Viral and Bacterial Genomes & DNA Technology

Viruses Tiny; much smaller than a bacteria Basic structure: – Nucleic acid (DNA or RNA) enclosed in a protein coat or capsid – May have an envelope derived from the host Rely on host cell for reproduction; usually host specific due to proteins and receptors

Types of Viruses

Viral Infections Lytic cycle – Kills the host cell by causing it to lyse – Injects genetic material into host and uses it to produce new viral proteins and make copies of DNA/RNA – New viruses are assembled and break open host cell to release new viruses

The Lytic Cycle After infection, bacterial viruses transcribe and translate their RNA, replicate their genomes, package them into capsids and lyse the cell

Lysogenic Cycle – Viral genetic material is injected into host cell and is incorporated into host cell genome Now called a prophage – Viral genome is copied every time host cell reproduces – Viral genome eventually exits the host genome and initiates lytic cycle

Retroviruses Type of RNA virus in which information flows backward – Reverse transcriptase transcribes DNA from RNA, which is then integrated into host (provirus) – Host can now transcribe viral DNA to assemble new viruses – How HIV works

Other Strange Infections Viroids – tiny, naked, circular molecules of RNA that infect plants Prions – misfolded proteins that convert normal proteins in the brain to prion version – Causes many degenerative brain diseases

Bacteria No nucleus One, double-stranded, circular DNA molecule in nucleoid region – Have no introns – Has an origin of replication May also contain plasmids – Small, self-replicating, disposable circles of DNA with small # of genes

Bacteria Reproduce by binary fission – Copy DNA and split into two identical cells – Begin replication at a single ori site and make DNA in both directions around the circle Genetic diversity/recombination accomplished by: – Mutations – Transformation – uptake of foreign DNA – Transduction – gene transfer by phages – Conjugation – one-way direct transfer of genes between two bacteria through a pilus (pili)

Binary Fission and Conjugation

Operons Series of genes that are turned on or off together; one promoter – many genes Three parts: – Operator- “on-off switch;” around promoter region and controls access of RNA polymerase – Promoter – binding location for RNA polymerase – Genes they control – entire sequence of DNA for the pathway

Operon Control Repressors – proteins that can bind to operator and block RNA polymerase, preventing transcription Can also be regulated by corepressors which help the repressor, inducers which inactivate the repressors, and activators which make it easier for RNA polymerase to bind

Genetic Engineering The direct manipulation of genes or DNA Has led to new biotechnology in which organisms or their parts are manipulated to make useful products Allows for the making of recombinant DNA – a DNA molecule made from two different sources

Process DNA must first be cut – Done with restriction enzymes, specifically restriction endonucleases – Are able to cleave or cut DNA at specific sites along the DNA strand – restriction sites Locate certain base sequences

Restriction enzymes Type I – make simple cuts across both DNA strands, near recognition site Not precise enough for manipulating DNA Type II – recognize a specific sequence and cleave at a particular site Used to make recombinant DNA Recognition sites are palindromes (same forwards and backwards) Causes staggered cuts which creates “sticky ends”

Creating Recombinant DNA The complementary sticky ends can be joined to a fragment from another DNA that is cut with the same enzyme DNA ligase helps to bond the new segments together to create a new recombinant DNA molecule

Recombinant DNA

Gel Electrophoresis In order to make use of the fragments made by cleaving DNA, the individual DNA segments must be separated Gel electrophoresis is the most common separation technique Takes advantage of the negative charge of DNA

How it works Restriction enzymes are used to cut DNA The fragments are loaded into a gel Electrical current is applied The DNA fragments will move through the gel towards the positive end of the charge Fragments move based on their size – Larger ones are slower The gel can be stained to visualize the fragments

Transformation Process in which genetic material can be transferred between cells (remember Griffith’s experiment) Can be used to create transgenic organisms – Recombinant DNA can be introduced into new cells and then be copied – The transformed cells can be used to make parts or all of an organism - transgenic

Cloning A genetically identical copy Molecular cloning involves isolating specific sequences or genes and making new copies of that sequence The cloning of recombinant DNA usually requires a vector to help insert the new DNA into a bacteria cell where it can be copied

cDNA Complementary DNA produced from an edited RNA transcript Requires reverse transcriptase Made so that the DNA sample contains no introns More efficient for expressing eukaryotic DNA in a bacteria

Vectors Something to carry the new DNA – Usually a plasmid or a phage Plasmids – small, circular DNA’s that are dispensable to a bacteria cell Phages – viruses that infect bacteria

Plasmid Vectors Used to clone small pieces of DNA Must have two components – Origin of replication to allow replication inside of the bacteria – Selectable marker – usually antibiotic resistance; allows presence of plasmid to be easily identified The plasmid is cut, new DNA is inserted, and the plasmid will be introduced into a bacteria cell by transformation – E. coli is often used for this process

Phage Vectors Are larger and can be used to copy larger sequences Most common is phage lambda Virus will infect host cell and use it to reproduce; new DNA is incorporated into host genome Phage genome is linear instead of circular

DNA libraries Using the processes of cloning smaller sequences, a collection of DNAs can be put together in a DNA library – Often a genomic library – a representation of the entire genome – DNA is fragmented and each fragment is inserted into vectors which are placed into host cells – Random collection of DNA fragments Each cell contains a single fragment in a plasmid or each phage contains a single fragment All cells or all phages together make up the library

Polymerase Chain Reaction (PCR) Much faster than cloning Can make billions of copies of a particular DNA segment without using cells Used when samples are very small DNA sample is incubated with a special DNA Polymerase, artificial DNA primers to flank the specific regions, and a supply of nucleotides

DNA Technology Using these and many other methods, biotechnology can be used for: – Diagnosing disease – Gene therapy – Medicine/pharmaceuticals – Forensics – Environmental issues – Agriculture