Lecture Notes - Genetics PDF

BIOL215 - Introductory Genetics Week 1 DNA as the Genetic Material DNA is the genetic material in the vast majority of organisms but there are some viruses that use RNA as their genetic material. Nucleotides are the building blocks of DNA composed of phosphate, sugar and a...

BIOL215 - Introductory Genetics Week 1 DNA as the Genetic Material DNA is the genetic material in the vast majority of organisms but there are some viruses that use RNA as their genetic material. Nucleotides are the building blocks of DNA composed of phosphate, sugar and a nitrogenous base (Adenine, Thymine, Cytosine, Guanine) Structurally DNA is a right-handed double helix with antiparallel strands of complementary bases. DNA has direction 5’ - 3’ There was an agreement that genetic material had to have 3 characteristics: ○ Must carry information in a stable form ○ Must replicate accurately; progeny = parent ○ Must be capable of change, in order to evolve The Search for Genetic Material Initially there was disagreement on what was the genetic material ○ Scientists accepted that the nucleus of calls contained heredity factors ○ Most believed proteins to be the source of inheritance ○ Did not believe DNA could be the genetic material: Only a polymer of 4 bases/nucleotides Proteins were far more complex View that protein in chromatin (rather than the DNA) was the hereditary material persisted until the mid 20th century Fred Griffith Experiment (1928) Provided a foundation of the discovery that DNA is the genetic material Was studying the infection patterns of the pathogen Streptococcus pneumoniae in mice Studied two major strains: ○ S-strain: colony on plate displayed ‘smooth’ appearance due to a polysaccharide on its capsular surface Provided protection from the host’s immune system Virulent ○ R-strain: ‘rough’ in appearance; lacking the polysaccharide Easily destroyed by host’s immune system Avirulent ○ Several variants of each major strain (IIS, IIIS, IIR, IIIR)  Occasionally IIS mutated to IIR but never to IIIR. IIIS mutated to IIIR, but never IIR. Conclusion of Griffith’s experiment: ○ Some heat stable component present in IIIS transformed IIR into IIIS ○ Whatever is transmitted must also be inherited, as recovered cells remained S when propagated on fresh plates Termed the transmitted substance the ‘transforming principle’ Avery, MacLeod & McCarty Experiment (1944) Repeated the Griffith experiment, but fractionated the ‘transforming principle’ in order to identify it Heat killed S cells, and then treated them chemically to purify the substance that transformed R cells into S cells Hershey & Chase Experiment (1953) The experiment that settled the argument ○ By this time, only two realistic contenders; protein & DNA Experimented with the T2 bacteriophage ○ Comprised of only protein & DNA ○ Cannot replicate on its own - relies on its host cell for everything ○ Infects E. coli ○ Very useful in the lab/can be grown in culture Genetic studies had shown that after entering a cell, the T2 ‘genetic elements’ directed the host to make new phage Followed the passing of genetic material from parent to offspring by differentially labeling DNA and protein by growing in media containing either 32P or 35S ○ 32P is primarily in the DNA, small amounts in protein compounds ○ 35S is primarily in the protein elements, small amounts in DNA Two experiments were performed ○ The page were grown in the presence of 32P/35S but bacteria were not  The only source of radiolabel is the phage Experiment 1: ○ Phage is allowed to infect bacterial culture ○ Following attachment, the culture is blended, physically removing the empty phage from the bacteria ○ This blended mixture is then centrifuged to separate the bacteria ○ The supernatant and the pellet are then analysed for the presence of radiolabels ○ Results: ○ Confirms that the only material to enter the cells is DNA ○ Thus must be genetic material Experiment 2: ○ Was similar, except that the phage are given time to reproduce in the bacteria ○ The offspring are then harvested, and examined for radioactivity ○ Results: When examined, small amounts of 32P are found in the new phage, but no 35S DNA being passed on to progeny, not protein Gierer & Schramm (1956)  Purified RNA from TMV Injected TMV RNA into tobacco leaves - lesions Digested TMV RNA with RNase - no lesions Conclusion: RNA was genetic material in TMV Fraenkel-Conrat & Singer (1957) Isolated 2 stocks that possessed different coat proteins One coat protein could encapsulate the other’s RNA and still function But the progeny viruses were always the type specified by the RNA, not the protein SUMMARY The 3 main experiments that established DNA as the genetic material Griffith’s Transformation Experiment provided foundational discovery of DNA as the genetic material Avery’s Transformation Experiment demonstrated the transforming principle was DNA but scientific community remained sceptical Hershey‐Chase Bacteriophage Experiment demonstrated DNA passed on to progeny not protein TMV is an example of an RNA based virus, and was involved in critical experiments that demonstrated RNA to be the genetic material DNA Packaging and Analysis What is a genome? The genome is the entire set of genetic instructions(DNA) found