Biochem 366 - Ch 05 - Molecular Genetic Techniques

Front 1

genotype

Back 1

of organism -genetic makeup; entire set of alleles for all the genes carried by an individual (“information”)

Front 2

phenotype

Back 2

phenotype of organism -all physical attributes or traits resulting from a given genotype

Front 3

alleles

Back 3

different forms or variants of a gene existing in populations

Front 4

wild type

Back 4

standard genotype; normal; non-mutant -generally denotes an allele present at a much higher frequency in a population

Front 5

wild type gene or allele

Back 5

genotype used as reference

Front 6

mutant gene or allele

Back 6

genotype that differs from wild type

Front 7

phenotype

Back 7

physical consequences resulting just from alleles under study

Front 8

diploidcells

Back 8

carry duplicate set of genes or chromosomes (somatic cells in higher eukaryotes)

Front 9

haploidcells

Back 9

carry a single set of genes or chromosomes (eggs and sperm in higher eukaryotes)

Front 10

homozygous

Back 10

diploid cell carries identical alleles, one on each homologous chromosome

Front 11

heterozygous

Back 11

diploid cell carries two different alleles, one on each homologous chromosome

Front 12

dominant mutation

Back 12

mutant phenotype observed in both heterozygous and homozygous genotype

Front 13

recessive mutation

Back 13

mutant phenotype observed only in homozygous genotype

Front 14

what do dominant mutations cause?

Back 14

dominant mutations often cause a gain-of-function(e.g., increase activity of encoded protein, confer new function on it, or lead to an altered spatial or temporal expression

Front 15

what do recessive mutations generally cause?

Back 15

recessive mutations generally cause partial or complete loss-of-function(e.g, remove part of or the entire gene, disrupt expression of the gene, or alter protein structure, and thus function, etc.)

Front 16

Regarding: dominant mutations that cause a loss of function
define haplo-insufficient genes

Back 16

both wild-type alleles are required
for function; loss of function in one allele is a dominant mutation

Front 17

Regarding: dominant mutations that cause a loss of function

Back 17

structural change in mutant protein interferes with function of wild-type allele (e.g., mutant subunit impairs assembly of multimericprotein) -similar to loss of function

Front 18

Point mutation

Back 18

alteration in gene sequence involving only a single bp

Front 19

Silent point mutation

Back 19

causing no change in amino acid sequence or protein activity

Front 20

Missensemutation

Back 20

substitution of one amino acid for another

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Nonsense mutation

Back 21

introducing premature stop codon

Front 22

Frame-shift mutation

Back 22

change in reading frame due to small
insertions or deletions

Front 23

Large alterations

Back 23

deletions, insertions, duplications,
rearrangements, etc.

Front 24

what two different mating types do haploid cells contain?

Back 24

a and alpha

Front 25

what are genetic screening procedures used for?

Back 25

to identify and isolate mutants

Front 26

what are the factors to consider regarding genetic screening procedures?

Back 26

1. HAPLOID vs DIPLOID organism
2. DOMINANT vs RECESSIVE mutation
3. LETHAT mutations vs CONDITIONAL mutations (mutant phenotype is displayed only under some conditions)

Front 27

why is genetic screening done easily for yeast?

Back 27

single cells (haploid or diploid) RAPIDLY GROW to form COLONIES on nutrient agar plates
essential genes can be studied using CONDITIONAL MUTATIONS (e.g. temperature sensitivity requirement for a specific metabolite, etc.)

Front 28

what are the five steps in cell division cycle mutants in yeast?

Back 28

1. add mutagen; distribute into smaller aliquots
2. incubate at 23 degrees C for 5 hours
3. plate out individual aliquots
4. incubate at 23
5. replica-plate and incubate
one will be grown at 23 while the other will be grown at 36. if there is no growth at 36 it is temperature sensitive

Front 29

genetic screening in diploid organisms requires what?

