Bio Test 4 Flashcards
PCR components
- DNA template, gene specific primer pairs, free nucleotides, Taq DNA ploymerase
- Thermocycle program
94C during PCR
opens two strands (denatures) the DNA template
55C during PCR
primers anneal (hybridize/connect) to the template, hydrogen bonds begin to form
72C during PCR
DNA synthesis (primer extension)
What does PCR do?
- exponential production of specific DNA fragments
- needs buffer containing Mg+2
- occurs about 30 times to get many copies
Knock out mice
- removing a genes function
- helicase gene exhibits premature aging
Procedure of knock out mice
- use mouse embryonic stem
- replace the DNA fragment of interest by recombination
- mosaic F1 (implant in animal)
- select for germ-line transmitted animal
genome
total genetic information carried by a cell or an organism
transcriptome
entire complement of RNAs produced by cell, differential expression comes into play
proteome
entire complement of proteins produces by a cell
proteomics
systematic study of the amounts, modifications, interactions, localization and functions of all or subsets of proteins at the whole-organisms, tissue, cellular and subcellular levels
characteristics of genomics
- construct genomic and cDNA libraries using recombinant DNA technique
- cDNAs are the complementary DNAs of mRNAs synthesized by reverse transcriptase enzyme from virus
genomic library
combination of recombinant plasmid and E.coli
cDNA
- reversely transcribed from mRNAs by reverse transcriptase enzyme
- single stranded cDNA can be replicated into double strand cDNA by DNA polymerase
- build transcriptome
DNA sequencing was developed by who?
Frederick Sanger
process of DNA sequencing
- use di-deoxynucleotides to stop the replication
- only one strand is used as template
- use one primer instead of two in PCR
- set up four reactions with regular nucleotides and single dideocynucleotide
- generate DNA fragments of different lengths
- run products on gel and read sequence from bottom to top of gel
process of automated DNA sequencing
- use fluorescence labeled dideoxynucleotides
- reaction is done in one tube
- data are read and assembled by computer
- sequencers provide accurate sequences up to 1000 bp long
procedure of genome sequencing project
- generate reocmbinant plasmids containing DNA fragments
- use automatic sequencer to obtain nucleotide sequences
- assembly of DNA clones by actual sequences
- annotation (to identify genes and their locations)
What are the findings of the human genome project?
- human genome contains 3.2 billion bp of DNA
- 1% actually encodes proteins
- ~50% DNAs are non-gene sequence repeated thousands of times
- genes are unequally distributed
- only have 30000 genes
- more different proteins than genes alternative splicing 300000 proteins vs 30000 genes
What are the four phases of the cell cycle?
G1
S
G2
M
what is interphase?
the collection of phases G1, S, G2
what does the G1 phase do?
primary growth, longest stage
what does the S phase do?
DNA synthesis
what does the G2 phase do?
second growth
what does the M phase do?
mitosis (nuclear division) and cytokinesis (cytoplasmic division)
cell cycle
- has four different phases
- cells have different cell-cycle times
- withdrawal from cell cycle: non-dividing cells
what does the G0 phase do?
resting state
cyclins
- expressed in a cyclic fashion
- no enzyme activity
- bind and activate Cdks
cell cycle checkpoints
proteins that check whether call is ready to move on to next phase
major cyclins and Cdks of vertebrates
S-Cdk complex
M-Cdk complex
G1-Cdk complex
S-Cdk complex
cyclin A pairs with Cdk2
M-Cdk complex
cyclin B pairs with Cdk1
G1-Cdk complex
cyclin D pairs with Cdk4 and 6
What are Cdks?
- cyclin-dependent protein kinases
- activated by cyclins
- contribute to their own eventual inactivation
G1 checkpoint
checks whether environment is favorable to begin S phase
G2 checkpoint
checks for damage and whether DNA is completely replicated
mitosis checkpoint
checks whether chromosomes are attached to spindles
activation of M-Cdk (as an example)
- cyclin B binds and activates Cdk1
- phosphorylated M-Cdk
- Cdc25 activates M-Cdk by dephosphorylation
- positive feedback phosphorylates more Cdc25
- activate APC
- complete M phase
inactivation of M-Cdk
- APC
- ubiquitylation of cyclin B
- degradation of cyclin B
- inactivation of M-Cdk
DNA damage inhibits what?
S-Cdk and G1/S-Cdk function
process of DNA damage that inhibits S-Cdk and G1/S-Cdk
- DNA damage
- kinases
- phosphorylate p53
- transcription of p21
- p21 protein binds and inhibits S-Cdk and G1/S-Cdk
What are the two types of cell death?
- necrosis
- apoptosis (programmed cell death)
necrosis
- osmotic imbalance
- influx of H2O into cell
- can burst the cell
apoptosis
- carried out by a family of proteases called caspases
- procaspases are activated through cleavage and reassembly
- main proteins that regulate the activation of procaspases are members of the Bcl2 family
- death signal goes to mitochondria first
what are the members of the Bcl2 family?
- Bax
- Bak
What do Bax and Bak do?
form channels on mitochondria causing release of cytochrome C which binds to adaptor protein and then activates first caspase (procaspase 9)
