Biol-K 324 Lec 4: Gene, Allele, Mutant

Front 1

Allele

Back 1

variants of a gene

Front 2

Mutant allele

Back 2

1. can be identified and studied using genetic and molecular genetic techniques

2. can be dominant or recessive

3. gain of function

Front 3

standard genotype

Back 3

wild type

Front 4

conditional mutations

Back 4

temperature sensitive alleles

Front 5

why are cells able to grow at 23 degrees, but are unable to grow at 36 degrees?

Back 5

higher temperature denature proteins, but can grow at lower temperature

Front 6

what are the low temperature and high temperature called?

Back 6

low temp: permissive temperature

higher temp: non- permissive temperature

Front 7

what kinds of genes did Leland Hartwell and colleagues find? what cell process do these genes control?

Back 7

1. found cell type mutants

2. found in small, rod-shaped genes

Front 8

recessive lethal mutations?

Back 8

inbreeding

Front 9

complementation analysis

Back 9

1. if alleles are in different genes, they survive (complementation)

2. if alleles are on different genes, offspring die when crossed together

Front 10

suppression and synthetic lethality

Back 10

**

Front 11

gene mapping

Back 11

1. recombination rates or crossover events that occur during meiosis can be calculated and used to create a genetic map

2. complete genomes are now available

Front 12

recombination

Back 12

1. cross over events

2. occur during meiosis

3. used to create a genetic map

4. less likely to occur if genes are close together on the chromosome

Front 13

recombinant DNA

Back 13

1. restriction enzymes

2. cloning

Front 14

centimorgan

Back 14

1. distance corresponding to 1 recombinant individual per 100 progeny

2. named after Thomas Hunt Morgan

Front 15

recombinant DNA technology

Back 15

1. the ability to manipulate and recombine DNA sequences

2. began with restriction enzymes and plasmids

3. allow people to manipulate DNA in various organisms to create mutants or characterize genetic changes

Front 16

restriction enzymes

Back 16

1. ex: E.coli

2. cuts on palindromes

3. leave "sticky ends"

Front 17

ligation

Back 17

linking DNA strands together via phosphodiester linkgage

Front 18

plasmid vectors: DNA cloning

Back 18

1. insertion of DNA fragments into plasmid allows that DNA to be carried in a bacteria

2. after ligating DNA together, plasmid is transformed into the bacteria and grown on selective (ampicillin-containing) media

Front 19

DNA cloning vectors required features

Back 19

1. replication origin

2. selection marker

3. region for DNA molecule insertion

Front 20

cDNA library

Back 20

1. combination of cloned cDNA fragments inserted into a collection of hosts cells

2. constitute some portion of the transcriptome of the organism

Front 21

library screening

Back 21

1. millions of clones are screened in a single screen for a cDNA

2. either DNA hybridization or protein expression are used to screen for clones

Front 22

yeast complementation screen

Back 22

1. used to test whether the mutations in two strains are in different genes

2. complementation will not occur if the mutation are in the same gene

Front 23

DNA gel electrophoresis

Back 23

1. separation and analysis of macromolecule

2. DNA, RNA , proteins and their fragments

3. based on size and charge

Front 24

Polymerase Chain Reaction (PCR)

Back 24

1. if the sequence from the ends of DNA region are known, then the intervening sequence can be amplified using PCR

2. uses a heat stable Taq polymerase

3. can be used for forensic assays, medical assays, for introducing mutations into DNA

Front 25

dideoxy-sequencing

Back 25

1. sues nucleotides at a small ration that lack a 3' hydroxyl group

2. chain of polymerized DNA is terminated by insertion of the ddNTP

3. ddNTP labeled with fluorescent tags

4. sequencing machine produces a tracing

5. sequence can be read

Front 26

What is southern blotting?

Back 26

DNA on a gel*?

Front 27

Northern blot?

Back 27

1. RNA is separated in agarose or polyacrylamide gel

2. RNAs transferred to a solid support membrane

3. RNAs are detected with a radioactive probe, like a Southern blot

Front 28

in situ hybridization

Back 28

probing RNA in tissues give spatial information

Front 29

why is RNA detected and not DNA in in situ hybridization?

Back 29

1. detects specific mRNA in morphologically preserved tissues

2. RNA-RNA hybrids are very thermostable and are resistant to digestion by RNases.

3. reduces possibility of background staining

Front 30

DNA Microarrays: gene chip

Back 30

1. RNA samples from different conditions are hybridized and compared using these arrays

2. technique allows comparison of thousands of genes rapidly and accurately

Front 31

expression of proteins

Back 31

1. bacterial expression of proteins can be used to obtain large amounts of protein

2. inducible expression is used because overexpression of proteins can be toxic in cells

Front 32

transfection

Back 32

1. transient transfection

2. stable transfection

Front 33

transient transfection

Back 33

plasmids replicate but are not segregated like chromosomes

Front 34

stable transfection

Back 34

plasmid DNA integrates into chromosomes and clones can be selected

Front 35

transduction

Back 35

RNA virus that integrates into chromosomes

Front 36

transduction

Back 36

when genes are introduced into cells using a virus

Front 37

tagging

Back 37

1. promoter-fusion

2. protein-fusion

Front 38

promoter-fusion

Back 38

a promoter drives expression in a cell type or during developmental stages

Front 39

protein-fusion

Back 39

tags a specific protein, and its distribution in cells can be followed

Front 40

what enzymes would you need for making a cDNA library?

Back 40

1. DNA ligase

2. restriction endonucleases

Front 41

PCR

Back 41

1. separate DNA

2. anneal primer

3. elongate