in a cell Viral genomes vary considerably, they can be: RNA or DNA Single or double-stranded Have different structures Different sizes Prokaryotic and Eukaryotic Genomes DNA is arranged in multiple linear chromosomes within the nucleus in most eukaryotic cells but exists primarily as a singular circular chromosome in prokayotic cells DNA in eukaryotes is packaged in an orderly way in the nucleus through tight binding around histone proteins DNA in prokayotes is found in the cytoplasm as a highly folded and supercoiled structure through the help from RNA and small DNA binding prototeins Packaging of DNA in Prokaryotes The chromosome is usually found condensed in a region of the cell known as the nucleoid DNA supercoiling is largely responsible for the ability of the chromosome to ‘fit’ in the cell The amount and type of supercoiling is controlled by a group of enzymes known as topoisomerases Looping further compacts the supercoiled chromosome tenfold The bacterial chromosome has 100+ looped domains held in place by unknown proteins and possibly some RNA molecules Packaging of DNA in Eukaryotes Chromosomes confined to the nucleus in eukaryotes DNA packaged with proteins into chromatin which is essentially identical in structure in all eukaryotes Histones play a crucial role in chromatin packing and are highly conserved among eukaryotes Histones are positively charged proteins that strongly adhere to negatively charged DNA and form complexes called nucleosomes Non-histones also play a role in stabilising compaction of DNA but are far less abundant and can vary markedly between cell types and organisms Genomic DNA Extraction Need to extract DNA to analyse or manipulate it Can extract DNA from a huge range of starting material ○ Cell extracts (research) ○ Tissue/blood (medicine) ○ Blood/saliva/semen/hair follicle (forensics) ○ Mosquito stomach contents, fossilized in amber (dinosaur theme-parks) Many commercially available kits Involves cell lysis and separation from cellular contents and proteins it might be associated with (particularly in the case of eukaryotes) Four steps are used to remove and purify the DNA from the rest of the cell: ○ Lyse cells with physical or chemical force to disrupt cell membrane (detergent) ○ Remove cell contents by treating with protease to destroy protein and RNase to destroy RNA and precipitate for removal by centrigugation ○ Precipitate DNA in the supernatant with ispropanol and wash in ethanol ○ Resuspend in water of buffer to concentrate ○ Centrifugation is performed between each step to pellet various components But how do we know it worked? ○ Check absorbance at 260/280 nm for approximate concentration and indication of purity ○ Run a sample on an agarose gel for visualision of extracted DNA against a marker Principles of Agarose Gel electrophoresis The agarose acts like a molecular sieve and the shorter fragments of DNA move more quickly through the gel A DNA marker with fragments of known lengths is usually run alongside your samples The DNA stain is stained for visualision with ethidium bromide or RedSafe and will appear as bands on the gel But genomic DNA is big and looks like a blob on an agarose gel ○ Chop it into smaller fragments using restriction enzymes = molecular scissors ○ These enzymes recognise and cut the sugar phosphate backbone at specific sequences Electrophoresis of Digested Genomic DNA Digestion of chromosomal DNA results in many fragments of varying size and can often appear as a smear when electrophoresed in agarose Restriction Enzymes Restriction enzymes catalyse double stranded DNA breaks Discovered in bacteria in 1962 as part of an enzymatic “immune system” ○ Recognises and destroys foreign DNA e.g. from bacteriophage infection ○ The bacterial DNA is modified and thus immune to cleavage by its own REs Each recognises a specific sequence - restriction site which is palindrome Sticky Ends Some restriction enzymes can cut DNA to produce two pieces of DNA with overhanging complementary ends The pieces can reanneal (stick back together) though hydrogen bonds between complementary bases Blunt Ends Some restriction enzymes can cut to produce two pieces of DNA with no overhangs The pieces can still reanneal (stick back together) Summary DNA is arranged in multiple linear chromosomes within the nucleus in most eukaryotic cells but exists primarily as a singular circular chromosome in prokaryotic cells DNA in eurkaryotes is packaged in an orderly way in the nucleus through tight binding around histone proteins to produce chromatin but in prokaryotes supercoils and loops to achieve compaction  Chromosomal DNA can be extracted using a simple multip-step procedure involving cell lysis, separation from protein and precipitation of DNA Restriction enzymes recognise specific DNA sequences and “cut” or fragment DNA at those sequences Agarose gel electrophoresis can be used to visualise DNA and mirgates from negative to positive electrode based on size with smallest travelling furthest DNA Sequencing & Analysis “Sanger sequencing dominated the research landscape until the early 21st century and lef to exceptional achievements, including the completion of a high quality, reference sequence of the human genome” Sanger Sequencing Also known