Mobile Genetic Elements + DNA Techniques

plasmids

extra-chromosomal circular double-stranded DNA found in bacteria, yeast, and fungi - aka vectors - small: less than 10, 000 bp - typically 2-50 copies per cell - have a single origin of replication (ori)--sequence which allows initiation of replication - often encode functions that impart a selective advantage to the host--can carry genes encoding antibiotic resistance, toxins, catabolism of unusual substrates - replicate using host machinery (independent of the chromosome)--normally do not integrate into the host genome - lack a protein coat and generally cannot move independently from cell to cell - can move from one cell to another through conjugation using an F pilus - can be used as vehicles/vectors--genes can be inserted into plasmids and amplified in bacterial; proteins an be expressed for research, pharmaceutical, industrial purposes

transduction

injection of forge in DNA by bacteriophage--phage genome may contain DNA from another bacterium

DNA purification

step 1: grow, isolate and lyse cells step 2: pellet cellular debris by centrifugation step 3: remove protein (perception with ammonium sulphate or TCA and centrifuge; or extract DNA with phenol) step 4: concentrate DNA by ethanol precipitation (DNA is insoluble in ethanol) or bind it to a matrix in column/tube (commercially available kits, most common approach) step 5: treat with ribonuclease to remove RNA - results in pure DNA but too many base pairs--usually want to study short fragments encoding proteins (genes)

insertional inactivation

the inserted DNA inactivates a gene in the vector by inserting into that gene (use antibiotic resistance genes) - allows cells which contain the recombinant DNA molecule to be identified.

sticky ends

the uneven ends of a double-stranded DNA molecule that has been cut with a restriction enzyme

PCR and SARS-CoV2

- RT-PCR is used to diagnose COVID-19 - genome is single stranded RNA--must first convert to DNA using viral reverse transcriptase (RT) enzyme and ddNTPs - RNA is the template, RT is there polymerase, dNTPs are added to make DNA - RT-PCRs use primers for the SARS-CoV2 genome; apply these to saliva or nasopharyngeal swap samples and the perform RT-PCR - more PT-PCR cycles needed to detect viral DNA, the lower the viral titre - only detects viral DNA, not infectious viral particles - treat it with fluorescence--more quickly fluorescence increases over threshold = more virus

restriction site

- a specific sequence on a DNA strand that is recognized as a cut-site by a restriction enzyme - can insert gene of interest contained between restriction sites into plasmids - usually palindromic--have the same 5'-3' sequence on both strands--cut at the same place on both strands - always has a 2-fold symmetry axis

recombinant vector

- a vector that contains an inserted fragment of DNA, such as a gene from a chromosome - ligase is used to join plasmid and. ligated fragment

bacterial vs. viral DNA

- bacterial DNA is methylated by a bacterial methyl's like methyltransferase (MTase)--methylation identifies DNA as bacterial DNA--will not be cleaved by bacterial RE; whereas, non-methylated foreign DNA (e.g., phage DNA) will be degraded

insertional inactivation using phenotypic markers

- can screen bacterial colonies looking for a phenotype--usually colour - insert the gene of interest into a gene that results in a colour change (e.g., lacZ gene forms beta-galactosidase which degrades the sugar X-gal to produce a blue product) - vector mixture is transformed into bacteria and grown on nutrient agar plates containing ampicillin and X-gal - only those cells carrying the vector will grow on amp (are ampR)--antibiotic selection - cells appear as colonies (started from a single cell)--thus are clones - colonies that appear blue carry the "wild type" vector; colonies that appear white have the lacZ gene disrupted by insertion of the gene of interest--these cannot produce beta-galactosidase and cannot hydrolyze X-gal - will need to determine sequence to know if gene the correct gene was inserted into the lacZ gene

3' overhang

- read 5' to 3' - sticky ends leave 3' overhang - less nucleotides on 3' end - 5' recesses - e.g., CTGCA/G

viral features

- type of nucleic acid and structure of viral chromosome - structure of coat - presence of membrane "envelope" - mode of entry into and exit from the host cell - site of mechanism of replication of genome

types of mobile genetic elements

- plasmids - viruses - transposable elements (transposons) - free DNA

5' overhang

- read 5' to 3' - sticky end leaves 5' overhang - less nucleotides on 5' end - 3' recesses - e.g., G/AATCC, A/AGCTT

insertion inactivation using antibiotic residence genes

- can uses antibiotic gene markers (e.g., tetracycline) to select for bacteria containing vector - insert the gene of interest (DNA insert) into a second antibiotic resistance marker (e.g., ampR) and growing the bacteria on a nutrient agar plate containing tetracycline (selects for the bacteria containing the vector) and with and without ampicillin - only cells that carry the vector with the intact tetR gene will grow on these plates (will produce a protein that protects the ribosome from tetracycline binding) - bacteria containing the vector with DNA insert in the ampR gene will grow on the ampicillin-minus plate but not on the plates with ampicillin as the insert disrupted the ampR gene--bacterial cannot make beta-lactamase and cannot degrade ampicillin--tells you which bacteria have the vector with the insert in the right place but not whether or not it is the correct insert--have to determine the sequence of the plasmid around the insertion site to show that

