Abstract:
A computational method has been developed to detect the conditions whereby gene expression control mechanisms will stop the transcription of RNA that would otherwise be used to form a connectron.

Description:
REFERENCE TO RELATED APPLICATION  
       [0001]    The present application is the subject of Provisional Application Serial No. 60/347,257 filed Jan. 14, 2002  
         [0002]    The present application is a continuation in part of U.S. patent application Ser. No. 09/866,925 filed May 30, 2001 entitled ALGORITHMIC DETERMINATION OF FLANKING DNA SEQUENCES THAT CONTROL THE EXPRESSION OF SETS OF GENES IN PROKARYOTIC, ARCHEA AND EUKARYOTIC GENOMES, incorporated herein by reference.  
         [0003]    The present application is an continuation in part of U.S. patent application Ser. No. 10/227,568 filed Aug. 26, 2002 entitled Determination of flanking DNA sequences that control the expression of sets of genes in the  Escherichia coli  K-12 MG1655 complete genome, incorporated herein by reference. 
     
    
     
       INTRODUCTION  
         [0004]    The connectron structure of a genome determines sets of four DNA sequences of minimum length of 15-bases (C1 and C2 which are in the 3′UTR of a gene, T1 which is on the 5′-side and T2 which is on the 3′-side of a set of genes). When some genes are transcribed into RNA the C1 and C2 sequences in the 3′UTR form the source of a connectron. In addition to binding to the T1 and T2 target sequences, the C1/C2 sequences can also bind to the DNA double-stranded sequences of other equivalent C1/C2 sequences that happen to lie elsewhere in the genome but in particular in the 3′UTR of other genes. When these triple-stranded RNA-DNA-DNA generalized Hoogsteen helices form, the translation of the DNA into RNA is halted and no additional C1/C2 connectron source sequences are produced. The lifetime of this interference RNA (iRNA) is proportional to length of the C1 and C2 sequences. Only the relative lengths of the lifetimes distinguish iRNAs from small temporal RNAs (stRNAs). This invention deals with the relationship between connectrons, iRNAs and stRNAs, as well as a program method for determining the iRNA and stRNA sequences with their associated lifetimes.  
         DEFINITIONS  
         [0005]    Interference RNA (iRNA)—Any sequence of RNA that can bind to a double-stranded DNA to form a triple-stranded generalized Hoogsteen helix.  
           [0006]    Small Temporal RNA (stRNA)—Any sequence of RNA that can bind to a double-stranded DNA to form a triple-stranded generalized Hoogsteen helix.  
         PRIOR ART  
         [0007]    A recent article in Science magazine (1) described interference RNA (iRNA) as the most important scientific breakthrough of 2002. This article provided a bibliography (references 2 to 15) that gives a good understanding of how scientists view the role of iRNA, stRNA and several other related RNAS (i.e. microRNA and small interfering RNA). None of these references mention the use of our patent pending invention of the tetradic relationship that we call a connectron nor do they mention the use of iRNA and stRNA in relationship to connectrons.  
         BRIEF DESCRIPTION OF THE OBJECT OF THE INVENTION  
         [0008]    The object of this invention is to provide a computational method that shows how the transcription of RNA that would otherwise be used to form a connectron can be stopped. 
       
    
    
     DESCRIPTION OF THE DRAWINGS  
       [0009]    The above and other objects, advantages and features of the invention will become more apparent when considered with the following specification and accompanying drawings and table wherein:  
         [0010]    [0010]FIG. 1 illustrates (a) Transcription and Editing. (b) Movement of the RNA through the Nucleus. (c) Connectron Formation. (d) Action of the DICER enzyme. (e) Binding of iRNA to double-stranded DNA of C1 and C2 sequences,  
         [0011]    [0011]FIG. 2 illustrates the overall layout of computer and program,  
         [0012]    [0012]FIG. 3 illustrates the process flow of computer program,  
         [0013]    [0013]FIG. 4 illustrates the determination of all C1/C2 matches and  
         [0014]    [0014]FIG. 5 illustrates the calculation of iRNA lifetimes. 
     