as the “Chain termination method” Developed by Fred Sanger in 1977 What you need: ○ Template DNA ○ Sequencing primer (just 1) ○ DNA polymerase ○ Nucleotides (dNTPs: dATP, dCTP, dGTP, dTTP) ○ Dideoxynucleotodes (ddNTPS) Sanger sequencing utilises dideoxy NTP (ddNTPs) they terminate the end and don’t allow a further chain, as no further nucleotides can be added The Sanger Sequencing Reaction This reaction needs to be performed for all 4 bases and results in fragments of varying lnegths Fragments are then separated by gel electrophoresis and analysed by autoradiograph What is the DNA sequence determined by this Sanger reaction? ○ Smallest fragments migrate the furthest in the gel Automated Sanger Sequencing Same principle but 4 fluorescent dideoxy-nucleotides used and only one reaction in automated machines attached to computers for data analysis Advantages: ○ Sequences millions of DNA fragments simultaneously to produce sequence data in megabases or gigabases ○ Cost per megabase of DNA sequenced in 2001 was US $5000. In July 2011 it was less that US $0.10 Sanger Method in more detail 1. The template DNA is first denatured into single strands using HEAT 2. An oligonucleotide (short DNA strand) called a “primer” is annealed to one of the two DNA strands a. The primer is usually 10-20 nucleotides long. It is designed by the investigator so that it’s 3’ end is next to the DNA sequence of interest 3. The primer “primes” DNA synthesis which is catalysed by a DNA polymerase enzyme Remember: DNA polymerase, requires a primer to begin DNA synthesis\ The 5’ to 3’ orientation ensures that the DNA made is complementary to the original sequence of interest 4. DNA polymerase + the four regular deoxynucleotide precursors are added: - dNTPs: dATP, dTTP, dCTP, dGTP 5. Plus small amount of ddNTPs; ddATP, ddTTP, ddGTP, ddCTP - has a 3’ H rather than a 3’ - OH on the deoxyribose sugar - also have fluorescent dyes that allow detection Important: This is what makes sanger different to a regular DNA polymerase reaction 6. DNA polymerase adds a nucleotide to the 3’OH at the ends of the primer 7. Two possibilities: a. Since most of the DNA precursors in the reaction are dNTPs, the probability is high that a dNTP will be used for the next extension step b. BUT there’s a small chance that a ddNTP is used.. And then the extended DNA chain has a 3’ H at the end, and DNA polymerase can’t add anymore nucleotides Detection ○ Fragments are then separated by gel electrophoresis A very sensitive type of electrophoresis in a very small capillary, and a laser eye at the end of the capillary detects the coloured fragments as they exit the capillary While the dyes emit similar colours, the computer converts the minor colour differences into a far more obvious difference; e.g A=green, G=black, T=red and C=blue End up with an output of coloured peaks, corresponding to each nucleotide position So now we have our DNA sequence, what do we do with it? Sequence Analysis There are many online tools for analysis of sequence data Some of the first and SIMPLE analysis you might do include ○ In silico sequence analysis Open reading frame and translation Determination of restriction enzyme sites ○ Database similarity searching An alignment with known/expected sequence to confirm correct Alignment with a database to determine identity or similarity to already published sequences In silico Sequence Analysis: RE sites Search for and map restriction sites within a sequence ○ Useful for cloning ○ Can provide expected data for comparison with electrophoresis result In silico Sequence Analysis: ORF’s Search for open reading frames and translated amino acid sequence ○ Prior to database similarity searching ○ Important when cloning for protein expression ○ Indentifying start and stop codons can help locate open reading frames = protein? Database Similarity Searching Looking for known sequences ○ Gene identification ○ Domain/motif identification ○ For functional or evolutionary studies Principle ○ Align the query sequences with each sequence in the database ○ Select those sequences with high alignment scores Summary DNA sequencing first achieved using Sanger’s chain termination method based of the incorporation of ddNTPS Automated sequencing based on same principle but 4 fluorescent ddNTPS in one reaction and automated machines attached to computers for data analysis Many online sequence analysis tools available for ○ RE analysis ○ ORF analysis ○ Database similarity searching Gene Amplifation and Analysis Amplifying DNA “Amplifying” DNA is making many, many more copies of DNA Especially important if working with small/limited amounts of sample This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence Target DNA can be amplified using the Polymerase Chain Reaction Application of PCR There are many application where PCR is beneficial/critical Recombinant DNA Technology relies on the ability to amplify the gene of interest for cloning What is PCR? Exponentially progressing synthesis of defined target DNA in vitro Can produce billions of copies of a specific sequence of target DNA Number of DNA molecules double every cycle of the reaction Normal PCR program: 30-40 cycles From two original matching DNA molecules can produce more than 1,000,000,000 (billion) new DNA molecules It is based on DNA replication but uses a heat-stable DNA polymerase: Taq Polymerase Uses a 3 stage cycle of heating and cooling DNA Assembly of the Reaction DNA template (genomic DNA,cDNA,etc) ○ Free from nucleases/materials that will interfere with the reaction Gene specific primers to suit application ○ Usually ~20bp, but can be much larger Heat stable polymerase ○ To maek the DNA… it needs to be able to withstand thermal cycling dNTPs ○ Incorporated into new chains Polymerase buffer ○ Enzyme specific, contains salts, co-factors (Mg2+) Three PCR Steps Step 1: Heat (95ºC) sample (denturation) to separate the double-stranded DNA to single strands Step 2: Cooling (45 - 55 ºC) the sample to allow single-stranded DNA primers to bind to the separated strands (annealing) Step 3: Heating (72 ºC) to allow Taq polymerase to extend the primers (5’ - 3’) to synetheise two new DNA strands (extension) = Two copies of original sequence The cycle is repeated many times to generate more copies of DNA Gene Analysis DNA and RNA probes play an important role in gene analysis A DNA/RNA probe in a small single stranded DNA/RNA sequence that is complementary to a known region of DNA/RNA The probe hybridises to target DNA Applications: ○ Identification (fingerprinting) ○ Gene expression analysis ○ DNA/RNA characterisation (mapping,relatedness) ○ Powerful research tool Southern Blot Hybridisation DNA fragments from an agarose gel are transferred to a filter by “blotting” and then detected using a homologous DNA probe which has been labelled. Diagnosis of Sickle Cell Anaemia Arises from a mutation in the B-globin gene which results in a different digestion profile with restriction enzyme Mstll Northern Blot Hybridisation Similar to Southern blotting but detects “gene expression” Rna is transferred to a membrane by “blotting” and then detected using a homologous DNA probe which has been radioactively or fluorescently labelled Useful for: ○ Detection of mRNA transcripts ○ Determining the stability of RNA ○ Investigation of transcriptional control Differential Gene Expression Gene expression pattern in a call or at a time is characteristic Virtually all differences in cell state or type are correlated with changes in mRNA levels of many genes ○ Developmental ○ Tissue/cell specific ○ Diseased ○ Environmental Expression patterns of many previously uncharcterised genes may provide clues to their possible function by comparison DNA Microarray The number of sequences from a variety of different organisms has increased so rapidly that there is a growing need for new, more powerful methods Microarrays allow the analysis of thousands of genes in a single experiment ○ Same principle also used to detect RNAs or DNA methylation markers, among others On the surface of the chip (2 cm2), there are thousands of tiny spots printed in designated positions (up to 65 000) Each spot contains a gene probe for known DNA sequences or genes. It can even detect a point mutation Summary PCR is a method of DNA amplification using heat stable polymerase and a 3 step cycling protocol PCR has many applications including recombinant DNA technology, disease diagnosis and fingerprinting Southern Blot can detect specific DNA fragments Northern Blot detects gene expression Microarray allows the analysis of thousands of genes in a single experiment and is based on hybridisation of a known probe with sample DNA Week 2 Bacteria and bacteriophages How DNA is organised in Bacterial Cells Bacterial versus Plasmid DNA Genomic DNA (Single Copy) Plasmid DNA (Multiple Copy) Genomic DNA is chromosomal DNA where Plasmid DNA is extra-chromosomal DNA in the genetic material is present bacteria and some yeasts, i.e. Plasmid It is primary DNA in all living organisms It is secondary DNA It is linear in eukaryotes whereas circular in It is circular prokaryotes As it encodes genetic information, it is much It is smaller larger than plasmid Genomic DNA is organised with proteins Plasmid DNA is not with histones called histones It contains essential genes which codes for It contains non-essential genes functional and structural proteins It can be transferred only through cell division It can be transferred through horizontal way within the same species of gene transfer between same or different species Bacteriophages Viruses that infect bacteria Attach to the outside of a host, and inject their genetic material into the host cell Rely on host cell to replicate The Genetic Material Varies in Phage Mostly dsDNA but can also be ssDNA, ssRNA or dsRNA Affects the way in which they replicate in the host Bacteria and their Viruses as Research Organisms Simple genetics ○ Single chromosome in bacteria Short reproduction cycles ○ Grow at exponential rate Can be studied in pure culture ○ A single species or mutant strain can be isolated Relatively cheap to culture ○ Basic media requirements Largely harmless Mutants are easily obtained Application to recombinant technology Bacteria and their Viruses as Research Organisms The ability of these to transfer genetic information between cells in a population provided early geneticists with: ○ The basis for chromosome mapping methodology  ○ With an improved understanding of genetic mutation and variation through