Back 29

inbreeding

Front 30

genetic screening in diploid organisms requires inbreeding because

Back 30

spontaneous or induced MUTATIONS is diploid organisms typically CHANGES ONLY ONE ALLELE(leads to heterozygous state)
RECESSIVE LETHAL mutations can be maintained in heterozygotes and are IDENTIFIED BY INBREEDING (homozygous progeny does not survive)

Front 31

what is non-permissive temperature?

Back 31

temperature at which the mutant phenotype is observed

Front 32

complementation analysis:
what is is?

Back 32

a classic methodology for determining whether independently isolated RECESSIVE MUTATIONS that produce the same phenotype in the SAME gene or in DIFFERENT genes

Front 33

complementation analysis:
can it be done on dominant mutants?

Back 33

NO

Front 34

complementation analysis:
this test can only be done in organisms that have?

Back 34

DIPLOID stages

Front 35

List 4 characteristics of plasmids

Back 35

1) Made of ds DNA and separate from a cell's chromosomal DNA

2) Occur naturally in bacteria and lower euks. (like yeast)

3) have parasitic or symbiotic relationship with host

4) Are duplicated before every cell division and copies segregate into each daughter cell.

Front 36

Define:

Restriction Enzymes

Back 36

Endonucleases produced by bacteria that recognize a 4-8 bp sequence and cleave there.

Front 37

Define:

Restriction Site

Back 37

The 4-8 bp sequence recognized by a restriction enzyme.

Front 38

Define:

Modification Enzyme

Back 38

Protects a bacteria's own DNA from its own restriction enzyme's by methylating some of the restriction site bases.

This prevents endonuclease from cutting the host DNA.

Front 39

Restriction Fragments:

Describe what is needed to bind blunt ends together.

Back 39

A blunt end can be ligated to any blunt end, but it requires higher concentrations of DNA and ligase.

Front 40

Fill in the blank:

The _____ (shorter/longer) the enzyme's recognition sequence, the _____ (more/less) frequently it will occur in a certain DNA sequence.

Back 40

...shorter..., ...more...

or

...longer..., ...less...

Front 41

How do you estimate how often a bp sequence will appear in a DNA sequence?

Back 41

4^x where 'x' is the length of the base pair sequence.

Example:

A 4 base-pair sequence will occur every 4^4 (256) base pairs.

Front 42

What kind of a bond does ligase form?

Back 42

Ligase forms phosphodiester bonds between DNA fragment ends.

Front 43

What 3 things does a cloning vector require?

Describe each component.

Back 43

Polylinker:
Also called Multiple Cloning site. Restriction enzyme sites in this region are unique in the vector (can be used for cloning).

ORI (Origin of Replication):
Origin where DNA replication is initiated by host enzymes.

Selectable Marker (Gene):
Something that will help you distinguish which vectors have been taken up by the host cell.
EXA: Amp^r - b-lactamase enzyme which confers resistance to ampicillin.

Front 44

what can double mutants reveal?

Back 44

the order in which proteins function

Front 45

what is the only requirement for obtaining useful informations from double mutants?

Back 45

they must have opposite effects on the output or the same regulated pathway.
pg 172

Front 46

what can genetic suppression reveal?

Back 46

interacting or redundant proteins

Front 47

Genetic suppression:
what does normal functioning of many cellular processes depend on?

Back 47

DEPENDS ON INTERACTIONS between different proteins (e.g, enzyme subunits)

Front 48

Genetic suppression:
for a complex composed of two non-identical protein subunits, what can a mutation in one subunit do?

Back 48

it can DISRUPT its ABILITY TO INTERACT with B(e.g. small structural change due to a point mutation)

Front 49

Genetic suppression:
in rare cases, the interaction can be restored by what?

Back 49

a SUPPRESSOR MUTATION in protein B (a compensatory mutation)

Front 50

Genetic suppression:
what does it tell your when the single mutants are abnormal but the double mutant is normal?

