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Cellular and Molecular Biology > Exam 2: Ch 5 DNA Clones and Recombination > Flashcards

Exam 2: Ch 5 DNA Clones and Recombination Flashcards

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1
Q

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

A

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

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2
Q

3 molecular genetic tools to answer the 3 questions

A

the gene that encodes the protein

a mutant cell line or organism that lacks the protein

source of the purified protein for biochemical studies

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3
Q

classical genetics

A

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

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4
Q

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

A

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

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5
Q

allele

A

naturally occurring alternate versions of a gene

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6
Q

mutagen

A

an agent that causes a heritable change in DNA sequence

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7
Q

genotype

A

particular set of alleles for all genes carried by an individual

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8
Q

wild-type

A

normal non-mutant allele

standard genotype

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9
Q

phenotype

A

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

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10
Q

saccharomyces cervisiae can exist in either _____ or _____ states

A

haploid, diploid

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11
Q

recessive alleles cause disorders why

A

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

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12
Q

dominant alleles cause disorders why

A

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

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13
Q

haplo-insufficient

A

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

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14
Q

commonly used mutagen

A

ethylmethane sulfonate EMS

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

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15
Q

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

A

DNA replication, cell divisions

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16
Q

how to avoid complexity in breeding experiments

A

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

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

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17
Q

if F1 progeny exhibit the mutant trait…

A

the mutant allele is dominant

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18
Q

if F1 progeny are wild-type…

A

the mutant allele is recessive

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19
Q

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

A

cross between haploid cells (a/alpha)

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

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20
Q

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

A

the cells undergo meiosis

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

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21
Q

genetic screens

A

procedures used to identify and isolate mutants

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

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22
Q

temperature sensitive mutations

A

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

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23
Q

permissive vs. nonpermissive

A

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

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24
Q

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

A

gene

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25
Q

genetic complementation

A

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

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26
Q

double mutants are useful in assessing…

A

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

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27
Q

ordering of biosynthetic pathway

A

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

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28
Q

ordering of signaling pathways

A

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

29
Q

suppressor mutations

A

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

30
Q

synthetic lethal mutations

A

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)

31
Q

genetic mapping

A

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

32
Q

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

A

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

33
Q

DNA cloning

A

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

34
Q

what is recombinant DNA

A

any DNA molecule composed of sequences derived from different sources

35
Q

DNA cloning process

A

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

36
Q

DNA library

A

a collection of DNA molecules each cloned into a vector molecule

37
Q

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

A

restriction enzymes

DNA ligases

both help produce recombinant DNA

38
Q

restriction enzyme

A

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

39
Q

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

A

modification enzyme

adds a methyl to prevent cleavage by restriction enzyme

40
Q

restriction enzyme mechanism

A

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

41
Q

inserting DNA fragments into vectors

A

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

42
Q

plasmid

A

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

43
Q

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

A

parasitic, symbiotic

plasmid DNA duplicated with each cell division like chromosomal DNA

44
Q

E. coli plasmids are used as ______

A

vectors

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

45
Q

plasmid transformation

A

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

46
Q

polylinker

A

synthetically generated sequence containing many different restriction sites

47
Q

genomic library

A

set of clones representing all DNA sequence in the genome

good for simple organisms, too complicated to represent complex organisms

48
Q

what percent of human genome represents protein coding DNA?

A

1.5%

49
Q

cDNA

A

complementary DNA–DNA copies of mRNA

synthesized and cloned into plasmid vectors to form a cDNA library

50
Q

generating cDNA libraries by cloning mRNA in vectors

A

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

51
Q

how to screen DNA libraries for a clone of interest

A

detection with an oligonucleotide probe

detect expressed protein

52
Q

hybridization

A

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

53
Q

using an oligonuleotide prode

A

DNA of interest is lysed and complementary oligonucleotide is added (radioactive or fluorescent)

DNA hybridized, and autoradiography is performed to visualize

54
Q

functional complementation

A

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

55
Q

shuttle vector

A

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

56
Q

important parts of a shuttle vector

A

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

57
Q

making shuttle vector yeast things

A

cut shuttle vector and ligate with yeast DNA

transform into E. coli

58
Q

functional complementation screen process

A

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

59
Q

gel electrophoresis allows separation of ____ DNA from cloned fragments

A

vector

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

60
Q

gel electrophoresis

A

technique used to separate DNA of different sizes

DNA is negative and moves toward positive electrode

smaller molecules move faster

61
Q

subcloning

A

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

62
Q

example use of subcloning

A

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

63
Q

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

A

complex mixture

64
Q

to use PCR, must know…

A

the nucleotide sequences are the ends of a particular DNA region

65
Q

how does PCR work

A

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

66
Q

tagging genes by insertion mutations

A

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

67
Q

tagging genes by insertion mutations example

A

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

68
Q

whole genome shotgun sequencing

A

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

69
Q

how does whole genome shotgun sequencing work

A

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

Cellular and Molecular Biology (34 decks)

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