Gene Regulation Flashcards
(41 cards)
1
Q
what makes cell types carry out different functions (3)
A
- cell type
- differentiated product
- specialized function
2
Q
how are cells regulated (2)
A
- cells have different proteins with different gene regulation
- regulation through transcription of genes and synthesis and function of protein post-transcriptionally
3
Q
how do we know that transcriptional regulation is a commonly used mechanism (3)
A
- differential distribution of transcripts within an organism
- transcription (not RNA degradation) is the principle mechanism for controlling transcript levels
- transcription factors are abundant and many are required for development
4
Q
what are the methods for detecting transcripts distribution (temporal and spatial) in an organism (5)
A
- RNA (Northern) blot
- RT-PCR
- In situ hybridization
- RNA sequencing
- Promoter-reporter gene fusion
5
Q
hybridization
A
- phenomenon where ssDNA or RNA molecules anneal to complementary DNA or RNA
6
Q
hybridization probe (2)
A
- fragment of DNA or RNA which may or may not be radioactively labelled
- can be used to detect presence of nucleotide sequences (DNA/RNA target) complementary to sequence in the probe
7
Q
RNA blot
A
- detection of RNA molecules in specific tissues
8
Q
advantages: RNA blot
A
- provides transcript size and abundance (location and intensity)
9
Q
disadvantages: RNA blot (2)
A
- time-consuming
- RNA is degraded easily
10
Q
reverse transcriptas PCR (RT-PCR)
A
- detects RNA transcripts by producing cDNA
11
Q
advantages: RT-PCR (2)
A
- fast
- sensitive
12
Q
disadvantages: RT-PCR (3)
A
- no information on transcript size
- crudely quantitative
- subject to artifacts/contamination
13
Q
why is RT-PCR not very quantitative
A
- after 30-40 cycles, amount of DNA product reaches a plateau that isn’t correlated with original amount of target DNA
14
Q
quantitative PCR (3)
A
- include problem with a reporter that fluoresces only when new DNA is synthesized
- amount of fluorescence measured reflects total amount of amplified DNA present
- analyze how fluorescence changed with each PCR cycle
15
Q
when do we visualize PCR in normal RT-PCR (2)
A
- after PCR cycles are complete; amplified DNA already at plateau
16
Q
when do we visualize PCR in quantitative RT-PCR
A
- measure CT: the number of PCR cycles it takes for detected fluorescence to be greater than threshold levels; before [plateau is reached
17
Q
advantages: in situ hybridization
A
- provides precise information on spatial distribution of gene transcript
18
Q
in situ hybridization
A
- detection of transcripts within a whole organism, organ, or tissue of interest
19
Q
disadvantages: in situ hybridization (3)
A
- difficult
- time consuming
- little information on amount of transcript
20
Q
advantages: RNA sequencing (3)
A
- sensitive
- quantitatively accurate
- provides information on all transcripts
21
Q
disadvantages: RNA-sequencing
A
- relatively expensive
22
Q
characteristics of reporter elements (2)
A
- easily detectable
- not normally expressed in model organisms
23
Q
restriction enzyme/endonuclease (2)
A
- enzyme that cuts DNA at or near restriction sites
- some cut DNA straight (blunt ends) and some cut staggered (sticky ends)
24
Q
restriction sites (2)
A
- specific recognition nucleotide sequences for restriction enzymes
- generally palindromic: sequence reads the same backward and forward
25
what is the purpose of restriction enzymes in bacterial cells (2)
- protect bacterial cells from foreign DNA
| - protect own DNA from restriction digestion by methylating recognition sites
26
how do scientists use restriction enzymes (2)
genetic analysis and cloning:
- fragment DNA to smaller pieces to make it easier to identify and characterize genes
- cut and paste DNA; restriction enzymes can be added to primers that are used to amplify genes
27
how can we estimate how many times a restriction enzyme will cut an x bases DNA
(1/4)^n x # of bases
| - n = number of bases in recognized sequence
28
vector
- DNA molecule used as a vehicle to artificially carry foreign material into another cell, where it van be replication/expressed
29
advantages: promoter-reporter gene fusions
- determines in which cells the gene of interest is transcribed
30
disadvantages: promoter-reporter gene fusions (2)
- time consuming
| - must know which sequences contain all the promoter regulatory sequences (enhancers)
31
which technique for transcript detection is: a genome-wide expression analysis
- RNA sequencing
32
which technique for transcript detection is: an analysis of expression of individual genes
- RNA blot
- RT-PCR
- in situ hybridization
- promoter-reporter gene fusions
33
how do we know there is differential distribution of transcripts within a developing organism
- mRNA transcripts for only a subset of genes are found in specific cell/tissue/organ type; only small amount are expressed in all cells
34
how do we know that transcription is the principle mechanism for controlling transcript levels
- promoter and enhancer regions of genes with specific patterns of transcript localization express reporter genes in the same way; differential transcript patterns should result from regulation of transcription
35
transcription factor (2)
- protein that influences RNA polymerase to increase or decrease transcription of a gene
- general TFs and specific TFs
36
general transcription factors (2)
- basal transcription factors
| - bind to core promoter and are required for transcription initiation
37
specific transcription factors
- bind to other sequences (enhancers, repressors) to regulate transcription of specific genes
38
how do we know that TFs are abundant and required for development (2)
- sequencing of entire genomes show that 10-20% of all genes encode TFs
- mutants disrupted in development identify genes that play key regulatory roles during development, and man encode TFs
39
control by transcription factors (4)
- influence expression of specific target gene by binding to enhancer elements in promoter region either alone or in complex
- target genes will be expressed only in cells containing TFs
- can work in combinatorial manner to generate different patterns of expression for different target genes in a multicellular organism
- activity of TFs is also controlled through space and time by: other TFs and the environment
40
what kind of evidence is needed to prove that gene Y is a target gene of TF X
- mutating X affects Y expression
| - X can bind to cis elements in the regulatory sequence of Y in vitro
41
cis elements
- DNA sequences that regulate transcription of nearby genes
