Genetics Exam 2

transposable elements (TEs)

transposable elements (TEs)

DNA segments that can move from one location to another

transpose

to move

transposition

nonhomologous recombination with insertion into DNA that has no sequence homology w/ the TEs

another position on the same chromosome, a plasmid, or a phage genome

(prokaryotes) a TE can move from one chromosome to

their transposition may affect the gene functions of the host

Why are TEs important to genetics?

affects the function of a gene (usually not good)

What does inserting into the coding sequence of a gene do?

affect its gene expression pattern (increase or decrease)

What does inserting into the promoter region of a gene do?

a high rate would kill the cell

why is transposition frequency usually low?

insertion sequences (IS)

the simplest TE found in prokaryotes

transposase

the gene for mobilization and insertion of DNA

inverted repeats (IR)

(define the borders) identical sequence at the terminals, in opposite orientations

transposons (Tn)

similar to IS, but carry additional genes (in addition to transposase), and are more complex, produce target site duplications (makes staggered cut at host DNA)

composite transposons, noncomposite transposons

what are the two types of prokaryotic transposons?

composite transposons

carry genes flanked on both sides by IS elements (ISL and ISR), which may be in direct or inverted orientation to each other. transposition of ___ results from the IS elements, which supply transposase and its recognition signals

noncomposite transposons

cary genes but do not terminate w/ IS elements, transposition enzymes (transposase and resolvase) are encoded in the central

transposase (enzyme)

catalyzes insertion of a Tn into new sites

resolvase

promotes a recombination (DNA exchange) event associated w/ transposition

by whether the element transposes via and RNA intermediate

How are the two classes of TE's in eukaryotes distinguished?

class I

(retroelements) an RNA intermediate is made from this element via txn, DNA is made from the RNA (via reverse rxn), DNA is then inserted into the target site and they remain permanently once inserted into the target (the copy # is big)

class II

(DNA elements) move directly from the donor site to the target site (can excise from the donor site and reverse the mutation)

Ty elements

(class I) encodes 2 proteins and contains 2 repeats (gag and pol, long terminal repeats)

gag

a structural protein that packages the RNA intermediate into a virus-like particle

pol

responsible for reverse txn and integration

long terminal repeats (LTR)

each contains a promoter and sequences recognized by pol

retrotransposition

(class I) transposition via an RNA intermediate

LINEs (long interspersed nuclear elements)

(class I) contains genes required for transposition (autonomous), can cause disease by inserting into genes (insertional mutagenesis)

SINEs (short interspersed nuclear elements)

(class I) do not encode enzymes needed for transposition, requires LINEs for mobilization (nonautonomous), do not have long terminal repeats, can cause disease by inserting into genes (insertional mutagenesis)

Ac/Ds elements

activator/dissociation

Ac

4563 bp long, short Irs and a transposase, an autonomous element bc it can move by itself

Ds

generated from Ac by deletions, contain IRs but no transposase, mutated versions of Ac, incapable of moving by themselves (nonautonomous), require Ac and its transposase for mobilization

DNA cloning

mass production of identical copies of a segment of DNA, usually done by replication in a suitable host and allows scientists to generate large amounts of pure DNA that can be further manipulated and studied

applications of cloned DNA

mapping genes, sequencing, mutating, transforming cells, making proteins, studying its function

procedure of DNA cloning

- isolate DNA

- use REs to cut DNA into manageable fragments

- ligate fragments into cloning vector

- introduce recombinant DNA into a host

- the host will replicate the DNA and pass copies

vector

artificially constructed DNA molecule that can replicate in a host organism

recombinant DNA

foreign DNA + vector = _______

recombinant DNA

DNAs from different sources, often from different organisms

restriction enzymes

recognizes and cuts a specific DNA sequence, most sites have symmetrical sequence (it reads the same on the top strand as it does on the bottom one), breaks phosphodiester bonds at points of cleavage, some make staggered cuts and others make flush cuts, almost all are found in bacteria and protect the bacterial host against invading viral DNA, "restrict" the viral infection

RE naming

genus, species, strain (optional), identified order

4, 6, or 8-bp

Most common RE's recognize palindromes of

the # of times the recognition sequence occurs

the # of cuts a RE makes in any given DNA molecule depends on what?

short, larger

base on probability, a ____ DNA sequence occurs more frequently than a ___ DNA sequence

1/4

what is the probability of a specific base?

(1/4)^n (n=# of bp in the recognition sequence)

the frequency of a particular restriction site = ?

because complementary ssDNAs anneal and are held together so that ligase can covalently link them

why are sticky ends useful in cloning?