    
     DESCRIPTION OF THE INVENTION  
       [0015]    As shown in FIG. 1, single-stranded RNA is produced when a gene is transcribed. The RNA transcript performs three roles. In role one, one or more copies of the RNA transcript may be edited to form the open reading frame mRNA for translation into protein. In role two, the single-stranded RNA can be used for connectron formation. In role three, other copies of the single-stranded RNA are cut into small fragment by the DICER enzyme. Characteristically the DICER enzyme cuts RNA into 21-base fragments. Two of these fragments are the C1 and the C2 sequences. These single-stranded RNA fragments then bind to the respective double-strand cognate DNA sequences to form two short triple-strand generalized Hoogsteen helices. The double-strand DNA sequences of C1 and C2 that are relevant are those that are in the 3′UTR of one or more genes. When the polymerase that is transcribing the double-stranded DNA into RNA comes to the C1 and C2 sequences that have the iRNA bound to them, the polymerase stops its transcribing action. The two generalized Hoogsteen helices act as a block to the formation of more single-stranded RNA of the C1 and C2 sequences. The Hoogsteen helices of both connectrons and iRNA have lifetimes that vary directly with the length of the generalized Hoogsteen helix. The effect of the iRNA (generalized) Hoogsteen helices is to prevent the formation of more C1-C2 RNA during the lifetime of these helices. The total systematic effect is that the first gene to express a particulate C1-C2 sequence inhibits all other genes with the same sequence from generating more C1-C2 sequenced RNA.  
         [0016]    This invention provides capabilities that are utilized in our application Ser. No. ______ filed contemporaneously herewith and entitles “Simulation of gene expression control using connectrons, interference RNAs (iRNAs) and small temporal RNAs (stRNAs) in prokaryotic, archea and eukaryotic genomes”. The iRNAs and stRNAs play a vital role in determining the simulation of cellular dynamics. This invention provides a way of utilizing iRNAs and stRNAs within the methodology of connectron control of gene expression.  
       EXAMPLE  
       [0017]    Connectron 350 is an example of a transient connectron. It is described in  E. coli  genomic patent application identified above as  
                                                                   C1/C2           T1-T2               Global_Id   Chromosome   Cl_Id   C2_Id   Chromosome   T1_Id   T2_Id   Connectron_Type                   350   1   26   26   1   321   346   transient                  
 
         [0018]    The C1/C2 source of the transient connectron 350 is represented in as  
                                                           Type   Num   Jobno   Chr   Start   Stop   Length   GeneName                   CNT   26   1   1   19.796   19.859   .064   --&gt; | | | | | | | | | | | | | |                  
 
         [0019]    The “Type” descriptor of this transient C1/C2 connectron source is “CNT”. The letter “N” indicates that the C1/C2 connectron source occurs on the negative strand of the double-stranded DNA of the genome. The letter “P” in this place would indicate a C1/C2 connectron source on the positive strand of the genomic DNA. The letter “T” in this descriptor indicates a “transient” connectron. Similarly, the letter “P” would indicate a permanent connectron that is shown in a later example. The “Start”, “Stop” and “Length” descriptors throughout these examples are given in kilo-bases (KB).  
         [0020]    Connectron 19340 is an example of a transient connectron. It is described in  E. coli  genomic patent application identified above as  
                                                                   C1/C2           T1-T2               Global_Id   Chromosome   Cl_Id   C2_Id   Chromosome   T1_Id   T2_Id   Connectron_Type                   19340   1   1260   1260   1   321   346   transient                  
 
         [0021]    The C1/C2 source of the transient connectron 19340 is represented in as  
                                                           Type   Num   Jobno   Chr   Start   Stop   Length   GeneName                   CPT   1260   1   1   1049.705   1049.769   .065   --&gt; | | |||| ||||||||||||||||                  
 
         [0022]    Connectron 23879 is an example of a transient connectron. It is described in  E. coli  genomic patent application identified above as  
                                                                   C1/C2           T1-T2               Global_Id   Chromosome   Cl_Id   C2_Id   Chromosome   T1_Id   T2_Id   Connectron_Type                   23879   1   1927   1927   1   321   346   transient                  
 
         [0023]    The C1/C2 source of the transient connectron 23879 is represented in as  
                                                           Type   Num   Jobno   Chr   Start   Stop   Length   GeneName                   CPT   1927   1   1   1976.526   1976.590   .065   --&gt; || |||||||||||||||||||||                  
 
         [0024]    Connectron 45018 is an example of a transient connectron. It is described in  E. coli  genomic patent application identified above as  
                                                                   C1/C2           T1-T2               Global_Id   Chromosome   Cl_Id   C2_Id   Chromosome   T1_Id   T2_Id   Connectron_Type                   45018   1   3424   3424   1   321   346   transient                  
 
