there is heterogeneity in human mrna start sites ( 1 ) ; 4060% of human genes are transcribed alternatively ( 2 ) , and 49% of multi - exon transcripts are accompanied by alternative splicing of the initial exon ( 3 ) .
transcription start sites might be altered in a variety of different cell types or affected by environmental conditions , such as methylation .
although an extensive collection of transcription start sites for a large number of human genes is available ( 4 ) , the frequencies of individual start sites are unclear .
there is a need for high - throughput technology to monitor the statistics of start site occurrences for a comprehensive understanding of the start site gene expression mechanism .
microarrays are unsuitable for this purpose because of their inability to detect novel start sites .
the serial analysis of gene expression ( sage ) method ( 5 ) has demonstrated its effectiveness at cataloging large quantities of expressed genes in cells or tissues from a variety of physiological , developmental and pathological states ( 611 ) .
the original sage generates short ( 10 + 4 bp ) nucleotide sequences , called tags , derived from the 3 ends of transcripts ; however , typical tags are too short to be uniquely identified with their corresponding genes .
this shortcoming was resolved using the longsage method ( 12 ) , a high - throughput means of profiling 21 bp tags , which are sufficiently long to be unambiguously identified with genes in most cases . however ,
existing sage methods are designed to monitor the 3 ends of transcripts , and the challenge was to extend the sage method so that it would be capable of capturing the novel 5 ends of transcripts and efficiently quantifying individual 5 end occurrences .
recently , hashimoto et al . ( 13 ) developed such a system for human cell lines , while shiraki et al .
the 5sage database stores a collection of data accumulated by using the hashimoto et al .
hashimoto et al . ( 13 ) have described the details of the method , and we present a brief summary here .
the method first profiles 21 bp tags by using a novel way of combining the oligo - capping method ( 15 ) , a modification of the oligo - capping method ( 16 ) and the longsage method ( 6 ) .
subsequently , these 5sage tags are aligned with the human genome to locate their positions , to begin a search for neighboring mrna start sites .
we found that 19  893 of 25  684 5sage tags in a human cell line , hek293 , were matched to the human genome .
of the 15  448 tags that hit a locus within the human genome , 85.896.1% of the 5sage tags were assigned to within 500 to + 200 nt of the mrna start sites in the refseq , unigene ( 17 ) and dbtss ( 4 ) databases , while 1774 tags were within the introns of known genes or uncharacterized regions , indicating possible novel start sites .
in the 5sage database server , users can browse transcription start sites and frequencies of individual genes by querying on the accession numbers of sequences in refseq , cluster identifiers in unigene or symbol names , such as hdac . to retrieve all the genes in the server , the word 
the user can impose additional conditions on the number of distinct start sites and the total frequency of 5sage tags monitored for individual genes of interest .
for instance , one can look for genes by monitoring five or more distinct start sites with 10 or more 5sage tag occurrences . in response to the query
clicking on each gene displays a window for browsing the transcription start sites ( figure 1 ) .
the  start site view initially displays the narrow , 150 bp region surrounding the transcription start site of the representation gene in refseq or unigene , while the  global view presents entire structures of individual transcripts that are helpful in comprehending alternatively spliced transcripts at a glance .
users can change the zoom magnification of each view independently by setting the ruler unit to an alternative base pair length .
the orange vertical lines depict transcription start points ; the depth of each orange line below the axis shows the frequency of the transcription start site .
the thick , green , horizontal lines are cpg islands , which are regions of 50 or more bp consisting of at least 50% g or c nucleotides .
for instance , figure 1 shows the transcription start sites of neurofilament 3 ( nef3 ) .
note the large number of start points detected for nef3 ; most are novel , and some start at the second or third exon .
transcription start site view also lists 5sage tags , their distances from the representative start site , their frequencies and their nearest expressed sequence tags .
we have performed long sage on the 3 ends of mrna in hek293 cells to validate the accuracy of our 5sage results ( 13 ) .
the total frequency of 3sage tags associated with the representative gene is also displayed with the 3sage tag sequences and distances from the start site .
3sage tags are sampled independently at random , the pearson correlation coefficient between the frequencies of 5sage and 3sage tags indicates moderate similarity ( 13 ) .
as on october 2004 , the 5sage database presents transcription start sites collected from human cell lines , hek293 and ramos .
start site information in other human cell lines is being collected for the analysis of start point variation in different tissues , and will be made available at the same website .
this work was supported in part by a grant - in - aid for scientific research on priority areas ( c ) from the ministry of education , culture , sports , science and technology of japan .
 to comprehensively identify transcription start sites and the frequencies of individual mrnas in human cell libraries , a method of 5 end serial analysis of gene expression ( sage ) was developed recently , which makes it possible to collect a large amount of start site information , and subsequently , we have established a related database server called 5sage . 
 this database displays the observed frequencies of individual 5 end sage tags and previously unknown transcription start sites in the promoter regions , introns and intergenic regions of known genes . 
 5sage will be useful for analyzing promoter regions and start site variation in different tissues , and is freely available at http://5sage.gi.k.u - tokyo.ac.jp/. 