RE

cuts DNA into fragments, breaks phosphodiester bonds

ligase

joins DNA fragments, forms phosphodiester bonds

ligation/forming of 2 DNA fragments

the basis of recombinant DNA formation and molecular cloning

Yes, bc they lack complementary sequences and a higher concentration of DNA and ligase is required

is it harder for blunt ended DNA to be ligated together?

plasmids

naturally occurring extrachromosomal DNA elements that can replicate within cells, nonessential, double stranded, circular, much smaller than the main chromosome

vectors

artificially constructed DNA molecules that can replicate in a host organism, used for cloning, most are derived from genetically engineered plasmids

ori, a selectable marker, and MCS

what does each E. coli plasmid vector contain?

ori (origin of replication)

a sequence required for DNA replication in the cell (where DNA unwinds and synthesis begins)

selectable marker

a gene that gives the cell a selectable phenotype

MCS (multiple cloning site)

a region containing unique RE sites (unique means it cuts only once in the vector), where a foreign DNA is inserted

genomic library

a collection of clones containing at least one copy of every DNA sequence in a genome, used to identify a cloned DNA containing a gene/sequence of interest

- partially digest the genomic DNA w a RE

- collect the desired size of fragments for cloning

- ligate fragments to a cut vector

- transform bacteria

- plate on selective medium and allow colonies to grow

- pick individual colonies to microtiter plate w/ 96 wells w/ a liquid medium

How does one make a genomic library?

cDNA (complementary DNA) library

a cell's mRNA molecules can be copied to make this, uncut ____ can be cloned to create a library representing only the genes being expressed in the cells at that time, they represent only the protein-coding region of the genome, smaller than genomic libraries, easier to work with, useful if seeking a gene that is active in a specific type of tissue, usually done as a prelude to genomic cloning

polylinker

multiple cloning site (region of RE sites)

dideoxy sequencing

based on DNA polymerase extending short oligos

DNA sequencing procedure

- dsDNA is heat-denatured to ssDNA and short oligo anneals to one strand and serves as primer

- a reaction mix is set up w ssDNA template, radioactive oligo primer, DNA polymerase, 4 deoxynucleotide precursors

- the reaction mix is divided into 4 tubes, each tube gets a different dideoxynucleotide added

- DNA polymerase adds nucleotides to the primer

- the 4 reaction mixes are denatured and run in adjacent lanes on a polyacrylamide gel

- sequence is read from bottom to top (5' to 3')

automated DNA sequencing

only once reaction mix is needed that contains all 4 ddNTPs (each tagged w a different color), DNA fragments generated are separate by electrophoresis in a single lane, gel is scanned by a laser device that determines which label is present at each position, sends info to computer, ouput is a series of colored peaks corresponding to the identity/position of nucleotides in a sequence

manual sequencing

uses same radioactive label in primer for all 4 rxns --> need 4 lanes

PCR (polymerase chain reaction)

technique to amplify DNA, produces many copies of a specific DNA sequence from a DNA mixture without having to clone the sequence in a host organism

- requires a heat resistant (95°C) DNA polymerase and a thermal cycler that automatically changes temp and incubation time

in vitro

within glass

in vivo

within living

PCR procedure

- denature dsDNA to ssDNA-->separate DNA strands

- anneal the 2 primers to the ssDNA (60°C)

- extend primers w/ DNA polymerase at 72°C

- repeat cycle of denaturation and primer annealing

- repeat the extension cycle, products the length of the target sequence begin to be produced

- repeat cycle of denaturation and primer annealing

- repeat the extension cycle, doubling the amount of target DNA w/ each round

- in 30 cycles millionfold amplification of the target occurs

allows primers to anneal

what does cooler temp do?

- amplifying DNA for cloning/subcloning (amplifying a mutant gene or amplifying a human gene using mouse primers)

- amplifying DNA from genomic DNA for sequencing w/o cloning

- disease diagnosis

- forensics in samples including trace amt of hair, blood, or semen

why is PCR useful?

related species have similar sequences

why can you use a mouse primer to amplify a human gene?

forensics

the analysis of legal evidence

limitations of PCR

- specific primers require a sequence info to be known

- can only amplify up to ~40 Kb

identifying genes

- open reading frame (ORF)

- promoters, kozak, poly(A) signal

- intron consensus

- model aided by full-length and partial cDNA sequences

BLAST (basic local alignment search tool)

online program allowing user to find homology between gene/protein of interest and database sequences, matches are often found in small stretches, similar proteins/domains often share the same functions

limits to BLAST

often genes have no matches or matches have no known function

experimental assigning of gene function

- obtain mutant via gene knockout (can't KO human, only lower organisms)

