Part C You perform another Y2H experiment with a library of cDNAs derived from h

Question

Part C You perform another Y2H experiment with a library of cDNAs derived from human T-cells cloned into the pPREY vector. (Why from T- cells?) As a control, you also transform your pBAIT and pPREY vectors into a second yeast strain containing the following control reporter (missing the Gal4 UAS) UAS Lac Z Reporter Gene Control Lac Z Reporter Gene 5(+3) 0 ( 13) Bait 3(± 2) 6(± 5) 103 13 Rev Rev Rev Rev Rev Rev 95(±5) 5 (5) 10 ( 11) 101 ( 8 1 ( 10) 6 (+ 8) 5( 2) 4(43 9 ( 5) 10 5(+3) Which Prey vector(s) encode a human protein that may interact with the HIV Rev protein? How can you identify the human protein? What does the control reporter tell you? Illustration of HIV life-cycle: integrase Nucleus 2 kb mRNAs

Explanation / Answer

A cDNA [ complementary DNA] is extracted from the T- cells because these are specially designed to recognise a specific antigen. The cDNA from eukaryotic cells are inserted into a prokaryotic cell .

The interaction of HIV rev protein with a structured region in env mRNA mediates the export of structural mRNA from the nucleus into the cytoplasm. In the absence of Rev, mRNAs of the HIV genes are retained in the nucleus, preventing their translation.

Rev is a 13-kDa protein that is composed of 116 amino acids. Rev's sequence contains two specific domains which contribute to its nuclear export and import. The protein typically performs its function as a tetramer.

HIV Rev protein are translated from completely processed mRNA transcripts while structural proteins are translated from incompletely spliced transcripts. Rev is needed to export incompletely spliced mRNAs in order to produce the viral structural protein.

Since Rev is absolutely necessary for HIV replication and it is expressed early on in infection, it has been suggested that Rev is a good target for antiviral therapies.

As for vectors which can encode a human protein that can react with HIV, it could be in genetically engineered bacteria like E.coli since it can encode full length proteins at high levels.

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