Transcript Cloning and Stem Cells

Cloning and Stem Cells
Stem Cells
• Cells that have not yet differentiated into their final
developmental stage and/or function.
• http://www.youtube.com/watch?v=NbjlG84cF1c
• Totipotent: Can be come any type of cell “totally potent”,
plant cells have more of this leading to “cuttings of leaves”
developing into whole plants. The fertilized egg and first 8
cells or so are totipotent. This means that a cell can be
removed from the 8 cell stage to do genetic testing and the
embryo can develop normally as the cells left are totipotent.
• Pluripotent: Can become more than one type of cell “plural
potency”. Stem cells in the bone marrow that will become
blood cells can become more than one kind of white or red
blood cell type.
• http://www.youtube.com/watch?v=rVh1FZcZZVk
Stem Cells characteristics
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Can divide indefinitely
Give rise to more than one mature cell type
Blood, liver, skin, intestinal lining
Can be cultured in petri dishes
Telomeres become worn down over divisions
so embryonic are more useful.
Embryonic vs. Stem cells
• Embryonic stem cells have divided less so there
chromosome ends (telomeres) are longer. This
leads to a much higher success level in culturing
and less likelihood of the stem cell line dying out.
• Placed in a petri dish with the proper nutrients
cells will develop into specific mature cell types: Ex
heart cells
• http://www.youtube.com/watch?v=nm7bVlk4wTo
• Possible uses: growing organs, growing neurons
and muscles, implanting into humans for
regeneration of organs, studying diseases
Cloning
• Creating cells, organs, or organisms that are exact
copies.
• Therapeutic vs. Reproductive
• Therapeutic makes organs and cells for
transplant, study, and treatment of affected
individuals.
• Reproductive makes a whole new organism such
as Dolly (the sheep), pigs for parts like heart
valves, and other farm animals to produce
products, drugs, and other needed molecules.
Steps to clone a mammal
• Need three organisms
• 1: donor of egg (nucleus
– Will be removed)
• 2: donor of cell with
– Nucleus to be put into
– Empty egg of #1
• 3: surrogate sheep that
– Will have implanted
– Embryo that will grow.
Review
• What type of cloning makes organs for transplant?
• How can you get stem cells in culture to develop into
one kind of mature cell?
• What cells in a human are totipotent?
• What are the protective ends of a chromosome that
shorten over time as each round of replication can’t
duplicate the complete DNA strand?
• What enzyme found in Cancer cells and gamete
producing cells can add to these ends to restore them
to their normal starting length?
• What is the term for the place where golgi bodies line
up to conduct cytokinesis in plant cells.
Control of gene expression
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Gene expression = transcription and translation
Transcription makes RNA from DNA
Translation makes protein from RNA
We must control the making of proteins so that
they are made in the proper amounts at the
proper time and in the proper cells. All
organisms have mechanisms for this.
– Pretranscriptional controls
– Posttranscriptional controls
– Post translational controls
Pretranscriptional
• Prokaryotic cells
– OPERON- a series of genes controlling the making of more than one
polypeptide using one promoter.
– Promoter-DNA where RNA polymerase binds to start
– Operator- DNA where repressor binds to stop transcription
– Operon- all of the DNA in this unit together
– Inducer- molecule that binds to repressor to make it “useless"
• Inducible operon: lac operon, is typically off but can be induced to
go on when a molecule is present that needs to be broken down
(lactose sugar present needs to make lactase enzyme)
• Repressible operon: trp operon, is typically on but can be repressed
to go off when the product of the reaction is already present
(tryptophan amino acid already present)
Lac operon
Trp operon
Review
• What are the chromosome pair in humans that are of
the same kind but not the same exact sequence called?
• What does the repressor in the lac operon bind to?
• What needs to bind to the represser for the lac operon
to be expressed?
• What are the two steps of gene expression?
• What is the inactivated X chromosome called?
• What are the proteins called that DNA binds to for
packaging?
• What “stage” are cells in that are no longer part of the
typical cell cycle such as adult neurons?
