19. Signal Transduction 2

Question 1

Describe how a mircoarray works.

Answer 1

Collections of oligonucleotides that each detect levels of a specific mRNA.
Red (experimental sample) versus green (control)
* black - no expression
* red - expressed higher in experimental sample
* green - expressed higher in control sample
* yellow - no difference (expressed in both)

Question 2

Explain heat maps.

Answer 2

Diagram that compares transcriptional changes
* clustering of cell mRNA expression based on similarity
* hierarchical clustering of specific gene (mRNA expression) based on similarity patterns across genes
* each row = one mRNA
* each column = one cellular mRNA sample

Question 3

DNA sequencing: Explain the dideoxy method.

Answer 3

Dideoxynucleotides terminate DNA synthesis bc of their lack of a 3’ hydroxyl group.
* DNA synthesis continues (in the presence of all 4 dioxynucleotides) until one of the ddG, ddA, ddT, or ddC is incorporated.
* products of the sequencing reaction are loaded onto a gel or into a tube
* seperation by electrophoresis

Question 4

Describe how Next Gen sequencing works.

Answer 4

• during sequencing, all 4 nucleotides are incorporated and they compete to become an addition to the sequencing strand (based on the template strand)
• each cycle adds one nucleotide
• wash
• cleave dye and terminating groups –> wash again
• repeat cycle

Question 5

Cyclic reversible terminators allow for…

Answer 5

fluorescent base calling, then continued elongation.

Question 6

Describe RNA-seq.

Answer 6

• uses next generation sequencing technology
• sequences cDNA that is made directly from mRNAs
• the number of reads is proportional to mRNA expression levels

Question 7

Describe ChiP-seq.

Answer 7

• uses NGS technology
• sequences DNA content of sheared immunoprecipitated chromatin
• number of reads is proportional to local chromatin modifications

Question 8

How do estogen receptors (nuclear receptors) mediate transcription responses?

Answer 8

Estrogen receptor alpha can activate transcription
* after a ligand binds, ligand-binded estrogen receptors have to travel into the nucleus
* transcription factors are activated
* can cause direct or indirect activation in the nucleus

Question 9

How are GCPRs involved in transcriptional signaling?

Answer 9

GCPRs are involved with NON-transcriptional signaling
* ligand binding initiates cascades of enzyme activation
* does not involve transcriptional (genetic) regulation of target genes