genetics exam 3

positional cloning / chromosome walk use

use for tightly linked genes. prepare genomic library, perform colony lift using known gene as probe, make new probe from end of first clone and rescreen until you find the desired gene, must have some way to recognize the gene

functional complementation use

use for temperature sensitive or fungi. prepare cDNA library from wild type in a plasmid of the organism, transform mutant and look for rescued organism

antibody probe use

use if there is a synthesized antibody. prepare cDNA library using an expression vector and perform colony
lift using antibody as probe

nucleic acid probe use

use if DNA sequence is known. screen genomic library by colony lift with gene as probe

degenerate primers use

use if protein / amino acid sequence is known. screen cDNA or genomic library by colony lift using degenerate primers synthesized based on the amino acid sequence of the protein as your probe

making a mouse

1. Infect ES cells from black mouse, using vector shown above

2. Select for recombinants in neomycin and ganciclovir

3. Verify that recombinants had deletion by Southern blot or PCR

4. Mix engineered ES cells w embryo from white mouse

5. Implant into pseudopregnant mouse to produce chimeric mice

6. Cross Chimeric X white to find mice that produce all black offspring

7. Cross hets to get homozygotes

maternal effect

phenotype of offspring is based on moms genotype

extranuclear inheritance

inheritance is determined by substances in the egg (all children of affected mother are affected)

pericentric inversion

includes centromere

paracentric inversion

does not include the centromere

deletion

removes part of a chromosome

Cri-du-chat syndrome

results from deletion of part of chromosome 5

Wolf-Hirschhorn syndrome

results from deletion of part of chromosome 4

duplication

part of chromosome duplicated

balanced chromosome complement

a complete set of chromosomes where no genetic material is lost or gained, even if the arrangement of the chromosomes is altered through structural changes like translocations or inversions

reciprocal translocation

non homologous chromosomes exchange DNA

Robertsonian translocation

special type of non-reciprocal translocation that fuses 2 acrocentric/telocentric chromosomes

• high frequency of spontaneous abortion and down syndrome

• short arms of chromosomes lost

inversion

part of chromosome “flipped”

endopolyploidy

having extra sets of chromosomes in some cells of the body

euploidy

correct number of chromosomes

monoploidy

orgamism or cell with only one copy of each chromosome

autopolyploid

composed of multiple sets of chromosomes from the same species

allopolyploid

composed of multiple sets of chromosomes from different species

• from cross fertilization between species

• offspring generally infertile because homologous chromosomes are too different for synapsis, cant go through meiosis

endoreduplication

replication without nuclear division

euploidy

a state where an organism or cell has the correct number of chromosomes, or a multiple of that number

aneuploidy

chromosome # differs from normal by less or more than a full chromosome set

polysomy

extra individual chromosome

trisomy

diploid with extra copy of 1 chromosome

• kleinfelter (XXY)

• trisomy 21 (down syndrome)

• trisomy 13 (patau syndrome)

• trisomy 18 (edwards syndrome)

triploid still have balance expression, but trisomy is unbalanced

monosomy

missing one copy of a chromosome

• turner syndrome (X0), sterile female

mitochondria (extranuclear inheritance)

refers to the passing of genetic information from mitochondria, which are organelles located outside the nucleus of a cell, from parent to offspring, meaning the genetic material is inherited from the cytoplasm rather than the nucleus itself; in most animals, this translates to solely inheriting mitochondrial DNA from the mother due to the egg contributing most of the cytoplasm to the zygote

• human myoclonic epilepsy with ragged-red muscle (MERRF)

chloroplasts

like mitochondria, include their own genome and are maternally inherited

endosymbiont

an organism that lives within the body or cells or another organism, usually in a mutualistic relationship

heteroplasmy

the presence of more than one type of mitochondrial DNA

homoplasmy

all copies of mitochondrial DNA are identical

wolbachia

• cytoplasmic endosymbiont in insects

• transmitted only by female

• causes male progeny to die, transforms male into female (able to reproduce without fertilization from male)

nucleoid

region in a prokaryote that contains all or most of the genetic material (not surrounded by a nuclear membrane)

conjugation

DNA transferred from donor to recipient with direct cell-cell contact

• DNA transferred as episome or plasmid

• plasmid - extrachromosomal circular DNA, contains F factor (fertility) and R factor (antibacterial resistance)

