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Biotechnology > 1 - Intro Lecture > Flashcards

1 - Intro Lecture Flashcards

1
Q

What are chromosomes composed of?

A

a centromere from which four arms protrude, and a telomere at each end

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2
Q

_______ helps to confer stability to the ends of the chromosome

A

telomere

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3
Q

____ pairs of autosomal chromosomes

A

22

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4
Q

___ sex determining chromosomes

A

2

XX (female)
XY (male)

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5
Q

Chromsomes are composed of ______ which is compactly folded mixtures of DNA and basic DNA-binding proteins

A

chromatin

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6
Q

Chromatin is composed of individually packaged units called ________

A

nucleosomes

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7
Q

How do nucleosomes appear as by electron microscopy ?

A

as beads on a thin string

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8
Q

Describe what a nucleosome consists of

A

octamer of basic DNA binding proteins called histones with a constant length of DNA wound around it

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9
Q

Each octamer consists of ?

A

two copies of histones- H2A, H2B, H3, and H4

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10
Q

Each nucleosome is separated from its neighbours by a length of DNA that is bound to _________

A

histone H1

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11
Q

What do nucleosomes do?

A

package DNA but also help to regulate gene expression

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12
Q

Compare prokaryotic and eukaryotic cells

A

Prokaryotic:

  • no nucleus
  • peptidoglycan

Eukaryotic:

  • nucleus
  • Golgi vesicles
  • mitochondria

slide 4

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13
Q

Describe the basic prokaryotic gene structure

A
  • no splice sites so no splicing
  • promoters are much less complex
  • a consensus sequence for binding RNA pol
  • sometimes a simple regulatory mechanism
  • no poly A signal so no poly A tail
  • half life of RNA controlled by other mechanisms
  • no 5’ methyl-guanosine cap
  • ribosomes bind to an RNA sequence called the shine-dalgarno sequence
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14
Q

Describe DNA replication

A
  • Synthesis is 5’ to 3’, the incoming nucleotide triphosphate is added to the 3’OH of the existing DNA strand
  • Diphosphate (PPi) isa good leaving group this along with hydrolysis of PPi to phosphate drives the reaction forward.
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15
Q

During DNA synthesis, the DNA polymerase makes a mistake about every _____ base pairs

A

10,000

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16
Q

The DNA polymerases have ______-________ activity which means they can replace improperly paired bases with the correct ones.

A

proof-reading

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17
Q

These DNa polymerases also have exonuclease activity so they can do what?

A

remove the mismatched base in addition to adding the right one

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18
Q

__________ cuts one strand of DNA, allowing it to twist around by one turn and then re-ligate. It does this repeatedly to relieve the torsional strain in the DNA created by the helices.

A

topoisomerase 1

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19
Q

The leading strand is synthesized continuously in the _______ direction.

A

5’ to 3’

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20
Q

The lagging strand is also synthesized in what direction?

A

5’ to 3’ (but in small discontinuous stretches known as Okazaki fragments)

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21
Q

A small RNA primer is made by ____ _______ to initiate synthesis. This is later degraded by RNase H followed by filling in DNA by DNA pol

A

RNA to primase

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22
Q

The fragments are joined by ______

A

ligase

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23
Q

What unwinds DNA?

What does unwinding cause?

A
  • helicase

- torsional strain on the DNA

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24
Q

Helicase unwinds DNA so ___ _________ can gain access to the DNA.

A

DNA polymerase

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25
Q

Replication starts at a specific sequence called the _____ of replication some times abbreviated ori

A

origin

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26
Q

The Lac operon is an example of a ___________

A

prokaryote

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27
Q

Lac operon:

What is the preferred carbon source?

A

glucose

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28
Q

Lac operon:

If glucose isn’t available, then ______ can be digested into galactose and glucose as a carbon source

A

lactose

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29
Q

Prokyarotic gene regulation:

When would there be no need to make genes for digesting lactose?

A

if lactose is not present or if sufficient glucose is

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30
Q

Prokyarotic gene regulation:

What does the lac operon control?

