Abstract:
Method of producing controls for use in gene expression analysis systems such as macroarrays, real-time PCR, northern blots, SAGE and microarrays. The controls are generated either from near-random sequence of DNA, or from inter- or intragenic regions of a genome. Ten specific control sequences are also disclosed. Also presented are methods of using these controls, including as negative controls, positive controls, and as calibrators of a gene expression analysis system.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims priority to U.S. provisional patent application serial No. 60/289,202, filed May 7, 2001; and No. 60/312,420, filed Aug. 15, 2001; the disclosures of which are incorporated herein by reference in their entireties. 
     
    
     
       REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISC  
         [0002]    The present application includes a Sequence Listing filed on one CD-R disc, provided in duplicate, containing a single file named PB0120.ST25.txt, having 32 kilobytes, last modified on May 6, 2002, and recorded on May 6, 2002. The Sequence Listing contained in said file on said disc is incorporated herein by reference in its entirety.  
         BACKGROUND OF THE INVENTION  
         [0003]    1. Field of the Invention  
           [0004]    The present invention relates to a method of using artificial genes as controls in gene expression analysis systems. More particularly, the present invention relates to a method of producing Controls for use in gene expression analysis systems such as macroarrays, real-time PCR, northern blots, SAGE and microarrays, such as those provided in the Microarray ScoreCard system.  
           [0005]    2. Description of Related Art  
           [0006]    Gene expression profiling is an important biological approach used to better understand the molecular mechanisms that govern cellular function and growth. Microarray analysis is one of the tools that can be applied to measure the relative expression levels of individual genes under different conditions. Microarray measurements often appear to be systematically biased, however, and the factors that contribute to this bias are many and ill-defined (Bowtell, D. L.,  Nature Genetics  21, 25-32 (1999); Brown, P. P. and Botstein, D.,  Nature Genetics  21, 33-37 (1999)). Others have recommended the use of “spikes” of purified mRNA at known concentrations as controls in microarray experiments. Affymetrix includes several for use with their GeneChip products. In the current state of the art, these selected genes are actual genes selected from very distantly related organisms. For example, the human chip (designed for use with human mRNA) includes control genes from bacterial and plant sources. Affymetrix sells mRNA corresponding to these genes for spiking into the labeling reaction and inclusion in the hybridization reaction.  
           [0007]    Each of the prior art controls includes transcribed sequences of DNA from some source. As a result, that source cannot be the subject of a hybridization experiment using those controls due to the inherent hybridization of the controls to its source. What is needed, therefore, is a set of controls which do not hybridize with the DNA of any source which may be the subject of an experiment. More desirably, there is a need for a control for gene expression analysis which does not hybridize with any known source.  
         SUMMARY OF THE INVENTION  
         [0008]    Accordingly, this invention provides a process of producing controls that are useful in gene expression analysis systems designed for any species and which can be tested to insure lack of hybridization with mRNA from sources other than the control DNA itself.  
           [0009]    The invention relates in a first embodiment to a process for producing at least one control for use in a gene expression analysis system. The process comprises selecting at least one non-transcribed (inter- or intragenic) region of genomic DNA from a known sequence, designing primer pairs for said at least one non-transcribed region and amplifying said at least one non-transcribed region of genomic DNA to generate corresponding double stranded DNA, then cloning said double stranded DNA using a vector to obtain additional double stranded DNA and formulating at least one control comprising said double stranded DNA.  
           [0010]    The present invention relates in a second embodiment to a process of producing at least one control for use in a gene expression analysis system wherein testing of said at least one non-transcribed region to ensure lack of hybridization with mRNA from sources other than said at least one non-transcribed region of genomic DNA is performed.  
           [0011]    The present invention in a third embodiment relates to said process further comprising purifying said DNA and mRNA, determining the concentrations thereof and formulating at least one control comprising said DNA or of said mRNA at selected concentrations and ratios.  
           [0012]    Another embodiment of the present invention is a control for use in a gene expression analysis system comprising a known amount of at least one DNA generated from at least one non-transcribed region of genomic DNA from a known sequence, or comprising a known amount of at least one mRNA generated from DNA generated from at least one non-transcribed region of genomic DNA from a known sequence. The present invention may optionally include generating mRNA complementary to said DNA and formulating at least one control comprising said mRNA, by optionally purifying said DNA and mRNA, determining the concentrations thereof and formulating at least one control comprising said DNA or of said mRNA at selected concentrations and ratios.  
           [0013]    Another embodiment of the present invention is a control for use in a gene expression analysis system wherein a known amount of at least one DNA sequence generated from at least one non-transcribed region of genomic DNA from a known sequence, a known amount of at least one mRNA generated from DNA generated from at least one non-transcribed region of genomic DNA from a known sequence is included, and the aforementioned control wherein, said DNA and mRNA do not hybridize with any DNA or mRNA from a source other than the at least one non-transcribed region of genomic DNA.  
           [0014]    The present invention, relates to a method of using said control, as a negative control in a gene expression analysis system by adding a known amount of said control containing a known amount of DNA, to a gene expression analysis system as a control sample and subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA and examining the control sample for the absence or presence of signal.  
           [0015]    Further, said controls can be used in a gene expression analysis system by adding a known amount of a said control containing a known amount of DNA to a gene expression analysis system as a control sample and subjecting the sample to hybridization conditions, in the presence of a said control containing a known amount of labeled complementary mRNA, and measuring the signal values for the labeled mRNA and determining the expression level of the DNA based on the signal value of the labeled mRNA.  
           [0016]    Additionally, said controls may be used as calibrators in a gene expression analysis system by adding a known amount of a said control containing known amounts of several DNA sequences to a gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs, each mRNA being at a different concentration and measuring the signal values for the labeled mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA.  
           [0017]    Also, the present invention relates to a method of using said controls as calibrators for gene expression ratios in a two-color gene expression analysis system by adding a known amount of at least one of said controls containing a known amount of DNA to a two-color gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled mRNAs for each DNA sample present and measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labelled mRNAs.  
           [0018]    A further embodiment of the present invention is a process of producing controls that are useful in gene expression analysis systems designed for any species and which can be tested to insure lack of hybridization with mRNA from sources other than the synthetic sequences of DNA from which the control is produced.  
           [0019]    One or more such controls can be produces by a process comprising synthesizing a near-random sequence of non-transcribed DNA, designing primer pairs for said at least one near random sequence and amplifying said non-transcribed DNA to generate corresponding double stranded DNA, then cloning said double stranded DNA using a vector to obtain additional double stranded DNA and formulating at least one control comprising said double stranded DNA.  
           [0020]    The process can also be used to produce at least one control for use in a gene expression analysis system wherein testing of said sequence of non-transcribed synthetic DNA to ensure lack of hybridization with mRNA from sources other than said sequence of non-transcribed DNA is performed.  
           [0021]    Additionally, mRNA complementary to said synthetic DNA can be generated and formulated to generate at least one control comprising said mRNA.  
           [0022]    DNA and mRNA can be subsequently purified, the concentrations thereof determined, and one or more controls comprising said DNA or said mRNA at selected concentrations and ratios be formulated.  
           [0023]    Another embodiment of the present invention is a control for use in a gene expression analysis system produced by the process comprises synthesizing a near-random sequence of DNA, designing primer pairs for said synthetic DNA and amplifying said DNA to generate corresponding double stranded DNA, then cloning said double stranded DNA using a vector to obtain additional double stranded DNA and formulating at least one control comprising a known amount of at least one said double stranded DNA or a known amount of at least one mRNA generated from said DNA, and optionally, wherein, said DNA and mRNA do not hybridize with any DNA or mRNA from a source other than said DNA sequence of non-transcribed DNA.  
