WO2001029210B1

WO2001029210B1 - A method for establishing connections between genes

Info

Publication number: WO2001029210B1
Application number: PCT/IL2000/000642
Authority: WO
Inventors: Ben-Zion Vider
Original assignee: Genena Ltd; Vider Ben Zion
Priority date: 1999-10-18
Filing date: 2000-10-12
Publication date: 2001-10-18
Also published as: IL132446A0; WO2001029210A1; AU7815500A

Abstract

Series of assays and recombinant constructs are presented hereinafter that establish the connections between the regulatory region of a first gene, and the product of a second gene. The regulatory region of the first gene is inserted into a recombinant construct, and linked downstream to it is the cDNA of the second gene and then the regulatory region of the second gene is inserted into a recombinant construct, and linked downstream to it is the cDNA of the first gene, and in every recombinant construct either the cDNA of the gene or the regulatory region is that of a cell cycle gene. The recombinant constructs are then transferred into the cells and the degree of activation of the regulatory elements are sorted out.

Claims

1 4AMENDED CLAIMS[received by the International Bureau on 27 May 2001 (27.05.01 ); original claims 1-14 replaced by new claims 1-18 (4 pages)]1 ) A method that categorize the function of an analyzed gene and the regulatory region of the analyzed gene as either related or not related to the cell cycle and to genes of the cell cycle by constructing special vectors that contain either the gene or the regulatory region of the gene fused respectively to a regulatory region of a cell cycle gene or to a cell cycle gene, and the vectors containing these elements are transferred into cells and the functional connections between these elements are studied by their degree of activation within the transfected cells.2) The method of claim 1 in which the relations between said one gene and said second gene regulatory element are defined by using the following recombinant vectors: Type I that contains the regulatory sequence of a first gene categorized as a cell cycle activator gene, and linked downstream to it is the cDNA of a second gene which is the analyzed gene A.Type II that contains the regulatory sequence of the analyzed gene A, and linked downstream to it is the cDNA of the first gene which is the cell cycle activator gene. Type in that contains the regulatory sequence of a third gene categorized as a cell cycle suppressor gene, and linked downstream to it is the cDNA of the gene which is the analyzed gene A.Type IV that contains the regulatory sequence of the analyzed gene A, and linked downstream to it is the cDNA of the third gene which is the cell cycle suppressor gene.3) The method of claim 2 in which the cell cycle activator gene is any one of the known cyclins, cyclin dependent kinases, or genes that can function as oncogenes.4) The method of claim 2 in which the cell cycle suppressor gene is any one of the known cyclin dependent kinase inhibitors, or genes that are responsible for apoptotic events of the cells or genes that can function as tumor suppressors.5) The method of claim 2 in which the regulatory region can be one or more of the following: a promoter, an enhancer, intron, 3' non translated cDNA, and regulatory regions within the gene coding sequence.6) The recombinant vectors of claim 2 each one of them containing a second regulatory region inserted in a different part of the construct, and the second regulatory region is identical to the first regulatory region and linked downstream to it is a reporter gene,7) The method of claims 1-6 that characterizes the vectors as retro viral vectors or adeno viral vectors suitable for transfection of mammalian cells, or other vectors suitable for 1 5transfection of other eukaryotic cells, which are designed for the analysis of interactions between suspected cis regulatory regions and a specific cell cycle gene product and thus contain the following unique elements: a. A multiple cloning site designed for the insertion of DNA segments suspected of acting as cis regulatory elements b. Downstream to the multiple cloning site a minimal promoter element c. Downstream to the minimal promoter element a sequence encoding for a gene which is a cell cycle gene.8) The vectors of claim 7 aimed for the analysis of interactions between a cell cycle regulatory region and a plurality of cDNAs thus contain the following unique elements: a. A regulatory region of a cell cycle gene b. Downstream to the regulatory region a minimal promoter element c. Downstream to the mmimal promoter element a multiple cloning site designed for the insertion of various cDNAs encoding for gene products candidate to interact with the regulatory region of 8a.9) A baseline vector for the vectors of claim 7 that analyzes the cis activity of DNA segments suspected of acting as regulatory elements and contains the multiple cloning site for the insertion of a DNA segment, downstream to it the minimal promoter element and downstream to it the reporter gene that measures the baseline activity of the cloned DNA segment.10) A baseline vector for the vectors of claim 8 that analyzes the cis activity of the cell cycle regulatory element and contains this element, downstream to it the minimal promoter element and downstream to it the reporter gene that measures the baseline activity of the regulatory elemen11 ) The method of the previous claims in which gene A or the regulatory region of gene A is that of a gene encoding a transcription factor.12) The method of claim 11 in which the transcription factor and their regulatory regions are those of genes that belong to the homeobox or MADSbox gene families.13) A method according to any of the previous claims in which in the vectors the gene sequence fused downstream to the minimal promoter element is in an anti-sense orientation thus enable the analysis of factors acting as inhibitors of the investigated regulatory region, and their insertion in anti-sense orientation result in a relief of such inhibition. 