Description of the Gene/Protein Characteristic Table
Features of the cloned DNA sequence
This section describes features of the nucleotide sequences of
cDNA clones actually characterized. Although the actual clones
contained an oligo(dT)-NotI adapter primer sequence and a SalI adapter
sequence at their 3'- and 5'-extremities, respectively, the nucleotide
sequences of these adapters are not shown here. This section is
intended to provide clone users with detailed information of clones,
which is not available from the public databases.
(1) Physical map
- The physical maps were constructed on the basis of the
sequence data of the cDNA clones. The horizontal scale
represents the cDNA length in kb. The ORFs and untranslated regions
are shown by solid and open boxes, respectively. The positions of
the first ATG codons are indicated by solid and open triangles
to indicate respectively those that lie within and outside
the confines of Kozak's rule. RepeatMasker, which is a program
that screens DNA sequences for interspersed repeats known to
exist in mammalian genomes, was applied to detect repeat sequences
in cDNA sequences (Smit, A. F. A. and Green, P., RepeatMasker at
). Alu sequences and other
repetitive sequences detected in this way are displayed
by dotted and hatched boxes, respectively.
(2) Restriction map
- Commercially available restriction enzymes
Roberts, R. J., Macelis, D.
"REBASE - restriction enzymes and methylases"
Nucleic Acids Res. 1998; 26: 338-350).
) are sorted according
to the number of the restriction sites present in the cDNA insert.
(3) Prediction of the protein coding region (GeneMark analysis)
- The graphic outputs of the GeneMark-RC analysis are displayed. Vertical
lines given in the graphs indicate the positions of termination codons.
If you would like to know more about the GeneMark-RC analysis,
please read the paper by Hirosawa et al.
(Hirosawa, M., Isono, K., Hayes, W., Borodovsky, M.
"Gene identification and classification in the Synechocystis genomic
sequence by recursive gene mark analysis" DNA Seq. 1997;
The GeneMark analysis gives the following warnings:
(a) Warning for N-terminal truncation of the coding region;
(b) Warning for spurious interruption of the coding region.
(4) Prediction of the genomic structure of the cDNA
- The cDNA sequence was subjected to BLAST search
(Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z.,
Miller, W., and Lipman, D.J.
Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs." 1997; Nucleic Acids Res 25: 3389-3402)
the human genome draft sequences in NCBI.
When a genomic fragment was found to be considerably similar to the cDNA
sequence (E-value = 0.0 and sequence identity is 90% or greater), the genomic
structure of the cDNA was assigned by
(Florea, L., Hartzell, G., Zhang, Z., Rubin, G.M., and Miller, W. "
A computer program for aligning a cDNA sequence with a genomic DNA sequence
1998; Genome Res. 8: 967-974)
on the genomic fragment.
(Burge, C. and Karlin, S. 1997; "
Prediction of complete gene structures
in human genomic DNA." J. Mol. Biol. 268: 78-94)
was also applied to detect the plausible gene structure on the genomic
fragment. The result of comparison of the gene structures deduced from
the cDNA and that predicted by GENSCAN were displayed in graphics.
Features of the predicted protein sequence
This section describes the features of the predicted protein sequence.
(1) FASTA homology searches against the nr database and Kazusa
human cDNA database
- Top 5 entries given the expectation value smaller than 0.001 in nr
database and Kazusa human cDNA databases (HUGE & NEDO) are shown. nr is a
non-redundant amino acid sequence database that was constructed in NCBI.
The numbers on the left and right
sides of a black line in the graphical overview indicate the lengths
(in amino acid residues) of the non-homologous N-terminal and
C-terminal portions flanking the homologous region (indicated by the
black line), respectively. The FASTA output and the multiple alignment
of these entries can be obtained by clicking.
(2) Analysis of Motifs, Profiles, and Membrane-spanning regions
The predicted protein sequences were examined for motifs present
in the PROSITE database.
Because weakly defined sequence motifs appear too many times in
the HUGE database and are, thus, unlikely to be informative,
the following motifs were excluded from the
analysis: amidation site; N-glycosylation site; cAMP- and
cGMP-dependent protein kinase phosphorylation site; casein kinase II
phosphorylation site; N-myristoylation site; protein kinase C
phosphorylation site; and tyrosine kinase phosphorylation site.
Profile entries in the PROSITE database were also searched for by using
pftools, a program
developed by Philipp Bucher in the Swiss Institute for Experimental
Domains in the Pfam database
(Sonnhammer, E. L. L., Eddy, S. R., Birney, E., Bateman, A.,
and Durbin, R.
"Pfam: multiple sequence alignments and HMM-profiles of protein domains"
Nucleic Acids Res 1998; 26, 320-322)
were searched for by using hmmer 2.1.
Membrane-spanning region were predicted by
(Hirokawa, T., Boon-Chieng, S., Mitaku, S.
"SOSUI: classification and
secondary structure prediction system for membrane proteins"
Bioinformatics 1998; 14:378-379).
RT-PCR ELISA is a combination of RT-PCR described above and the
following quantification of the products by ELISA.
RT-PCR ELISA was carried out essentially as described in the
instruction of a PCR ELISA (DIG labeling) kit (cat. no. 1 636 120;
Boehringer Mannheim Biochemica).
External control reactions using the authentic plasmid allowed mRNA
levels to be expressed as equivalent amounts of the authentic plasmid
DNA (fg) per ng of poly(A)+ RNA
(the starting material of RT-PCR).
For at-a-glance screening, the mRNA levels are displayed by color
codes using the digit-color conversion panel shown in the figure
(unit: fg of equivalent plasmid DNA / ng of poly(A)
Because this expression pattern was obtained from a single run of
RT-PCR ELISA, the expression profile has a chance to include
significant run-to-run variations. Accordingly, the expression
profiles should be used only for the screening of genes on the basis
of tissue specificity.
If more accurate quantitative expression profiles are required, more
statistically reliable approaches should be employed
(e.g., multiplication of RT-PCR-ELISA measurements, DNA chip analysis,
RNA blot analysis, etc.).