           [0024]    The present invention, additionally, relates to a method of using said controls containing a known amount of DNA, as a negative control in a gene expression analysis system including adding a known amount of said control containing a known amount of DNA to a gene expression analysis system as a control sample, and subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA and examining the control sample for the absence or presence of signal.  
           [0025]    Further, said controls may be used in a gene expression analysis system wherein a known amount of a said control containing a known amount of DNA is added to a gene expression analysis system as a control sample and subjecting the sample to hybridization conditions in the presence of a said control containing a known amount of labeled complementary mRNA and measuring the signal values for the labeled mRNA and determining the expression level of the DNA based on the signal value of the labeled mRNA.  
           [0026]    The present invention, also relates to a method of using said controls as calibrators in a gene expression analysis system including adding known amounts of a said control containing known amounts of several DNAs to a gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs, each mRNA being at a different concentration and measuring the signal values for the labeled mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA.  
           [0027]    The present invention, additionally, relates to a method of using said controls as calibrators for gene expression ratios in a two-color gene expression analysis system comprising adding a known amount of at least one of said controls containing a known amount of DNA to a two-color gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled mRNAs for each DNA sample present and measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labeled mRNAs.  
           [0028]    Further embodiments and uses of the current invention will become apparent from a consideration of the ensuing description. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0029]    The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which like characters refer to like parts throughout, and in which:  
         [0030]    [0030]FIG. 1 presents the control nucleotide sequences of YIR1;  
         [0031]    [0031]FIG. 2 presents the control nucleotide sequences of YIR2;  
         [0032]    [0032]FIG. 3 presents the control nucleotide sequences of YIR3;  
         [0033]    [0033]FIG. 4 presents the control nucleotide sequences of YIR4;  
         [0034]    [0034]FIG. 5 presents the control nucleotide sequences of YIR5;  
         [0035]    [0035]FIG. 6 presents the control nucleotide sequences of YIR6;  
         [0036]    [0036]FIG. 7 presents the control nucleotide sequences of YIR7;  
         [0037]    [0037]FIG. 8 presents the control nucleotide sequences of YIR8;  
         [0038]    [0038]FIG. 9 presents the control nucleotide sequences of YIR11;  
         [0039]    [0039]FIG. 10 presents the control nucleotide sequences of YIR19;  
         [0040]    [0040]FIG. 11 presents the nucleotide sequences of YIR1s used in a spike mix;  
         [0041]    [0041]FIG. 12 presents the nucleotide sequences of YIR2s used in a spike mix;  
         [0042]    [0042]FIG. 13 presents the nucleotide sequences of YIR3s used in a spike mix;  
         [0043]    [0043]FIG. 14 presents the nucleotide sequences of YIR4s used in a spike mix;  
         [0044]    [0044]FIG. 15 presents the nucleotide sequences of YIR5s used in a spike mix;  
         [0045]    [0045]FIG. 16 presents the nucleotide sequences of YIR6s used in a spike mix;  
         [0046]    [0046]FIG. 17 presents the nucleotide sequences of YIR7s used in a spike mix;  
         [0047]    [0047]FIG. 18 presents the nucleotide sequences of YIR8s used in a spike mix;  
         [0048]    [0048]FIG. 19 presents the nucleotide sequences of YIR11s used in a spike mix; and  
         [0049]    [0049]FIG. 20 presents the nucleotide sequences of YIR19s used in a spike mix. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0050]    The present invention teaches Controls for use in gene expression analysis systems such as microarrays. Many have expressed interest in being able to obtain suitable genes and spikes as controls for inclusion in their arrays.  
         [0051]    An advantage of the Controls of this invention is that a single set can be used with assay systems designed for any species, as these Controls will not be present unless intentionally added. This contrasts with the concept of using genes from “distantly related species.” For example, an analysis system directed at detecting human gene expression might employ a  Bacillus subtilis  gene as control, which may not be present in a human genetic material. But this control might be present in bacterial genetic material (or at least, cross hybridize), thus it may not be a good control for an experiment on bacterial gene expression. The novel Controls presented here provide an advantage over the state of the art in that the same set of controls can be used without regard to the species for the test sample RNA.  
         [0052]    The present invention employs the novel approaches of using either non-transcribed genomic sequences or totally random synthetic sequences as a template and generating both DNA and complementary “mRNA” from such sequences, for use as controls. The Controls could be devised de novo by designing near-random sequences and synthesizing them resulting in synthetic macromolecules as universal controls. Totally synthetic random DNA fragments are so designed that they do not cross-hybridize with each other or with RNA from any biologically relevant species (meaning species whose DNA or RNA might be present in the gene expression analysis system). The cost of generating such large synthetic DNA molecules can be high. However, they only need to be generated a single time. Additionally, fragment size can be increased by ligating smaller synthetic fragments together by known methods. In this way, fragments large enough to be easily cloned can be created. Through cloning and PCR sufficient quantities of DNA for use as controls can be produced and mRNA can be generated by in vitro transcription for use in controls.  
         [0053]    A simpler approach is to identify sequences from the non-transcribed regions of genomic DNA from an organism, and use these as a template for synthesis via PCR (polymerase chain reaction). Ideally, sequences of around 1000 bases (could range from 500 to 2000 bases) are selected based on computer searches of publicly accessible sequence data. The criteria for selection include:  
         [0054]    1. The sequence must be from a non-transcribed region (intergenic or intronic region); and  
         [0055]    2.The sequence must not have homology with or be predicted to hybridise with any known/published gene or expressed sequence tag (EST).  
         [0056]    PCR primer pairs are designed for the selected sequence(s) and PCR is performed using genomic DNA (as a template) to generate PCR fragments (dsDNA) corresponding to the non-transcribed sequence(s) as the control DNA. Additional control DNA can be cloned using a vector and standard techniques. Subsequently, standard techniques such as in vitro transcription are used to generate mRNA (complementary to the cDNA and containing a poly-A tail) as the control mRNA. Standard techniques are used for purifying the Control DNA and Control mRNA products, and for estimating their concentrations.  
         [0057]    Empirical testing is also performed to ensure lack of hybridization between the Control DNA on the array and other mRNAs, as well as with mRNA from important gene expression systems (e.g., human, mouse, Arabidopsis, etc.).  
         [0058]    The above approaches were used to generate ten control sequences from intergenic regions of the yeast  Saccharomyces cerevisiae  genome. Specifically, using yeast genome sequence data publicly available (http://genome-www.stanford.edu/Saccharomyces/), intergenic regions approximately 1 kb in size were identified. These sequences were BLAST&#39;d and those showing no homology to other sequences were identified as candidates for artificial gene controls. Candidates were analyzed for GC-content and a subset with a GC-content of ≧36% were identified. Specific primer sequences have been identified and synthesized. PCR products amplified with the specific primers have been cloned directly into the pGEM™-T Easy vector (Promega Corp., Madison, Wis.). Both array targets and templates for spike mRNA have been amplified from these clones using distinct and specific primers.  
         [0059]    To maximize the chances of identifying 10 control sequences, a greater number of intergenic regions have been cloned for testing. All candidate sequences were spotted on glass microarray slides and hybridized with each candidate spike mRNA independently to identify those that cross-hybridize. Ten candidates exhibiting specific hybridization were chosen to form the specific set of controls. When used as controls, all of the ten yeast intergenic regions (YIRs) were generated by PCR with specific primers (Table 1), using 5 ng of cloned template (plasmid DNA) and a primer concentration of 0.5 μM in a 100 μl reaction volume, and cycled as follows: 35 cycles of  
                                                           TABLE 1                           Primers used for amplification of controls.                Target   Forward Primer   Reverse Primer                    YIR1   TTCGTTGGATTGAGTAAGAA   SEQ ID NO: 21   GCACTTCTAGTAAGCACATG   SEQ ID NO: 31                   YIR2   GCGAATAACCAAAACGAGAC   SEQ ID NO: 22   GCACTAAACTAAAACCGTGA   SEQ ID NO: 32               YIR3   TGTTTTTGCTATATTACGTGGG   SEQ ID NO: 23   CCAGCGAACACAATTCAAAA   SEQ ID NO: 33               YIR4   TTTCGGTAGTGAGATGGCAG   SEQ ID NO: 24   TGTACCACTTTTGCACCATA   SEQ ID NO: 34               YIR5   TTAGTTTGGAACAGCAGTGT   SEQ ID NO: 25   GTTTCCTCGCTCATACCCTA   SEQ ID NO: 35               YIR6   AATGAGTTACCGTCTGTTAC   SEQ ID NO: 26   AGTAAAGTCATGGTGGATTG   SEQ ID NO: 36               YIR7   TCCTAGAGTAGCGATTCCCC   SEQ ID NO: 27   GCACCTATCGTCATTGTCTT   SEQ ID NO: 37               YIR8   TAGTTGGAGGTTGGTGAGTA   SEQ ID NO: 28   CTTCAACTCGTACGTGATGG   SEQ ID NO: 38               YIR11   CCATTCATATCATTTAGTGC   SEQ ID NO: 29   CCATTCCAGTTCATATTGAA   SEQ ID NO: 39               YIR19   GATTTAATACAGTACCTTTCTTCGC   SEQ ID NO: 30   CCACTTTGATGGACTATTATGTATG   SEQ ID NO: 40                  
 
