other proteins in promoter or tata box

specific transcription factor

proximal control elements located close to the promoter

enhancers

distal control elements that may be far away from a gene or located in an intron

activators

protein binding to an enhancer
to start gene transcription
bound activators make mediator proteins interact w promoter proteins

combinatorial control for gene activation

combination of control elements activate transcription only when w appropriate activator proteins
ex. liver and lens cells

post transcriptional regulation rna processing
alternative rna splicing

different mrna molecules are produced from same primary transcription
depending on which rna segments are treated as exons or introns

rna degregation

lifespan of mrna molecules in the cytoplasm determines protein synthesis
eukaryotic mrna lives longer than prokaryotic

initiation of translation

selected mrnas can be blocked by regulatory proteins binding to mRNA sequences
translation initiation factors are activated in an egg after fertilization

protein processing and degregation

after translation

noncoding rnas play mult roles in controlling gene expression

only a small fraction of dna codes for proteins

_______control _____

noncoding rnas control gene expression

________fraction of dna codes for __
_ fraction of dna transcribed into _

small fraction of dna codes for proteins (rrna,trna)

big fraction of dna transcribed into noncoding rnas

regulate _ at 2 points:

noncoding rnas regulate gene expression at 2 points: mrna translation, chromatin configurations

expression of single genes in situ hybridization

uses labeled complementary dna

complementary dna cdna

made by reverse transcription of the mrna and represents only part of the genome/subset of genes transcribed into mrna

another name for reverse transcriptase

polymerase cain rx rt-pcr

application of reverse transcriptase

compares gene expression
in diff embryonic stages

expression of mult genes

microarrays

embryotic development of an organism

zygote to organism

3 parts of embryotic development of an organism

cell division
cell differentiation
morphogenesis

cell division

large # identical cells thru mitosis

cell differentiation

cells become specialized in structure function

morphogenesis

"creation of form" or organism's shape

determination

irreversible series of events leads to cell differentiation

cytoplasmic determinants

maternal substances in egg distributed unevenly in early cells of embryo
dividing zygote causes different cytoplasmic determinants and gene expression

induction

cells triggered to differentiate
cell-cell signals are molecules produced by 1 cell influ neighboring cells
ex. growth factors

cloning organisms

produces 1+ organisms genetically identical to the "parent" that donated single cell
ex. carrot cell

cloning plants totipotent cell

cells that give rise to differentiated cell and generate a new organism

single differentiated cells from root

incubated in culture medium and able to grow into complete adult plants

shows that differentiation is

not necessarily irreversible in plants like it is in animals

cloning animals nuclear transplantation

nucleus of unfertilized egg cell or zygote is removed and replaced w nucleus of differentiated cell

experimetns w frog embryos

transplanted nucleus often support normal development of egg

the _____ the _______, the _____ the ____ of normally developing ________

the older the donor nucleus, the lower the percentage of normally developing tadpoles

problems w animal cloning

only small percentage of cloned embryos developed normally to birth
and need reversal of epigenetic changes
ex. acetylation and methylation of dna
w/o it genes can't be expressed correclty to develop

stem cell

unspecialized cell w all genes "turned on"
differentiate into specialized cells of one or more types

embryonic stem cells

isolated from embryos at the blastocyst stage are able to differentiate into all cell types

adult body stem cells

replace nonreproducing specialized cells

the aim of stem cell research is supply cells for the repair of damaged or diseased organ

reproductive cloning of human embryos is unethical
therapeutic cloning produces embryonic stem cells that could lead to medical benefits

proto oncogene

starts cell division

tumor supression gene

inhibits cell div

_____ in this gene that _____ protein prod may contribute to ______

mutations in this gene that decrease protein prod may contribute to cancer

translocation,if it ends up near an active promoter

transcription may increase

amplification of a proto-oncogene

increases the number of copies of the gene

point mutations

causes an increase in gene expression can create a hyperactive protein

tumor supressor genes

help prevent uncontrolled cell growth

tumor supressor proteins

repair damaged dna
control cell adhension
inhibit cell cycle in cell signaling pathway

_____ in this gene that _____ protein prod may contribute to ______

mutations in this gene that decrease protein prod may contribute to cancer

ras gene

proto-oncogene for cell cycle

_____ can lead to prod of _____________ and _______ cell div and occur in _________

mutations can lead to prod of hyperactive ras protein and increase cell div and occur in 30% of cancers

p53 gene

tumor suppressor gene