Patents held by the Myles Thaler Center

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Patents held by the Myles Thaler Center

Myles Thaler Center

United States Patent
5,880,276
Hammarskjold, et al.
March 9, 1999

Purified retroviral constitutive transport enhancer elements that enhance nucleocytoplasmic transport of mRNA, and methods of making and using the elements

Abstract

A novel retroviral nucleotide sequence comprising a constitutive transport enhancer which functions to transport mRNA transcripts from the nucleus to the cytoplasm of a cell, wherein the mRNA transcript is either differentially spliced, alternatively spliced, incompletely spliced, or unspliced. Additionally disclosed are methods of using the constitutive transport enhancer to screen agents for antiviral activity against rev-dependent HIV proteins by expressing the proteins in a rev-negative subgenomic construct containing the enhancer either in the presence of absence of the agent. Additionally, disclosed are methods of making a constitutive transport enhancer by isolating a sequence to be tested for constitutive transport enhancer activity, inserting the sequence into a vector containing a DNA sequence not normally transported from the nucleus to the cytoplasm so that both the enhancer and DNA molecule are transcribed as part of a functional mRNA transcript in a mammalian cell, introducing the recombinant vector into a mammalian cell, assaying for the nucleocytoplasmic transport of mRNA corresponding to the DNA molecule, and isolating and purifying the sequence having constitutive transport enhancer activity.


Inventors:

 

Assignee:

 

Appl.No.:


Filed:


Hammarskjold; Marie-Louise (Earlysville, VA); Rekosh; David (Earlysville, VA); Bray; Molly (Charlottesville, VA); Hunter; Eric (Birmingham, AL)


The Research Foundation of the State University of NY
(Amherst, NY); University of Alabama at Birmingham Research Foundation (Birmingham, AL


638975


April 25, 1996

Current U.S. Class:
536/24.1; 435/91.1; 536/23.1

Intern'l Class:

C07H 021/04;C07H 021/02; C12P 019/34

Field of Search:

435/172.3,320.1 536/24.1

References Cited [Referenced By]

Other References


 

Bray et al., 1994, Proc. Natl. Acad. Sci. USA 91:1256-1260.

Reeck et al., 1987, Cell 50:667.

Lewin, 1987, Science 237:1570.

 

Primary Examiner: Adams; Donald E.

Assistant Examiner: Parkin; Jeffrey S

Attorney, Agent or Firm: Hodgson, Russ, Andrews Woods & Goodyear LLP


Goverment Interests


This invention was made with government support under grant numbers AI-25721 and AI-25784, awarded by the National Cooperative Drug Discovery Group; and grant numbers AI-27290, AI-30399, and AI-33319, awarded by the National Institutes of Allergy and Infectious Disease. The government has certain rights in this invention.


Parent Case Text


This application is a continuation-in-part of U.S. application Ser. No. 08/246,987 filed May 20, 1994 now U.S. Pat. No. 5,585,263, the disclosure of which is incorporated herein by reference


Claims


We claim:

1. A purified subgenomic constitutive transport enhancer element comprising the nucleic acid sequence of SEQ ID NO:2, or an analogous nucleic acid molecule obtained from simian retrovirus type 1 (SRV-1) or simian retrovirus type 2 (SRV-2), wherein the constitutive transport enhancer element functions in cis to enhance the nuclear to cytoplasmic transport of a heterologous mRNA transcript when present in said transcript.

2. A recombinant vector comprising:

(a) a cis-acting constitutive transport enhancer element according to claim 1;

(b) a mammalian-expressible promoter; and

(c) a DNA molecule to be expressed in a mammalian cell, wherein the DNA molecule is transcribed into mRNA which is differentially spliced, alternatively spliced, incompletely spliced or unspliced, the DNA molecule and the constitutive transport enhancer element are present in a correct orientation and transcribed into a functional mRNA transcript, and the constitutive transport enhancer element enhances the nuclear to cytoplasmic transport of said mRNA transcript.

3. The recombinant vector according to claim 2, wherein said constitutive transport enhancer element consists of a nucleotide sequence disclosed in SEQ ID NO:2.

4. The recombinant vector according to claim 2 wherein said DNA molecule is transcribed into a rev-dependent HIV transcript, wherein the vector is rev-negative, and wherein said nuclear to cytoplasmic transport occurs in the absence of Rev.

5. The recombinant vector according to claim 4, wherein the promoter is the HIV LTR.

6. A cell which contains the recombinant vector of claim 3.

7. A cell which contains the recombinant vector of claim 4.

8. A cell which contains the recombinant vector of claim 5.

9. A method of using a constitutive transport enhancer element to screen for agents that interfere with the expression or function of an HIV protein expressed in a rev-dependent manner comprising:

(a) expressing in mammalian cells an HIV protein from a rev-negative subgenomic construct, wherein the construct contains the constitutive transport enhancer element according to claim 1 in a correct orientation, and an HIV gene which is transcribed into a rev-dependent transcript, wherein the mRNA transcript from said construct contains RNA sequences for the constitutive transport enhancer and for the gene, and wherein expression is under the control of a promoter, and in the presence or absence of said agent;

(b) quantitating the relative amount of expression or function of the HIV protein in the assay in the presence and in the absence of the agent; and

(c) comparing the relative amounts from step (b) to identify anti-viral activity of that agent against the HIV protein.

10. The screening assay according to claim 9, wherein the promoter is the HIV LTR.

11. A process of isolating and purifying a constitutive transport enhancer element (CTE), wherein the constitutive transport enhancer element functions in cis to enhance the nuclear to cytoplasmic transport of a heterologous mRNA transcript when present in said transcript comprising the steps of:

(a) isolating a retroviral or cellular genomic sequence being screened for CTE activity;

(b) inserting the sequence into a vector to form a recombinant vector, wherein said sequence is present in a cis orientation relative to a heterologous DNA molecule that is transcribed into a heterologous mRNA which is differentially spliced, alternatively spliced, incompletely spliced or unspliced, wherein said heterologous mRNA is not transported into the cytoplasm in the absence of a sequence containing CTE activity;

(c) introducing the recombinant vector into mammalian cells;

(d) culturing the mammalian cells;

(e) assaying the cultured cells for expression of the DNA molecule by detecting the presence of, heterologous mRNA transcripts in the cytoplasm of said cells or the production of a protein encoded by the DNA molecule, wherein detection of such expression indicates that the sequence comprises a constitutive transport enhancer element; and

(f) isolating and purifying the sequence from the recombinant vector.

12. A modification of the process according to claim 11, wherein said modification comprises the additional steps of:

(a) generating fragments of the sequence identified from step (f) of claim 11 containing the constitutive transport enhancer element;

(b) inserting said fragments into a vector to form a recombinant vector, wherein said fragments are present in a cis orientation relative to a heterologous DNA molecule that is transcribed into a heterologous mRNA which is differentially spliced, alternatively spliced, incompletely spliced or unspliced, wherein said heterologous mRNA is not transported into the cytoplasm in the absence of a sequence containing CTE activity;

(c) introducing the recombinant vector into mammalian cells;

(d) culturing the mammalian cells;

(e) assaying the cultured cells for expression of the DNA molecule by detecting the presence of heterologous mRNA transcripts in the cytoplasm of said cells or the production of a protein encoded by the DNA molecule wherein detection of such expression indicates that the sequence comprises a constitutive transport enhancer element; and (f) isolating and purifying the sequence from the recombinant vector.

13. A process of making a cis acting constitutive transport enhancer element according to claim 1, wherein said process is selected from the group consisting of enzymatic amplification and chemical synthesis