Molecular Bio. Exam 2

clone

a molecule, cell, or organism that was produced from another single entity

what made cloning possible?

1. restriction enzyme (endonuclease)

2. plasmid DNA vectors

endonuclease (restriction enzyme)

• DNA cutting enzymes (molecular scissors) by cleaving phosophodiester bonds that join adjacent nucleotides in a DNA strand

• primarily found in bacteria (and in some archaea)

• binds, recognizes, and cuts specific DNA sequences (restriction site)

  • each restriction site is a palindrome

• 4-6 bp cutters since they recognize restriction sites with a sequence of 4-6 nucleotides

plasmid DNA vectors

• circular form of self-replicating DNA; can be manipulated to carry and clone other pieces of DNA

• are extrachromosomal; present in the bacterial cytoplasm in addition to the main bacterial chromosome

• small, 1-4 bp in size and self replicating

• typically high copy (40-50 in the bacterial cell)

vectors

pieces of DNA that can accept, carry, and replicate other pieces of DNA

W. Arber et al

• proposed that restricted phage growth occured because bacteria contained enzymes that could cut viral DNA

• bacteria lack immune systems, but these enzymes provide protective mechanism

H. Smith

• isolated and characterized the first restriction enzyme, HindIII (isolated from Haemophilius influenzae)

• took enzymes from bacteria that could cut viral, plasmid DNA

What is the purpose of restriction enzymes in bacteria? (native use)

• act as their immune system by protecting them from bacteriophages by restricting phage replication

• the methylated regions of bacterial DNA prevent the restriction enzymes from “cutting” / self-digesting its own chromosomal DNA

  • phages do not methylate their DNA, so they are susceptible to degradation by endonuclease (although some have evolved to methylate their DNA)

Sticky and blunt ends

• restriction enzymes cut DNA create ends that are sticky or blunt

• sticky (cohesive) ends are overhanging single stranded ends

• blunt ends are double stranded ends

Which enzymes are preferred for cloning, those that create blunt ends or sticky ends

sticky ends are preferred for cloning since DNA fragments with sticky ends can be easily joined together because they base pair with each other by forming weak hydrogen bonds

Berg

• founded recombinant DNA technology by joining together DNA from E. coli + DNA from a primate virus, SV40

• first, isolated chromosomal DNA from E. coli and DNA from SV40

• next, cut both with EcoRI

• then, added ligase to the mixture of digested DNAs (binds fragments)

• finally, created a hybrid molecule of DNA (recombinant)

Cohen

• focused on small circular pieces of DNA called plasmids

• focused on replication, transfer, and resistance to antibiotics

• worked with Boyer to develop the first plasmid vector for cloning purposes (pSC101)

what is the gold standard plasmid backbone

PUC18

steps of creating a recombinant molecule

1. restriction enzyme cuts double stranded DNA at restriction sequences

2. DNA fragments with cohesive ends produced

3. two fragments of DNA cut with the same endonuclease come together by base pairing

4. joined fragments form linear or circular molecule

5. ligase unites backbone to form recombinant DNA

RAC (recombinant DNA advisory committee)

evaluate recombinant technology and establish guidelines for research

effective transformation process

• Cohen et al

• inserting foreign DNA into bacteria

electroporation

apply brief pulse of high voltage electricity to create tiny holes in the bacteria wall that allow the DNA to enter

transformation

the process by which a bacterial cell takes up foreign DNA (plasmid) from its surrounding

chemical transformation of bacterial cells

1. bacterial cells treated with calcium chloride (calcium chloride will reduce repulsion between DNA and bacterial surface)

2. plasmid DNA added while cells are chilled on ice

3. mixture is heat shocked (heat shock helps DNA cross bacterial envelope)