A

the expression of genes lac Z, Y, and A

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31
Q

Prokyarotic gene regulation:

What dose Lac I do?

A

Produces the lac repressor which binds to the operator in the absence of lactose. If lactose is present it binds to the repressor and prevents it from binding to the operator

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32
Q

Prokyarotic gene regulation:

What does RNA polymerase do?

A

Binds to the promoter. If the repressor is bound to the operator RNA polymerase cannot transcribe lac Z, Y and A

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33
Q

Prokyarotic gene regulation:

What does CAP do?

A

Binds the cAMP receptor protein which helps RNA polymerase bind to the promoter

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34
Q

Prokyarotic gene regulation:

What does the cAMP receptor protein do?

A

Binds cAMP and only then will it bind to the CAP site

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35
Q

Prokyarotic gene regulation:

The [cAMP] increases as [glucose] _______

A

decreases

**inverse relationship

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36
Q

make sense of slide 13

A

OKAY

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37
Q

Gene regulation in _______ is more complicated than in bacteria.

A

eukaryotes

38
Q

Eukaryotic gene regulation:

Does RNA polymerase bind to the promoter by itself?

A

it does not

39
Q

Eukaryotic gene regulation:

A group of proteins must also bind to the promotor (along with RNA polymerase), what are they?

A
  • transcription factors
  • co-activators
  • co-repressors
  • depending on the proteins that are bound expression can be higher or lower
40
Q

Eukaryotic gene regulation:

A Cis acting element is a DNA sequence that a DNA binding protein (_____ acting element) will bind it

A

Trans

41
Q

What are the promoters region of a eukaryotic gene?

A

the TATA box
the GC box
the CCAAT box

42
Q

What is the consensus sequence of the TATA box?

A

TATAAAA

43
Q

What binds to the TATA box?

A

Transcription factor IID (TFIID)

44
Q

What does TATA-binding on the TATA box do?

A

bends the DNA and forces the helix to open slightly, probably allowing better access to RNA polymerase

45
Q

What is the consensus sequence of the GC box?

A

GGGCGG

46
Q

GC box:

The transcriptional activator ____ binds to this region

A

Sp1

47
Q

What is the consensus sequence of the CCAAT box?

A

GGCCAATCT

48
Q

What do enhancers do?

A

bind activators and repressors

49
Q

Where are enhancers located?

A

some distance from the gene

50
Q

Describe enhancer sequence

A

can be relative to the promotor or flipped in the opposite orientation

51
Q

Activators bound at an _______ can work together to switch on a gene

A

enhancer

52
Q

What does the 5’ methyl-guanosine cap do in RNA processing ?

A

Composed of a methyl-guanosine nucleotide covalently attached to the 5’ phosphate of the first nucleotide of the mRNA

  • recognition signal for ribosome
  • signal to export out of nucleus
  • increases half life of mRNA
53
Q

What does the Poly A tail do ?

A
  • It is a specific mRNA sequence AAUAAA is recognized by the enzyme polyadenylate polymerase
  • Cleave the primary transcript 11-30 bases at 3’ end
  • Add a stretch of 200-250 adenosines (increases mRNA half life and nuclear export)
54
Q

What is RNA splicing?

A

The process of removing introns from pre-mRNA to form mRNA is called RNA splicing or just splicing

55
Q

RNA splicing is a 2-step process: Describe it

A
  • The cleavage of the 5’ intron/exon junction occurs simultaneously with the formation of a new phosphodiester bond between the phosphate at the 5’ end of the intron and an adenine residue just within the 3’ end of the intron
  • Another trans-esterification reaction occurs, in which the phosphodiester bond at the 3’ intron/exon junction is cleaved and the two cleaved axons are joined.
56
Q

One of the largest known human genes is Titin, which has more than 175 ____

A

exons

57
Q

In mammalian cells, intron removal largely depends on specific features of individual introns, what are some?