recombination Horizontal Gene Transfer (HGT) is transfer between different species ○ Common in bacteria HGT mapping techniques including recombination mapping, transformation, and transduction contributed to the production of some very detailed chromosomal maps for bacteria Horizontal Gene Transfer HGT has played a significant role in the evolution of bacteria ○ Transfer of genes encoding survival advantages like antibiotic resistance genes or genes conferring enhanced pathogenicity Bacteria that Cause Disease Bacteria that can cause disease are known as pathogenic bacteria They encode virulence determinant that establish disease in a host. For example the ability to: ○ Adhere to eukaryotic membranes ○ Invade host cells ○ Resist phagocytosis ○ Lyse eukaryotic cells ○ Damage host tissue ○ Trigger the production of a cascade of host immune-modulating molecules Determinant that Promote Colonisation The ability to use motility and other means to ○ Contact host cells and disseminate within a host ○ Combat cillary action of host epithelial surfaces The ability to adhere to host cells and resist physical removal ○ Pili ○ Afimbrial adhesions The ability to invade host cells ○ Production of enzymes for digestion of host membranes The ability to evade host defences ○ Capsule and slime production Determinants that Damage Host Bacterial products that damage host cells damage and interrupt normal cell function There are 3 reasons for toxin production ○ Disrupt ethithelial cells and therefore gain access to the body ○ Disrupt the cells of the immune system ○ Disrupt host cells to promote nutrient release There are two types of toxins ○ Endotoxins - part of the outer portion of the cell wall of gram-negative bacteria. They are liberated when the bacteria die and the cell wall breaks apart ○ Exotoxins - are produced inside mostly gram-positive bacteria as part of their growth and metabolism. They are then secreted or released following lysis into the surrounding medium Understanding the Genetics of Pathogenic Bacteria is Crucial to Treatment and Prevention of Disease Bacteria in Human Health While there are bacteria in the environment or living in our gut that cause disease there are also a multitude of bacteria that benefit our health = microbiome Roles of the Human Microbiome Most of the microbes that inhabit our body supply crucial ecosystem services that benefit the entire host-microbe system and ○ Help digest our food ○ Regulate our immune system ○ Protect against other bacteria that cause disease ○ Produce vitamins including B vitamins, viamtines b12, thiamine and riboflavin and vitamin K, which is needed for blood coagulation Links to many diseases, obesity and even depression Key Learning Concepts Know the difference between plasmid and bacterial DNA Understand the diversity of genetic material in phage Understand what makes bacteria and bacteriophage suitable as research organisms Understand the role of horizontal gene transfer in the evolution of bacteria Types of virulence determinants in pathogenic bacteria  Have an understanding of how virulence determinant can aid in the development of treatment and prevention strategies ○ What a likely target for vaccine development is and why? Know the role of the microbiome in human health Horizontal Gene Transfer Transformation Unidirectional transfer of extracellular DNA into cells - recipients are called Transformants Bacterial transformation first observed by Frederick Griffith in 1928 in Streptococcus pneumonia Two types of strep - with and without protective capsule ○ “Rough” R-Strain - no capsule - non-pathogenic/avirulent ○ “Smooth” S-Strain - with capsule - pathogenic/virulent Griffith’s Experiment ○ Bacteria are ‘transformed’ ○ Can be conducted in vitro ○ Bacteria are altered to enable them to take up DNA ○ Engineered information ○ Grithiths observations; capsule provides protection against phagocytosis Transformation: Competence Bacteria are naturally competent Competent: a physiological state permitting efficient uptake of macromolecular DNA Bacteria species (including pathogens) very in their ability to take up DNA Artificial Transformation Majority of bacteria are not naturally competent for artificial transformation Competence can be induced with chemical or electric shock Results in temporary pores in the membrane for uptake of foreign DNA Competency and transformation are important tools in recombinant DNA technology/cloning Fate of Transformed DNA Horizontal Gene Transfer: Conjugation A process in which there is unidirectional transfer of genetic information through DIRECT cellular contact between donor and recipient bacterial cell Conjugation needs physical contact between donor and recipient bacterial cell Receipients that have received a piece of donor DNA - Trnasconjugants Conjugation is mediated by - conjugation pili Lederberg Tatum Experiment: Evidence for Mating in Bacteria Conjugation was discovered in 1946 (Johua Lederberg and Edward Tatum) They studied 2 E.Coli Strains that differed in their nutritional requirements ○ Strain A: met, bio, thr+, leu+, thi+ ○ Strain B: met+, bio+, thr, leu, thi Auxotrophic - cells cannot make certain nutrient (in this case strain a: methionine biotin) Prototrophic - cells are able to synthesise