restriction endonucleases

- different restriction enzymes recognize different restriction sites, produce different products - used to cut our a gene of interest from a genome and insert it into a vector cut with the same enzyme - enzymes that recognize a short DNA sequence (a restriction site) and cut both DNA strands - produced by bacteria to protect against viral infection--REs degrade viral genomes--natural function if to cleave foreign DNA--only works on non-methylated areas - often leave "sticky ends" or "overhangs"--overhangs can be 5' or 3'and can base pair to other DNA cut by the same restriction endonuclease (cuts are complementary)

metabolic activities

- e.g., iron acquisition, utilization of new carbon sources, nitrogen fixation

gene organization in eukaryotes

- each gene has its own set of regulatory sequences, which are bound by proteins that regulate expression of that gene

drug (antibiotic) resistance

- gene encoding an enzyme that can inactivate a drug (e.g., tetracycline resistance genes provide ribosome proteins that are resistant to tetracycline) - gene encoding a variant protein which is unaffected by a drug (i.e., can substitute for the protein that is inactivated by a given antibiotic but it is not itself inactivated) - gene encoding an efflux pump that actively exports the antibiotic from the bacterium

lysogenic phage

- genome becomes integrated (lysogenizes) into the host cell chromosome (via recombination) - forms a prophage that is replicated along with the bacterial (or other host) chromosome - passed on to daughter cells

lytic phage

- genome does not integrate into the host chromosome - replicated extra-chromosomally - assembled phage often lyse their host cell the they are released--kills cell

mechanism of restriction endonucleases

- hydrolyze a phosphodiester bond within each DNA strand - leaves an exposed 5'-PO4 on one site and an exposed 3'-OH on the other side of each strand

viruses

- infectious DNA or RNA-containing elements that possess a protein coat that allows them to move from cell to cell--replicate using host proteins, some use some of their own proteins (e.g., polymerases), which are synthesized in the host - small intracellular parasites (30-300 nm in diameter) - require host metabolic and biosynthetic machinery to propagate - can infect cells of plants and animals - infect bacteria = bacteriophage or phage - contain either an RNA or DNA genome surrounded by a protective, virus-encoded protein coat that allows them to move from cell to cell - multiplication is often lethal to the cell---can overtake cellular machinery for their own replication - often cause disease (e.g., polio, influenza, HIV, SARS-CoV2

polymerase chain reaction (PCR)

- instead of cutting a gene of interest out of a genome it can be closed out by PCR - amplifies specific DNA sequences in vitro extremely fast, extremely sensitive--requires very little template DNA - requires knowledge of partial nucleotide sequences - amplified DNA can be closed into a vector or sequenced or used as a diagnostic

DNA ligase and REs

- joins DNA ends--forms phosphodiester bonds--reverses the action of endonucleases - requires ATP, a 3'-OH end and a 5'-phosphate end - works most efficiently when sticky ends overlap because the ends are brought together by the base pairing - can also count blunt ends--ends bind to active site of enzyme, brought together by enzyme - DNA ligase is not sequence specific

benefits of PCR

- leads to exponential amplification of selected DNA fragment - beneficial for: 1. isolation of known fragment from a complex genome or from the genomes of many individuals 2. cloning of a gene/gene fragment for protein expression (to study the protein/manufacture it) 3. diagnostic tool for a particular virus or bacteria (e.g., SARS-CoV2) 4. forensics--identifying DNA at crime scene 5. study of relatedness 6. studies of molecular evolution 7. can detect rare sequence 8. isolation of multigene families--use primers that hybridized to conserve regions

virulence factors

- may contribute to or be essential for the virulence of a pathogenic bacterium, toxins, secretion systems, pili, other virulence factors - e.g., Bacillus anthraces and clostridium tetanus toxins are encoded on plasmid genes

transposable elements (transposons)

