Exam 2: Ch 5 DNA Clones and Recombination Flashcards
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
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
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
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
allele
naturally occurring alternate versions of a gene
mutagen
an agent that causes a heritable change in DNA sequence
genotype
particular set of alleles for all genes carried by an individual
wild-type
normal non-mutant allele
standard genotype
phenotype
all the physical attributes or traits of an individual that are a consequence of the genotype
saccharomyces cervisiae can exist in either _____ or _____ states
haploid, diploid
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
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
haplo-insufficient
dominant mutation that causes a loss of function because not enough gene product is made
commonly used mutagen
ethylmethane sulfonate EMS
chemically modifies guanine bases in DNA leading to conversion of GC to AT
meiosis consists of one round of _____ ____ followed by two separate _____ ________
DNA replication, cell divisions
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
if F1 progeny exhibit the mutant trait…
the mutant allele is dominant
if F1 progeny are wild-type…
the mutant allele is recessive
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
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
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
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
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
complementation tests determine whether different recessive mutations are in the same ______
gene
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
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
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
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
suppressor mutations
a mutation in 1 protein is suppressed by a second mutation in a different protein
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)
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
____ recombination occurs between two genes on the same chromosome the closer together they are
less, they are linked (same chromosome and close together)
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
what is recombinant DNA
any DNA molecule composed of sequences derived from different sources
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
DNA library
a collection of DNA molecules each cloned into a vector molecule
which two enzymes allow insertion of DNA fragments into cloning vectors?
restriction enzymes
DNA ligases
both help produce recombinant DNA
restriction enzyme
endonuclease that recognizes restriction sites (4-8bp) that are commonly palindromic and cuts the DNA at these sites
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
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
inserting DNA fragments into vectors
DNA fragments with either sticky or blunt ends can be inserted into complementary vector DNA by DNA ligases
plasmid
circular dsDNA separate from chromosomal DNA in bacteria and lower eukaryotes like yeast
plasmids exist in a ____ or ______ relationship with their host cell
parasitic, symbiotic
plasmid DNA duplicated with each cell division like chromosomal DNA
E. coli plasmids are used as ______
vectors
replication initiated at ORI (replication origin) and inserted DNA is replicated too
plasmid transformation
when E. coli cells take up the plasmid containing recombinant DNA
polylinker
synthetically generated sequence containing many different restriction sites
genomic library
set of clones representing all DNA sequence in the genome
good for simple organisms, too complicated to represent complex organisms
what percent of human genome represents protein coding DNA?
1.5%
cDNA
complementary DNA–DNA copies of mRNA
synthesized and cloned into plasmid vectors to form a cDNA library
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
how to screen DNA libraries for a clone of interest
detection with an oligonucleotide probe
detect expressed protein
hybridization
ability of complementary ssDNA or ssRNA molecules to zip up via base pairing
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
functional complementation
screen a DNA library for a cloned gene that expresses a protein that complements a recessive mutation to fix it
shuttle vector
a plasmid capable of replication in E. coli and yeast cells
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
making shuttle vector yeast things
cut shuttle vector and ligate with yeast DNA
transform into E. coli
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
gel electrophoresis allows separation of ____ DNA from cloned fragments
vector
cut recombinant DNA clone with same restriction enzyme used to produce it
gel electrophoresis
technique used to separate DNA of different sizes
DNA is negative and moves toward positive electrode
smaller molecules move faster
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
example use of subcloning
replace the normal promoter with a segment of DNA containing a different promoter
the polymerase chain reaction (PCR) amplifies a specific DNA sequence from a _______ ________
complex mixture
to use PCR, must know…
the nucleotide sequences are the ends of a particular DNA region
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
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
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
whole genome shotgun sequencing
fastest and most cost-effective method for sequencing long stretches of DNA and most genomes (including human)
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
