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Gene order in yeast Ty element

Consider the following artificially created yeast Ty element:

See attached file for full problem description.

Upstream from the element has been placed the very strong PGAL promoter which is induced by galactose added to the media. The transcript traverses the whole element (large dashed arrow). Downstream from the normal Ty Open Reading Frames was inserted the yeast HIS3 gene, including its own promoter (small dashed arrow shows transcript), but in reverse orientation. Inserted into the middle of HIS3, and therefore blocking its expression, is an Intervening Sequence (IVS) from another gene. The IVS orientation corresponds to the P GAL transcript (left-to-right).

This construct was placed on a plasmid and transformed into a his3 mutant yeast strain. The transformed strain remained a his auxotroph. However, when grown on solid media containing galactose (but no his), a small number of His+ colonies appeared and grew out of the his- background.

a) What caused the appearance of His+ colonies? How was HIS3 expressed if the gene (as shown) has an IVS disrupting its reading frame?

b) Do you think that the His+ cells require the continued presence of the original Ty containing plasmid to be His+?

c) What would the His phenotype be of a cell transformed with an identical plasmid but with the IVS reversed in direction?

d) What would the His phenotype be of a cell transformed with an identical plasmid but with the HIS3-IVS DNA reversed in direction? (That is, HIS3 now transcribed in the left to-right direction, but the IVS oriented right-to-left).

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Solution Preview

Upon insertion of the plasmid into the yeast genome it is subject to transcriptional and translational processing. In the presence of galactose the promoter will be activated to transcribe the pre-mRNA.

Gene: P(gal)-5'UTR-TyORF-siH-IVS-siH-3'UTR

premRNA 5'UTR-TyORF-siH-IVS-siH-3'UTR

mRNA 5'UTR-TyORF-siH-siH-3'UTR

protein product Ty (and some His if ribosome attaches to His AUG)

Splicing of the pre-mRNA will remove the IVS from the His transcription unit. IVS normally contain splice acceptor/donator sequence that allows them to be spliced when in the same orientation of the transcript. Normal translation will produce large amounts of Ty protein. There is also the possibility that the ribosome will attach to the His ...