A
  • How well the splice signal sequences match the consensus splice site sequences
  • Intron length
  • Other regulatory sequences located within the pre-mRNA such as the polypyrimidine tract
58
Q

Describe polycistronic mRNA

A
  • mRNA encode more than one protein

- the information for each arranged in tandem

59
Q

Describe the Shine-Dalgarno box in mRNA

A
  • a specific ribosome-binding sequence

- adjacent to the start site for each protein

60
Q

Briefly describe what you need for the making of protein drugs

A
  • the gene
  • a vector to carry the gene (usually a plasmid)
  • a way to get the gene into the vector
  • a way to get the vector with the gene into the microorganisms or cultured mammalian cells
  • a way to select for the microorganisms or cultured mammalian cells that have the vector with the gene
  • a way to control the expression of the gene once it is in the microorganisms or cultured mammalian cells
  • a way to lyse the cells and purify the protein drug away from other contaminating proteins made by microorganisms or cultured mammalian cells
61
Q

Get the gene from the human cell by making what is known as ____

A

cDNA

62
Q

Amplify the gene from cDNA using ______ ______ _____

A

polymerase chain reaction (PCR)

63
Q

What is a plasmid?

A

A vector for transferring gemes

64
Q

Describe a plasmid

A

A long sequence of circular double stranded DNA

  • Place to put gene
  • Selection mechanism
  • Method of self replication
  • A promoter (place to initiate translation)
65
Q

______ may be used to transfer genes to mammalian, yeast, insect and bacterial cells

A

Vectors

66
Q

Plasmids are put into bacteria and yeast by a process known as __________, in insect and mammalian cells this is known as transfection

A

transformation

67
Q

What is antibiotic selection?

A

This allows to select for cells that have the plasmid

Ex. if we are using bacterial cells we will use an antibiotic resistance gene such as B-lactamase. If we grow the bacteria in the presence penicillin only the bacteria that have the plasmid and hence express the gene can grow.

68
Q

What is an affinity tag?

A

Not always present

  • Expressed as part of the protein of interest
  • Allows us to capture the protein we expressed to purify it
69
Q

What is a protease cleavage site?

A

Not always present
-A specific proteolytic cleavage site within the expressed protein usually 4 to 5 amino acids long to cut off the affinity tag to produce a protein free of the affinity tag

70
Q

Once the protein is expressed, it can be purified with ______ _____

A

affinity resin

71
Q

What is a multiple cloning region?

A

a short sequence with multiple restriction sites

72
Q

What are restriction sites?

A

specific sequences that are specifically cleaved by restriction enzymes

73
Q

The restriction sites in the multiple cloning region only appear _____ in the whole plasmid

A

ONCE

74
Q

What is sub cloning?

A

the process of putting the gene of interest into a plasmid, can be done with restriction enzymes

75
Q

_______ expression system is the most popular system of expression recombinant proteins

A

bacterial

76
Q

______ expression system is a good alternative when a bacterial expression system is not adequate

A

yeast

77
Q

Proteins expressed in yeast will have most of the _______ post-translational modification

A

eukaryotic

78
Q

______ cells recognize most mammalian protein-targeting sequence, thus they can express most mammalian proteins

A

Insect

79
Q

Insect cell vectors are called ________ vectors

A

Baculovirus

80
Q

Insect cells grow more _______ than bacterial and yeast cells

A

slowly

81
Q

________ cells provide the best expression system for generating recombinant eukaryotic proteins

A

mammalian

82
Q

Mammalian cells grow _____

A

slowly

83
Q

Advantages of bacteria expression system

A

Easy, fast, can produce a large quantity of protein rapidly

84
Q

Disadvantages of bacteria expression system

A

No post-translational modifications

85
Q

Advantages of yeast expression system

A

Some post-translational modifications, faster than mammalian

86
Q

Disadvantages of yeast expression system

A

handling may be difficult

87
Q

Advantages of insect cell expression system

A

Large quantities of proteins, some post-translational modifications, secreted and tagged forms

88
Q

Disadvantages of insect cell expression system

A

Difficult to handle large plasmids

89
Q

Advantages of mammalian cell expression system

A

post-translational modifications

90
Q

Disadvantages of mammalian cell expression system

A

Slow, more complex, expensive

Biotechnology (9 decks)

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