• determine cell shape
• locomotion
• intracellular transport of organelles
• seperation of chromosomes during mitosis
• NOT cytokinesis

colchicine, colcemid

binds tubulin, inhibit MT polymerization, blocks mitosis

colchicine promotes depolymerization because concentration of tubulin will be too low

specific for rapidly dividing cells

binds tubulin and inhibits MT poymerization

GTP binds to B tubulin, and when GTP is hydrolized to GDP shortly after binding, the affinity is weakened, favoring depolimerizatio

properties of microtubles

• forms stable arrangements to support cell projections
• growth is favored at plus end
• used for vescicle transport
• in vitro, both ends show growth
• dimer subunits

• found in the PCM
• acts as a seed for MT assembly

dyenin

• motor protein
• moves toward negative end of microtubules (toward centrosome)
• abundant in cilia
• helps position the golgi apparatus

• fundamental structure of flagella and cilia
• central pair of MT surrounded by 9 outer pairs
• pairs: one complete, A, (13 protofilaments) and one incomplete, B, (11 protofilaments)
• nexin connects outer pairs together
• radial spoke connects outer MT to the inner pair
• dyenin is motor protein

the minus ends of the microtubules are anchored here

structurally similar to a centriole (9 triplet organization)

individual microtubules alternate between cycles of growth and shrinkage

important for remodling of the cytoskeleton during cytokinesis

• cap ends of microtubules
• track growing microtubules towards a specific location
• dissassemble either by severing or increasing depolorization

1. cell growth

2. DNA replication

3. distribution of duplicated chromosomes

4. cell division

chromosomes condense

cytoskeleton forms mitotic spindle

nuclear envelope disappears and reforms

cytokinesis

seperation of chromosomes

1 hour

Cell metabolically active, growth continues

11 hours

2n

DNA replication

cannot detect number of chromosomes

 8 hours

cell growth

proteins synthesized

scan for mistakes

4n

4 hours

Early Prophase

chromosomes condense

centromere proteins bind to form kinetochore

formation of mitotic spindle

centrosomes migrate

nuclear membrane breakdown

prometaphase

microtubules connect to the kinetochores

balance chromosomes along metaphase plate

when the chromosomes are fully isoloated

connection between chromatids broken

chromosomes seperate

cells start to divide

nuclear envelope reforms

microtubules repolarize

chromosomes decondense

cytokinesis

chromosomal microtubule

not attached, but stabalized by overlapping

push centrosomes apart using kinesin

extend towards the periphery

freely exposed positive ends

associates with dynein at cell membrane

seperation of sister chromatids via kinetochore during mitosis

chromotids move along the MT in the minus direction, driven by dyenien

seperation of centrosomes during mitosis

elongation of polar microtubules

overlapping polar microtubules slide past eachother due to kinesins

also pull apart by astral MTs by cytoplasmic dyenin

Regulation

Lac operon - no lactose

Lactose repressor translated from lacI and binds to lac promotor sequence preventing translation

negative regulation

lac operon - presence of lactose

• repressor gene translated and binds to promotor
• lactose binds to repressor and causes an allosteric change and repressor can no longer bind
• free tranlsation of the lac genes

lac z

b galactosidase - catalyzes the hydrolysis of lactose to glucose and galactose

provides the feedback that shuts off the expression of the lac operon

lac a

• as the concentration of glucose decreases, cAMP increases
• cAMP binds to CRP
• the complex binds to the lac promotor and largely enhances the binding of RNA polymerase
• MAXIMAL expression

• lac inhibits the repressor
• but cAMP levels are low, so the binding of RNA polymerase is inefficient

post transcriptional regulation

• at level of MRA stability - degrade mRNA, lac mRNA degraded from 3' end, so lac a, lac y, lac z
• level of translocation - based of strength and availability of shine delgardo sequence
• rate of translation

• acellular
• do not regulate transport of substances in and out of them by a membrane
• do not perform metabolic functions
• obligate intracellular parasites

linear, double strand DNA

1. attachment
2. penetration
3. early gene expression
4. replication
5. late gene expression
6. assembly
7. release

interaction of receptor on host

glycoprotein and capsid protein

virus recognizes signaling molecules

viral life cycle - penetration

• phage injects only nucleic acid
• sometimes entire virus enters - fusion(virus has membrane) OR endocytosis(virus could be naked or have membrane)

viral life cycle - early gene expression

• viral genes that lie adjacent to the promoter attracts the host RNA polymerase to transcribe
• shuts down host transcription
• stimulates viral genome replication
• stimulates viral gene transcription (late gene expression)

viral life cycle - replication

proteins translated during early gene expression stimulate late gene transcription

produces capsid proteins needed for assembly and enzymes needed to release virus

• phage lyses host cells
• animal viruses bud off

neither positive or negative sense

makes RNA via dsDNA intermediate using reverse transcriptase

phage dna forms using nucleotides from digested host DNA

trascribes phage dna and tranlates viral RNA

cells lyse and release newly formed phage, host is destroyed

• Reverse transcriptase synthesizes cDNA from viral RNA
• complementary strand synnthesized from reverse transcriptase
• viral RNA degrads
• new dsDNA integrated into host genome
• foreign DNA replicated over and over by the host
• translation of viral RNA
• protease cleaves long protein chains

The close approximation of two cells during which they exchange genetic material - DNA from one bacteria to another

• ds DNA
• circular
• outside of chromosome
• have an ori - replicate independently from cell chromosomes
• can be present in multiple copies
• enable bacteria to live in inhospitable environments
• resistnance to antibiotics
• allow conjugation

• ori
• some way to select for vector in cell (?)
• some way to distinguish vector alone from vector and insert - polylinker and reporter

gene libraries

all the cloned DNA fragments generated by action of a restriction endonuclease on a genome or chromosome

includes total chromosomal DNA - which includes introns, so proteins cannot be produced directly

1. harvest tissue

2. isolate mature mRNA

3. use reverse transcriptase to make RNA - DNA hybrid

4. degrade RNA

5.  use DNA polymerase to synthesize complementary strand

6. insert cDNA ito vector, transform into host cells

1.  Select - kill all cells that did not take in a vector, insert or not

2.  Screen - cells with an insert usually do not have a reporter gene

3. Probe - use a small part of known protein sequence as a probe to see out target sequences