         [0060]    94° C. 20 sec., 52° C. 20 sec., 72° C. 2 min., followed by extension at 72° C. for 5 min.  
         [0061]    All YIR control mRNAs for the spike mix are generated by in vitro transcription. Templates for in vitro transcription (IVT) are generated by amplification with specific primers that are designed to introduce a T7 RNA polymerase promoter on the 5′ end and a polyT (T21) tail on the 3′ end of the PCR products (see Table 2). Run-off mRNA is produced using 1 μl of these PCR products per reaction with the AmpliScribe system (Epicentre, Madison, Wis.). IVT products are purified using the RNAEasy system (Qiagen Inc., Valencia, Calif.) and quantified by spectrophotometry.  
         [0062]    [0062]FIG. 1 through FIG. 10 presents the nucleotide sequences of the ten YIR controls, while FIGS. 11 through 20 presents the nucleotide sequences of the ten YIRs (‘s’ for spike mix) as used in a spike mix. The primer sequences used for amplifying the controls were listed in Table 1, the primer sequences used for amplifying spike mix templates were listed in Table 2. These sequences are further presented in the Sequence Listing, incorporated herein by reference in its entirety, as follows:  
         [0063]    SEQ ID NO: 1-8  
         [0064]    nt, control nucleotide sequences YIR1 through YIR 8;  
         [0065]    SEQ ID NO: 9  
         [0066]    nt, control nucleotide sequence YIR11;  
         [0067]    SEQ ID NO: 10  
         [0068]    nt, control nucleotide sequence YIR19;  
                                                           TABLE 2                           Primers used for amplification of in vitro           transcription targets.            Template   Forward Primer   Reverse Primer                    YIR1   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 41   TTTTTTTTTTTTTTTTTTTTTGAA   SEQ ID NO: 51               CGACTCACTATAGGGAGAAATGTC   TACTTCCACTTTGGTGC           GATACTGTGTTACG               YIR2   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 42   TTTTTTTTTTTTTTTTTTTTTAAT   SEQ ID NO: 52           CGACTCACTATAGGGAGATTTCTT   ATGCGGCTGCGCTAAAA           TTTCCCTATTTCTCACTGG               YIR3   GCATTAGCGGCCGCGPAATTAATA   SEQ ID NO: 43   TTTTTTTTTTTTTTTTTTTTTAGT   SEQ ID NO: 53           CGACTCACTATAGGGAGAACTGTA   CGGTAATTTCTTTCTGG           TATAAAAGAGGACTGC               YIR4   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 44   TTTTTTTTTTTTTTTTTTTTTCCA   SEQ ID NO: 54           CGACTCACTATAGGGAGAATAATA   CCATGACGTCATTAACTTAAAT           ACTTCTGGCTTTTCGC               YIR5   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 45   TTTTTTTTTTTTTTTTTTTTTTTT   SEQ ID NO: 55           CGACTCACTATAGGGAGAAGATAC   AAAGGTATCATCCCTGT           CGTCCTTGGATAGA               YIR6   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 46   TTTTTTTTTTTTTTTTTTTTTGCC   SEQ ID NO: 56           CGACTCACTATAGGGAGATTGGGA   GGACCTTTCAAGCATAA           CGGTTTTTGCACTAAGAA               YIR7   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 47   TTTTTTTTTTTTTTTTTTTTTCAT   SEQ ID NO: 57           CGACTCACTATAGGGAGATTCGCG   AATTAGGGGTTCTGATA           TATTCTTACATCTT               YIR8   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 48   TTTTTTTTTTTTTTTTTTTTTCAT   SEQ ID NO: 58           CGACTCACTATAGGGAGACCAGAT   GTTAGACTGAAAGCAAA           TGCTTACAAAAGAA               YIR11   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 49   TTTTTTTTTTTTTTTTTTTTTATT   SEQ ID NO: 59           CGACTCACTATAGGGAGATTATGG   AAATCTCGGCTAGCCAC           CTACTTTTCATTCC               YIR19   GCATTAGCGGCCGCGAAATTAATA   SEQ ID NO: 50   TTTTTTTTTTTTTTTTTTTTTAGC   SEQ ID NO: 60           CGACTCACTATAGGGAGAGCTAGG   ATAAAACCTCAGCTTTA           ATCTATATGCGAAT                  
 
         [0069]    The following examples demostrate how these Control DNA and Control mRNA are then used as controls in microarray gene expression experiments:  
         [0070]    1. Control DNA included in the array, but for which no complementary artificial mRNA is spiked into the RNA sample, serves as a negative control;  
         [0071]    2. Several different Control DNA samples may be included in an array, and the complementary Control mRNA for each is included at a known concentration, each having a different concentration of mRNA. The signals from the array features corresponding to these Controls or Calibrators may be used to construct a “dose-response curve” or calibration curve to estimate the relationship between signal and amount of mRNA from the sample;  
         [0072]    3. In two-color microarray gene expression studies, it is possible to include different, known, levels of Control mRNA complementary to Control DNA in the labeling reaction for each channel. Comparing the ratio of signals for the two dyes from that gene can be compared to the ratio of concentrations of the two Control mRNA molecules. This can serve as a test of the accuracy of the system for determining gene expression ratios.  
         [0073]    4. Mixtures of several different Control mRNA species can be prepared (spike mixes) at known concentrations and ratios to simplify the experimental protocol while providing a comprehensive set of precision and accuracy information. Table 3 demonstrates one embodiment of this concept. The presence of the dynamic range controls (those included in the labeling reaction at a ratio of 1:1) allows the user to determine the sensitivity of the system. They are also useful for demonstrating the precision of the normalisation method used. For the ratio controls, individual mRNAs are spiked into the two labeling reactions at different concentrations, such that a specific sequence is represented at different levels in each color.  
         [0074]    The above examples illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed.  
         [0075]    Those skilled in the art having the benefit of the teachings of the present invention as set forth above, can effect numerous modifications thereto. These modifications are to be construed as being encompassed within the scope of the present invention as set forth in the appended claims. Table 3. Suggested Control mRNA spike mix composition for two-color gene expression ratio experiments.  
                                                                                             Target   Conc. In mix               Cy3:Cy5   (pg/5μl mix)   Relative                Control   Ratio   Cy3   Cy5   abundance*                       YIR1s   1:1   33 000   33 000     3.3%           YIR2s   1:1   10 000   10 000      1%           YIR3s   1:1    1 000    1 000     0.1%           YTR4s   1:1     330     330    0.033%           YIR5s   1:1     100     100    0.01%           YIR6s   1:1      33      33   0.0033%           YIR7s   1:3    1 000    3 000   NA           YTR8s   3:1    3 000    1 000   NA           YTR11s    1:10    1 000   10 000   NA           YIR19s   10:1    10 000    1 000   NA                                  
 
