Control of Gene Expression

CloningS0matic cell nuclear transfer

Researchers clone animals by nuclear transplantation SOMATIC CELL NUCLEAR TRANSFER: A nucleus of

an egg cell is replaced with the nucleus of a somatic cell from an adult

Thus far, attempts at human cloning have not succeeded in producing an embryo of more than six cells Embryonic development depends on the control of

gene expression (aka protein synthesis)

Somatic Cell Nuclear Transfer

In reproductive cloning, the embryo is implanted in a surrogate mother. The goal is to produce a cloned offspring.

In therapeutic cloning, the idea is to produce a source of embryonic stem cells.

Stem cells can help patients with damaged tissues.

Stem cells are NOT specialized in structure and function, therefore they can take on the role of damaged cells in damaged tissues.

Reproductive vs. Therapeutic Cloning

Removenucleusfrom eggcell

Donorcell

Add somaticcell fromadult donor

Grow in culture to producean early embryo (blastocyst)

Nucleus fromdonor cell

Implant blastocystin surrogate mother

Remove embryonic stem cells from blastocyst andgrow in culture

Clone of donoris born(REPRODUCTIVEcloning)

Induce stemcells to formspecialized cellsfor THERAPEUTICuse

Dolly the Sheep

Reproductive vs. Therapeutic Cloning

Genetic Regulation in Prokaryotes

Proteins interacting with DNA turn prokaryotic genes on or off in response to environmental changes.

The process by which genetic information flows from genes to proteins is called gene expression. Our earliest understanding of gene control came

from the bacterium E. coli (Reminder: Bacteria are prokaryotes.)

Proteins turn genes on or off

Cellular Differentiation & the Cloning of Eukaryotes

Differentiation yields a variety of cell types, each expressing a different combination of genes

In multicellular eukaryotes, cells become specialized as a zygote develops into a mature organism Different types of cells make different kinds of

proteins. Different combinations of genes are active in

each type.

Cellular Differentiation

Differentiated cells may retain all of their genetic potential. (Even if turned off, the gene is still present.)

Most differentiated cells retain a complete set of genes In general, all somatic cells of a multicellular

organism have the same genes. So a carrot plant can be grown from a single

carrot cell.

Retain Genetic Potential

The cloning of tadpoles showed that the nuclei of differentiated animal cells retain their full genetic potential

Early Nuclear Transfer

Tadpole (frog larva)

Intestinal cell

Frog egg cell

Nucleus

Nucleus

UV

Nucleusdestroyed

Transplantationof nucleus

Eight-cellembryo

Tadpole

The first mammalian clone, a sheep named Dolly, was produced in 1997

Dolly provided further evidence for the developmental potential of cell nuclei.

Dolly again

Connection: Reproductive cloning of nonhuman mammals has applications in basic research, agriculture, and medicine. Scientists clone farm animals with specific sets

of desirable traits. Piglet clones might someday provide a source

of organs for human transplant.

Applications of Nonhuman Mammalian Cloning

Connection: Because stem cells can both perpetuate themselves and give rise to differentiated cells, they have great therapeutic potential

Adult stem cells can also perpetuate themselves in culture and give rise to differentiated cells But they are harder to culture than embryonic

stem cells. They generally give rise to only a limited range

of cell types, in contrast with embryonic stem cells.

Therapeutic Cloning

Differentiation of embryonic stem cells in culture

Culturedembryonicstem cells

Different cultureconditions

Different types ofdifferentiated cells

Heart muscle cells

Nerve cells

Liver cells

DNA packing in eukaryotic chromosomes helps regulate gene expression.

A chromosome contains a DNA double helix wound around clusters of histone proteins.

DNA packing tends to block gene expression.

Gene Regulation in Eukaryotes

DNAdoublehelix(2-nmdiameter)

Metaphase chromosome

700nm

Tight helical fiber(30-nm diameter)

Nucleosome(10-nm diameter)

Histones

“Beads ona string”

Supercoil(200-nm diameter)

In female mammals, one X chromosome is inactive in each cell.

