Biotech Test 2 Set 1

Front 1

Bacteriophage vectors vs. plasmid vectors (2)

Back 1

1)bacteriophage can splice it's genome into bacterial chromosome
2)bacteriophage can hold 20kb of DNA (as opposed to 10kb)

Front 2

Genetically engineered bacteriophages have...

Back 2

2 BamHI sites in their genome

Front 3

20kb of DNA can be removed from a genetically engineered bacteriophage w/o...

Back 3

affecting its ability to infect a cell

Front 4

Weaknesses of using a prokaryotic host cell for a vector (2) and why are these weaknesses HUGE?

Back 4

1)Can't perform post translational modifications (like glycosylation)
2)Can't excise introns

These fxns are needed for biotech drugs that are glycoproteins which require glycosylation

Front 5

Yeasts are used instead of E. coli sometimes b/c...(2) (and ex of a yeast used)

Back 5

1)rapidly replicate
2)can perform post-translational modifications like glycosylation

S. cerevisiae

Front 6

Yeasts as hosts for a vector weaknesses? (3)

Back 6

1)expensive to maintain in culture
2)OVER glycosylate proteins
3)can't excise introns

Front 7

Yeasts and prokaryotes both require ____ to be a vector

Back 7

cDNA

Front 8

Use of higher eukaryote cells as vector hosts (adv(2) and 2 types of cells used

Back 8

1)can remove introns
2)can do post translational modifications (glycosylations)

1)Chinese Hamster Ovary cells
2)Baby Hamster Kidney cells

Front 9

Weakness of using eukaryote cells as vector hosts (3)

Back 9

1)hard to maintain in culture
2)high cost
3)do not replicate very rapidly

Front 10

Diabetes Type 1 (3)

Back 10

1)pancreas does NOT make insulin
2)juvenile diabetes
3)insulin-dependent diabetes

Front 11

Diabetes Type 2 (3)

Back 11

1)usually happens via obesity
2)insulin independent
3)cells can't effectively bind insulin

Front 12

Symptoms of DM2 (3)

Back 12

1)thirsty
2)"sweet" urine
3)excessive urination

Front 13

Steps to make insulin (2)

Back 13

1)Preproinsulin cleaved to proinsulin and signal peptide upon entering B cells
2)proinsulin cleaved to insulin, c-peptide, and dipeptides

Front 14

____ guides preproinsulin to B cells

Back 14

signal peptide

Front 15

Insulin consists of...

Back 15

an A and B chain

Front 16

What joins A and B chain of insulin together

Back 16

2 INTERdisulfide bonds
1 INTRAdisulfide bonds

Front 17

Problems w/ porcine/bovine insulin

Back 17

P-differed by 1 AA on B chain, so ppl got allergic rxns

B-differed by 2AAs on the A chain so allergic rxns

Front 18

How is insulin stored in a B-cell

Back 18

Stabilized by Zn it forms a hexamer

Front 19

Insulin must be ____ to be active in the body

Back 19

MUST BE A MONOMER, not the hexamer form stored in the B-cells

Front 20

Gilbert's method of cloning the insulin gene (6)

Back 20

1)preproinsulin cDNA isolated from induced tumors in rat pancreas
2)Add poly C tails to cDNA
3)Utilize PstI restriction site in ampicillin in pBR322
4)Add poly G tails to opened ampicillin resistance site
5)mix rat cDNA w/ pBR322 and T4 ligase
6)pBR322 vector w/ rat insulin gene is created

Front 21

Problem w/ GILBERT's method

Back 21

created hybrid protein (proinsulin/pencillinase), but was SECRETED

Front 22

GILBERT's protein was proven to be secreted via...

Back 22

Found it in periplasm via Ig method, shocking membrane releases hybrid protein from periplasm

Front 23

Rutter and Goodman's method of cloning insulin gene (7)

Back 23

1)Make cDNA w/ reversetranscriptase from mRNA of human insulin
2)Remove mRNA w/ alkali/RNAaseH
3)use reverse transcriptase to duplicate strand
4)cut "hairpin" w/ s1 nuclease
5)Add HindIII linkers by blunt-end ligation
6)Cut gene and pBR322 plasmid to produce sticky ends
7)mix gene, plasmid, and T4 ligase and vector is created

Front 24

S1 nuclease...

Back 24

ONLY attaches to one stranded DNA

Front 25

Problem w/ Rutter/Goodman method

Back 25

PROTEIN NOT SECRETED

Front 26

Genetech difference from Gilbert and Goodman?

Back 26

didnt use cDNA just synthesized GENES for insulin A/B chains

Front 27

Basic steps of Genetech's insulin cloning? (6)

Back 27

1)A and B gene synthesized separately but all this is done to both genes
2)link gene to B-gal gene using a methionine
3)Insert gene (A or B and B-gal) into EcoRI site of pBR322, compromizing TCN resistance
4)Insert vector into E. coli and harvest protein
5)Split A/B from B-gal using cyanogen bromide (cuts the methionine connection)
6)Mix A/B chain together

Front 28

1st insulin on the market was...

Back 28

Humilin

Front 29

Human insulin (2 types and host cell used)

Back 29

1)Humilin (E. coli)
2)Novolin (S. cerevisiae)

Front 30

Lispro insulin
a)brand name
b)rate of axn
c)how different from human insulin

Back 30

1)Humalog
2)rapid acting (15-30min)
3)has a lys and pro switched on B chain

Front 31

Aspart insulin
a)brand name
b)rate of axn
c)how different from human insulin

Back 31

1)Novolog
2)rapid acting (5-10min)
3)Asp where pro should be

Front 32

Glargine insulin
a)brand name
b)rate of axn
c)how different from human insulin

Back 32

a)Lantus
b)ultra long lasting (24-48hr)
c)2 extra Arg on B and Gly where Asn is on A

Front 33

Why is Lantus long lasting?