         [0076]    [0076] 
     
       
       
         1 
         
           
             60  
           
           
             1  
             1000  
             DNA  
             Saccharomyces cerevisiae  
           
            1 

ttcgttggat tgagtaagaa aatgtcgata ctgtgttacg tttgcaagga aaagatttag     60 

ttgcgattag ccattcattc ttgtggaaac ttctttaaaa agggatggcg atggagtact    120 

tatgtccaat tatgaagtca aatttatttt caaaccgtta cacgtagatt attttcctgc    180 

agtggtaagt atctttgaag tgttgaaagt tttttggcac atattttttt gcggatgtgg    240 

gcctgagttt cctgttagaa acaaagatat gcttaaaact aaataacatt ggaaattagg    300 

gcatagtctt caatgttata cttaaacatc acagcaggag attgagatga ttgaaagaat    360 

ggtgcaagaa atgattcatt aacattcttc caagttttgc aatatttgca agtattacta    420 

tcagacttta gttgaagtga ctatgctatt accaaatttc actggagcca gaaaaataaa    480 

gatcacttag agacaaagaa aagtaacatc ttcaacataa gccggagctc aaaagtagga    540 

aatcggataa gaaacttgat tctgttattt caagtgattt tttgctgtat cgcccacgtt    600 

cttgttgtag atgttttgta gatgtgggac cgaaagtaag tgaacagtga agtaaacaga    660 

ataggattct aaaaaagagc ttataaactg ttctttaaaa ttttttcttt cgtgaatgtc    720 

ctcgagctat ctcaaagaaa acgaaatctt cattcaactt aagtgtaggt attatttgct    780 

gtttcatcaa atgcggcacc aaagtggaag tattctagtc agttagtttt tcattgtgag    840 

gaattgatat gtcgttctct gatagaacca cgctaagttg gtgttcaatt ttttgcaatt    900 

cggtagtaat tatgccttgc aacatgtttt attcttttat agtgtgatac cgtcaatatg    960 

atgatttgcg gtggagcatg catgtgctta ctagaagtgc                         1000 

 
           
             2  
             1000  
             DNA  
             Saccharomyces cerevisiae  
           
            2 

gcgaataacc aaaacgagac tactttttac cattacaacc attttctttt tccctatttc     60 

tcactggttg acagaaatca gtgtgctatc atcctaccat atgcgctaaa cttattgtct    120 

ttctcctcct agagatgctg tattccatgc atattctgaa cgatgggttg gtgtttttat    180 

caagcaaggt taatcacatg gcgtggcttg ctccacacat cagtagaaaa cgcataccgc    240 

agcggaatcc ttaaataata agtgatttta ctgttcatca actacaatcg gactctttca    300 

caattaccct tcttgttttc cacatttact gttaaatgaa gggatgtaca gaaggcttag    360 

gaaaacctgt gctgaatact ggatggacac tgcattccca cagtgaaact tttatagata    420 

cactgtcagt tattttcgaa ctttcatcaa gttgctgagt tttagtatcc ctttgcctta    480 

gctatatgtt tgaatgagca aaatatttgc aatgtctcta gctttcttga aatattggtt    540 

tatattgagg gcttggtaag atttcaaatt tcactttgaa atactcagga gaaaaatcat    600 

gctcttttga taatttggtg actaaacata cataaaacag tttaattttg ggtggtaatg    660 

gctgtgtgac tagctataga aagaaaaaaa ttaaaaaaaa aaaaaaaaat caagtagttc    720 

ctgcactgcg acgtccatta tagcattatg aattggtccc tgatttacgc atgcgataaa    780 

ctatttttag cgcagccgca tattatccga gaataacttc cgacataaga aaattcgcag    840 

aaaatagata aaaaactgct cttggcattc ttcacttcct ctattacaca ctgtgtcata    900 

ccacaatcat ctcacagtat gtatttgtat gtttatacat gctataacgt aaaacaatgt    960 

agaatatata tctaaatacc tcacggtttt agtttagtgc                         1000 

 
           
             3  
             969  
             DNA  
             Saccharomyces cerevisiae  
           
            3 

tgtttttgct atattacgtg ggttttttat ttatactgta tataaaagag gactgcaata     60 

gcacaagatt aagatagaat ggcttcaaac agccgccttt tatacatatt ggtaaaagct    120 

cgcgaatcgc accatatccc ttatcctgta atcaaatcga tctaggtgca gatacagatc    180 

aattcataaa aagaaattga agcaccagtt tatcactact acactatctt tttctttttt    240 

tttttttttt gcgaagtttc gccctttgtt caatatcact tgataagttg tgggcttttt    300 

ctgtcactca ttcggcttaa aaagtattcg ttcttttgtg ttttatgaaa agggaacgtg    360 

atataaaaaa acatcctttg gtgtgggaca tgggcttttg tttagagaat ggttatcact    420 

accgccccca cccttgaaag ccacagaaaa tgaaaaagta tgtgaataag gtgtgaactc    480 

tataacattt tggccaaatg ccacagccga tctgcatatt ccaatggaca taatgcaaca    540 

acaattgatg tcacattctc ttacacactt cgattggtcc gtacgtagta ctttttacat    600 

aactgactca ggcgtttcct tcattgaaat gctcatctat tgccaagtac atagaatcca    660 

cagtgcatag gtttatgaga tgcttggaag atgtacgatc gcctgcacta tattagtata    720 

ttttttcagg ctttacaaaa ccagaaagaa attaccgact gtaatactta atttccatga    780 

ttttaatcgt atggtccgtg aggaaagagg aattttaggt aaaaaaaaac ctttgtctat    840 

caaaacataa aagaaaagaa aaaaattaaa ttgaataagt cagcttttta gcatgaccac    900 

agtaataata gtaatacgat atcagcatga gctgctaaac attaagaatt tttgaattgt    960 

gttcgctgg                                                            969 

 
           
             4  
             1037  
             DNA  
             Saccharomyces cerevisiae  
           
            4 

tttcggtagt gagatggcag ttcgaggggt tttttattca aaataataac ttctggcttt     60 

tcgcttttat atagcagaaa aaaaagccgt cgaggcgcgc gcgttcatgc aatggctcag    120 

taacctcggg atagaaaaag ggcaacaatg ttgagctatt ttaggcacag aaactttact    180 

attcgaaaag ggcatccatt tcatttccga ttttctatct agctcactcg ataatcgtaa    240 

tagtactttt ataaaacttt agtgcgggta ctgtgagagt gtgccgtaac tttggtttac    300 

atttaaggtg cgaccagcaa tgtcactact tttacaacaa ccgccatatg gctcgagaat    360 

ttcattatca catggaatgc ctgtgacaaa actgtgtaaa tatctaatag aaattagatg    420 

tagctgtcac aaatatttac acaggaaaga gcctgtccta cgagtatctt acatgaagat    480 

tcatagaacc aatttacttg cgaatgtgaa caacctttca acatcatttc aataccattt    540 

tccctcctta tgtttggtgt cactgtaaag cggatcaaag caaaacatag aggtacggtg    600 

gtgctaagat catgcatgac ctctgggtaa ttactacttc tcccgcttgt tttgagattc    660 

tgtatataaa tatttcaaac aaaaggatag agcgcggatg gcaggcctta tagtaaaagt    720 

tattcgtttt aatcatgtgt cagtatgaga ttctatgaca atagtatgag aagatagggt    780 

gaagtaaaag tatctgtatg actatagagt gcagttatat tacaatatat tgaatagatc    840 

ataatggtat gacgatatta aggaacattt aagttaatga cgtcatggtg gtatagatac    900 

gcaattgagt gtgtttatgt attattgttg aaaagtagaa tatttttatg tttaggtgat    960 

tttgatgata tttttatgta atattgacat aagtgcatat aaattgagtg gttagtatat   1020 

ggtgcaaaag tggtaca                                                  1037 

 
           
