Exam 2: Ch 5 DNA Clones and Recombination

Question 1

3 questions a molecular cell biologist asks about a newly discovered protein

Answer 1

what is the function in the context of a living cell

what is the biochemical function of the purified protein

where is the protein located

Question 2

3 molecular genetic tools to answer the 3 questions

Answer 2

the gene that encodes the protein

a mutant cell line or organism that lacks the protein

source of the purified protein for biochemical studies

Question 3

classical genetics

Answer 3

isolation of a mutant that is defective in a process of interest

genetic methods used identify and isolate affected gene

isolated gene used to produce a large quantity of the protein

study when and where the protein is expressed in an organism

Question 4

genetic analyses of mutants defective in a particular process can reveal 3 things

Answer 4

new genes required for the process to occur

the order in which gene products act in the process

whether proteins encoded by different genes interact with each other

Question 5

allele

Answer 5

naturally occurring alternate versions of a gene

Question 6

mutagen

Answer 6

an agent that causes a heritable change in DNA sequence

Question 7

genotype

Answer 7

particular set of alleles for all genes carried by an individual

Question 8

wild-type

Answer 8

normal non-mutant allele

standard genotype

Question 9

phenotype

Answer 9

all the physical attributes or traits of an individual that are a consequence of the genotype

Question 10

saccharomyces cervisiae can exist in either _____ or _____ states

Answer 10

haploid, diploid

Question 11

recessive alleles cause disorders why

Answer 11

inactivates affected gene leading to partial or complete loss of function

remove part or the entire gene, disrupts expression, or alters the structure of the encoded protein

Question 12

dominant alleles cause disorders why

Answer 12

mutation that causes some kind of gain of function

increase activity of encoded protein, confer a new function to it, lead to inappropriate spatial or temporal patterns of expression

Question 13

haplo-insufficient

Answer 13

dominant mutation that causes a loss of function because not enough gene product is made

Question 14

commonly used mutagen

Answer 14

ethylmethane sulfonate EMS

chemically modifies guanine bases in DNA leading to conversion of GC to AT

Question 15

meiosis consists of one round of _____ ____ followed by two separate _____ ________

Answer 15

DNA replication, cell divisions

Question 16

how to avoid complexity in breeding experiments

Answer 16

begin with true-breeding strains (homozygous for genes being examined)

mate true-breeding mutant to true-breeding wild-type to produce heterozygous F1 generation

Question 17

if F1 progeny exhibit the mutant trait…

Answer 17

the mutant allele is dominant

Question 18

if F1 progeny are wild-type…

Answer 18

the mutant allele is recessive

Question 19

how to determine if mutant alleles are dominant or recessive in S. cervisiae

Answer 19

cross between haploid cells (a/alpha)

if heterozygote a/alpha diploid exhibits mutant trait, its dominant

Question 20

what happens when a/alpha diploids of S. cervisiae are placed under starvation conditions

Answer 20

the cells undergo meiosis

make 4 haploid spores, two of type a and two of type alpha

Question 21

genetic screens

Answer 21

procedures used to identify and isolate mutants

depend on whether the organism is haploid or diploid, and if the mutation is recessive or dominant

Question 22

temperature sensitive mutations

Answer 22

isolated in bacteria and lower eukaryotes

ex. single missense mutation could cause the mutant protein to have reduced thermal stability so it’s fully functional at 1 temp and denatured at another

Question 23

permissive vs. nonpermissive

Answer 23

permissive: temp at which the mutant phenotype is not observed even though the mutant allele is present
nonpermissive: temp at which the mutant phenotype is observed

Question 24

complementation tests determine whether different recessive mutations are in the same ______

Answer 24

gene

Question 25

genetic complementation

Answer 25

the restoration of the wild-type phenotype by mating of two different mutants if two recessive mutations are in the same gene, then the offspring have the mutant phenotype b/c neither allele provides a functional copy if the mutations are in different genes, a wild-type allele of each gene will be present and the offspring is normal

Question 26

double mutants are useful in assessing...

Answer 26

the order in which proteins function biosynthetic pathways where a precursor is converted via one or more intermediates to a final product signaling pathways that regulate other processes and involve the flow of info rather than chem. intermediates

Question 27

ordering of biosynthetic pathway

Answer 27

biosynthesis of trp in bacteria enzymes required catalyze conversion of one of the intermediates in the pathway to the next (trp operon) the order of action of different genes for these enzymes was deduced by examining which intermediates accumulated in each mutant when each enzyme was mutated

Question 28

ordering of signaling pathways

Answer 28

two mutations have opposite effects on the output of the same regulated pathway commonly, 1 mutation represses expression of a reporter gene even when the signal is present and the other mutation results in reporter gene expression in the absence of signal

Question 29

suppressor mutations

Answer 29

a mutation in 1 protein is suppressed by a second mutation in a different protein

Question 30

synthetic lethal mutations

Answer 30

the deleterious effect of a mutation is greatly exacerbated by a second mutation in a related gene can help reveal nonessential genes (if the gene is inactivated, the product is still made by a different gene)

Question 31

genetic mapping

Answer 31

studies designed to determine the position of a gene on a chromosome used to locate the gene that is affected by a mutation of interest

Question 32

____ recombination occurs between two genes on the same chromosome the closer together they are

Answer 32

less, they are linked (same chromosome and close together)

Question 33

DNA cloning

Answer 33

allows researchers to prepare large numbers of identical DNA obtain small regions of an organism's DNA that make up specific genes link the DNA fragment to a vector DNA molecule that can replicate within a host cell

Question 34

what is recombinant DNA

Answer 34

any DNA molecule composed of sequences derived from different sources

Question 35

DNA cloning process

Answer 35

vector + DNA fragment = recombinant DNA replication of recombinant DNA within host cells (rep. of inserted DNA + vector) isolation, sequencing, and manipulation of purified DNA fragmenting

Question 36

DNA library

Answer 36

a collection of DNA molecules each cloned into a vector molecule

Question 37

which two enzymes allow insertion of DNA fragments into cloning vectors?

