Theme 3- Module 4

Question 1

What is in situ hybridization used for?

Answer 1

To identify which embryonic cells/tissues express a gene that play an important role in the embryonic development of the organism

Question 2

What does in situ hybridization do?

Answer 2

Finds WHERE the corresponding mRNA that is transcribed from that gene can be found during development in the intact organism

Question 3

How does in situ hybridization work?

Answer 3

Fluorescently labelled short single-stranded segment of DNA or RNA binds/hybridizes in a complementary fashion to the target mRNA molecule

Question 4

What can we do to attain a better understanding of temporal differences that may exist in gene expression during development?

Answer 4

Carry out and compare (spatial) situ hybridizations throughout the different stages of development

Question 5

What is the advantage of using DNA microarray techniques instead of in situ hybridizations?

Answer 5

With DNA microarrays, you can examine the expression of thousands of genes at once

In situ hybridization is useful to assess the gene expression levels of a FEW genes of interest

Question 6

How do DNA microarrays work?

Answer 6

Set up glass slides that have tiny spots containing a known DNA sequence or gene

DNA molecules act as probes to detect gene expression

Question 7

What is the transcriptome?

Answer 7

The set of mRNA transcripts that are expressed by various genes

Question 8

What are oligonucleotides?

Answer 8

Short fragments of nucleic acids

Question 9

How can we find out which genes are involved in transformation of a normal cell to a tumour cell?

Answer 9

Grow both cell types in culture

Isolate the gene products or mRNA that is transcribed by these two cell types

Undertake a DNA microarray analysis.

Question 10

Once the mRNA has been isolated from normal cells and tumour cells, what are they used for in the proceeding DNA microarray analysis?

Answer 10

They serve as templates for making complementary cDNA molecules to the mRNA

Question 11

Which enzyme is responsible for making complementary cDNA molecules to the mRNA?

Answer 11

Reverse transcriptase enzyme

Question 12

What do they use to make it easier to identify the different cDNA during the microarray analysis?

Answer 12

Different coloured fluorescent nucleotides that become part of the newly synthesized cDNA molecule

Question 13

Describe the main steps of preparing cancerous and normal DNA for microarray analysis

Answer 13

Isolate mRNA from cancer cells and normal cells

Reverse transcription to cDNA with fluorescent nucleotides

Combine equal amounts of labelled cDNA from normal cells and from cancer cells

Apply mixture to microarray chip

Test for hybridization

Question 14

How can you know a particular gene is active based on the microarray analysis?

Answer 14

Active genes produce many molecules of mRNA

We will therefore have more labelled cDNA molecules available after reverse transcription

(which will be able to hybridize to the DNA on the microarray chip and produce bright spots)

Question 15

Why is it important to have an equal mixture of the labelled cDNA of both the normal epithelial cells and breast carcinoma cells?

Answer 15

They need to compete for the synthetic complementary DNA fragments in the spots of the microarray chip.

Question 16

Normal cDNA were labelled with green and cancer cDNA were labelled with red.

If a fluorescent spot is green for example, that means:

a) the specific gene in that spot is upregulated in normal cells and downregulated in carcinoma cells.
b) the specific gene in that spot is downregulated in normal cells and upregulated in carcinoma cells.

Answer 16

a) the specific gene in that spot is upregulated in normal cells and downregulated in carcinoma cells.

Question 17

Why are the spots brighter if the gene is more active?

Answer 17

If the gene is active, it produces more mRNA, which leads to brighter spots.

Question 18

If there is no fluorescence at all in a particular spot, what does that indicate?

Answer 18

That the gene may be inactive

Question 19

How can computational software provide further insight into groups of genes that may participate in common cellular processes at common times?

Answer 19

It can further analyze the data from multiple microarrays and cluster genes that show similar expression patterns

Question 20

What is the purpose of short, noncoding regulatory double stranded RNA molecules?

Answer 20

To activate RNA interfering machinery which then interferes with mRNA translation

Question 21

Give two examples of small regulatory RNA

Answer 21

microRNA

siRNA

Question 22

How are both types of small regulatory RNA processed?

Answer 22

Transcribed from protein-encoding genes

Form hairpin loops due to complementary base-pairing

Loops are then cleaved (via enzymes) into smaller, single stranded mRNA fragments

Becomes incorporated as part of a (or RISC complex)

Question 23

After activating the RISC complex, how do microRNAs inhibit translation?

Answer 23

Contain sequences that are complementary to specific target

Will bind in a NON-EXACT manner to the target mRNA sequence and the proteins within the RISC complex then inhibit translation

Question 24

What does the RISC complex stand for?

Answer 24

RNA-induced silencing complex

Question 25

After activating the RISC complex, how do siRNAs inhibit translation?

Answer 25

Bind to the complementary sequence (EXACT) by an association with the RISC complex and induce the cleavage of the target mRNA

(cleavage destabilizes the target mRNA)

Question 26

The length of time in which a protein functions in a cell can be limited by a process referred to as __________

Answer 26

Selective degradation

Question 27

What do proteasomes do?

Answer 27

They break peptide bonds and as a result degrade unneeded or damaged proteins into smaller amino acids that can be recycled

Question 28

During protein degradation, what are target proteins tagged with?

Answer 28

Polyubiquitin chain

small ubiquitin proteins

Question 29

The tagging process in protein degradation requires:

a) ATP
b) GTP

Answer 29

a) ATP

Question 30

What are the three main steps of ubiquitination?

Answer 30

1) Activation of ubiquitination
2) Conjugation to the target substrate protein
3) Ligation

Question 31

The passage of the target protein through the proteasome requires:

a) ATP
b) GTP

Answer 31

a) ATP