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

• 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• 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• 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