• mating involves transfer of F (transfer of F+ to F-)

episome

genetic element that can exist free in cell or integrated into chromosome

merodiploid

a bacterial cell that has two copies of a portion of its chromosomal complement

transformation

take up of free DNA from environment

• may be in form of plasmid or free dna

• allows spread of drug resistance between strains

lytic

infection ruptures cell to release new phage particles

lysogenic

virus integrates into bacterial chromosome, transmitted to daughter cells

virulent phage

phage only capable of lytic growth

temperate phage

phage that are only capable of lysogeny

transduction

transfer of bacterial DNA by phage

specialized transduction

only certain portion of bacterial chromosome can be transferred between bacteria

generalized transduction

ability to transduce any bacterial genes

restriction endonuclease

enzymes that bind and cut DNA at specific sequences

genomic DNA vs. cDNA

Genomic DNA- complete DNA sequence of an organism, including both coding and non-coding regions

cDNA (complementary DNA) is a synthetic DNA molecule created from mRNA, containing only the coding regions of expressed genes

plasmid

a genetic structure in a cell that can replicate independently of the chromosomes,

southern blot definition

a lab technique used to study DNA. Using agarose gel electrophoresis, genes are separated by size.

northern blot

gel electrophoresis used to study RNA

SDS-PAGE

western blot

SDS page used to separate proteins that are transferred to a membrane, incubated with a protein-specific antibody that is detected in some way

PCR

Next generation DNA sequencing

site-directed mutagenesis

uses PCR to mutate a specific nucleotide in a plasmid vector. After cell replicates, some of the replication enzymes will have “fixed” the non-matching base pair by adding the new matching nucleotide.

reporter transgene

a gene that is introduced into an organism to report the expression of another gene or protein (lacZ - blue, gfp - fluorescent green, luciferase - produces light)

fuse regulatory sequences to reporter

transgenic organism

organisms that have had genes from other species introduced into their genome

genetically modified plant

transgenic plants

can be mutated to be herbicide resistant or to have Bt toxin that kills the insects that eat it

could have increased yield, improved crop, and reduce herbicide and pesticide use

no evidence of harm to human health, but may cause decreased biodiversity

gene therapy

a medical technique that treats or prevents disease by altering a person's genes

adult stem cell

some tissues have stem cells to replace damaged cells

less potential than the other 2 types due to the fact that it may not be able to differentiate, also can’t culture large numbers

embryonic stem cell

derived from 4-5 day old embryo, typically from infertility clinics

pluripotent (can become any type of cell)

induced pluripotent stem cell

introducing 4 genes into differentiated cells can cause them to revert to a stem like state

has more potential than adult stem cells, has extended proliferation like embryonic stem cells, can be derived from self so no chance of organ rejection

reverse genetics

general approach of moving from a cloned gene to identifying a mutation in that gene

gene knock out

used to study genes by assessing what happens when they are mutated/not functioning.

RNAi

a pathway that triggers degradation of mRNA that corresponds to the dsRNA that was introduced.

Can be used to mimic the effect of a mutation without actually causing one by causing the targeted gene to not be able to synthesize any protein.

CRISPR-Cas9

a technique involving the use of a bacterial defense mechanism that uses stored viral RNA to clip DNA that is complementary to the guide RNA. We can synthesize any RNA to target specific DNA.

Can be used to correct mutations in chromosomes, or create mutations (knock out dominant mutant allele)

not ready for use in humans due to off target events

prophage

a viral genome integrated into a host cells chromosome

basic organism cloning steps

obtain eggs, remove nucleus, fuse enucleated cells with somatic cells, let develop