             5  
             950  
             DNA  
             Saccharomyces cerevisiae  
           
            5 

ttagtttgga acagcagtgt agataccgtc cttggataga gcgctggaga tagctggtct     60 

caatctggtg gagtaccatg ggacaccagt gatgactcta gtgacttgat cagcgggaat    120 

accagtcaac atagtggtga aatcaccgta gttgaaaaca gcttcagcaa tttcaactgg    180 

gtaagtttca gttggatgag cagcttggaa catatagtat tcagccaaat gagctctgat    240 

atctgagacg tagacaccta attcgaccag gttaactctt tcgtcagagg gagataaagt    300 

agtggtggct ggggcagcag cgacaccagc agcaatagca gcgacaccag caacaattga    360 

agttagtttg accatttttt tcgattgaac ttttgtagat ctttttagtg aagatgtgag    420 

ctcactcgaa tgtaaataac aatgccaaat tgtcggaaag agttaatcaa agctgctcta    480 

tttatatgcc gttttttaat aagcgacgga cgaacagata aattgttgaa tagctatttc    540 

actgctgata tttctcttac ttgggctccc ctatcccata ctcttcacca ctacaaatat    600 

gcagttgccc tttcttcaac aatgcttttt ttatagatct cgtatacgga tccgcgcctt    660 

tgtactacct atatcttatt atgatatata caggagcaca ggaatgttcg gtacagggat    720 

gataccttta aaggaagttt tggcatgcct tgacaacttc aattaatctt tggccaagaa    780 

aatgaaccag aaatcaaatt ttattctgtg ccctctgaac gagggcaata tccaatgttt    840 

gacactaaac ggttgtcagg agaaaaattg aatgtttccc aaatcagaaa cattaaaatc    900 

cctctatatg atcagaggag tcgtacctgt tagggtatga gcgaggaaac               950 

 
           
             6  
             982  
             DNA  
             Saccharomyces cerevisiae  
           
            6 

aatgagttac cgtctgttac ttttgggacg gtttttgcac taagaacaga cgagtttacg     60 

gttatcctca acaagcaagc aagtatttgc taatctagat gccattccga atcattactc    120 

atacgttact attgagagat gttttacaat agatgagaag aatacaatgt ccagagctcc    180 

tggtatgcta gagtgcatat tccaggtctt attcgaatca tatcataccg tccatttcaa    240 

caatggtgaa atgtggtcca catatatcag aaatcttaac atttagtgag gagagccagt    300 

agaaaaatgt gcgcaagcgg aaagaagtca ttcacagaca cgtttaacaa aacaccacca    360 

cagcagcttt gtctcttgat tctgatcagt ttgccatcga agaagcaaaa ttgtggtgtt    420 

atttttttca aacaaaactt ttttggcaac agcagttttc ttctggatat ttgtacttta    480 

tcatccaacc gatgaaagct ggtttcctgt caacctacat ttaaatggcc cgtacttctt    540 

caaaaccgct agataagcaa attaacccaa cttttgagcg tcctaaattc cccttggctc    600 

agaagactcg ttaatatggg aagtttaagt cctaccatat aatcaaattg gaagctttct    660 

gtgttcgaat ggctattcta accgctgggc tattaatcag aggggaagtg aaatgaccga    720 

gacgtattat acgtcatgtt gacatcaaca atttaaggaa aaaaataaaa aaaagcaatg    780 

aaaaagggtt tttttaagtt gaagaccctt ttcaaatata tgttgctttg aattgtatct    840 

accgtctcgt ttcttctgct ttaccgtttt tttttgcctt ctttagatat gtcttttatg    900 

cttgaaaggt ccggctttaa tgcattcatc taaacgtagt attcctattt ttgaactgct    960 

accaatccac catgacttta ct                                             982 

 
           
             7  
             1010  
             DNA  
             Saccharomyces cerevisiae  
           
            7 

tcctagagta gcgattcccc ttcgcgtatt cttacatctt cgaagagaac ttctggtgta     60 

agtataataa atattatagc tctatcgaat ggtgcaatta tttaccaaat tctcaatagg    120 

aatccataat actacatacg atactaatat tctagtattt ttatacttat tatttctttt    180 

ttattacacc agcaatcgtt gcaaattatc ttctgataga atttctgagg gtatcctaaa    240 

cttatgccat tttcttggac tgtaaatcat acttggatgt tgtgcattag tcaataatcg    300 

gttcttgttc caacgattac atgtaaatga agggagaaat aattatggta aatcatgcgg    360 

cggtcctttt ggtgatgcag tatccatagt cactacataa caatcttagt caccttgtat    420 

tgattcacca cataatcctg cagagcccgc tatgtcctta atctgcgcga taactctcct    480 

acccctgaat tttgagagcg ccatagcaaa ccgataaagc tggcacaatt aaaggtatcg    540 

gtgttgtcag aattaggtgc ctcctgcttt tttttttttc ctgctcttat atccgttata    600 

tccgaatgat ttttatcgct tgtttaaaaa atactttccc gatatatata tatagtctcc    660 

ctttaaattt gtttccggta agtttttaac accaataaat gaaaagaaat gactacggtg    720 

atgaatatga gccgcgcatt gaatcaggtt atgtaagtat cagaacccct aattatgatg    780 

tcactcttac ccttcgatgg ctaagcggcg actgggatgc cgggaaaagc tctacaaatc    840 

tactaaaaaa gtcaaatata cagctgtaaa cttctttcct cgtctacatc atggtaacga    900 

ttgttcaatc tttacttcgt gtcttttttt ttttctatgt actttctatt ccaacctatg    960 

tgaagactaa aattcacctt agtaaacgta aagacaatga cgataggtgc              1010 

 
           
             8  
             951  
             DNA  
             Saccharomyces cerevisiae  
           
            8 

tagttggagg ttggtgagta ccagattgct tacaaaagaa tagcgagcca acatttgctc     60 

tgcctcaggc ctcttggtgc tgcttgaaga ctcatcttat atggcttttg tatgtcatga    120 

tttgttcttg tacattatgt gttgatatta aacaaattga tttttttttt tttgcgatag    180 

caagcagata atgaaagaga caaggacttg gaacatccga taagactgcg ccgatatcga    240 

tcttacagtc cttcccttgt gtcatgactt tcggaaaagc atcctcgtcg actggtagtt    300 

tgctgtctgt cacgtgctga agggtctgat acattttttt aaagataaga gacggggttt    360 

acccttcgga ggactaagcg agatctccaa gtaaagatct cgcttatcaa gaaagcagcc    420 

aagtgtggaa cgtccttttt tttggtttca aaaagatatt caacagttta cactgcagct    480 

ttaattgcct caaaaggata tcatgaggtg atctagggtc agaagggaaa gattacagca    540 

tcttgagttg aatcacatct gcaaaaggtg gtattattga cgttgctctt ccttaatgga    600 

aactcatggg gtttggaaag gaggtgcggt aatctatttt tttcgaacac aaaacctaac    660 

cttgaaaaga aactgtccaa tttcattgaa cttacctcag aacgggccgg agtctttgct    720 

ttcagtctaa catggtctaa tttcttcgaa aagcttcatt taattgttag actgtggttt    780 

tacaaggaaa aaaccagtgc tatactgaag cgatacccag aactaattac cttgtgtgac    840 

gattcggctc agcgaaacgg acatggtaaa attgggaatt tgaaagcagg cagcagcctt    900 

gtacagcgac atgacgatag gtttagaatc cccatcacgt acgagttgaa g             951 

 
           
