Exam 2: Ch 5 DNA Clones and Recombination Flashcards Preview

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Flashcards in Exam 2: Ch 5 DNA Clones and Recombination Deck (69):
1

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

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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3 molecular genetic tools to answer the 3 questions

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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classical genetics

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

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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allele

naturally occurring alternate versions of a gene

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mutagen

an agent that causes a heritable change in DNA sequence

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genotype

particular set of alleles for all genes carried by an individual

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wild-type

normal non-mutant allele

standard genotype

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phenotype

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

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saccharomyces cervisiae can exist in either _____ or _____ states

haploid, diploid

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recessive alleles cause disorders why

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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dominant alleles cause disorders why

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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haplo-insufficient

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

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commonly used mutagen

ethylmethane sulfonate EMS

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

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

DNA replication, cell divisions

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how to avoid complexity in breeding experiments

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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if F1 progeny exhibit the mutant trait...

the mutant allele is dominant

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if F1 progeny are wild-type...

the mutant allele is recessive

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

cross between haploid cells (a/alpha)

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

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

the cells undergo meiosis

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

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genetic screens

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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temperature sensitive mutations

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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permissive vs. nonpermissive

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

gene

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genetic complementation

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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double mutants are useful in assessing...

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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ordering of biosynthetic pathway

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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ordering of signaling pathways

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

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suppressor mutations

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

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synthetic lethal mutations

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)

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genetic mapping

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

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

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

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DNA cloning

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

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what is recombinant DNA

any DNA molecule composed of sequences derived from different sources

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DNA cloning process

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

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DNA library

a collection of DNA molecules each cloned into a vector molecule

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

restriction enzymes

DNA ligases

both help produce recombinant DNA

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restriction enzyme

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

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

modification enzyme

adds a methyl to prevent cleavage by restriction enzyme

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restriction enzyme mechanism

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

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inserting DNA fragments into vectors

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

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plasmid

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

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

parasitic, symbiotic

plasmid DNA duplicated with each cell division like chromosomal DNA

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E. coli plasmids are used as ______

vectors

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

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plasmid transformation

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

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polylinker

synthetically generated sequence containing many different restriction sites

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genomic library

set of clones representing all DNA sequence in the genome

good for simple organisms, too complicated to represent complex organisms

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what percent of human genome represents protein coding DNA?

1.5%

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cDNA

complementary DNA--DNA copies of mRNA

synthesized and cloned into plasmid vectors to form a cDNA library

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generating cDNA libraries by cloning mRNA in vectors

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

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how to screen DNA libraries for a clone of interest

detection with an oligonucleotide probe

detect expressed protein

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hybridization

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

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using an oligonuleotide prode

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

DNA hybridized, and autoradiography is performed to visualize

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functional complementation

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

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shuttle vector

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

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important parts of a shuttle vector

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

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making shuttle vector yeast things

cut shuttle vector and ligate with yeast DNA

transform into E. coli

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functional complementation screen process

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

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gel electrophoresis allows separation of ____ DNA from cloned fragments

vector

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

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gel electrophoresis

technique used to separate DNA of different sizes

DNA is negative and moves toward positive electrode

smaller molecules move faster

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subcloning

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

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example use of subcloning

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

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

complex mixture

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to use PCR, must know...

the nucleotide sequences are the ends of a particular DNA region

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how does PCR work

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

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tagging genes by insertion mutations

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

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tagging genes by insertion mutations example

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

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whole genome shotgun sequencing

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

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how does whole genome shotgun sequencing work

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