all nutrients Minimal medium - contain the minimum nutrients for possible colony growth, generally without the presence of amino acids Davis U-tube Do cells need to be in physical contact? Placed strains A and B in a liquid medium on either sides of the U-tube Separated by a filter with pores too small to allow bacteria to move through Cells plated out on minimal medium No prototrophic colonies observed Conclusion: cell to cell contact is a must for conjugation Conjugation - Transfer of the F plasmid 1. Donor cell attaches to a recipient cell with its pilus 2. Pilis may draw cells together 3. One strand of F Plasmid DNA plasmid DNA transfers to the recipient 4. The recipient synthesizes a complementary strand to become an F+ cell with a pilus; the donor synthesises a complementary stand, restoring its complete plasmid High-Frequence Recombination Hƒr cells Any plasmid that can integrate into the chromosome is known as an episome The F plasmid can integrate into the chromosome These strains are able to transfer chromosomal genes, not just plasmid genes Such strains known as Hƒr strains (high frequency recombination) Transfer of chromosomal DNA in Hƒr cells 1. The F+ cell 2. Integration of F by crossing-over 3. Conjugation of Hƒr with F- 4. Integrated F factor is nicked, and nicked strand transfers to the recipient cell, bringing bacterial genes with it 5. Transferred strand is copies, and donor bacterial genes are appearing in the recipient Horizontal Gene Transfer: Transduction The process by which a virus transfers genetic material from one bacteria to another Genetic recombination Genes from a host cell are incorporated into a bacteriophage and then carried to another host cell when the bacteriophage initiates another cycle of infection 1. Phage infects the donor bacterial cell 2. Phage DNA and proteins are made, and the bacterial chromosome is broken down into pieces 3. Occassionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA 4. A phage carrying bacterial DNA injects a new host cell, the recipient cell 5. Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells Gene Transfer Agents GTA’s are DNA containing virus-like particles that are produced by some bacteria Encoded by a cluster of head, tail and DNA packaging genes strongly resembling those of bacteriophage DNA fragments are packaged and injected into cells and therefore mediating HGT Gene Transfer Agents The maintenance of these genes over hundred of billions of years in some cases supports the original theory that their pesistence is attributable to the benefits arisong from the recombination they promote But the cost of cell death to release GTA’s is more recently believes to be greater than the benefits so the theory is being questioned and the search is underway for other factors providing benefit Plasmids What is a plasmid Small, circular, double-stranded DNA molecules that replicate indepentally found naturally in bacteria and some yeasts Can be infectious (self-transmissible via conjugation) Can integrate into the main chromosome - episome Have been modified for use in genetic engineering Elements of a Plasmid Origin of replication (ori) ○ Species specific sequence needed for the plasmid to replicate in host Origin of transfer (oriT) - optional ○ Sequence needed for the transfer of the plasmid from one bacterium to another during conjugation Restriction sites Various genes with promoters ○ Virulence genes ○ Antibiotic resistance genes ○ Many others ?? Plasmid Types Fertility (F) plasmids ○ Carry instructions for conjugation Resistance (R) Plasmids ○ Carry genes for resistance to one or more antimicrobials Bacteriocin plasmids ○ Carry genes for proteinaceous toxins calls bacteriocins ○ Bacteriocins can kill bacterial cells of the same or similar species that lack the plasmid Virulence plasmids ○ Carry instructions for structures, enzymes or toxins that can enable a bacterium to become a pathogen Resistance Plasmids Antibiotic resistance genes carried naturally by many bacterial species A single plasmid can carry the genes to resist different antimicrobials in addition to genes that encourage spread of the plasmid Typicaaly transferred by conjugation Responsible for multi-drug resistant pathogenic strains Bacteriocin Plasmids Genes for bacteriocin production are carried on plasmids Involves attachment to specific cell receptors in a narrow host range They have bactericidal mode of action Play a major role in prevention of human disease, bacterial infection and contribute to maintaining healthy gut microflora Virulence Plasmids Many pathogenic bacteria carry genes for virulence on plasmids Typically large (>40 kb) low copy number plasmids Encode genes that help bacteria infect humans, animals, or even plants by a variety of mechanisms These virulence factors can ○ Be toxins that damage or kill animal cells ○ Help bacteria to attach to and invade animal cells ○ Protect bacteria against retaliation by the immune system Plasmids for use in recombinant DNA technology Have certain features that make them suitable for: ○ Gene clonin ○ Genetic engineering ○ Recombinant protein expression Can be used in bacteria, eukaryotic cells and plants Features dependent on host and purpose Multiple cloning site (MCS) ○ Region of dna