14) The method of claim 13 in which the anti-sense oriented sequence is that of a gene family conserved region, and the regulatory element attached upstream to it is that of a cell cycle gene.15) A method according to claims 1-13 that identifies connections between sequences within the coding region of genes suspected of acting as cis-regulatory elements, especially coding regions of conserved sequences of gene family members, and a cell cycle gene, thus design a construct that is composed of the following constituents:1 ) an upstream gene family conserved segment acting as the regulatory element.2) minimal promoter region is linked downstream to the regulatory region of item 15.1.3) full cDNA of the heterologous gene which is a cell cycle gene that is linked downstream to the minimal promoter region.16) A method that uses the vectors of claims 7 and 9 in order to analyze the possible role of any DNA segment suspected of acting as cis regulatory element and its interaction with cell cycle genes and includes the following steps: a. Preparing large amount of the baseline vector of claim 9. b. Inserting into the baseline vector of claim 9 various DNA segments suspected of having cis regulatory activity thus forming a baseline vector library. c. Transferring the recombinant library of item b into the appropriate host cells for example mammalian cells and screening for cells bearing the recombinant construct and expressing the reporter gene. d. Identifying the insert DNA sequences and inserting them into either the cell cycle bearing gene vectors of claim 7, thus foπning a cell cycle related recombinant library. e. Co-transfecting the cells, which already contain the baseline recombinant construct of item b with the recombinant construct library of item d and measuring the reporter gene activity produced by the baseline construct.17) A method that uses the vectors of claims 8 and 10 in order to analyze the possible interaction of a cell cycle cis-acting regulatory element with other genes and includes the following steps: a. Preparing large amount of the baseline vector of claim 10. b. Transferring the vector of item a into the appropriate host cells for example mammalian cells and screening the expression of the reporter gene. c. Inserting into the vector of claim 8 plurality of cDNAs of a selected cDNA expression library. 1 7d. Co-lransfecting the cells, which already contain the baseline vector of item b with a recombinant construct library of item 17c and measuring the reporter gene activity produced by the baseline construct. 18) A kit according to any of the previous claims containing; the recombinant vector with an already inserted regulatory region of a cell cycle gene, or the cDNA of a cell cycle gene, and all the materials necessary for analyzing the functional connections between the genes, and the instructions to carry out the assays. 1 8STATEMENT UNDER ARTICLE 19 (1)The subgroup of the anti-sense oriented genes' sequences is an additive, complementing the suggested method, and it is described in c kirns 13-14.According to part of what was claimed the method categorized the function of an analyzed gene as either related or not related to the regulatory regions of genes of the cell cycle and vice versa. The vector is introduced into the cells and if and when there is an auto regulatory positive feedback between the gene product and the regulatory element, it results in an increased expression of the gene product, and it can be measured by various means. This augmentation of transcription can be direct or indirect.In the entire described constructs the transcripts that are transcribed are those of the genes and if the gene product has a positive effect on the regulatory region it results in the increased expression. Actually when an inhibitory effect of a gene product on the regulatory region is suspected it can not be detected efficiently.We now suggest the use of anti-sense transcripts, which are generally inhibiting the expression of their native genes, as the downstream transcript. Thus if the native gene is suspected of inhibiting a regulatory element in a cell then attaching to that regulatory element the anti-sense sequence of such a gene product will result in decreased inhibition and actually the activation of the regulatory region, thus actually forming a positive auto regulatory loop.Further advantage is gained out of the use of an anti-sense sequence, as not a large fragment is required in order to neutralize the effect of the gene. It is even further intensified when the anti-sense sequence is derived from the conserved region of a gene family. Then according to the degree of conservation chosen as the anti-sense sequence, the blocking of many genes can be obtained. If such a conserved anti-sense oriented sequence is positioned downstream to a regulatory region, it intensifies the general tendency of such a gene family toward the regulatory sequence. If many members of a gene family in the native situation inhibit the transcription from the specific regulatory element, then inhibiting their expression will result in the opposite phenomena.Thus the possible role of any gene family and especially those encoding for transcription factors, toward regulatory regions can be studied. The regulatory region according to the scope of the present invention is that of a cell cycle gene.Beside the aspect of anti-sense sequences several other additions and modifications were made: 1 9

1. A new claim No 1 replaces the original one offering a better definition of the scope of the invention with its related aspects.

2. New claims No 7-10 and 15-17 replaces the original claims No 7-9 and 13. They provide more detailed explanation of the technical steps related to the construction of the various vectors, claims No 7-10, and then their usage in the various situations and types of cells, claims 15-17.

3. More attention was drawn to the use of a minimal promoter element between the regulatory element and the expressed gene (especially claims 7-10), as in many instances regulatory elements can not activate the transcription machinery by themselves.