BMSC 210 Post Midterm 1

-proteins that are needed at the same levels in a cell at all times

-to prevent the production of unneeded proteins

• -specificity provided by AA side chain interactions with phosphate backbone of DNA
• -major groove of DNA is main site of protein binding
• -generally look for inverted repeats for regulatory proteins

• -proteins composed of two identical polypeptides
• -interact specifically with inverted repeats

• -class of protein domain 
• -first helix for recognition
• -second for stabilization

• -structure that binds zinc ion
• -typically 2-3 zinc fingers to bind to DNA

• -spaced every 7 AA
• -does not directly interact with DNA

• -catalyze specific reaction on DNA (transcription by RNA polymerase)
• -binding to block transcription (negative regulation)
• -binding to activate transcription (positive regulation)

• -greatly influenced by environment
• -presence or absence of specific small molecules
• -interactions between small molecules and DNA binding proteins

-regulatory mechanism that stops transcription

• -preventing the synthesis of an enzyme in response to a signal
• -generally affects anabolic enzymes (arginine biosynthesis)

• -production of an enzyme in response to a signal
• -typically affects catabolic enzymes (lac operon)
• -enzymes only made when needed=no wasted energy

-substance that induces enzyme synthesis

-substance that represses enzyme synthesis

-collective term covering all inducers and repressors

• -effectors affect transcription indirectly 
• -they bind to allosteric repressor proteins
• -this activates them and they bind to area near DNA promoter called the operator

-cluster of genes arranged in a linear fashion whose expression is under the control of a single operator

-downstream of the promoter

-inhibitory, which makes it a negative control

• -a regulator protein that activates the binding of RNA polymerase to DNA
• -maltose catabolism in E Coli

• -at activator binding site
• -NOT OPERATOR (like in negative control)

-help RNA polymerase recognize the promoter

• -multiple operons controlled by the same regulatory protein
• -exist for positive control (maltose)
• -exist for negative control (arginine)

• -recognize specific DNA sequences and then cut there
• -common in prokaryotes
• -rare in eukaryotes

• -pentameric with separate restriction (R), methylation (M) and DNA sequence recognition (S) subunits
• -cut DNA at RANDOM far from recognition sequences

• -first REases to be discovered and purified 
• -no lab use as they cut at random (unlike type 2)
• -originally thought to be rare, now known as common

• -cleave DNA within their recognition sequence and most useful REase for specific DNA manipulation
• -recognize inverted repeats (palindromes)
• -sequence usually 4-8BP long
• -form sticky or blunt ends
• -protect cell from foreign DNA invasion (virus)
• -used in gene cloning

• -large, combination and restriction enzyme
• -cleave outside recognition sequence, 25 BP away
• -require 2 sequences in opposite orientations within same DNA molecule to get cleavage, therefore rarely give complete digests

• -recognize modified, typically methylated DNA
• -McrBC and Mrr systems in E Coli

• -protect cell's DNA from REases
• -usually a methylation of DNA
• -type 1,2 and 3 have separate modification enzymes

• -separates DNA molecules based on size
• -gels usually agarose (a polysaccharide)
• -Nucleic acids move through electrified gel to positive end(attract - DNA backbone)
• -small molecules move farther

-can be used to stain gel in electrophoresis so DNA can be seen under UV light

-map of the location of RE cuts on a segment of DNA

-allows creation of color coded gene map based on where RE cut and electrophoresis based on stained color movements

-base pairing of single strands of DNA or RNA from two different sources to give hybrid double helix

-segment of single stranded DNA used in hybridization with predetermined identity

-hybridization where DNA is in the gel and probe is RNA or DNA

-RNA is in the gel

• 1-isolation and fragmentation of source DNA 
• 2-Insertion of DNA fragment into cloning vector
• 3-introduction of cloned DNA into host organism

• -isolation and fragmentation of source DNA
• -source DNA can be genomic DNA, RNA or PCR amplified fragments
• -genomic DNA must first be RE digested