- study mutant phenotype (can't KO essential genes, silence them by RNAi instead)

transcriptomics

whole-genome study of gene expression

- microarray or

- sequencing of all RNA transcripts

gene expression

when/what/how much genes are expressed globally

proteomics

global study of protein expression and function

- expression

- modifications or

- protein-protein interaction

by only the plate # and the well #, no other info on a particular clone unless the entire genome has been sequenced

How is each clone in a DNA library specified?

screening expressed sequences, looking for a gene that's active in a specific type of tissue, when you've already isolated the protein of interest and have raised an antibody against that protein

when is a cDNA library useful?

cDNA screening

- construct a cDNA library w/ an expression vector

- cDNA library is made from the recombinant DNA vectors using transformed bacteria and microtiter plates

- library replicas are printed on membrane filter, then grown on the filter

- cells are lysed "in situ" w/ proteins bound to filter

- filter is incubated w/ radioactively labeled antibody and placed on xray filmfor 1-12 hrs

- colonies w/ antibody bound will emit radioactive atoms and will be visible as dark spots on the film

- once identified, a cDNA clone can be used to make radioactive ssDNA (probe) and isolate the complete gene from a genomic library

expression vector

a vector that allows expression (txn & tln), contains all the necessary sequences for expression

in situ

in place, in their original positions

screening a genomic library

- similar to screening a cDNA library (instead the filter is incubated w/ labeled ssDNA which forms hybrids w/ complementary ssDNA molecules bound to the filter)

- if a clone contains a DNA that binds to the radioactive DNA probe, it's detected by an autoradiogram

when part of the DNA sequence is known, radioactive ssDNA made from a cDNA clone will recognize a genomic DNA clone

why is genomic library screen useful?

identifying DNA in libraries using heterologous probes

- probe made from a cloned equivalent gene from another organism

-if the gene is highly conserved or the species is closely related, the probe should be able to anneal the gene of interest

heterologous

from a different species

identifying DNA libraries using oligonucleotide probes

- if a protein has been purified, and some of the aa sequence are known, deduce DNA sequence from the aa sequence

- synthetic ssDNA can be made, labeled, and used as a probe for library screening

- degeneracy in genetic code--> mixture of oligonucleotides must be prepared--> may detect false positives and correct clone, try to pick the least degenerated region

identifying genes by complementation of mutations

- well defined mutants may be used to clone genes by complementation, in which cloned genes overcome a defect in the mutant

- a genomic library is made from the WT yeast strain

- the library is transformed into a yeast strain w/ 2 mutations, one to allow selection of vector-containing transformants, the other a mutation in the gene for which the WT copy is sought

- only those containing the arg1+ clone can grow on minimal medium

restriction mapping

determines the order and distances of RE cut sites in a segment of DNA

method of restriction mapping

cloned DNA can be cut w/ various RE's, electrophoresed on an agarose gel and visualized w/ EtBr

procedure of restriction mapping

- starting material: many copies of DNA molecule

- do a series of single & double RE digests on the DNA

- make a gel in an electrophoresis casting tray

- each RE digest mixture is loaded in a lane of the gel

- DNA is stained w/ EtBr, which binds to DNA & fluoresces under UV light

- the gel is photographed, and the size of each fragment is determined by comparison w the marker fragments

subcloning and confirming results of a cloning experiment

what is restriction mapping useful for?

subcloning

moving a segment of a clone to another vector

ensure the resulting plasmid contains the desired DNA in the proper orientation

how does one confirm the results of a cloning experiment?

southern blot

- used to determine the location of restriction sites in a genome region of interest, useful for cloning

- DNA fragments are transferred from a gel to a filter and then hybridized with a probe

southern blot procedure

- genomic DNA samples are cut w different REs

- each sample is electrophoresed in a lane of agarose gel

- DNA is denatured to single strands, and DNA is transferred to a membrane filter, paper towels soak up buffer and DNA move w the buffer from the gel onto the filter (where they stick)

- the DNA fragments on the filter are arranged just as they were in the gel

- labeled probe is added to the filter, where it will hybridize w/ any complementary DNA fragments that were on the original gel

- analyze results

western blot

detects protein

eastern blot

detects protein modification

-determining the copy # of a gene in a genome

-comparing homologous genes in different species

- screening individuals for a disease gene which is associated w a RE cutting pattern

-etc

why is southern blot useful?