LATG Chapters 8 & 9 Molecular Biology and Genetics

Molecular Biology …is the study of biology at the molecular level …focuses specifically on DNA, RNA, and protein …is a tool used to study genetics

Some Definitions Genetics …is the study of how genes interact (with each other AND their environment) to produce the inherited characteristics that we see every day Genome …the entire collection of genes an organism has.

More Definitions: Genotype = the genetic makeup of an organism –Every person (every mouse, every cow) has two copies of each gene, one from each parent “Homozygous normal” = two normal copies (aka Wildtype) “Heterozygote” = one normal & one abnormal copy “Homozygous abnormal” = two abnormal copies (in transgenics, aka “Knock-Out”

There’s still more... Phenotype = the physical features of an organism (i.e., tall/short; red/white etc) Mutation = any change in the DNA of a gene Genetic Engineering...is the term used to describe the manipulation of the genetic make-up of an organism

Where is your DNA located?

Where is your DNA? DNA is in the nucleus of the cell, on structures called chromosomes. Chromosomes are made of genes Genes are made of DNA

Structure of DNA DNA is a long string (polymer) of 4 bases These bases universal! –A = Adenosine –T = Thymine –C = Cytosine –G = Guanine The order (sequence) of the bases is what makes one gene different from another gene.

DNA Structure, cont’d A -- T C -- G GATTCC CTAAGG DNA exists as a double helix (twisted ladder) Each rung of the ladder is a base pair

How do cells transmit their genetic information? Replication = the process by which a cell duplicates its DNA. When a cell divides,one copy gets passed onto the new cell

How do cells interpret the information in the DNA? Transcription: the process by which the DNA code is “read”. –DNA is transcribed into RNA in the nucleus

Transcription RNA has 4 bases: Adenine A=U Guanine G=C Cytosine Uracil*** (Uracil is used instead of Thymine) Unlike DNA, RNA is single-stranded

Translation Occurs outside the nucleus, via ribosomes RNA is “read” in groups of three bases called “codons.” Each codon corresponds to an amino acid

Information Flow in the Cell

Techniques Extraction of DNA Restriction Digestion PCR Electrophoresis Southern blotting

Extraction of DNA Enzymes “digest” cell walls and release DNA into solution Add phenol to remove proteins Spin to separate DNA from proteins Add ethanol

Restriction enzymes These are enzymes that cut (digest) DNA at specific sites (sequences). Examples: –Eco RI only cuts the sequence …GAATTC… –Pst 1 only cuts the sequence …CTGCAG Because everyone’s DNA is comprised of the same 4 nucleotides (A,T, C, G), you can attach one species to another...

Cloning (using RE)

Polymerase Chain Reaction (PCR) Based on the fact that A=T and C=G Need only a tiny bit of DNA ***Must know some of the sequence of the gene of interest Three simple steps: heat, add primers etc, cool solution so bases will bind, repeat! Can amplify a piece of DNA a million-fold!

PCR

Detection of DNA Use agarose gels Gel acts like a filter: DNA separates by size Stain the gel with a dye to make the DNA to make it fluoresce under UV light.

Blotting Southern Blot –DNA cut with enzymes –transferred to membrane –hybridized to probe that is specific to gene of interest –exposed to film –if gene is present, you will see a band on the film

Types of Blots Southern blotting -- DNA Northern blotting -- RNA Western blotting -- protein

Genetic Engineering of Mice –Two types Transgenic - a gene is added via pronuclear injection –This is used to “overexpress” a gene –ex: Alzheimer’s and Beta-amyloid Targeted Mutation (aka “KO”) - a gene altered then added to the genome using ES cells –This is used to delete a gene –ex: ERKO--estrogen receptor KO mice

Transgenic mice Created through pronuclear injection Need 4 groups of mice –superovulated females –stud males –vasectomized males –pseudopregnant females

The Mice Superovulated females --given hormone injections to make them release more eggs than usual (30-60) Stud Males --are mated with the s.o.females so that a lot of embryos are produced Pseudopregnant females --a female is mated to a sterile male so that her body will produce hormones that prepare it for pregnancy

Procedure

Transgenic Mice Transgene can integrate ANYWHERE in the mouse genome. Integrates in 1-several hundred copies Must screen pups (PCR) to determine which pup have the transgene, and will pass it on. Must observe transgenic mice carefully to observe phenotype

Phenotype Depends on the gene you’re overexpressing Sometimes it’s obvious, sometimes it’s not. Must observe these mice carefully

Knockout Mice Knockout mice --a gene is deleted Similar to transgenic mice, must carefully observe for phenotype Need the same 4 groups of mice, plus ES cells ES cells = embryonic stem cells –totipotent

Embryonic Stem Cells Are from the inner cell mass of a blastocyst can develop into any part of the body “totipotent”

KnockOut Mice Culture ES cells from white (129) mice and target gene using electric current Mate black mice; insert “white” ES cells into black blastocyst Pups = chimeras (black and white) Mate chimera to black mouse. If white pups are produced, targeted gene has been passed on!

Transgenic Animals and You You are very important!! These mice are expensive/time- consuming to make You must carefully observe the animals for phenotype changes –strange gait, spinning, too fat, too thin, scaly skin…

Good Luck!