• -insertion of DNA fragment into cloning vector
• -most vectors derived from plasmids or viruses 
• -DNA generally inserted in vitro

• -enzyme that joins 2 DNA molecules
• -works with sticky or blunt ends

• -introduction of cloned DNA into host organism
• -transformation is often used to get recombinant DNA into host

-mixture of cells containing a variety of genes

-gene libraries made by cloning random genome fragments

• 1-add radioactive DNA or RNA probes to lysed bacteria and denatured DNA then wash away anything not radioactive
• 2-partially lyse cells then add antigen and attempt to detect blood serum antibodies

• -De Novo synth of 100 BP oligonucleotides
• -multiple oligonucleotides can be ligated together
• -can be used for primers, probes and in-site directed mutagenesis

-produce mutations at random

• -performed in vitro
• -used to access specific AA in protein

-process of cutting DNA fragment out and replacing it with synthetic DNA fragment

• -when cassettes are inserted into the middle of the gene
• -causes knockout mutations

• -proteins that are easy to detect and assay
• -LacZ, Luciferase, GFP

• -small size
• -easy to isolate DNA
• -independent ORI
• -get multiple copies of cloned gene per cell
• -selectable markers

• -modified ColE1 plasmid 
• -common cloning vector
• -contains ampicillin resistance and LacZ genes
• -contains polylinker within LacZ gene

• -white colonies have foreign DNA, blue do not
• -LacZ is inactivated by foreign DNA, inactivated LacZ cannot process Xgal=blue does not develop

-genetic element that cannot replicate independently of a living host

• -virus particle
• -extracellular form of a virus
• -exists outside host
• -facilitates transmission from one host cell to another 
• -contain nucleic acid genome surrounded by protein coat and in some cases other layers of material

-on the basis of the hosts they infect

• -protein shell that surrounds the genome of a virus particle
• -made up of a highly repetitive and precise pattern around nucleic acids

• -subunit of the capsid
• -smallest morphological unit visible with electron microscope

-complete complex of nucleic acid and protein packed in the virion

• -virus that contains additional layers around the nucleocapsid
• -lipid bilayer with embedded proteins 
• -envelope makes initial contact with host cell
• -

• -helical (rod shaped)
• -icosahedral (spherical)

• -rod shaped viruses 
• -length of virus determined by length of nucleic acid 
• -width of virus determined by size and packaging of protein subunits
• -tobacco mosaic virus

• -spherical viruses 
• -most efficient arrangement of subunits in a closed shell
• -human papillomavirus

• -contains rigid spikes of haemagglutinin and neuraminidase that form characteristic halo projections around negatively stained virus particles
• -binds sialic acid on host membranes

• -virions composed of several parts, each with separate shapes and symmetries
• -bacterial viruses contain complicated structures (icosahedral heads and helical tails)

• -lysozyme
• -nucleic acid polymerases 
• -neuraminidases

• -make hole in cell wall
• -lyses bacterial cell

• -enzyme that cleaves glycosidic bonds
• -allows liberation of viruses from cell

-number of infections units per volume of fluid

• -analogous to the bacterial colony 
• -one way to measure virus infectivity

• -clear zones that develop on lawns of host cells
• -lawn can be bacterial or tissue culture
• -each plaque results from infection by a single virus particle

• -quantitative virology 
• -number of plaque forming units almost always lower than direct counts by electron microscopy due to inactive virions and conditions not appropriate for infectivity

• 1.attachment (adsorption)
• 2.entry(penetration)
• 3.synthesis
• 4.assembly
• 5.release

• -one step growth
• -latent period (eclipse and maturation)
• -burst size (number of virions released)

host cell that allows the complete replication cycle of a virus to occur

• -very complex
• -virions attach to cells via tail fibers that interact with polysaccharides on E. Coli cell envelope
• -tail fibers retract
• -tail core makes contact with E. Coli cell wall
• -lysozyme like enzyme forms small pore in peptidoglycan 
• -tail sheath contracts and viral DNA passes into cytoplasm