- mobile DNA elements that lack a coat and can be excised from one part of the host genome and insert into another part via recombination - aka jumping genes - "jump" or transpose from one site on the chromosome to another random site within the cell - range in size from 100s to 10,000 bp - present in multiple copies per cell encode multiple proteins including transposes--enzymes that catalyze transposition - events are tightly regulated, occur infrequently--random transposition can be lethal to cell - found in all types of cells - can spread from one cell to another, carried on plasmids--can confer new properties on the accepter cell (e.g., antibiotic resistance)

transposition

- moves transposable elements from one site to another random site in the genome - a form of genetic recombination

gene organization in prokaryotes

- multiple protein-coding genes are usually encoded in an operon, under the control of one set of regulatory elements

free DNA

- obtained from lysed bacteria and can be taken up by other related bacteria

cell properties that can be conferred by plasmid genes

- offer survival advantage - drug (antibiotic) resistance - virulence factors - metabolic activities

using PCR to introduce restriction sites

- used for cloning - primers used in PCR can be used to incorporate restriction sites at each end of the PCR product - ends do not need to be complementary to the DNA sequence being amplified - because restriction sites are one primer, they become part of the PCR product - restriction site primer allow the PCR products to be digested with an appropriate REs and then cloned into a complementary site in a vector

bacteriophage

- viruses that infect bacteria - phage genomes are comparable in size to plasmids (and can be much larger) - phage genomes and plasmids possess many of the same properties--suggest close evolutionary relationship - phage genome encodes viral coat proteins that allow it to be "packaged" and released from the host cell to infect another host cell--plasmids are only found intracellularly

conjugation

1. compatible strains (F+ and F-) join via a conjugative (F) pilus 2. conjugative bridge forms 3. plasmid material is transferred from F+ donor cell to F- recipient cell

DNA cloning

1. digest/cut the vector DNA at restriction endonuclease sites using sequence-specific REs--produces single-stranded DNA sticky ends 2. excise the gene from the chromosome with the same REs so that its ends are complementary to the sticky ends of the vector--more commonly the gene is generated using polymerase chain reaction and the correct RE sites are added to the gene at the same time--PCR product is then digested with REs 3. mix the RE-digested gene and vector--their complementary ends will base pair together--ligate with DNA ligase--product = recombinant DNA 4. transfer recombinant DNA from the test tube to a host cell using transformation--host cell amplifies the vector by replicating it and proliferating, resulting in many new bacteria and many copies of the vector 5. select or identify host cells that contain the recombinant DNA (usually antibiotics)

plasmids as cloning vectors

1. excision using endonuclease called restriction enzymes--cut at a specific internal sites throughout the genome 2. polymerase chain reaction (PCR) - also be synthesized commercially - genes are inserted into plasmid by permeabilizing bacterial membranes - bacteria are allowed to grow in nutrient rich conditions--produces billions of copies of the plasmid--can then be extracted using commercially available kits - bacteria can be grown under conditions that induce expression of the gene insert to produce large quantities of the recombinant protein for research, pharmaceuticals, etc.

essential components of cloning vectors

1. having one or multiple site for cloning in the gene of interest - these are restriction endonuclease (RE) sites that can be treated with an endonuclease that cuts at a specific site to open up the vector and insert the foreign DNA--cloning vectors have several restriction endonuclease sites clustered together--multiple cloning site (MCS) 2. have a regulatory region to control gene expression (e.g., promotor for the lac operon) 3. have a selectable marker, usually a gene that encodes for antibiotic resistance (e.g., AmpR)--allows you to select bacteria that carry the vector--only these will grow on media containing that antibiotic 4. have an origin of replication so that the plasmid can be replicated with the bacterial chromosome (Can have a few or 100s of copies of a plasmid)

types of horizontal gene transfer

1. transformation 2. conjugation 3. transduction

selective advantage of cell properties through plasmid genes

acquisition of new genes can allow the bacterium to adapt to a new environment, resist antibiotics, colonize hosts

blunt ends

cuts are straight through both DNA strands at the line of symmetry - no overhang - not as useful as it doesn't have sticky ends and can't base pair with a complementary end of a plasmid - e.g., GTT/AAC

recombination

integrating DNA or RNA into the "host" genome that are then replicated and expressed along with the host cell genes

mobile genetic elements

nucleic acid segments (DNA or RNA) that can move in and out of cells or between different regions of the cell genome, imparting new functions and diversity - some exhibit recombination - some replicate independently of the host genome - ALL require a host for replication--exploit host cells machinery and metabolism to propagate - can be powerful tools to study normal cell machinery

horizontal gene transfer

occurs when mobile genetic elements are passed from one cell to another - usually occurs between closely related organisms

steps of PCR

three steps: 1. heat to separate strands 2. hybridization of primers--annealing occurs and cooler temperatures--temperature depends of melting temperature of primers - synthetic oligonucleotides (DNA primers) that are designed to be complementary to the DNA sequences flanking the region of interest 3. DNA synthesis of primers--polymerase will continue to extend the daughter strand until the template ends - primers become part of the DNA strand that has be synthesized from them--no formation of Okazaki fragments - products become templates and are finite in size - length of the products will be defined by the forward and reverse primers--after many rounds these will be the dominant PCR products - products increase exponentially over time 2^x

transformation

uptake of naked DNA (linear or plasmid)