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 ). 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 (REBASE; 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; 8(1-2): 17-29).

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) against 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 SIM4 (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.

Features of the predicted protein sequence

This section describes the features of the predicted protein sequence.

(1) Revision of the cloned DNA sequence before prediction of the protein sequence

When necessary, the cDNA sequences of the isolated clones were revised by direct RT-PCR/sequencing experiments in order to eliminate artifact(s) signaled by the GeneMark-RC analysis (see "Features of the cloned DNA sequence"). Therefore, some of the predicted protein sequences are not derived from the cloned cDNA sequence but from the revised cDNA sequence. Hence, it should be noted that some cDNA clones do not carry the identical sequences to those deposited to GenBank/EMBL/DDBJ databases because they are the experimentally revised ones. To avoid any confusion, this section states whether or not the predicted protein sequence is obtained by conceptual translation of the revised cDNA sequence. The revised sequences by the RT-PCR/sequencing experiments can be accessible by clicking. As far as possible, the nature of the revision is also described (deletion, insertion, nonsense mutation, or removal of retianed intronic sequence).

(2) FASTA homology searches against the nr database

Top 5 entries given the expectation value smaller than 0.001 in nr database are shown. "nr" stands for the non-redundant amino acid sequence database that has been constructed by 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.

(3) Analysis of Motifs, Domains, and Membrane-spanning regions

The predicted protein sequences were examined for motifs present in the InterPro database. Because weakly defined sequence motifs appear too many times in general 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.

Motifs/Domains in the InterPro database were searched for by InterProScan. (Zdobnov, EM, and Apweiler, R. InterProScan--an integration platform for the signature-recognition methods in InterPro" Bioinformatics 2001; 17:847-848).

Membrane-spanning region were predicted by SOSUI (Hirokawa, T., Boon-Chieng, S., Mitaku, S. "SOSUI: classification and secondary structure prediction system for membrane proteins" Bioinformatics 1998; 14:378-379).

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