containing several unique restriction sites for insertion of foreign DNA fragments (cloning) Selectable marker ○ Cells with the plasmid can be easily distinguishable from calls that lack the plasmid, commonly a gene for resistance to an antibiotic Origin of replication ○ Sequence needed for the plasmid to replicate in host Example : pHSG398 pHSG398 is a common cloning vector available commercially Has an origin of replication  Carried the chloramphenicol resistance gene (Cmr) The MCS has sites for EcoRI and BamHI The MCS sits within the lacZ gene for selection of clones are ligation and transformation Plasmid Extraction Relies on the differential denaturation and re-annealing of plasmid DNA compared to chromosomal DNA Solution 2: NaOH and SDS = alkaline lysis ○ Detergent and high pH, lyses the cells denatures and precipitates chromosomal DNA ○ Plasmid DNA remains relatively soluble Solution 3: Na Acetate ○ Neutralises the alkaline pH ○ Precipitates proteins and forms SDS-protein complex ○ Chromosomal DNA creatures and aggregates with proteins Centrifugation ○ Pellets the protein-chromosomal aggregates ○ Plasmids will be present in the supernatant ○ These plasmids can be precipitated with ethanol Electrophoresis of Plasmids Analysis of DNA (plasmid) extracts is usually done by agarose gel electrophoresis Additional Features of Plasmid Many additional features added to plasmids for the purpose of expression and purification Dependant on downstream application/purpose Viruses Small obligate, sub-microscopic particles - range from very small to large Parasites - cannot replicate without infecting another (host) organsim Cause many infections of humans, animals, plants and bacteria Most viruses have a very narrow host range ○ Smallpox virus only injects humans ○ TMV only infects plants Comprised of genes wrapped in a protein coat Can also have a lipid envelope Have extremely small and efficient genomes - cant code for a wide range of proteins ○ Most simple virus encode only 4 proteins ○ More complex virus encode 200 proteins ○ They dont encode enzymes for energy production or protein synthesis Three viral shapes; Helical, Polyhedral, Complex Genetic Material of Viruses Contain a single type of nucleic acid - either DNA or RNA (but not both) May be linear and segmented or singular and circular Much smaller than genomes of cells Proteins encoded by the coronovirus genome The RNA genome of SARS-Cov-2 encodes 29 proteins Four structural proteins ○ E & M proteins form the viral envelope ○ N binds to the virus’s RNA genome ○ S protein binds to human receptors The non-structural proteins get expressed as two long polypeptides ○ Includes the main protease(Nsp5) and RNA polymerase(Nsp12) Replication in DNA Viruses dsDNA genomes can be replicated using replication machinery of the cell No intermediates requires DNA converted to mRNA for translation to produce regulatory proteins New Viral DNA and new vial proteins packaged for release Replication in Retroviruses Reverse transcriptase makes a DNA copy of the RNA degrading the DNA at the same time Then uses this DNA strands as a template to complete a DNA double helix The DNA then enters the nucleus and integrates into the chromosomal DNA of the host - becoming a PROVIRUS The proviral DNA is transcribed into viral RNA fragments and translated into viral proteins New capsids are assembled around viral RNA fragments and reverse transcriptase The nucleocapsid “bud” from the plasma membrane as complete virus Mutation in Viruses Viral mutations occur at a rate much greater than the cells they infect Virus mutations create genetic variation RNA viruses, like the flu and measles, are more prone to changes and mutations compared with DNA viruses, such as herpes,smallpox and human papillomavirus Based on work with bacteriophages, single-strand DNA viruses tend to mutate faster than double-stranded DNA viruses Some persistent infections, notable HIV, mutations emerge at an extremely high rate because of the very high replication rate and the high error rate of the virion reverse transriptase (approximately 1 base error per 10000 nucleotides transcribed) Role of Viruses in Cancer Some viruses carry copies of oncogenes as part of their genomes’  Some influence the expression of proto-oncogenes or suppressor genes already present in host Specific viruses are known to cause around 15% of human cancers Burkitts lymphoma 0 the endemic type of burkitt lymphoma is almost always linked to a previous infection with the Epstein-Barr virus. Virus that causes glandular fever Hodgkins disease 0 infectious mononucleosis an infection caused by Epstein-Barr virus Kaposi’s sarcoma - found in people with AIDS caused by infection with a virus called the Kaposi sarcoma association herpesvirus (same family as EBV) Cervical cancer - HPV sexually aquired - Ian frazer vaccine has halved cervical cancer rates Week 4 Recombinant DNA Why Clone Genes? To facilitate study and application of interested genes Products of Recombinant DNA Technology Hormone and cytokines ○ Interferon, interleukin ○ Insulin ○ Human growth hormone Vaccines ○ Hepatitis ○ Whooping cough ○ AIDS ○ Cancers Agriculture ○ Pest & Herbicide resistant plants ○ Nutrient-enriched plants Gene therapy ○ Cystic fibrosis Genome editing ○ Discovering functions of genes Recombinant DNA Technology Step 1: Construction of a recombinant DNA molecule Isolate (purify) the DNA molecule containing the gene of interest DNA is isolated from the organism which contains the gene of interest Examples ○ Plasmid dna from bacteria ○ Genomic dna from bacteria ○ Genomic dna from eukaryotes Cloning Vectors There are different kids of cloning vectors Example: Plasmids Viruses/phages Artificial chromosomes RE digestion and ligation Phage T4 DNA ligase is the most common one used Can ligate both blunt and sticky-end DNAs In nature, DNA ligases are involved in DNA replication and repair For certain purposes, such as protein expression, the DNA (gene) must be inserted in the correct orientation Cloning with 2 sticky ends Sticky ends must be compatible Cloning is directional Insert-vector ligation is efficient Recognistion sites of ligated restriction enzymes are intacts Cloning with 2 different but compatible ends Sticky ends must be compatible Cloning is directional Insert-vector ligation is efficient Vector self-ligation is low Recognition sites of original enzymes may be destroyed after ligation Cloning with 1 sticky end and 1 blunt end Directional cloning is maintained Ligation of the blunt ends may be less efficient Directional cloning with PCR and REs Engineer RE cut sites sequences to the ends of the sequences of interest to match the sequences in the vector in the correct direction Directional Cloning w/ PCR & Recombination Engineer homologous sequences to the ends of the sequences of interest to match the sequences in the bector in the correct direction Gibson Assembly (isothermal assembly reaction) Uses 3 common molecular biology enzymes: 5’ exonuclease, polymerase and ligase 5’ Exonuclease digest the 5’ end of double-stranded DNA fragments to generate 3’ single-stranded overhangs In a reaction mixture including other DNA fragments that has 20-40 bp of homology at their ends, the resulting complementary “sticky ends” will find each other and anneal Polymerase fill in any remaining regions of single-stranded DNA Ligase fuses the nicks resulting in a single DNA fragment Major benefit: allows for simple assembly of multiple fragments of DNA in the chosen orientation, without the need for any unwanted sequence at the junctions Recombineering Recombineering is in vivo genetic engineering, also known as homologous recombination-mediated genetic engineering “In vivo” is within a bacterial cell, usually E.coli or S.enterica Highly efficient and precise method for genetically engineering DNA in vivo Can be used for gene ○ Replacements ○ Deletions ○ Insertions ○ Inversions ○ Single and multiple point mutations  ○ Cloning and gene/protein tagging is also posible Genetic modifications are catalysed by bacteriophage recombination proteins produced within the bacterium Does not rely on specific sequences, such as restriction enzyme sites These phage functions are able to recombine DNAs containing short, 50 base, homologies Alternative model for ssDNA recombination ○ As an oligo enters the cell, it is coated by the phage annealase Rebß protein Which protects it and mediates annealing with complementary ssDNA ○ The Redß-coated recombining oligo anneals And joined to the lagging-strand at the DNA replication fork ○ If the changes within this oligo escape MMR, following another round of replication, the mutation becomes permanent Step 2: Introducing into a host cell Many ways, e.g. ○ Transformation of plasmid ○ Conjugation of plasmid ○ Transduction of bacteriophage or cosmid ○ Transposition of transposon Artificial Transformation Majority of bacteria are not naturally competent Competence can be induced with chemicals or electric shock Results in temporary pores in the membrane for uptake of foreign DNA Competency and transformation are important tools in recombinant DNA technology/cloning Step 3: Identification of a clone carrying the gene of interest by selection or screening Positive selection E.g. antibiotics resistance or auxotophy complementation Disadvantage ○ Empty vector satisfies selection condition ○ You dont select against vector without desired insert Negative Selection Ccd operon codes a toxin-antitoxin system ccdB → toxin ccdB ○ Acts as a DNA gyrase poison, locking up DNA with gyrase with broken double-stranded DNA, causing cell death ccdA→ anti-toxin ccdA Cells without ccdA die due to ccdB toxicity Colony PCR 1. Design primers to detect your insert 2. Run a PCR reaction using the supernatant of lysed bacteria as template 3. Run in AGE to analyse product size Primer Design Pros Cons Insert specific Simple yes or no test results Doesnt tell you the orientation of your insert or if the insert is in your plasmid Have to make a new primer pair for each insert Backbone-specific Provides information about Doesnt provide information the size of the insert and if its about the orientation of the in the plasmid insert Can be used to screen clones that were created with the same backbone because primers are plasmid dependent Orientation-specific Tell you the orientation of the You have to make a new insert primer pair for each insert Tell you if the insert is present since primers are insert in the plasmid dependent Great for blunt end cloning