             9  
             952  
             DNA  
             Saccharomyces cerevisiae  
           
            9 

ccattcatat catttagtgc ttatggctac ttttcattcc tcaattattg taaattgacc     60 

atcttaatta tatttctgat attgagtagg tggacttcat tagtattttt acaaatatta    120 

tcaccttctt atgtaggatt agcattacat accctctaat taaaaaaagt taacattaat    180 

tacattttaa aaaaaattgt aatagtatga tagtaggacc tgacagccat ttgaataagg    240 

tttcgagtgc tttaacgttc cactgatttt atgtagttca tatgggggtt agtctggttt    300 

gaggaggaga atttcaggga agcagtggcc gttgaatctc cctgtagggc gctgattatt    360 

tttatcctaa taatccaaaa atgacaatgt caataaagaa aacttaccga gttctgtgaa    420 

tttctcccta aaaaattact aattatacct gggcgagttt tgaactcttt ggcaaataaa    480 

cttggggtaa acctttcgat tataaagacg ttactgctca aaaatgtgta gaagcataag    540 

gagatattct ctcgtatgtt taattggagt tggctttttt ggactctgaa gtttgagtat    600 

gggaggggaa gtaatcgaga ttagattccc tgatgttcac atatggggat aaagaatgct    660 

ttttgggata tgattgtttc tttccgtcgt tacggttgta ggtgcaacga attgcgtaag    720 

ggtggctagc cgagatttaa tgacgacgca aaagggaata actgtgacag gaagatgaat    780 

tcacaaagtt tataaaaaga aagggcgatg cactgctaca tggttgaaca aggcactaca    840 

taattcacag cttgtagctt gtaaataaaa agagcattca cgcgatatac gattttcaat    900 

gatcactcta agaggaacgg cgaaaaatag aattcaatat gaactggaat gg            952 

 
           
             10  
             967  
             DNA  
             Saccharomyces cerevisiae  
           
            10 

gatttaatac agtacctttc ttcgctagga tctatatgcg aatatatcac atatgtaaat     60 

tataagctca tcgcaaaacc aaaaaaaaaa aaattttcaa taatttttca ctaatcttca    120 

aaaacaaatg gggtaacccg tacaagagtt attaaaaccc aaaatgacaa aatcgcgaca    180 

attcaatcct acttaattag caataacata ctagcggtag agctactatc acatgttgaa    240 

ccttgaatgc tcaattcatt gtactcaata ctgctatcaa aagaaaaaaa atgtattaat    300 

tatattcttg tcaaaatcaa ttttacacta taagaggaaa atgttcttca gtcctagtaa    360 

cattagtttt ctccctttgc tagagacttt acataatatc ctagaaggta aaattcgata    420 

atacagcagt aaagtcgtat attggtagca atccttggtg acgctgactt tttttttttg    480 

taattttatt gtttagttca tgataaaaaa cttcaaatca cttttaatct ggtagacaga    540 

gaaaacaaat cgaaacgaaa atagagaact acgaataaaa aaatataagt ggagaagatc    600 

gtcactacgc attaaacaat attgatcgct caatgccagt actgcgcgta aaagtttagt    660 

aacttaacga tttaggcaca atttgagaaa aatttcgccc tgcagtaagt atgttattca    720 

gtacgatata aagctgaggt tttatgctgg caacgttcag attttttagg ttatcagcaa    780 

tgttaaaata ttaaatagga tacttttatt gtttgagacc accctcaatg ccagatatgt    840 

taaacgcttt tttctggagt gaggtatcat agaaaaaggc tcgagtacat caagcactta    900 

aaggttcaac actctactgt tacttcttta agctaagcta ttcatacata atagtccatc    960 

aaagtgg                                                              967 

 
           
             11  
             795  
             DNA  
             Saccharomyces cerevisiae  
           
            11 

aatgtcgata ctgtgttacg tttgcaagga aaagatttag ttgcgattag ccattcattc     60 

ttgtggaaac ttctttaaaa agggatggcg atggagtact tatgtccaat tatgaagtca    120 

aatttatttt caaaccgtta cacgtagatt attttcctgc agtggtaagt atctttgaag    180 

tgttgaaagt tttttggcac atattttttt gcggatgtgg gcctgagttt cctgttagaa    240 

acaaagatat gcttaaaact aaataacatt ggaaattagg gcatagtctt caatgttata    300 

cttaaacatc acagcaggag attgagatga ttgaaagaat ggtgcaagaa atgattcatt    360 

aacattcttc caagttttgc aatatttgca agtattacta tcagacttta gttgaagtga    420 

ctatgctatt accaaatttc actggagcca gaaaaataaa gatcacttag agacaaagaa    480 

aagtaacatc ttcaacataa gccggagctc aaaagtagga aatcggataa gaaacttgat    540 

tctgttattt caagtgattt tttgctgtat cgcccacgtt cttgttgtag atgttttgta    600 

gatgtgggac cgaaagtaag tgaacagtga agtaaacaga ataggattct aaaaaagagc    660 

ttataaactg ttctttaaaa ttttttcttt cgtgaatgtc ctcgagctat ctcaaagaaa    720 

acgaaatctt cattcaactt aagtgtaggt attatttgct gtttcatcaa atgcggcacc    780 

aaagtggaag tattc                                                     795 

 
           
             12  
             762  
             DNA  
             Saccharomyces cerevisiae  
           
            12 

tttctttttc cctatttctc actggttgac agaaatcagt gtgctatcat cctaccatat     60 

gcgctaaact tattgtcttt ctcctcctag agatgctgta ttccatgcat attctgaacg    120 

atgggttggt gtttttatca agcaaggtta atcacatggc gtggcttgct ccacacatca    180 

gtagaaaacg cataccgcag cggaatcctt aaataataag tgattttact gttcatcaac    240 

tacaatcgga ctctttcaca attacccttc ttgttttcca catttactgt taaatgaagg    300 

gatgtacaga aggcttagga aaacctgtgc tgaatactgg atggacactg cattcccaca    360 

gtgaaacttt tatagataca ctgtcagtta ttttcgaact ttcatcaagt tgctgagttt    420 

tagtatccct ttgccttagc tatatgtttg aatgagcaaa atatttgcaa tgtctctagc    480 

tttcttgaaa tattggttta tattgagggc ttggtaagat ttcaaatttc actttgaaat    540 

actcaggaga aaaatcatgc tcttttgata atttggtgac taaacataca taaaacagtt    600 

taattttggg tggtaatggc tgtgtgacta gctatagaaa gaaaaaaatt aaaaaaaaaa    660 

aaaaaaatca agtagttcct gcactgcgac gtccattata gcattatgaa ttggtccctg    720 

atttacgcat gcgataaact atttttagcg cagccgcata tt                       762 

 
           
             13  
             726  
             DNA  
             Saccharomyces cerevisiae  
           
            13 

actgtatata aaagaggact gcaatagcac aagattaaga tagaatggct tcaaacagcc     60 

gccttttata catattggta aaagctcgcg aatcgcacca tatcccttat cctgtaatca    120 

aatcgatcta ggtgcagata cagatcaatt cataaaaaga aattgaagca ccagtttatc    180 

actactacac tatctttttc tttttttttt ttttttgcga agtttcgccc tttgttcaat    240 

atcacttgat aagttgtggg ctttttctgt cactcattcg gcttaaaaag tattcgttct    300 

tttgtgtttt atgaaaaggg aacgtgatat aaaaaaacat cctttggtgt gggacatggg    360 

cttttgttta gagaatggtt atcactaccg cccccaccct tgaaagccac agaaaatgaa    420 

aaagtatgtg aataaggtgt gaactctata acattttggc caaatgccac agccgatctg    480 

catattccaa tggacataat gcaacaacaa ttgatgtcac attctcttac acacttcgat    540 

tggtccgtac gtagtacttt ttacataact gactcaggcg tttccttcat tgaaatgctc    600 

atctattgcc aagtacatag aatccacagt gcataggttt atgagatgct tggaagatgt    660 

acgatcgcct gcactatatt agtatatttt ttcaggcttt acaaaaccag aaagaaatta    720 

ccgact                                                               726 

 
           