4. plasmid DNA enters bacterial cells and is replicated and express their genes

What is chemical transformation inefficient

• only a small amount of the bacterial cells take up plasmid DNA

• even among cells exposed to plasmids, not every cell successfully takes one up

• some cells that take up DNA may not survive or maintain the plasmid

electroporation

a brief pulse of high voltage electricity used to create tiny holes in the bacteria cell that allow the DNA to enter; inefficient, but more efficient than chemical transformation

selection of recombinant bacteria

• selection - facilitates the identification of recombinant bacteria while preventing the growth of nontransformed bacteria and bacteria that contain plasmid without foreign DNA

• antibiotic selection (transformant selection) - plate transformed cells on plates containing different antibiotics to identify recombinant bacteria and non transformed bacteria

recombinant vs transformant

• transformant - any host cell that has taken up external (exogenous) DNA from the environment

• recombinant - cell or molecular that contains DNA from two or more different sources (such as a vector carrying a foreign gene of DNA)

antibiotic selection

• method of selecting recombinants

• transformed cells are put on a plate containing different antibiotics to identify recombinant bacteria and non-transformed bacteria

  • cloning vectors typically carry an antibiotic resistance gene, so only bacteria that took up the plasmid will survive and grow (which will also remove nontransformants)

• the antibiotic resistance is a selection marker to identify transformants markers

Blue White Screening

• method of selecting recombinants

• DNA is cloned into the restriction site in the lacZ gene (when its prevented by an inserted gene, the lacZ gene cannot produce functional beta gal)

• When X gal (artifical lactose) is added to the plate, if functional lacZ is present = blue colony

• Mon-functional lacZ = white colongy = clone = genetically identical bacterial cells each containing copies of recomb. plasmid

what was the first human protein expressed via recombinant techniques

insulin

how was the first human protein, insulin, expressed

clone human insulin DNA sequence into a plasmid and the bacteria cells were then used to synthesize the protein product of the cloned gene; able to generate alot of pure protein with this technique

what was the second recombinant protein produced

hHG (human growth hormone); clinically used if insufficient growth hormone

can all human proteins be cloned in E. coli?

no, larger / complex human proteins would not be good due to post translational modifications

practical features of DNA cloning vectors

• size: small enough to be separated from chromosomal of host bacteria (most often E. coli)

• origin of origin of replication (ori) - this site for DNA replication allows plasmids to replicate independently of host chromosome

• high copy number associated with plasmid - normal numbers of most endogenous plasmids are relatively low (<12). most desirable cloning plasmids are high copy number

promoter

needed for induction of expression

multiple cloning site (MCS)

• where plasmid is cleaved by endonuclease

• polylinker site; stretch of DNA with recognition sequences for numerous restriction enzymes

  • engineered into the plasmid so that digestion of the plasmid with RE does not result in loss of a fragment of DNA, but instead the circular plasmid becomes linearized with cut

  • MCD provides great flexibility in the range of DNA fragments that can be cloned into a plasmid because it is possible to insert fragments generated cy cutting with different enzymes

draw a vector with vector components

selectable marker genes

• genes allow for the selection and identification of bacteria that have been transformed with a recombinant plasmid, when compared to non-transformed cells. some of the most common include, ampicillan resistance (amp) and tetracycline resistance (tet)

• also selects for recombinants also possible using selection marker, such as lacZ gene

RNA polymerase promoter sequence

• used for RNA transcription in vivo and in vitro by RNA polymerase

• in vivo this allows cells to make RNA from cloned genes, thus the expression of the protein

• in vitro the transcribed RNA can be used to syntehsized RNA “probes” to aid in study of gene expression

DNA sequencing primer sequences

these sequences permit nucleotide sequencing of cloned DNA fragments that have inserted into the plasmid

TOPO cloning

• method that allows the insertion of a gene into a vector without the need for restriction enzymes or ligases

• uses the topoisomerase I enzymes for ligation

  • topoisomerase I is bound to the vector; function in place of ligase, allowing the insertion / connection of DNA fragments

• used almost exclusively for cloning of PCR products

Advantage of TOPO cloning

• speed of simplicity

• high efficiency

• no need for restriction enzymes digestion

ccdB gene for cloning

• a gene encoding a toxin that inhibits bacterial DNA gyrase

• when incorporated into a cloning vector, it becomes part of the negative selection (of recombinants) process

