MolBio_Sp04_pres_05.ppt

Viruses Important pathogens Viruses are essential for cellular research Gene delivery agents Introduce viral genetic material into host cells Viral genes direct multiplication and assembly Progeny virus escape from cell Process repeats Virus Obligate intracellular parasite parasitic genome Requires host cell to multiply Virion Morphologies Icosahedral - 20 equivalent faces Helical - filaments Enveloped - surrounded by membrane Complex - bacteriophages (phages) Helical and Icosahedral Enveloped Complex Bacteriophage Viral Multiplication Cycle Five stages Adsorption - attach to host (receptor) Penetration - genome enters cell Replication - synthesis of components Assembly - progeny virions assemble Escape - progeny leaves host (lysis or budding) Diversity of Viruses Categorize by genome Class I - dsDNA, herpesvirus Class II - ssDNA, parvovirus Class III - dsRNA, rotavirus Class IV - (+)ssRNA, poliovirus Class V - (-)ssRNA, orthomyxovirus (flu) Class VI - Retroviruses (+)ssRNA to dsDNA, HIV) Retroviruses 3.15 Very different multiplication cycle RNA DNA, reverse transcription Recombinant DNA Technology Techniques have revolutionized study of cells Isolate, amplify, sequence, and manipulate any gene from any organism Also called molecular cloning Insert gene of interest into a vehicle called a vector (usually plasmid DNA) Transform (introduce) plasmid into E. coli or other cell for multiplication, expression and analysis Plasmid - Vector for Cloning 3.20 Small, circular DNA that replicates independently in the cell Three features antibiotic resistance marker origin of replication region to insert foreign DNA Preparing Insert and Vector DNA Gene of interest must be cut out of chromosome Plasmid DNA must be cut for insertion of gene Special enzymes called restriction endonucleases will do both Restriction Endonuclease Cleaves DNA at recognition sequence Made by bacteria “restrict” invading DNA (usually viral) Recognition is usually a palindrome 5’ - GAATTC - 3’ 3’