             14  
             849  
             DNA  
             Saccharomyces cerevisiae  
           
            14 

ataataactt ctggcttttc gcttttatat agcagaaaaa aaagccgtcg aggcgcgcgc     60 

gttcatgcaa tggctcagta acctcgggat agaaaaaggg caacaatgtt gagctatttt    120 

aggcacagaa actttactat tcgaaaaggg catccatttc atttccgatt ttctatctag    180 

ctcactcgat aatcgtaata gtacttttat aaaactttag tgcgggtact gtgagagtgt    240 

gccgtaactt tggtttacat ttaaggtgcg accagcaatg tcactacttt tacaacaacc    300 

gccatatggc tcgagaattt cattatcaca tggaatgcct gtgacaaaac tgtgtaaata    360 

tctaatagaa attagatgta gctgtcacaa atatttacac aggaaagagc ctgtcctacg    420 

agtatcttac atgaagattc atagaaccaa tttacttgcg aatgtgaaca acctttcaac    480 

atcatttcaa taccattttc cctccttatg tttggtgtca ctgtaaagcg gatcaaagca    540 

aaacatagag gtacggtggt gctaagatca tgcatgacct ctgggtaatt actacttctc    600 

ccgcttgttt tgagattctg tatataaata tttcaaacaa aaggatagag cgcggatggc    660 

aggccttata gtaaaagtta ttcgttttaa tcatgtgtca gtatgagatt ctatgacaat    720 

agtatgagaa gatagggtga agtaaaagta tctgtatgac tatagagtgc agttatatta    780 

caatatattg aatagatcat aatggtatga cgatattaag gaacatttaa gttaatgacg    840 

tcatggtgg                                                            849 

 
           
             15  
             712  
             DNA  
             Saccharomyces cerevisiae  
           
            15 

agataccgtc cttggataga gcgctggaga tagctggtct caatctggtg gagtaccatg     60 

ggacaccagt gatgactcta gtgacttgat cagcgggaat accagtcaac atagtggtga    120 

aatcaccgta gttgaaaaca gcttcagcaa tttcaactgg gtaagtttca gttggatgag    180 

cagcttggaa catatagtat tcagccaaat gagctctgat atctgagacg tagacaccta    240 

attcgaccag gttaactctt tcgtcagagg gagataaagt agtggtggct ggggcagcag    300 

cgacaccagc agcaatagca gcgacaccag caacaattga agttagtttg accatttttt    360 

tcgattgaac ttttgtagat ctttttagtg aagatgtgag ctcactcgaa tgtaaataac    420 

aatgccaaat tgtcggaaag agttaatcaa agctgctcta tttatatgcc gttttttaat    480 

aagcgacgga cgaacagata aattgttgaa tagctatttc actgctgata tttctcttac    540 

ttgggctccc ctatcccata ctcttcacca ctacaaatat gcagttgccc tttcttcaac    600 

aatgcttttt ttatagatct cgtatacgga tccgcgcctt tgtactacct atatcttatt    660 

atgatatata caggagcaca ggaatgttcg gtacagggat gataccttta aa            712 

 
           
             16  
             893  
             DNA  
             Saccharomyces cerevisiae  
           
            16 

ttgggacggt ttttgcacta agaacagacg agtttacggt tatcctcaac aagcaagcaa     60 

gtatttgcta atctagatgc cattccgaat cattactcat acgttactat tgagagatgt    120 

tttacaatag atgagaagaa tacaatgtcc agagctcctg gtatgctaga gtgcatattc    180 

caggtcttat tcgaatcata tcataccgtc catttcaaca atggtgaaat gtggtccaca    240 

tatatcagaa atcttaacat ttagtgagga gagccagtag aaaaatgtgc gcaagcggaa    300 

agaagtcatt cacagacacg tttaacaaaa caccaccaca gcagctttgt ctcttgattc    360 

tgatcagttt gccatcgaag aagcaaaatt gtggtgttat ttttttcaaa caaaactttt    420 

ttggcaacag cagttttctt ctggatattt gtactttatc atccaaccga tgaaagctgg    480 

tttcctgtca acctacattt aaatggcccg tacttcttca aaaccgctag ataagcaaat    540 

taacccaact tttgagcgtc ctaaattccc cttggctcag aagactcgtt aatatgggaa    600 

gtttaagtcc taccatataa tcaaattgga agctttctgt gttcgaatgg ctattctaac    660 

cgctgggcta ttaatcagag gggaagtgaa atgaccgaga cgtattatac gtcatgttga    720 

catcaacaat ttaaggaaaa aaataaaaaa aagcaatgaa aaagggtttt tttaagttga    780 

agaccctttt caaatatatg ttgctttgaa ttgtatctac cgtctcgttt cttctgcttt    840 

accgtttttt tttgccttct ttagatatgt cttttatgct tgaaaggtcc ggc           893 

 
           
             17  
             757  
             DNA  
             Saccharomyces cerevisiae  
           
            17 

ttcgcgtatt cttacatctt cgaagagaac ttctggtgta agtataataa atattatagc     60 

tctatcgaat ggtgcaatta tttaccaaat tctcaatagg aatccataat actacatacg    120 

atactaatat tctagtattt ttatacttat tatttctttt ttattacacc agcaatcgtt    180 

gcaaattatc ttctgataga atttctgagg gtatcctaaa cttatgccat tttcttggac    240 

tgtaaatcat acttggatgt tgtgcattag tcaataatcg gttcttgttc caacgattac    300 

atgtaaatga agggagaaat aattatggta aatcatgcgg cggtcctttt ggtgatgcag    360 

tatccatagt cactacataa caatcttagt caccttgtat tgattcacca cataatcctg    420 

cagagcccgc tatgtcctta atctgcgcga taactctcct acccctgaat tttgagagcg    480 

ccatagcaaa ccgataaagc tggcacaatt aaaggtatcg gtgttgtcag aattaggtgc    540 

ctcctgcttt tttttttttc ctgctcttat atccgttata tccgaatgat ttttatcgct    600 

tgtttaaaaa atactttccc gatatatata tatagtctcc ctttaaattt gtttccggta    660 

agtttttaac accaataaat gaaaagaaat gactacggtg atgaatatga gccgcgcatt    720 

gaatcaggtt atgtaagtat cagaacccct aattatg                             757 

 
           
             18  
             714  
             DNA  
             Saccharomyces cerevisiae  
           
            18 

ccagattgct tacaaaagaa tagcgagcca acatttgctc tgcctcaggc ctcttggtgc     60 

tgcttgaaga ctcatcttat atggcttttg tatgtcatga tttgttcttg tacattatgt    120 

gttgatatta aacaaattga tttttttttt tttgcgatag caagcagata atgaaagaga    180 

caaggacttg gaacatccga taagactgcg ccgatatcga tcttacagtc cttcccttgt    240 

gtcatgactt tcggaaaagc atcctcgtcg actggtagtt tgctgtctgt cacgtgctga    300 

agggtctgat acattttttt aaagataaga gacggggttt acccttcgga ggactaagcg    360 

agatctccaa gtaaagatct cgcttatcaa gaaagcagcc aagtgtggaa cgtccttttt    420 

tttggtttca aaaagatatt caacagttta cactgcagct ttaattgcct caaaaggata    480 

tcatgaggtg atctagggtc agaagggaaa gattacagca tcttgagttg aatcacatct    540 

gcaaaaggtg gtattattga cgttgctctt ccttaatgga aactcatggg gtttggaaag    600 

gaggtgcggt aatctatttt tttcgaacac aaaacctaac cttgaaaaga aactgtccaa    660 

tttcattgaa cttacctcag aacgggccgg agtctttgct ttcagtctaa catg          714 

 
           
