Bio Test 4

Question 1

PCR components

Answer 1

• DNA template, gene specific primer pairs, free nucleotides, Taq DNA ploymerase
• Thermocycle program

Question 2

94C during PCR

Answer 2

opens two strands (denatures) the DNA template

Question 3

55C during PCR

Answer 3

primers anneal (hybridize/connect) to the template, hydrogen bonds begin to form

Question 4

72C during PCR

Answer 4

DNA synthesis (primer extension)

Question 5

What does PCR do?

Answer 5

• exponential production of specific DNA fragments
• needs buffer containing Mg+2
• occurs about 30 times to get many copies

Question 6

Knock out mice

Answer 6

• removing a genes function

- helicase gene exhibits premature aging

Question 7

Procedure of knock out mice

Answer 7

• use mouse embryonic stem
• replace the DNA fragment of interest by recombination
• mosaic F1 (implant in animal)
• select for germ-line transmitted animal

Question 8

genome

Answer 8

total genetic information carried by a cell or an organism

Question 9

transcriptome

Answer 9

entire complement of RNAs produced by cell, differential expression comes into play

Question 10

proteome

Answer 10

entire complement of proteins produces by a cell

Question 11

proteomics

Answer 11

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

Question 12

characteristics of genomics

Answer 12

• construct genomic and cDNA libraries using recombinant DNA technique
• cDNAs are the complementary DNAs of mRNAs synthesized by reverse transcriptase enzyme from virus

Question 13

genomic library

Answer 13

combination of recombinant plasmid and E.coli

Question 14

cDNA

Answer 14

• reversely transcribed from mRNAs by reverse transcriptase enzyme
• single stranded cDNA can be replicated into double strand cDNA by DNA polymerase
• build transcriptome

Question 15

DNA sequencing was developed by who?

Answer 15

Frederick Sanger

Question 16

process of DNA sequencing

Answer 16

• 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

Question 17

process of automated DNA sequencing

Answer 17

• 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

Question 18

procedure of genome sequencing project

Answer 18

• 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)

Question 19

What are the findings of the human genome project?

Answer 19

• 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

Question 20

What are the four phases of the cell cycle?

Answer 20

G1
S
G2
M

Question 21

what is interphase?

Answer 21

the collection of phases G1, S, G2

Question 22

what does the G1 phase do?

Answer 22

primary growth, longest stage

Question 23

what does the S phase do?

Answer 23

DNA synthesis

Question 24

what does the G2 phase do?

Answer 24

second growth

Question 25

what does the M phase do?

Answer 25

mitosis (nuclear division) and cytokinesis (cytoplasmic division)

Question 26

cell cycle

Answer 26

- has four different phases - cells have different cell-cycle times - withdrawal from cell cycle: non-dividing cells

Question 27

what does the G0 phase do?

Answer 27

resting state

Question 28

cyclins

Answer 28

- expressed in a cyclic fashion - no enzyme activity - bind and activate Cdks

Question 29

cell cycle checkpoints

Answer 29

proteins that check whether call is ready to move on to next phase

Question 30

major cyclins and Cdks of vertebrates

Answer 30

S-Cdk complex M-Cdk complex G1-Cdk complex

Question 31

S-Cdk complex

Answer 31

cyclin A pairs with Cdk2

Question 32

M-Cdk complex

Answer 32

cyclin B pairs with Cdk1

Question 33

G1-Cdk complex

Answer 33

cyclin D pairs with Cdk4 and 6

Question 34

What are Cdks?

Answer 34

- cyclin-dependent protein kinases - activated by cyclins - contribute to their own eventual inactivation

Question 35

G1 checkpoint

Answer 35

checks whether environment is favorable to begin S phase

Question 36

G2 checkpoint

Answer 36

checks for damage and whether DNA is completely replicated

Question 37

mitosis checkpoint

Answer 37

checks whether chromosomes are attached to spindles

Question 38

activation of M-Cdk (as an example)

Answer 38

- 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

Question 39

inactivation of M-Cdk

Answer 39

- APC - ubiquitylation of cyclin B - degradation of cyclin B - inactivation of M-Cdk

Question 40

DNA damage inhibits what?

Answer 40

S-Cdk and G1/S-Cdk function

Question 41

process of DNA damage that inhibits S-Cdk and G1/S-Cdk

Answer 41

- DNA damage - kinases - phosphorylate p53 - transcription of p21 - p21 protein binds and inhibits S-Cdk and G1/S-Cdk

Question 42

What are the two types of cell death?

Answer 42

- necrosis | - apoptosis (programmed cell death)

Question 43

necrosis

Answer 43

- osmotic imbalance - influx of H2O into cell - can burst the cell

Question 44

apoptosis

Answer 44

- 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

Question 45

what are the members of the Bcl2 family?

Answer 45

- Bax | - Bak

Question 46

What do Bax and Bak do?

Answer 46

form channels on mitochondria causing release of cytochrome C which binds to adaptor protein and then activates first caspase (procaspase 9)