• is a poison / toxin that motivates the E. coli to keep and maintain the vector

• empty vectors express the gene and those cells are killed; insertion of exogenous DNA results in disruption of the gene, and plasmids are no longer lethal

Function of ccdB in the TOPO system

• the presence of ccdB gene that only bacteria with recombinant plasmids survive (no empty vectors)

• insertion of a gene into the vector interrupts ccdB gene (insertion mutation that inhibits expression), thus preventing toxicity

TOPO cloning with ccdB gene

• Plasmid vector - contains the ccDB gene and the TOPO cloning site

• steps in the process:

  • insert preparation - amplify / cut / synthesize the gene of interest with compatible ends

  • topoisomerase reaction - the gene is inserted into the TOPO vector; which possesses the attached topoisomerase enzyme

  • transformation - the recombinant plasmid is introduced into E. coli cells

  • selection - only cells with an insert (which disrupts ccdB expression) will survive

  • colony growth - clones with the recombinant plasmid are selected fro further analysis

advantage of using ccdB and TOPO vector system

• speed: no need for restriction renzyme digestion or ligation steps

• higher efficiency: direct ligation of insert into vector increases the cloning success rate

• reduced background: negative selection using ccdB gene helps reduce false positives

positive selection

only bacteria that have incorporated the plasmid (with or without the insert) will survive due to antibiotic resistance gene

negative selection (ccdB)

if the plasmid does not contain an insert (or incorrect insert), the ccdB gene will be expressed, and bacterial growth will be inhibited

How to identify recombinants of interests

• molecular cloning requires some method of screening recombinant colonies

• traditionally this was accomplished via restriction enzyme digestion

the steps of colony PCR

1. use / design primers to detect the presence of your insert

2. set up a standard PCR reaction (primers, dNTPs, polymerase) using the supernatant of lysed bacteria (plasmid should be present) as template

3. analyze PCR products (amplicons) on a gel to analyze product size

colony PCR

fast and cost effective technique used in molecular biology to verify whether a cloned DNA fragment has been correctly inserted into a vector

Process of colony PCR

1. colony picking: a colony (conformed recombinant) is selected from the plate

2. cell lysis: picked colony is added directly to PCR mixture; initial heating step used to lyse cells and release the plasmid

3. PCR amplification: primers are designed to flank the region of interest (inserted gene). these primers help amplify the target sequence if the insert is present in the plasmid

4. analysis of results: products are analyzed via agarose gel electrophoresis to confirm the size of the amplified fragment. if the insert is present, you will observe a band at the expected size

how are recombinants of interest identified

• vector-specific primers

• insert (target gene) - specific primers

• a combination of those listed above can be used to determine the specific orientation of the insert

gene

• a segment of DNA used to make a functional product; the functional product is either RNA or polypeptide

• first step of gene expression is transcription

transcription

• transcribing nucleic acids of DNA to nucleic acids of RNA

• the copying of a double stranded DNA sequence into a single stranded RNA sequence

structural genes

• encode the amino acid sequence of a polypeptide (primary structure of protein)

• are the blue print of functional RNAs (mRNA)

• the synthesis of functional proteins determine an organisms traits

central dogma

the path from gene to trait; DNA to RNA to protein

what defines the beginning and end of a gene and regulates the level of RNA synthesis

DNA base sequences

gene expression

the overall process by which the information within a gene is used to produce a functional product which can, in concert with environmental factors, determine a trait

template strand

the DNA strand that is transcribed into RNA

coding strand

strand carrying the code that is being conveyed

transcription factors

recognize promoter and reguluatory sequences and help control transcription

codons

specifcy the amino acid sequence of the protein

ribosome binding site

in bacteria, position the ribosome for translation

the 3 stages of transcription

initiation, elongation, termination

RNA transcript

single stranded RNA molecule produced when RNA pol copies a DNA sequence

RNA transcripts from —- genes are not translates

nonstructural

promoters

• sequences that promote gene expression

• direct the exact location for the initiation of transcription

• consensus sequence

• most promoter region is labeled with negative numbers (there is no base numbered 0), while the bases to the right are numbered in a positive direction (+1 is the 1st base transcribed)

