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Generated: September 23, 2019

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Claims for Patent: 5,691,137

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Summary for Patent: 5,691,137
Title: Methods of screening candidate agents for biological activity using yeast cells
Abstract:Methods of identifying and screening for agents with biological activity against target molecules using yeast cell cultures are described.
Inventor(s): Rosbash; Michael (Newton Lower Falls, MA), Stutz; Francoise (Brookline, MA)
Assignee: Brandeis University (Waltham, MA)
Application Number:08/297,808
Patent Claims:1. A method of screening a candidate agent for biological activity against HIV-1 Rev protein comprising:

a) contacting a yeast double transformant with a candidate agent to be tested for activity against HIV-1 Rev protein, said yeast double transformant produced by introducing into a yeast strain which lacks the gene encoding a copper chelator protein:

i) a reporter construct comprising regulatory control sequences operably linked to a DNA sequence encoding a copper chelator protein and a DNA sequence encoding the Rev Response Element, wherein the DNA sequence encoding the copper chelator protein is interrupted with a synthetic intron, and the intron sequence is out of frame with the DNA sequence encoding the copper chelator protein; and

ii) a recombinant plasmid comprising regulatory sequences operably linked to a DNA sequence encoding the HIV-1 Rev protein,

said yeast double transformant being maintained in culture medium containing low copper concentration;

b) increasing the copper concentration in the culture medium, and determining the growth rate of the yeast double transformant; and

c) comparing the growth rate of the yeast double transformant grown in the presence of the candidate agent with the growth rate of the yeast double transformant grown under similar conditions but in the absence of the candidate agent to determine if the candidate agent has activity against HIV-1 Rev protein, wherein if the candidate agent has activity against HIV-1 Rev, the growth rate of the yeast double transformant cultured in the presence of the candidate agent will be greater than the growth rate of the yeast double transformant grown in the absence of the candidate agent.

2. The method of claim 1 wherein the yeast strain is Saccharomyces cerevisiae.

3. A method of screening a candidate agent for biological activity against HIV-1 Rev protein comprising:

a) contacting a yeast double transformant and a yeast control strain, said yeast control strain encoding a selectable marker, with a candidate agent to be tested for activity against the HIV-1 Rev protein, said yeast double transformant produced by introducing into a defective yeast strain which lacks the gene encoding a copper chelator protein:

i) a reporter construct comprising regulatory control sequences operably linked to a DNA sequence encoding a copper chelator protein and a DNA sequence encoding the Rev Response Element, wherein the DNA sequence encoding the copper chelator protein is interrupted with a synthetic intron, and the intron sequence is out of frame with the DNA sequence encoding the copper chelator protein; and

ii) a recombinant plasmid comprising regulatory sequences operably linked to a DNA sequence encoding the HIV-1 Rev protein, said yeast double transformant and yeast control strain being maintained in culture medium containing low copper concentration;

b) increasing the copper concentration in the culture medium of both strains and determining the growth rate of the yeast double transformant and the yeast control strain; and

c) determining the ratio of the growth rate of the yeast double transformant and the yeast control strain to determine if the candidate agent has specific activity against the target molecule, wherein a candidate agent that has specific activity against the target will enhance the growth ratio of yeast double transformant to yeast control.

4. A method of claim 3 wherein the yeast strain is Saccharomyces cerevisiae.

5. A method of screening a library of DNA sequences encoding peptides to determine the biological activity of one or more of the peptides encoded by the DNA sequences against HIV-1 Rev protein, comprising:

a) introducing into a yeast strain which lacks a gene encoding a copper chelator protein:

i) a reporter construct comprising regulatory control sequences operably linked to a DNA sequence encoding a copper chelator protein and a DNA sequence encoding the Rev Response Element, wherein the DNA sequence encoding the copper chelator protein is interrupted with a synthetic intron; and

ii) a recombinant plasmid comprising regulatory sequences operably linked to a DNA sequence encoding HIV-1 Rev protein,

the introduction of the reporter construct and the recombinant plasmid thereby producing yeast double transformants;

b) introducing into the yeast double transformants of step a) plasmids containing DNA sequence inserts obtained from a random library of DNA sequences which are expressed in the yeast;

c) maintaining said yeast double transformants expressing said library of DNA sequences of step b) under permissive culture conditions;

d) altering the culture conditions to restrictive growth conditions and maintaining the yeast double transformants expression said library of DNA sequences of step c) for sufficient time to kill or severely retard the growth of the yeast double transformants expressing said library of DNA sequences in which the activity of the HIV-1 REV is not inhibited;

e) harvesting the yeast double transformants expressing said library of DNA sequences which grow more rapidly under restrictive conditions; and

f) determining the nucleotide sequences of said DNA sequence inserts present in the yeast double transformants.

6. A reporter gene construct which is expressed in yeast comprising:

a) a promoter sequence and transcription initiation site sufficient to direct transcription of a gene sequence in yeast;

b) a nucleotide sequence encoding CUP1, wherein a synthetic intron sequence containing a consensus 5' splice site, branchpoint and 3' splice site sequence interrupts the CUP1 coding sequence, said intron sequence being out-of-frame frame with the CUP1 coding sequence;

c) a nucleotide sequence encoding the HIV-1 Rev Response Element; and

d) a transcription termination sequence, wherein said sequences are operably linked for expression of the reporter gene construct in yeast.

Details for Patent 5,691,137

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04 ➤ Sign Up Brandeis University (Waltham, MA) 2037-11-20 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04 ➤ Sign Up Brandeis University (Waltham, MA) 2037-11-20 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04 ➤ Sign Up Brandeis University (Waltham, MA) 2037-11-20 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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