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Genetics > Week 6 > Flashcards

Week 6 Flashcards

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1
Q

Central question in the regulation of gene expression

A

How does a cell or organism with the same initial DNA sequence/genotype exhibit different phenotypes

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2
Q

Differential expression is due to

A

Environmental conditions; different cell types

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3
Q

Bacteria

A

Uncompartmented; no organelles

mRNAs polycistronic (generally)

Coupled transcription and translation

mRNA primary transcripts not spliced (generally)

One RNA polymerase

Bare DNA

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4
Q

Eukaryotes

A

Compartmented; nucleus cytoplasm etc.

mRNAs monocistronic (generally)

Uncoupled transcription and translation

mRNA primary transcripts spliced and modified

Multiple RNA polymerases

Chromatin

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5
Q

Polycistronic mRNA

A

A single mRNA encodes multiple polypeptides (in viruses the DNA is expressed as polycistronic in a eukaryote)

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6
Q

Transcription/translation eukaryotes

A

mRNA must be processed before export from the nucleus and translation

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7
Q

Central Dogma

A 
Gene
Transcription
Transcript
Translation
Protein
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8
Q

Bacteria: Transcription Regulation Question

A

Is the gene the same structure?

Is the gene transcribed?

Is the transcript initiated?

Is the transcript terminated?

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9
Q

Bacteria: Transcript Regulation Question

A

How stable?

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10
Q

Bacteria: Translation Regulation Question

A

Is the transcript translated?

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11
Q

Bacteria: Protein Regulation Question

A

Is the protein active

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12
Q

Eukaryotes: Transcription Regulation Question

A

Is the gene the same structure?

Is the transcript initiated?

Where is the transcript initiated?

Where does the transcript end?

How was the transcript spliced?

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13
Q

Eukaryotes: Transcript Regulation Question

A

How stable?

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14
Q

Eukaryote: Translation Regulation Question

A

Is the transcript translated?
Where is the transcript?
Is the transcript exported from the nucleus?

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15
Q

Eukaryote: Protein Regulation Question

A

Is the protein active?

Where is the protein?

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16
Q

Is the transcript present or not?

A

Transcript detection/accumulation

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17
Q

What is the structure of the transcript?

A

Transcript analysis/sequencing

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18
Q

Is the protein expressed?

A

Protein detection/antibody activity

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19
Q

Transformer gene and female development: transcription

A

Transcription of the tra gene occurs in both males and females, the transcript is present in both cells.

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20
Q

Transformer gene and female development: translation

A

Is TRA mRNA translated in females only?

YES I detect TRA protein in females

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21
Q

Structure of tra transcripts

A

Male transcript has extra RNA sequence information that introduces a stop codon in the mRNA.

In females the splicing of the mRNA results in mRNA that lacks this sequence that is present in the male transcript resulting in no premature truncation.

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22
Q

Transcription steps

A

1-RNAP binds to the DNA forming a closed complex: regulates the rate at which the gene is transcribed/amount of transcript produced

RNAP binds to a promoter in both bacteria and eukaryotes

2-RNAP forms an open complex; melted DNA bubble

3-Elongation; RNAP transcribes the gene

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23
Q

Transcription DNA elements: Promoters

A

Promoter in bacteria: -35/-10, RNAP directly binds

Promoter in eukaryotes: TATA; 30 bp upstream of transcription start

24
Q

Transcription DNA elements: Regulatory Sequence

A

Rate of transcription are regulated by regulatory sequence

positive and negative regulatory sequences

25
Q

Bacteria: regulatory sequence

A 
Activator sequence (positive)
Operator (negative)
26
Q

Yeast: regulatory sequence

A

UAS (positive)

Operator/Silence (negative)

27
Q

Humans/Mice: regulaory sequence

A

Enhancer

Silencer

28
Q

Differences between eukaryotic and prokaryotic promoters

A

eukaryotic promoters are not sufficient for transcription in eukaryotes

a transcription unit with a promoter is generally going to be transcribed in prokaryotes

28
Q

Differences between eukaryotic and prokaryotic promoters

A

eukaryotic promoters are not sufficient for transcription in eukaryotes

a transcription unit with a promoter is generally going to be transcribed in prokaryotes

29
Q

Promoter only

A

Transcription in prokaryote

No transcription in eukaryotes

30
Q

Silencer/operator and promoter

A

no transcription in both prokaryotes and eukaryotes

31
Q

activator/enhancer and promoter

A

transcription in eukaryotes and prokaryotes

32
Q

activator/enhancer, silencer/operator and promoter

A

no transcription in prokaryotes and eukaryotes

33
Q

Transcription factors

A

DNA-binding proteins that recognize specific DNA sequences

When bound to DNA they affect the rate of initiation of transcription

DNA-binding protiens recognize specific DNA sequences by specific amino-acid base pair interactions

DNA binding can be influenced by the binding of small molecules.