Back 50

it provides evidence that the two proteins interact

Front 51

Mapping gene positions by recombination:
where does GENETIC RECOMBINATION take place?

Back 51

before the 1st meiotic cell division in germ cells when the replicated chromosomes of a homologous pair align with each other

Front 52

what is CROSSING OVER?

Back 52

crossing over between the tow genes at meiosis results in two parental gametes (germ cells)

Front 53

how can FREQUENCY OF RECOMBINATIONS be measured?

Back 53

by diploid offspring (or haploid in yeast)

Front 54

what does synthetic lethality reveal?

Back 54

protein interactions

Front 55

define synthetic lethality

Back 55

a mutation if one of two interacting proteins leads to a partial defect but a mutation in both causes a severe defect

Front 56

what is the second type of synthetic lethality?

Back 56

can also be caused by mutations in NONESSENTIAL genes that encode proteins in REDUNDANT PATHWAYS (either pathway provides the same function)

Front 57

Plasmid Cloning Vectors:

Why is it important for the recognition sites on the polylinker to be unique (i.e. no restriction site appears more than once)?

Back 57

Because if there was more than 1 of a certain recognition site, the restriction enzyme would cut in more than 1 place.

Also, you don't want your polylinker to have a recognition site that has a sequence of DNA that is common in some other part of the vector.

Front 58

Cloning Procedure:

What are 2 methods that help allow for plasmid DNA uptake?

Back 58

1) Exposure to CaCl₂ and a heat pulse destabilizes bacterial wall enough to create transient pores to allow plasmid in.

2) Electroporation:
Shock cell wall, which creates transient pores to allow plasmid in.

Front 59

What percentage of the human genome represents protein coding sequences?

Back 59

1.5%

Front 60

How do you separate mRNA from a mixture of all types of RNA?

Describe the procedure.

Back 60

Affinity Chromatography

In an oligo-dT (poly-T) column, the poly-A tail on the 3' end of an mRNA molecule will bind, thus allowing the other types of RNA (rRNA, tRNA) to filter through.

After this step, you can wash with an alkaline solution to get the mRNA to be released from the column.

Front 61

What are 4 characteristics of Genomic Libraries?

Back 61

1) Should (if it is a good one) represent the whole genome.

2) Contains coding (exon) and noncoding (introns, promoters, etc.) fragments/sequences.

3) Genome can be isolated from any type of tissue or cell in the animal.

4) Can be more specific (e.g. genomic DNA library of a certain type of cell line; e.g. Mouse 3T3 fibroblasts)

Front 62

What are 4 characteristics of a cDNA library?

Back 62

1) Should (if it is a good one) represent the whole mRNA population present at the time of RNA isolation.

2) Represents genes that are expressed *at that time* *in that place* (cell type, tissue type, etc).

3) It is more specialized than a genomic library.

4) Contains coding sequences and 5' & 3' UTR's.

Front 63

Define:

Hybridization

Back 63

i.e. annealing or renaturation

Formation of h-bonds between complimentary nucleic acid strands in solution

Front 64

Define:

T_m

Back 64

Melting temperature

The temperature at whichi 50% of complimentary nucleic acid strands in solution are in ds h-bonded form, and the other 50% in ss form.

Front 65

Define:

Shuttle vector

Back 65

A vector that is constructed so that it can propagate in 2 different host species.

e.g. Plasmids that can replicate in both Ecoli and yeast cells.

Front 66

Define:

(genetic) Transformation

Back 66

The genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign genetic material (DNA).

Front 67

Describe the Blue-White Screening test.

Back 67

A molecular technique that allows for the detection of successful ligations in vector-based gene cloning.

Your DNA of choice is ligated onto a vector. The vector is then transformed into a cell and grown on an agar plate in the presence of X-GAL.

Cells that contain vectors that successfully had the DNA strand ligated into them will turn WHITE.

Cells that contain vectors that did NOT successfully have the DNA strand ligated into them will turn BLUE.