An extreme example of DNA packing in interphase cells is X chromosome inactivation

Female Mammals

EARLY EMBRYO

Cell divisionand

X chromosomeinactivation

X chromosomes

Allele fororange fur

Allele forblack fur

TWO CELL POPULATIONSIN ADULT

Active X

Inactive X

Orange fur

Inactive X

Active X Black fur

Genetic Biotechnology

Since ancient times, humans have bred animals and plants to increase the likelihood of certain desirable traits. Example: Hunting dogs or larger fruits

Two methods are used: Hybridization: crossing different parent organisms with different

forms of a trait to produce an offspring with a specific trait Example: hybrid rice that produces greater yield and another hybrid rice

that contains greater nutritional properties Disadvantage: Time-consuming and expensive

Inbreeding: crossing closely related parent organisms who have been bred to have desired traits to ensure that the traits are passed on Example: German shepherd dogs Disadvantage: Undesirable recessive traits are often passed down with

the desirable traits.

Selective Breeding

A process by which an organism’s DNA is manipulated in order to insert the DNA of another organism (creates recombinant DNA) Purpose: Incorporate the desirable traits of one

organism into another organism Example: Bioluminescent trait – A type of jellyfish

contains a protein (GFP: green fluorescent protein) that causes it to glow. Scientists insert the DNA that codes for GFP into the DNA of mosquito larvae so that they will glow. Mosquito populations can be controlled as the larvae are more easily located.

Produces “genetically modified organisms”

Genetic Engineering

Selective breeding and genetic engineering require scientists to use special tools or processes to manipulate DNA. Restriction Enzymes: cut DNA into smaller

fragments with “sticky ends” that allow it to connect to other fragments of DNA

Gel Electrophoresis: electrical currents separate DNA fragments based on size allowing fragments to be sorted and studied

DNA Tools

Useful in genetic engineering where DNA of one organism is inserted into the DNA of another organism (recombinant DNA)

Example: EcoRI restriction enzyme cuts a GAATTC sequence

Restriction Enzymes

Restriction enzymes are found naturally in bacteria cells. The

bacteria developed the enzymes to fight against viruses. They

chop up the viral DNA that gets inserted into their cells.

Electrical currents run through the DNA samples that have been cut into fragments. Smaller fragments travel more quickly from the – electrode to the + electrode.

Gel Electrophoresis

Separated fragments can be studied or combined with other fragments to

create recombinant DNA.

When a particular new DNA sequence has been developed in recombinant DNA, bacteria cells are used to make multiple copies. The bacteria cells are heated which causes pores to open in their

cell walls. The recombinant DNA moves through the pores into the bacteria

cell. As the bacteria cell replicates, the recombinant DNA is replicated,

too.

Purpose: to create many copies of the desirable sequence that can be used in genetically-modified organisms

Gene Cloning

Learning the sequence of DNA fragments can allow scientists to understand the function of certain sequences of bases.

Restriction enzymes are used to cut large DNA strands into shorter fragments.

Dyes are used to color known bases, and the known colored known bases bind to the unknown sequence. The unknown sequence can be read by following complementary base-pairing rules.

DNA Sequencing

Purpose: create copies of selected DNA segments when the sample size of DNA is too small to run all of the needed tests

Uses a thermal cycler to separate the DNA double helix and DNA polymerase enzymes to create copies of the selected segments.

Extremely useful in forensic science and medicine

Polymerase Chain Reaction

Purpose: Insert beneficial genes into a needy organism

How? A mutated gene is located on a chromosome. A “normal” gene is inserted into a chromosome to

replace the dysfunctional one using a “viral vector.” The virus infects a cell and injects its genetic material

including the “normal” gene. The cell begins replicated the “normal” gene, and the mutation is corrected.

Gene Therapy