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 attB2-dT adapter primer sequence and an attB1 adapter sequence at their 3'- and 5'-extremities, respectively (Ohara O, Nagase T, Mitsui G, Kohga H, Kikuno R, Hiraoka S, Takahashi Y, Kitajima S, Saga Y, Koseki H. " Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method. " DNA Res. 2002; 9: 47-57) , 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 longest ORF predicted by GeneMark 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 ). SINE 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 mouse genome draft sequences in EBI. 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.

GENSCAN (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.

(5) Comparison of structure with the corresponding human KIAA cDNA

Each mKIAA cDNA in the ROUGE database is a mouse homologue of human KIAA cDNA. Three types of alignment were prepared:
i) DNA sequence based, ii) AA sequence based and iii) Physical map.

i) DNA sequence-based alignment was made by comparing the DNA sequences between mKIAA and KIAA cDNAs by GAP program in the GCG package. The longest coding region predicted by GeneMark was determined as a CDS for each of mouse and human KIAA cDNAs in this alignment. However, when the CDS positions were not identical between the mouse and human KIAA cDNAs on the aligned DNA sequences, the mouse cDNA sequence was translated based on the human CDS information. Thus the mouse amino acid sequence here may not be identical to the protein sequence used for the protein sequence analysis below (Features of the predicted protein sequence).

ii) To construct AA sequence-based alignment, we translated every CDS of mKIAA and KIAA cDNA sequences according to the GeneMark analysis at first. All of the predicted CDS whose length were longer than 150 bp were translated. Then the amino acid sequences were compared and aligned between mouse and human. When the sequence identity exceeded 50%, we aligned the cDNA sequences of the corresponding regions between mouse and human based on the amino acid sequence alignment.

iii) In the comparable physical map, the corresponding CDS between mouse and human KIAA DNA which was obtained by ii) was connected by thine lines. The longest and other CDSs were colored in dark and light blue, respectively. The conservation of polyA signal obtained from i) was also indicated. When the positions of polyA signal were conserved between mouse and human KIAA cDNAs, they were colored in red, otherwise, they were colored in green. If another polyA signal was found upsteream from -35-bp region in either human or mouse KIAA cDNA, and the position was conserved between the two species, it was colored in orange as an indication of possible alternative polyA signal.

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 ROUGE database

Top 5 entries given the expectation value smaller than 0.001 in nr database and ROUGE 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.

(2) 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 the ROUGE 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.

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).

How to obtain mKIAA clone(s)
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