         [0025]    The C1/C2 source of the transient connectron 45018 is represented in as  
                                                           Type   Num   Jobno   Chr   Start   Stop   Length   GeneName                   CPT   3424   1   1   3581.763   3581.827   .065   --&gt; || | | |||| |||||||||||                  
 
         [0026]    These four connectrons are driven by four C1/C2 instances that share the same 64-base sequence as shown in bold below.  
                                   C1/C2   26     GCATGACAAAGTCATCGGGCATTATCTGAACATAAAACACTATCAATAAGTTGGAGTCATTACC                     C1/C2   1260     GCATGACAAAGTCATCGGGCATTATCTGAACATAAAACACTATCAATAAGTTGGAGTCATTACCG                 C1/C2   1927     GCATGACAAAGTCATCGGGCATTATCTGAACATAAAACACTATCAATAAGTTGGAGTCATTACCC                 C1/C2   3424     GCATGACAAAGTCATCGGGCATTATCTGAACATAAAACACTATCAATAAGTTGGAGTCATTACCG            
 
         [0027]    All of the data for the transient connectron 350 are pulled together in the following table that is the “terse” description of the connectron.  
                                                                                                                                                                                                                                                                                                                                                     Connectron Relationships                Global_Id   Type                       350   transient                        Control Sequences                Direction   Chromosome   C1/C2_Id   Start   Stop   Length                       negative   1   26   19.859   19.796   .064                        Trigger Gene            Name   COG_Id   Start   Stop   Length                    insb_1   COG1662   .508   19.811   .698                    Target Sequences                Direction   Chromosome   T1_Id   Start   Stop   Length                       negative   1   321   279.118   278.386   .733                            T2_Id   Start   Stop   Length                       346   290.589   289.833   .757                        Controlled Genes            Local_Id   Chromosome   Group   Name   COG_Id   Direction   Start   Stop   Length                    1   1   Group0058   insb_2   COG1662   positive   278.402   279.099   .698       2   1   Group0059   yagb   —   positive   279.609   281.207   1.598       3   1   Group0059   yaga   COG1425   negative   281.207   280.053   1.155       4   1   Group0060   yage   COG0329   positive   281.481   284.392   2.911       5   1   Group0060   yagf   COG0129   positive   282.425   284.392   1.968       6   1   Group0061   yagg   COG2211   positive   284.619   287.623   3.004       7   1   Group0061   yagh   —   positive   286.013   287.623   1.611       8   1   Group0062   yagf   COG1414   positive   287.628   289.529   1.901       9   1   Group0062   argf   COG0078   negative   289.529   288.525   1.005                    Controlled Connectrons            Local_Id   Chromosome   C1/C2_Id   Direction   Start   Stop   Length                    1   1   327   negative   279.335   279.136   .200       2   1   337   negative   287.273   287.259   .015       3   1   339   negative   287.296   287.282   .015       4   1   342   negative   288.502   288.471   .032       5   1   345   negative   290.589   289.833   .757                  
 
         [0028]    When gene insb (COG1662) is transcribed, the C1/C2 sequence is produced in the 3′UTR. Depending on how the DICER enzyme works there can be many different fragments. A few such fragments are shown below  
         [0029]    First example of a DICER cut of C1/C2 26 
                             GCATGACAAAGTCATCGGGCA TTATCTGAACATAAAACAC TATCAATAAGTTGGAGTCATT                
 
         [0030]    Second example of a DICER cut of C1/C2 26 
                             CATGACAAAGTCATCGGGCAT TATCTGAACATAAAACACT ATCAATAAGTTGGAGTCATTA                
 
         [0031]    Third example of a DICER cut of C1/C2 26 
                             ATGACAAAGTCATCGGGCATT ATCTGAACATAAAACACTA TCAATAAGTTGGAGTCATTAC                
 
         [0032]    A given operation of the DICER enzyme will produce one of these examples or similar examples. The iRNA fragments will then bind as triple-stranded helices to the equivalent sequences in the C1/C2 instances 1260, 1927, and 3424. When the genes associated with these C1/C2 sequences transcribe, the polymerase will find the sequence instances 1260, 1927 and 3424 blocked by triple-stranded generalized Hoogsteen helices formed by the iRNA from C1/C2 26.  
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