Eukarytoic pretranscriptional control
• Eukaryotic cells
– Promoter- Where the RNA polymerase binds
– ENHANCERS- Activator proteins bind to enhancer DNA at a point away from
the promoter which then bends the DNA to allow it to bind to the promoter
and to allow the RNA polymerase to bind.
– SILENCERS- section of DNA where repressor proteins can bind to stop
transcription
– TRANSCRIPTION FACTORS- molecules that bind to the promoter which allows
the RNA polymerase to attach more easily. They bind to the TATA box on the
promoter.
– WRAPPING UP OF CHROMATIN
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DNA is wrapped around histone proteins to form “beads” called nucleosomes.
Euchromatin: loosely wrapped and ready for transcription
Heterochromatin: tightly wound and not ready for transcription
DNA methylation: adding CH3 methyl groups makes DNA unaccessible
Histone Acetylation: adding C2H3O2 acetyl groups to the histone proteins that the DNA
is bound to makes them loosen their grip and transcription can go faster.
Post Transcriptional
• Eukaryotic cells only as no nuclear membrane in
prokaryotic cells means it will start translation even before
transcription is completed.
• Primary transcript of RNA is made into functional mRNA
transcript. Controlling this process controls transcription.
– GTP cap on 5’ end (phosphate end) of mRNA to allow it to
attach to the ribosome
– Poly A tail on 3’ end (sugar end) of the mRNA to help it make its
way through the small nuclear pores
– Spliceosome (small nuclear riboproteins snrps) remove introns
and splice together exons
– Sometimes exons can be altneratively spliced to create a different protein in
translation ABCD sections can be ADBC
Review
• What is added to the sugar end of a primary
transcript? (3’ end)
• What is cut out by snrps?
• What process is started by the binding of a
molecule to the promoter?
• What is mRNA called before it is modified to
become funcitonal?
• What phase of meiosis does synapsis happen?
• What binds to silencer DNA?
• How many chromosomes are there in anaphase?
mRNA modications to become
functional
Translational controls
• Breakdown of mRNA: timing varies; red blood
cells mRNA very long lasting as they need to
make hemoglobin for life and that is almost their
entire job so no control is needed.
• Initiation of translation: Sometimes need
molecule to help assemble tertiary structure such
as heme group in hemoglobin with its Iron.
• Protein activation: Golgi: polypeptides need to
be altered to make them functional.
• Protein breakdown: after the final protein is
made it can be degraded or last longer depending
on function.
Animal body development
• Gradient of regulatory proteins leads to segmentation of all
animals.
• Homeotic genes: master switches then determine which
segment will become what.
• Homeboxes: a short string of nucleotides within the
homeotic gene that can trigger a group of genes that lead
to an appendage or major organ to develop.
• One homeotic gene triggers the growth of an eye and if it is
broken no eye develops. The homeotic eye master switch
can be transplanted from humans to mice to trigger eye
development in the mouse. The homeobox genes in the
mouse are still the same so it is a mouse eye and not a
human eye.
Review
• What are the master switches called that determine
the segment development of animals?
• What are the segments of DNA that repressors bind to
in the lac and trp operon called?
• What are the molecules that bind to the TATA box of
the promoter in eukaryotic cells called?
• Where in the cell does translation take place?
• How many tetrads would line up during metaphase II?
• What type of cell would likely want to have long lasting
mRNA?
Nondisjunction
• Chromsomes fail to divide properly during
meiosis so a fertilized egg ends up with an
extra (trisomy) or missing (monosomy)
chromosome.
• Ex. Down syndrome where a person has an
extra 21st chromosome so they have 47 total.
Most problems lead to death, but the sex
chromosomes have more survival.
Sex Chromosome nondisjunction
• Turner syndrome: XO O represents missing
chromosome: female, webbing, short and
undeveloped, very serious.
• Klinefelter’s syndrome: XXY Male with some
female secondary characteristics not as
severe.
• XXX superfemale: no issues
• XYY maybe more aggressive, but not too
significant