34
Q

Lac Operon

A

transcribed form one promoter and the transcript encodes three proteins:

Beta galactosidase (Z): enzyme
Permease (Y): transporter
Galatosidase acetyltransferase (A)

Polycistronic

35
Q

Lac operon regulatory sequences

A

P and O are part of the gene regulatory sequence of Lacz

seperate laci gene encodes the lac repressor

36
Q

LacZ-/Oc

A

LacZ: recessive lf, no beta galactosidase in glucose/lactose media

Oc: dominant, gf, beta galactosidase is always present in the glucose/lactose media

37
Q

laci-/laci-sr

A

laci: recessive, betagalactosidase is always present

laci-sr: dominant, betagalactosidase is not present

38
Q

Operator constuitive allele

A

Operator is knocked out such that the lac repressor cannot find the operatory site and bind to prevent transcription

39
Q

Trans-acting

A

Diffusable factor can bind to DNA and regulate transcription

The conclusion of an intergenic complementation analysis

40
Q

Cis-acting

A

the conclusion of an intragenic analysis of gf and lf of alleles

41
Q

The experiment showing trans-acting

A

Basic complementation analysis (cis/trans test)

trans: different DNA molecules; wt lacz and lf laci are placed next to each other; lf lacz and laci

we see normal gene expression

function wt copies are placed in cis to each other function normally

42
Q

The experiment shows a trans-acting conclusion

A

The wild type Laci gene encodes a factor which will be a a protein that is able to diffuse and interact with the lacZ gene

43
Q

cis-acting analysis

A

cis-acting refers to cis-dominance which is the interaction between two mutant alleles in the same locus/gene. To observe cis-dominance, one allele is a dominant gain of function and the other allele is a recessive loss of function; they have the opposite phenotypes.

44
Q

cis-acting steps

A

place wt operator cis to lacz lf

place gf operator cis to wt lacz

oc allele is dominant to lacz allele when trans

Oc can’t have an effect on the WT coding region on another molecule, it can only affect the expression of a gene on the same molecule.

wt allele is dominant when oc and lf lacz are cis to each other; normal expression

45
Q

How can we transcription occurring the cell

A

take advantage of splicing. Placed a block of DNA that encodes the MS2 binding sites found in RNA and bound by an RNA binding protein

the ms2 binding site is placed in the intron of the ftz gene such that when the nascent primary transcipt is formed it contains the ms2 binding site

after splicing the ms2 binding site is rapidly degraded.

the presence of a ms2 binding protein fused to a red fluorescent protein detects the nascent transcrip

46
Q

Reporter genes

A

reporter gene expression is easy to assay (green fluorescence)

reporter genes can reproduce the expression of a gene

use of reporter genes requires that you use an organism where it is possible to reintroduce DNA (transform)

47
Q

Easy to assay+recapitulates gene expression patterns

A

Fuse the regulatory region of a gene to a reporter gene that is easy to assay

in frame fusion; ATG and the regulatory sequence

48
Q

Identify cis-regulatory sequences

A

mutate the regulatory region and look at how expression of the reporter gene is expressed

49
Q

Genetic analysis of RNA cis-acting elements

A

Introduce a change in DNA, that when transcribed results in the production of an RNA that has a sequence change that may or may not affect the regulation of the expression of the protein from this transcript.

50
Q

transformer gene and female development

A

tra mRNA is longer in males and results in no protein

tra mRNA is shorter due to alternative splicing results in active gene product

51
Q

SXL expression

A

in males there is no SXL

in females there is SXL expressed

51
Q

SXL expression

A

in males there is no SXL

in females there is SXL expressed

52
Q

Alternative splicing

A

alternative 3’ splice sites used in the processing of the tra transcripts

earlier 3’ ss is used in males,

sxl protein in females binds to sxl binding site forcing the spliceosome complex to bind to a 3’ downstream, the male stop codon is spliced out and active protein product is produced (tra)

53
Q

Translation regulation

A

A mature mRNA of the same structure is present, but in one condition it is translated and in another it is not translated

54
Q

Regulation of hunchback mRNA and protein expression

A

hunchback mRNA is present in the whole cell

HB protein is only present in the anterior end

Nanos is present at the posterior end of the cell blocking hunch back expression

HB protein expressed at the posterior end results in no abdomen

55
Q

What does Nanos recognize in HB mRNA to suppress translation?

A

Nanos requires NRE in order to suppress translation of the HB mRNA (nanos regulatory element)

pumillio another protein expressed in the entirety of the cell and binds to the NRE, pumillio then binds nanos

Pumilio binds NRE both at the anterior and posterior end of the embryo, no nanos at the anterior end, HB mRNA is translated and we get HB protein.

Genetics (16 decks)

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