Back 33

All AAs added are basic, they increase half-life due to precipitation in blood forming depots

Front 34

How is insulin synthesized today? (2 and source)

Back 34

1)mRNA to cDNA of proinsulin and put in E.coli
2)treat w/ enzymes to release pro part
3)soure mRNA got from pancreatic cancer

Front 35

Clinical uses of HGH (5)

Back 35

1)dwarfism
2)HGH deficiency in kids/adults
3)growth failure in kids due to renal insufficiency
4)Turner's Syndrome
5)Prader-Willi Syndrome

Front 36

Commercially available recombinant HGH products (2)

Back 36

1)Somatrem
2)Somatotropin

Front 37

Somatotropin
a)generic name
b)company produced by
c)difference from human HGH
d)problems...

Back 37

a)rHGH
b)Eli Lilly
c)nothing
d)none

Front 38

Most current formulations of rHGH are...

Back 38

lyophilized (freeze dried) that are reconstituted prior to injection

Front 39

Other dosage form of rHGH

Back 39

2 different liquid formulations available

Front 40

Why is HGH being used now more often in the elderly

Back 40

to decr fat and incr lean

Front 41

FSH is classified as... (2)

Back 41

1)glycoprotein hormone
2)gonadotropin that acts @ male and female gonads

Front 42

Clinical uses of rFSH (3)

Back 42

1)stimulate spermatogeneis in men w/ hypogonadotropic hypogonadism
2)Ovarian hyperstimulation in women who are unresponsive to clomiphere
3)FERTILITY OF MEN AND WOMEN

Front 43

rFSH
a)host cells used
b)purity

Back 43

a)chinese hamster ovary
b)97%

Front 44

2 rFSH products on the market

Back 44

1)Folitropin alpha
2)Folitropin beta

Front 45

Folitropin alpha brand names (2)

Back 45

1)Gonal-F
2)Seronois A

Front 46

Folitropin beta brand names (2)

Back 46

1)Puregon
2)Follistrim

Front 47

Coronary clots are formed from...(2)

Back 47

1)aggregated platelet cells
2)insoluble fibrin which represents matrix of the clot

Front 48

Thrombolytic agents effect on the clot (2)

Back 48

1)bind to fibrin of clot
2)lead to fibrin degradation products which are soluble and hence lyse the clot

Front 49

First recombinant human tPA activator

Back 49

Alteplase (Activase)

Front 50

tPA fxn (2)

Back 50

1)conversion of plasminogen to active plasmin by hydrolysis of Arg560-Val561 peptide bond of the plasminogen
2)plasmin degrades the insoluble fibrin clot into soluble degradation product

Front 51

tPA characteristics (5)

Back 51

a)high affinity for fibrin
b)strong plasminogen activator
c)tPA enhances activation of plasminogen by 600x in presence of fibrin
d)CLOT SPECIFIC
e)less SEs than other thrombolytics b/c so clot specific

Front 52

rtPA
a)produced by... (2)

Back 52

a)cDNA cloning technology
b)expressed in CHO cells b/c glycosylated protein

Front 53

tPA is synthesized by...and properties of the chemical structure (5)

Back 53

a)vascular endothelial cells

b)single chain protein
c)glycosylated
d)serine protease activity
e)17 disulfide bonds
f)Asp117 critical for its liver metabolism

Front 54

tPA structural domains and which 2 bind fibrin (5)

Back 54

1)finger domain***
2)growth factor domain
3)kringle domain1
4)kringle domain2***
5)serine protease domain

Front 55

Clinical usage of Alteplase (rtPA) (3)

Back 55

1)treat AMI
2)acute massive pulmonary embolism
3)ischemic stroke

Front 56

Adverse rxns of alteplase (rtPA) (6)

Back 56

1)bleeding (internal/superficial)
2)co-use of other anti-coagulants further incr bleeding
3)mild hypersensitivity rxns
4)n/v
5)fever
6)hypotension

Front 57

2nd generation of recombinant thrombolytic agents are...(2)

Back 57

1)analogues of alteplase
2)made b/c t1/2 of alteplase is less than 5min due to Asp117 n-glycosylation

Front 58

Disadvantages of alteplace in AMI treatment (2)

Back 58

1)rapid clearance by liver means you have to give it by infusion
2)complicated administration scheme

Front 59

tPA analogues produced b/c... (3)

Back 59

1)prolong half-life allowing a single bolus admin
2)higher level of fibrin specificity to decr bleeding
3)resistance to endogenous tPA inactivtor

Front 60

2 alteplase analogs

Back 60

1)reteplase
2)tenecteplase

Front 61

Reteplase difference from alteplase (5)

Back 61

1)nonglycosylated deletion mutation
2)missing kringle1, finger, growth factor domain
3)expressed in E.coli b/c its non-glycosylated
4)longer half-life
5)greater thrombolytic POTENCY

Front 62

Clinical usage of Reteplase (3)

Back 62

1)imporved ventricular fxn following an AMI
2)reduction of CHF
3)decr mortality from AMI

Front 63

Alteplase ADR's

Back 63

1)bleeding