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Biochem > Regulating Gene Expression > Flashcards

Regulating Gene Expression Flashcards

1
Q

In regards to the cDNA library problem, if one wants to express a eukaryotic protein in E. coli most eukaryotic genes will not work. Why?

A

The genes contain introns.

note: the way around this problem is to make cDNA using reverse trasncriptase

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2
Q

The first step in creating the cDNA library is isolating the total ___ from a cell

A

mRNAs

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3
Q

In the second step of the cDNA library, the mRNAs are converted to cDNA by reverse transcriptase and DNA polymerase I. Isolated mRNA serves as a ___ for copying the RNA into DNA

A

primer

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4
Q

This nicks the RNA, leaving segments as primer for DNA polymerase I

A

RNase H

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5
Q

This fills in the blanks, removes primers, and fills again

A

DNA pol I

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6
Q

This makes the DNA blunt ended

A

T4 poly

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7
Q

Adding linkers prepares the cDNA for ___ into a vector

A

cloning

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8
Q

How could one isolate a gene for a particular protein with an unknown nucleotide sequence?

A

synthesize an oligodeoxynucelotide probe which would be complimentary to one of the strands of the cloned gene

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9
Q

Which oligonucleotides should be used when making the probe?

A

sequences that contain AAs with the least degenerate codons (such Trp and Met; Asn and Lys have minimum wobble)

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10
Q

The Radioactive-antibody test. Describe process

A
  1. a plastic disk coated with anti-insulin antibody is touched to the surface of colonies on a petri dish
  2. insulin molecules from an insulin-producing colony bind to antibodies
  3. radioactivity adheres to the disk at the positions of insulin-producing colonies
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11
Q

PCR permits as high as ___ fold increase in the concentration of a single DNA molecule

A

10^10

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12
Q

This PCR technique produces a fingerprint of an organism that would allow identification of disease causing organisms. - With the set of short, random primers, different organisms will produce unique PCR products visualized by gel electrophoresis

A

RAPD analysis (random amplified polymorphic DNA)

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13
Q

In regards to use of PCR in genetic testing, ___ can be designed to detect specific ___ in affected patients

A

primers; mutations

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14
Q

PCR is so sensitive that only one or few copies of target DNA are required. True or false?

A

true

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15
Q

The DNA polymerase enzyme is isolated from ___ bacteria so that it is able to resist denaturation even in boiling water

A

thermophilic (heat-loving)

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16
Q

These are proteins which are found in relatively constant amount in cells. Their rates of synthesis and degradation are relatively constant in a given cell type regardless of the metabolic state of the cell

A

constitutive proteins

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17
Q

These are proteins which are usually found in low concentrations in a given cell type but whose concentration can be increased markedly in response to the proper stimulus

A

inducible proteins

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18
Q

True or false? eukaryotic cells contain much less DNA than is found in prokaryotic cells

A

false; contain much more

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19
Q

___ ____ are most important for regulation in eukaryotes

A

transcriptional activators

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20
Q

____ is an inhibitor of dihydrofolate reductase, an enzyme indirectly important to the synthesis of thymidine and thus DNA (anticancer drug)

A

Methotrexate

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21
Q

Cancer cells can develop resistance to methotrexate by increasing the number of genes for DHFR to 40 to 400 copies per cell. These extra copies recombine to somehow form ____ ____ chromosomes

A

double minute

note: these extrachromosomal elements are inherited in an unstable fashion

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22
Q

These are called “jumping genes”

A

transposons

23
Q

True or false? Although humans have many transposons in their genomes, there is little to fear because many are inactive and they jump infrequently

A

true

note: however, transposons have been found to cause mutation

24
Q

Transposons in bacteria sometimes contain ____ ____ genes and can “jump” onto plasmids which can be taken up by sensitive strains as one mechanism for resistance transfer

A

antibiotic resistance

25
Q

In regards to transcriptional activators, the activator sequences, known as ____ are further activated by tissue-specific proteins/hormones

A

enhancers

26
Q

Enhancers are ___ acting DNA sequences. they activate transcription only in the ____ where they reside

A

cis; chromosome (DNA molecule)

27
Q

enhancers are often effective in either orientation. true or false?

A

true

28
Q

Enhancers only affect transcription nearby where they are located. true or false?

A

false; can affect transcription thousands of bps away

29
Q

enhancers affect the transcription of any gene in their vicinity. true or false?

A

true

30
Q

How do enhancers work?

A

transcriptional activators bind to the enhancer and the enhancer stimulates RNA polymerase transcription of specific genes

31
Q

This is a 72 bp sequence which can enhance the transcription of many genes. An experiment with this enhancer shows that permissive enhancers can stimulate any promoter in the vicinity

A

SV40

32
Q

This enhancer is stimulated by zinc and increases the transcription of the gene for the metal-binding protein. An experiment with this enhancer shows that certain enhancers are not specific for a given promoter

A

Metallothionein

33
Q

Cloning this enhancer with the polymer large T antigen caused pancreatic tumors in transgenic mice. Showed that enhancers can be tissue specific.

A

pancreatic B-cell tissue enhancer

34
Q

Albumin has an enhancer which binds a liver specific transcription factor. This explains why albumin is synthesized exclusively by the ___

A

liver

35
Q

Small regions of proteins make direct contact with the DNA and bind by ___ bonds and ___ ___ ___ forces

A

hydrogen; van der walls

36
Q

In regards to types of binding, these proteins consist of at least 2 connected coils of an alpha-helix with one of the coils lying flush in a major groove and the other turned away about 90 degrees

A

helix-turn-helix

37
Q

What type of binding domain to Lac Repressor proteins contain?

A

helix-turn-helix

38
Q

In regards to binding domains, the estrogen receptor contains several ___ ___

A

zinc fingers

39
Q

Zinc finger domains bind to the ___ groove of DNA

A

major

40
Q

In regards to binding domains, this is a modified or smaller region of a much larger DNA binding protein. A leucine at every 7th position in the “zipper” alpha helix.

A

leucine zipper

41
Q

The leucine zipper holds the DNA binding ___-___ domains in the correct position for interaction with DNA

A

alpha-helical

42
Q

The transcription factor ___-___ __ binds to the major groove of DNA through multiple interactions which bonds or loops in the protein structure. Shows similarities to Ig light chain antigen binding domains

A

NF-Kappa Beta

43
Q

Jak-STAT is a signaling pathway vital for the function of ____

A

cytokines

44
Q

JAKs are ___ kinases which phosphorylate specific tyrosines on STATs. STATs are a family of transcription factors which migrate to the ___ after being phosphorylated to stimulate transcription of genes which mediate cytokine function

A

tyrosine; nucleus

45
Q

Loss of gene for STAT 1 causes loss of response to what?

A

viral or bacterial infection

46
Q

Loss of STAT 4 causes what?

A

no TH1 cell function

47
Q

Loss of STAT 5 causes what?

A

no breast development or lactation

48
Q

Post-trancriptional controls appear to play a role in regulating which mRNAs actually reach the ____

A

cytoplasm

49
Q

Regulation of this could be a control point in mRNA synthesis

A

splicing

50
Q

Certain messages are unstable because the message contains an extra ___-___ rich sequence near the 3’ end

A

A-U

51
Q

What is masked RNA?

A

mRNA that is stored in the cell and not translated - repressor proteins bind to it

52
Q

What do Micro-RNAs do?

A

small RNA transcripts which regulate the levels of translation of messages.

note: they form double-stranded segments which signal nuclease degradation or block translation

53
Q

True or false? Micro-RNAs degrade the message

A

true

Biochem (38 decks)

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