             19  
             721  
             DNA  
             Saccharomyces cerevisiae  
           
            19 

ttatggctac ttttcattcc tcaattattg taaattgacc atcttaatta tatttctgat     60 

attgagtagg tggacttcat tagtattttt acaaatatta tcaccttctt atgtaggatt    120 

agcattacat accctctaat taaaaaaagt taacattaat tacattttaa aaaaaattgt    180 

aatagtatga tagtaggacc tgacagccat ttgaataagg tttcgagtgc tttaacgttc    240 

cactgatttt atgtagttca tatgggggtt agtctggttt gaggaggaga atttcaggga    300 

agcagtggcc gttgaatctc cctgtagggc gctgattatt tttatcctaa taatccaaaa    360 

atgacaatgt caataaagaa aacttaccga gttctgtgaa tttctcccta aaaaattact    420 

aattatacct gggcgagttt tgaactcttt ggcaaataaa cttggggtaa acctttcgat    480 

tataaagacg ttactgctca aaaatgtgta gaagcataag gagatattct ctcgtatgtt    540 

taattggagt tggctttttt ggactctgaa gtttgagtat gggaggggaa gtaatcgaga    600 

ttagattccc tgatgttcac atatggggat aaagaatgct ttttgggata tgattgtttc    660 

tttccgtcgt tacggttgta ggtgcaacga attgcgtaag ggtggctagc cgagatttaa    720 

t                                                                    721 

 
           
             20  
             725  
             DNA  
             Saccharomyces cerevisiae  
           
            20 

gctaggatct atatgcgaat atatcacata tgtaaattat aagctcatcg caaaaccaaa     60 

aaaaaaaaaa ttttcaataa tttttcacta atcttcaaaa acaaatgggg taacccgtac    120 

aagagttatt aaaacccaaa atgacaaaat cgcgacaatt caatcctact taattagcaa    180 

taacatacta gcggtagagc tactatcaca tgttgaacct tgaatgctca attcattgta    240 

ctcaatactg ctatcaaaag aaaaaaaatg tattaattat attcttgtca aaatcaattt    300 

tacactataa gaggaaaatg ttcttcagtc ctagtaacat tagttttctc cctttgctag    360 

agactttaca taatatccta gaaggtaaaa ttcgataata cagcagtaaa gtcgtatatt    420 

ggtagcaatc cttggtgacg ctgacttttt ttttttgtaa ttttattgtt tagttcatga    480 

taaaaaactt caaatcactt ttaatctggt agacagagaa aacaaatcga aacgaaaata    540 

gagaactacg aataaaaaaa tataagtgga gaagatcgtc actacgcatt aaacaatatt    600 

gatcgctcaa tgccagtact gcgcgtaaaa gtttagtaac ttaacgattt aggcacaatt    660 

tgagaaaaat ttcgccctgc agtaagtatg ttattcagta cgatataaag ctgaggtttt    720 

atgct                                                                725 

 
           
             21  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            21 

ttcgttggat tgagtaagaa                                                 20 

 
           
             22  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            22 

gcgaataacc aaaacgagac                                                 20 

 
           
             23  
             22  
             DNA  
             Saccharomyces cerevisiae  
           
            23 

tgtttttgct atattacgtg gg                                              22 

 
           
             24  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            24 

tttcggtagt gagatggcag                                                 20 

 
           
             25  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            25 

ttagtttgga acagcagtgt                                                 20 

 
           
             26  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            26 

aatgagttac cgtctgttac                                                 20 

 
           
             27  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            27 

tcctagagta gcgattcccc                                                 20 

 
           
             28  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            28 

tagttggagg ttggtgagta                                                 20 

 
           
             29  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            29 

ccattcatat catttagtgc                                                 20 

 
           
             30  
             25  
             DNA  
             Saccharomyces cerevisiae  
           
            30 

gatttaatac agtacctttc ttcgc                                           25 

 
           
             31  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            31 

gcacttctag taagcacatg                                                 20 

 
           
             32  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            32 

gcactaaact aaaaccgtga                                                 20 

 
           
             33  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            33 

ccagcgaaca caattcaaaa                                                 20 

 
           
             34  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            34 

tgtaccactt ttgcaccata                                                 20 

 
           
             35  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            35 

gtttcctcgc tcatacccta                                                 20 

 
           
             36  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            36 

agtaaagtca tggtggattg                                                 20 

 
           
             37  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            37 

gcacctatcg tcattgtctt                                                 20 

 
           
             38  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            38 

cttcaactcg tacgtgatgg                                                 20 

 
           
             39  
             20  
             DNA  
             Saccharomyces cerevisiae  
           
            39 

ccattccagt tcatattgaa                                                 20 

 
           
             40  
             25  
             DNA  
             Saccharomyces cerevisiae  
           
            40 

ccactttgat ggactattat gtatg                                           25 

 
           
             41  
             62  
             DNA  
             Saccharomyces cerevisiae  
           
            41 

gcattagcgg ccgcgaaatt aatacgactc actataggga gaaatgtcga tactgtgtta     60 

cg                                                                    62 

 
           
             42  
             67  
             DNA  
             Saccharomyces cerevisiae  
           
            42 

gcattagcgg ccgcgaaatt aatacgactc actataggga gatttctttt tccctatttc     60 

tcactgg                                                               67 

 
           
             43  
             64  
             DNA  
             Saccharomyces cerevisiae  
           
            43 

gcattagcgg ccgcgaaatt aatacgactc actataggga gaactgtata taaaagagga     60 

ctgc                                                                  64 

 
           
             44  
             64  
             DNA  
             Saccharomyces cerevisiae  
           
            44 

gcattagcgg ccgcgaaatt aatacgactc actataggga gaataataac ttctggcttt     60 

tcgc                                                                  64 

 
           
             45  
             62  
             DNA  
             Saccharomyces cerevisiae  
           
            45 

gcattagcgg ccgcgaaatt aatacgactc actataggga gaagataccg tccttggata     60 

ga                                                                    62 

 
           
             46  
             66  
             DNA  
             Saccharomyces cerevisiae  
           
            46 

gcattagcgg ccgcgaaatt aatacgactc actataggga gattgggacg gtttttgcac     60 

taagaa                                                                66 

 
           
             47  
             62  
             DNA  
             Saccharomyces cerevisiae  
           
            47 

gcattagcgg ccgcgaaatt aatacgactc actataggga gattcgcgta ttcttacatc     60 

tt                                                                    62 

 
           
             48  
             62  
             DNA  
             Saccharomyces cerevisiae  
           
            48 

gcattagcgg ccgcgaaatt aatacgactc actataggga gaccagattg cttacaaaag     60 

aa                                                                    62 

 
           
             49  
             62  
             DNA  
             Saccharomyces cerevisiae  
           
            49 

gcattagcgg ccgcgaaatt aatacgactc actataggga gattatggct acttttcatt     60 

cc                                                                    62 

 
           
             50  
             62  
             DNA  
             Saccharomyces cerevisiae  
           
            50 

gcattagcgg ccgcgaaatt aatacgactc actataggga gagctaggat ctatatgcga     60 

at                                                                    62 

 
           
             51  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            51 

tttttttttt tttttttttt tgaatacttc cactttggtg c                         41 

 
           
             52  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            52 

tttttttttt tttttttttt taatatgcgg ctgcgctaaa a                         41 

 
           
             53  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            53 

tttttttttt tttttttttt tagtcggtaa tttctttctg g                         41 

 
           
             54  
             46  
             DNA  
             Saccharomyces cerevisiae  
           
            54 

tttttttttt tttttttttt tccaccatga cgtcattaac ttaaat                    46 

 
           
             55  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            55 

tttttttttt tttttttttt ttttaaaggt atcatccctg t                         41 

 
           
             56  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            56 

tttttttttt tttttttttt tgccggacct ttcaagcata a                         41 

 
           
             57  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            57 

tttttttttt tttttttttt tcataattag gggttctgat a                         41 

 
           
             58  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            58 

tttttttttt tttttttttt tcatgttaga ctgaaagcaa a                         41 

 
           
             59  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            59 

tttttttttt tttttttttt tattaaatct cggctagcca c                         41 

 
           
             60  
             41  
             DNA  
             Saccharomyces cerevisiae  
           
            60 

tttttttttt tttttttttt tagcataaaa cctcagcttt a                         41