Plasmid +1 -789 Luc -419 Luc -309 = HLuc -239 Luc --199 Luc --149 Luc -123 Luc -59 = Luc -15%D Luc Luc Luc + + + 2 4 6. 10 34 Relative Luciferase Activity PCD3Y-789. PCD3Y-419, PCD3Y-309. PCD3Y-239. PCD3Y-199, PCD3Y-149, PCD3Y-123, PCD3Y-59. PCD3Y-15. Parent Plasmid, Parent Plasmid. with non-CD3Y Active Promoter +1 Transcribed Region Upstream Regulatory Sequences CD3Y Gene Plasmid Introduce Plasmids into T Lymphocytes Location in Which CD3Y Sequences Are Inserted Luciferase Gene Luciferase Production Poly (A) Signal T Lymphocytes with Plasmids Plasmid

Human Anatomy & Physiology (11th Edition)
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CD3 is a signaling protein that is typically found only in the plasma membrane of immune system T lymphocytes. CD3 is composed of several different polypeptides, including a gamma chain, CD3γ. Scientists analyzed the promoter of the CD3γ chain gene for regulatory sequences that might have positive or negative effects on expression of the gene.

The scientists cloned fragments of the CD3γ gene that included the first transcribed nucleotides plus up to 789 nucleotides of upstream regulatory sequences into plasmids in which the gene for the firefly enzyme luciferase immediately follows the fragments. The plasmids were then introduced into a line of T lymphocytes (Figure 1), and the cells were allowed to grow for a short while. Because the regulatory sequences of the CD3γ gene immediately precede the luciferase gene in the plasmids, the activity, either positive or negative, of the regulatory sequences affected the amount of luciferase gene expression by the T lymphocytes. Luciferase catalyzes a reaction that results in the release of lightand is responsible for the bioluminescence (light flashes) of fireflies. By quantifying the bioluminescence, or luciferase activity, in the cells, the scientists were able to determine the effects of each CD3γ gene fragment cloned into the plasmids (Figure 2) on expression of the gene.

(a) Identify both the cellular component and the location of the component that is responsible for producing the luciferase protein from mRNAs transcribed in the plasmid-containing T lymphocytes. Explain what dictates to the lymphocytes the correct order in which amino acids should be linked to form the luciferase protein.

Plasmid
+1
-789
Luc
-419
Luc
-309 =
HLuc
-239
Luc
--199
Luc
--149
Luc
-123
Luc
-59 =
Luc
-15%D
Luc
Luc
Luc
+
+
+
2
4
6.
10 34
Relative
Luciferase
Activity
PCD3Y-789.
PCD3Y-419,
PCD3Y-309.
PCD3Y-239.
PCD3Y-199,
PCD3Y-149,
PCD3Y-123,
PCD3Y-59.
PCD3Y-15.
Parent Plasmid,
Parent Plasmid.
with non-CD3Y
Active Promoter
Transcribed Image Text:Plasmid +1 -789 Luc -419 Luc -309 = HLuc -239 Luc --199 Luc --149 Luc -123 Luc -59 = Luc -15%D Luc Luc Luc + + + 2 4 6. 10 34 Relative Luciferase Activity PCD3Y-789. PCD3Y-419, PCD3Y-309. PCD3Y-239. PCD3Y-199, PCD3Y-149, PCD3Y-123, PCD3Y-59. PCD3Y-15. Parent Plasmid, Parent Plasmid. with non-CD3Y Active Promoter
+1
Transcribed Region
Upstream
Regulatory
Sequences
CD3Y Gene
Plasmid
Introduce
Plasmids into
T Lymphocytes
Location in
Which CD3Y
Sequences
Are Inserted
Luciferase Gene
Luciferase
Production
Poly (A)
Signal
T Lymphocytes
with Plasmids
Plasmid
Transcribed Image Text:+1 Transcribed Region Upstream Regulatory Sequences CD3Y Gene Plasmid Introduce Plasmids into T Lymphocytes Location in Which CD3Y Sequences Are Inserted Luciferase Gene Luciferase Production Poly (A) Signal T Lymphocytes with Plasmids Plasmid
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