Gene Regulation

Question 1

what makes cell types carry out different functions (3)

Answer 1

• cell type
• differentiated product
• specialized function

Question 2

how are cells regulated (2)

Answer 2

• cells have different proteins with different gene regulation
• regulation through transcription of genes and synthesis and function of protein post-transcriptionally

Question 3

how do we know that transcriptional regulation is a commonly used mechanism (3)

Answer 3

• 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

Question 4

what are the methods for detecting transcripts distribution (temporal and spatial) in an organism (5)

Answer 4

1. RNA (Northern) blot
2. RT-PCR
3. In situ hybridization
4. RNA sequencing
5. Promoter-reporter gene fusion

Question 5

hybridization

Answer 5

• phenomenon where ssDNA or RNA molecules anneal to complementary DNA or RNA

Question 6

hybridization probe (2)

Answer 6

• 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

Question 7

RNA blot

Answer 7

• detection of RNA molecules in specific tissues

Question 8

advantages: RNA blot

Answer 8

• provides transcript size and abundance (location and intensity)

Question 9

disadvantages: RNA blot (2)

Answer 9

• time-consuming

- RNA is degraded easily

Question 10

reverse transcriptas PCR (RT-PCR)

Answer 10

• detects RNA transcripts by producing cDNA

Question 11

advantages: RT-PCR (2)

Answer 11

• fast

- sensitive

Question 12

disadvantages: RT-PCR (3)

Answer 12

• no information on transcript size
• crudely quantitative
• subject to artifacts/contamination

Question 13

why is RT-PCR not very quantitative

Answer 13

• after 30-40 cycles, amount of DNA product reaches a plateau that isn’t correlated with original amount of target DNA

Question 14

quantitative PCR (3)

Answer 14

• 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

Question 15

when do we visualize PCR in normal RT-PCR (2)

Answer 15

• after PCR cycles are complete; amplified DNA already at plateau

Question 16

when do we visualize PCR in quantitative RT-PCR

Answer 16

• measure CT: the number of PCR cycles it takes for detected fluorescence to be greater than threshold levels; before [plateau is reached

Question 17

advantages: in situ hybridization

Answer 17

• provides precise information on spatial distribution of gene transcript

Question 18

in situ hybridization

Answer 18

• detection of transcripts within a whole organism, organ, or tissue of interest

Question 19

disadvantages: in situ hybridization (3)

Answer 19

• difficult
• time consuming
• little information on amount of transcript

Question 20

advantages: RNA sequencing (3)

Answer 20

• sensitive
• quantitatively accurate
• provides information on all transcripts

Question 21

disadvantages: RNA-sequencing

Answer 21

• relatively expensive

Question 22

characteristics of reporter elements (2)

Answer 22

• easily detectable

- not normally expressed in model organisms

Question 23

restriction enzyme/endonuclease (2)

Answer 23

• enzyme that cuts DNA at or near restriction sites

- some cut DNA straight (blunt ends) and some cut staggered (sticky ends)

Question 24

restriction sites (2)

Answer 24

• specific recognition nucleotide sequences for restriction enzymes
• generally palindromic: sequence reads the same backward and forward

Question 25

what is the purpose of restriction enzymes in bacterial cells (2)

Answer 25

• protect bacterial cells from foreign DNA

- protect own DNA from restriction digestion by methylating recognition sites

Question 26

how do scientists use restriction enzymes (2)

Answer 26

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

Question 27

how can we estimate how many times a restriction enzyme will cut an x bases DNA

Answer 27

(1/4)^n x # of bases

- n = number of bases in recognized sequence

Question 28

vector

Answer 28

• DNA molecule used as a vehicle to artificially carry foreign material into another cell, where it van be replication/expressed

Question 29

advantages: promoter-reporter gene fusions

Answer 29

• determines in which cells the gene of interest is transcribed

Question 30

disadvantages: promoter-reporter gene fusions (2)

Answer 30

• time consuming

- must know which sequences contain all the promoter regulatory sequences (enhancers)

Question 31

which technique for transcript detection is: a genome-wide expression analysis

Answer 31

• RNA sequencing

Question 32

which technique for transcript detection is: an analysis of expression of individual genes

Answer 32

• RNA blot
• RT-PCR
• in situ hybridization
• promoter-reporter gene fusions

Question 33

how do we know there is differential distribution of transcripts within a developing organism

Answer 33

• mRNA transcripts for only a subset of genes are found in specific cell/tissue/organ type; only small amount are expressed in all cells

Question 34

how do we know that transcription is the principle mechanism for controlling transcript levels

Answer 34

• 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

Question 35

transcription factor (2)

Answer 35

• protein that influences RNA polymerase to increase or decrease transcription of a gene
• general TFs and specific TFs

Question 36

general transcription factors (2)

Answer 36

• basal transcription factors

- bind to core promoter and are required for transcription initiation

Question 37

specific transcription factors

Answer 37

• bind to other sequences (enhancers, repressors) to regulate transcription of specific genes

Question 38

how do we know that TFs are abundant and required for development (2)

Answer 38

• 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

Question 39

control by transcription factors (4)

Answer 39

• 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

Question 40

what kind of evidence is needed to prove that gene Y is a target gene of TF X

Answer 40

• mutating X affects Y expression

- X can bind to cis elements in the regulatory sequence of Y in vitro

Question 41

cis elements

Answer 41

• DNA sequences that regulate transcription of nearby genes