TATA box (pribnow box)

• specific short sequence element that is cirtically important for promoter recognition

• noncoding area in the promoter region between -35 and -10

• the most commonly occurring bases

why are their differences in the promoter sequence between organisms / bacterial genes

• the differences in the promoter sequence allow for specificity

• to identify different promoters but still allow machinery to identify

RNA polymerase

• the enzyme that catalyzes the synthesis of RNA; is held onto the strand by beta prime

RNA polymerase holo enzyme

• two parts: core enzyme and sigma factor

• core enzyme; five subunits

  • a2 and a1 hold subunits together

• sigma factor; gives affinity to different promoters

• will assemble at promoter region

initiation

• RNA polymerase holoenzyme binds loosely to the DNA

• scans along DNA until encountering a promoter region

  • recognizes the -35 to -10 regions

• binding of the RNA polymerase to the promoter forms the closed complex

• the open complex is formed when the TATA box in the -10 region is unwounds

• a short RNA strand is made and the sigma factor is released and marks the ends of the initiation

elongation

• the core enzyme now slides down the DNA to synthesize an RNA strand

• the open complex formed by the action of RNA pol is about 17 bases long; behind the open complex, the DNA rewinds back into a double helix

• RNA transcript is synthesized using the DNA strand as a template

  • the DNA strand used is the template or antisense strand

  • the opposite DNA strand is the coding strand

• on avg. the rate of RNA synthesis is about 43 nucleotides per second

termination

• the end of RNA synthesis

• the short RNA-DNA hybrid of the open complex is forced to separate, which releases the newly made RNA as well as the RNA pol

• E. coli has 2 different mechanisms for termination: rho-dependent and rho-indepdent

rho-dependent termination

• requires a protein known a rho, an ATP dependent RNA helicase

• Rho binds to a specific C-rich, G-poor sequence on the newly synthesized RNA called a rut (rho utilization) site

• Rho moves along the RNA in the 5’→ 3’ direction, using ATP to power its movement

• when RNA pol pauses at a termination region, Rho catches up to the pol

• Rho unwinds the RNA-DNA causing the newly synthesized RNA to be released

• transcription terminates and RNA pol dissociated from the DNA

rho-independent

• does not require rho protein or another termination factor

• the DNA sequences that produce an RNA with inverted repeats, followed by a region reich in adenine bases

• this inverted repeats cause the newly synthesized RNA to fold into a stable stem-loop (hairpin) structure

• the RNA pol pauses when it reaches the hairpin

• the weak A-U pairs between the RNA and DNA template destabilize the RNA-DNA hybrid

• the newly synthesizes RNA dissociates from the DNA, and RNA polymerase is released

transcription in eukaryotes

• many of the basic features of gene transcription are similar in bacteria and eukaryotes

• eukaryotic transcription is considerably more complex due to:

  • eukaryotic cells are large and structurally complex

  • requires more genes / elaborate patterns of gene expression

  • multicellularity adds another level of regulation

    • different cell types express different sets of genes

    • genes must be expressed in the correct cells at the appropriate time

Eukaryotic polymerases

• nuclear DNA is transcribed by three different RNA polymerases

• RNA pol I - trancribes all rRNA genes (except for 5S and rRNA)

• RNA pol II - transcribes all structural genes (thus synthesizes all mRNAs); transcribes some snRNA genes

• RNA pol III - transcribes all tRNA genes and the 5S rRNA gene

eukaryotic promoter sequences

• more variable and often more complex than those of bacteria

• for structural genes: regulatory elements, TATA box, transcriptional start site

• core promoter - relatively short and consists of the TATA box and transcriptional start site