Answer 37

restriction enzymes DNA ligases both help produce recombinant DNA

Question 38

restriction enzyme

Answer 38

endonuclease that recognizes restriction sites (4-8bp) that are commonly palindromic and cuts the DNA at these sites

Question 39

for each restriction enzyme, bacteria produce a _____ enzyme that protects the host bacterium's own DNA from cleavage

Answer 39

modification enzyme adds a methyl to prevent cleavage by restriction enzyme

Question 40

restriction enzyme mechanism

Answer 40

generates fragments with a single-stranded tail at both ends (sticky ends), or a blunt end (flush) fragments with sticky tail or blunt ends are complementary to all other fragments produced by same restriction enzyme

Question 41

inserting DNA fragments into vectors

Answer 41

DNA fragments with either sticky or blunt ends can be inserted into complementary vector DNA by DNA ligases

Question 42

plasmid

Answer 42

circular dsDNA separate from chromosomal DNA in bacteria and lower eukaryotes like yeast

Question 43

plasmids exist in a ____ or ______ relationship with their host cell

Answer 43

parasitic, symbiotic plasmid DNA duplicated with each cell division like chromosomal DNA

Question 44

E. coli plasmids are used as ______

Answer 44

vectors replication initiated at ORI (replication origin) and inserted DNA is replicated too

Question 45

plasmid transformation

Answer 45

when E. coli cells take up the plasmid containing recombinant DNA

Question 46

polylinker

Answer 46

synthetically generated sequence containing many different restriction sites

Question 47

genomic library

Answer 47

set of clones representing all DNA sequence in the genome good for simple organisms, too complicated to represent complex organisms

Question 48

what percent of human genome represents protein coding DNA?

Answer 48

1.5%

Question 49

cDNA

Answer 49

complementary DNA--DNA copies of mRNA synthesized and cloned into plasmid vectors to form a cDNA library

Question 50

generating cDNA libraries by cloning mRNA in vectors

Answer 50

must separate mRNA from other RNA using poly A tails reverse transcriptase makes a DNA strand complementary to mRNA (opposite of transcription) restriction enzymes make sticky ends in ds cDNA and in plasmid vectors, then ligase joins them plasmid vectors transformed into E. coli

Question 51

how to screen DNA libraries for a clone of interest

Answer 51

detection with an oligonucleotide probe detect expressed protein

Question 52

hybridization

Answer 52

ability of complementary ssDNA or ssRNA molecules to zip up via base pairing

Question 53

using an oligonuleotide prode

Answer 53

DNA of interest is lysed and complementary oligonucleotide is added (radioactive or fluorescent) DNA hybridized, and autoradiography is performed to visualize

Question 54

functional complementation

Answer 54

screen a DNA library for a cloned gene that expresses a protein that complements a recessive mutation to fix it

Question 55

shuttle vector

Answer 55

a plasmid capable of replication in E. coli and yeast cells

Question 56

important parts of a shuttle vector

Answer 56

ARS-origin of DNA replication in yeast CEN-yeast centromere which separates plasmid during yeast cell division URA3-enzyme for uracil synthesis that marks a yeast mutant

Question 57

making shuttle vector yeast things

Answer 57

cut shuttle vector and ligate with yeast DNA transform into E. coli

Question 58

functional complementation screen process

Answer 58

transform yeast with a temp-sensitive mutant with shuttle vector remove uracil, so only colonies with plasmid URA3 survive incubate at nonpermissive temp so only colonies with wild-type gene survive

Question 59

gel electrophoresis allows separation of ____ DNA from cloned fragments

Answer 59

vector cut recombinant DNA clone with same restriction enzyme used to produce it

Question 60

gel electrophoresis

Answer 60

technique used to separate DNA of different sizes DNA is negative and moves toward positive electrode smaller molecules move faster

Question 61

subcloning

Answer 61

inserting fragmented vector DNA (after electrophoresis) into a plasmid vector and transformed into E. coli used to rearrange parts of genes into new, useful configurations

Question 62

example use of subcloning

Answer 62

replace the normal promoter with a segment of DNA containing a different promoter

Question 63

the polymerase chain reaction (PCR) amplifies a specific DNA sequence from a _______ ________

Answer 63

complex mixture

Question 64

to use PCR, must know...

Answer 64

the nucleotide sequences are the ends of a particular DNA region

Question 65

how does PCR work

Answer 65

denature dsDNA and hybridize complementary ssDNA in a controlled fashion uses high temp functioning enzyme, Taq DNA polymerase and 2 primers flanking sequence of interest at the end, you get a ton of copies of the DNA sequence of interest

Question 66

tagging genes by insertion mutations

Answer 66

PCR amplifies a tagged gene from the DNA of a mutant strain produce mutations by inserting a known DNA sequence into the genome of an experimental organism using mobile DNA elements

Question 67

tagging genes by insertion mutations example

Answer 67

use of modified fruit fly mobile DNA element, P element insertion of P element causes a mutation with an interesting phenotype, then PCR performed on sequences adjacent to insertion site avoid cloning large #s of DNA fragments and having to screen to detect the fragment for the mutated gene of interest

Question 68

whole genome shotgun sequencing

Answer 68

fastest and most cost-effective method for sequencing long stretches of DNA and most genomes (including human)

Question 69

how does whole genome shotgun sequencing work

Answer 69

sequencing random clones from a genomic library each segment sequences ~10 times segments assembled using a computer algorithm that aligns the sequences using regions of overlap