• 1941
• one gene; one enzyme
• neurospora

• gene exchange
• 1946
• E. Coli

genome?

gene

codon

operon?

• All genetic info a cell has
• a piece of DNA that encodes for a protein
• 3 nucleotide sequence that encodes for a particular AA
• operates many genes simultaneously ( 1 promotor=many genes)

1. operator
2. promotor
3. ribosomal binding site
4. start codon
5. ORF
6. stop codon
7. Terminator

1. bases (ATGC)
2. deoxyribose sugars
3. phosphate

pyrimidine bases

purine bases

pyr - CTU

Pur - GA

major - makes bps visible to extracellular proteins to detect sequence

minor - RNA pol binds to it

key enzymes fxn: DNA polymerase

Ligase

Helicase

single stranded binding proteins

Primase

• DNA pol - read template and add new nucleotides to 3' end in 5' to 3' fashion
• lig - removes primer and connects okazaki fragments
• helicase - unzips DNA for replication
• ssbp - makes sure DNA doesnt reconnect
• creates RNA primer for DNA pol to attach

• transcribes DNA to RNA in a 5' to 3' matter
• doesnt need primer as it binds promotor of gene
• detaches once terminator is reached

role of sigma factor in transcription?

• helps RNA pol bind to promotor by helping recognition and initiation

• key enzyme in translation
• make sure proper AA binds to proper tRNA

1. RNA threads through ribosome
2. tRNA lines up its anticodon with the complementary codon and bind to large subunit of ribosome
3. peptide bonds made between adjacent AA's on tRNAS
4. tRNA's are released

DNA and priimary RNA

removed in mature mRNA

1. missense - change in bp that causes a change in AA 
2. nonsense - change in bp that encodes a stop signal
3. samesense(silent)- change in bp that causes no change

1. insertion - bp is inserted
2. inversion - part of sequence is flipped
3. deletion - bp is removed

1. spontaneous - DNA replication
2. induced - chemicals, radiation

1. chemicals - base analogs, base modifiers
2. radiation - x-rays, UV (cause thymine dimers)
3. biological mutagens (transposons, insertion sequences)

• photoreactivation - uses enzyme photolyase which recognizes dimer and breaks it apart
• excision - uses enzyme UVrABC to nick DNA downstream of dimer and have DNA pol fix DNA and have DNA ligase seal up nick
• SOS repair - fast repair that is very error prone

1. make a master plate
2. innoculate velvet with same pattern of bacteria
3. place velvet on defined media with sole glucose energy source
4. see what colonies grow and don't, those that dont grow are histidine defficient (His auxotroph)
5. grow on enriched media plate (to isolate)

• tests for bacterial mutagenicity
• hisitidine auxotrophs are put on minimal glucose media and a test compound is put on the plate. an increase in growth around the innoculating disk shows an increase in mutation therefore the test compound is a mutagen.

Homologous recombination

site-specific recombination?

HR - requires long regions of homology and the RecA protein. single or double strand can be exchanged.

Site Sp - requires only a short region of homology and  doesnt need the RecA protein.

1. transformation
2. conjugation
3. transduction

• donor cell releasing free DNA into environment to be absorbed by recipient cell.
• 1928 - Griffiths exp - Smooth and rough colonies. mouse injected with: R = alive; S=dead; R + dead S=dead. Smooth colony DNA transformed into R DNA.

1. transforming DNA binds to cell
2. Binding protein sheds off one strand creating ssDNA
3. Uptake of ssDNA- RecA proteins bind
4. DNA is incorporated into chromosome via homologous recombination
5. cell division = one transformed and one not

• treat with cold CaCl2 or electroporation

• strain improvement
• gene mapping
• genetically engineering bacteria

• gene transfer of a plasmid by direct cell to cell contact.
• discovered by lederburg and Tatum in 1946 at UW-Madison in E. coli

• extra chromosomal DNA that contains an origin
• 1 to 1000 kbps
• 1 to 500 copies/cell
• unessential genes to cell viability

replication of Plasmid? 2 types

• theta replication - like bacterial chromosome, may be bi or unidirectional
• rolling circle - DNA rolled off like a spool and both replicated. (gram +)

• conjugable plasmids - contain tra operan and second replicating origin (oriT)
• non-conjugable - lack the tra operon and oriT
• mobilizing - contain oriT but lack the tra operan. can transfer if somewhere else in genome.
• Resistance transfer factors - carry resistance antibiotics

• p for plasmid
•  researchers initials
• number plasmid made

1. donor and recepient cells attach via sex pilus
2. pilus retracts and cells fuse
3. enzyme encoded by tra operon nicks plasmid at oriT
4. one strand moves through to the other cell
5. replicate via rolling circle replication
6. after entire plasmid is transfered cells separate

1. same as reg conjugation until
2. F-Plasmid performs recombination incorporating itself into chromosome
3. portion of F plasmid and donor DNA moves into recipient cell before broken apart
4. complementary DNA is synthesized
5. F plasmid and donor DNA recombine with recipient DNA creating a recombinant F- cell 

• can go in and out of chromosome
• same steps as plasmid conjugation

1. gene mapping - Hfr conjugation
2. strain improvement
3. transfer of recombinant DNA

• intracellular obligate parasite
• not a cell
• carries genetic information
• capable of replication in cells

• while running Koch's postulates some infectious causing agents were unfilterable so they were called filterable viruses. thought to be small bacteria
• Tobacco mosaic virus (TMV) in 1892

• ss(+) or ds DNA 
• ss(+ or -) or ds RNA
• + encodes for a protein
• - needs to be complemented to make + strand

• made of numerous proteins
• filamentous or icosahedral
• complex have tails and fibers

• outer layer of viroid made of lipids and glycoproteins

1. attachment
2. penetration
3. synthesis (replication)
4. assembly 
5. release

1. attachment
2. injection
3. incorporation into DNA
4. cell division

• contain ssRNA 
• reverse transcriptase creates DNA version that is incorporated into host genome

• ssRNA
• corona virus

viroid?

prion?

• v - ssRNA no capsid or genes
• p - infectious protein that causes CWD, mad cow, CJD

1. DNA of host cell is in phage instead of viral DNA.
2. DNA is injected in new host cell
3. DNA is recombined with host chromosome

• phage DNA circulizes and detaches from host DNA
• phage DNA replicates
• phages are synthesized
• cell lyses and releases phage
• rare - part of host DNA is exchanged for phage DNA (could incorporate a gene)

recognize a 6bp sequence that they cut at to create sticky ends

name by first letter in genus and first two letters in species E. coli = Eco RI

1. plasmid
2. bacteriophage
3. cosmid
4. Yeast artificial chromosome

• allowed us to clone much bigger pieces of DNA
• human genome project

1. isolate DNA with genes you want to clone
2. buy a plasmid 
3. cut both with same restriction enzyme
4. mix and add DNA lyses
5. transfer into competent E. coli
6. plate LB + amp + Xgal

1. apply heat to target genes 
2. apply primers and DNA pol
3. primer extension
4. repeat
5. gene copies are made

• new vaccines
• gene therapy
• bioremediation
• agriculture
• protein synthesis