genes and gene expression

Question 1

T or F: genes required for normal cell processes will be expressed in every cell type

Answer 1

true

Question 2

T or F; some genes will only be expressed in specific cells

Answer 2

true

Question 3

T or F: the level of expression in genes is the same across different cell types

Answer 3

false; the level of expression of genes is different from one cell type to another

Question 4

what does DNA microarray assay help us determine

Answer 4

allows us to look at all of the genes in an organism that are expressed at a given time

Question 5

what is cDNA and how is it made

Answer 5

cDNA is complementary DNA and it’s made by making use of reverse transcriptase

Question 6

describe the process of DNA microarray assay

Answer 6

mRNA is isolated and cDNA is made and then flourescently labeled. The cDNA is applied to the spots and the unbound cDNA is rinsed away, therefore where we see flourescence in the tray is which genes are expressed

Question 7

what is a more popular approach than DNA microarray assay to see which genes are expressed at a given time

Answer 7

RNA sequencing (aka RNA-seq)

Question 8

describe RNA-seq

Answer 8

mRNAs are isolated and cut intro fragments, then they’re reverse transcribed into cDNAs, then the cDNAs are sequenced. then the sequences are mapped on a computer and the data will sow which genes are expressed

Question 9

why would we use reverse transcriptase-polymerase chain reaction? (RT-PCR)

Answer 9

helps us determine whether a specific gene is being expressed + when

Question 10

describe the process of RT-PCR

Answer 10

isolate mRNA from the organism at dif stages of embryonic developent, produce cDNAs, carry out PCR, run the PCR products on a gel, and the presence of product will indicate what stage the gene is expressed at

Question 11

what do we use to quantitate the level of gene expression for an individual gene

Answer 11

quantitative RT-PCR aka qRT-pCR

Question 12

describe the process of qRT-PCR

Answer 12

same procedure as for RT-PCR, except that a florescently-labeled probe that binds to dsDNA (double-stranded DNA from PCR) is added to the PCR. a thermocycler that measures florescence will monitor the reaction

Question 13

how do we determine when and where a gene is expressed

Answer 13

by using reporter gene constructs

Question 14

give examples of reporter gene constructs

Answer 14

luciferase, GFP, E coli beta-galactosidase

Question 15

what is Pax6

Answer 15

a gene required for the development of the eye and also certain regions of the brain, spinal cord, and pancreas

Question 16

describe the process of using reporter gene construct for the Pax6 gene

Answer 16

plasmid is microinjected into a pronucleus of a fertilized mouse egg, and then the promoter-reporter gene construct becomes incorporated into the genome. the fertilized egg is then implanted into the uterus of a pseudopregnant mouse = transgenic mouse embryo. embryo is removed after 10.5 days, then incubated with X gal (makes blue precipitate), so after we can see that Pax6 is present where we see blue

Question 17

what technique is used to get info on the time and place that an mRNA is produced (other than reporter gene constructs)

Answer 17

in Situ hybridization

Question 18

what question does in Situ hybridization give us an answer to

Answer 18

where genes are expressed during a certain stage of embryonic development

Question 19

describe the process of in situ hybridization

Answer 19

fix drosophila so RNA is exposed in an intact form, incubate with a labeled cDNA or RNA probe (florescent tag), wash off any unbound probe, then look at it under a florescent microscope

Question 20

gene expression is usually controlled at the level of _______

Answer 20

transcription initiation