Prediction of the Coding Sequences of Unidentified Human Genes. XVII. The Complete Sequences of 100 New cDNA Clones from Brain Which Code for Large Proteins in vitro

To provide information regarding the coding sequences of unidentified human genes, we have conducted a sequencing project of human cDNAs which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unknown human genes, named KIAA1444 to KIAA1543, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.4 kb and 2.6 kb (856 amino acid residues), respectively. Database searches of the predicted amino acid sequences classified 53 predicted gene products into the following five functional categories: cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. It was also revealed that homologues for 32 KIAA gene products were detected in the databases, which were similar in sequence through almost their entire regions. Additionally, the chromosomal loci of the genes were determined by using human-rodent hybrid panels unless their chromosomal loci were already assigned in the public databases. The expression levels of the genes were monitored in spinal cord, fetal brain and fetal liver, as well as in 10 human tissues and 8 brain regions, by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

In December of 1999, the complete DNA sequence cov-Currently, we have focused our sequencing efforts on ering almost the entire region of the euchromatic part the unidentified genes encoding large proteins in human of human chromosome 22 was reported. 1 Moreover, in brain since these gene products appear to play important the near future, a working draft sequence, covering at roles in the central nervous system. 3 ' 4 As an extension least 90% of the human genome, is expected to become of the preceding studies, we herein report the predicted available as a result of international collaboration of hu-coding sequence of 100 new cDNA clones which have the man genome sequencing. However, it is difficult to con-potential to code for large proteins in vitro. In addition clusively identify genes only from the genomic sequence to the specific features of the newly predicted protein seby any current computer programs because of compli-quences annotated by the database search, the expression cated and extensive splicing events during transcription profiles and the chromosomal locations of these 100 new and our limited knowledge of the sequence signals which genes are also reported. The growing catalog of genes define the transcriptional start and termination sites, encoding large proteins should provide a wealth of in-Therefore, it is still important to collect full-length cD-formation concerning the primary structures of proteins NAs and analyze their sequences, not only for utiliza-present in the human brain that are difficult to identify tion for functional studies but also for interpretation of by conventional methods of gene discovery, the gene structure, even alter the human genome sequence becomes available. Considering this situation, x Sequence Analysis and Prediction of Proteinwe have been making efforts to accumulate information Coding Regions in cDNA Clones on the coding sequences of unidentified human genes. 2 1451  1452  1453  1454  1455  1456  1457  1458  1459  1460  1461  1462  1463  1464  1465  1466  1467  1468  1469  1470  1471  1472  1473  1474  1475  1476  1477  1478  1479  1480  1481  1482  1483  1484  1485  I486  1487  1488  1489  1490  1491  1492  1493   Accession  number"   AB040877  AB040878  ABO40879  AB040880  AB040881  AB040882  AB040883  AB040884  AB040885  AB040886  AB04O887  AB040888  AB040889  AB040890  AB040891  AB040892  ABO4O893  AB040894  AB040895  AB040896  AB04O897  AB040898  AB040899  AB040900  AB040901  AB04O9O2  AB040903  AB040904  AB040905  AB040906  AB040907  AB04O908  AB040909  AB040910  AB040911  AB040912  AB040913  AB040914  AB040915  AB040916  AB040917  AB040918  AB040919  AB040920  AB040921  AB040922  AB040923  AB040924  AB040925   a) Accession numbers of DDBJ, EMBL and GenBank databases, b) Values excluding poly (A) sequences, c) Chromosome numbers identified by using GeneBridge 4 radiation hydrid panel unless specified. The actual primer sequences and the PCR conditions used for the radiation hybrid mapping are accessible through the World Wide Web at http://www.kazusa.or.jp/huge. The chromosomal locations highlighted by asterisks were fetched from the UniGene database. The chromosomal locations highlighted by sharp were referred from the GeneBank database because the sequences of the cDNA clones could be found in the genomic sequences whose chromosome numbers were assigned. The actual primer sequences and the PCR conditions used for the radiation hybrid mapping are also accessible through the World Wide Web site referred above, d) Chromosome number determined by using CCR human-rodent hybrid panel, e) cDNA and ORF lengths were revised by direct analysis of the RT-PCR products, f) Nucleotide sequences were determined after subcloning of the internal Not I-digested fragment. Therefore, cDNA length of these genes represented those of internal Not I-digested fragment, g) cDNA clones were selected by analysis of 5'-end single-pass sequences by the computer-assisted method.  Definition semaphorin G -mouse brain-enriched guanylate kinase-associated protein 2 mRNA, complete cds. -rat DOC4 mRNA, complete cds. -mouse membrane-associated phosphatidylinositol transfer protein mRNA. complete cds. -mouse ephnn type-A receptor 8 precursor (EC 2.7 1.112) -mouse RanBPM, complete cds -human tyrosinc phosphalase TDM mRNA, complete cds -rat serineythreonine kinase MARK1 -rat GTPase activating protein rotund -Drciiopliila melanogasler semaphorin Via mRNA, complete cds -mouse neuroligin 3 mRNA, complete cds. -rat APICAL-Iike protein -human probable calcium-transporting ATPase 3 (EC 3.6.1.38) -Succharomvces cerevisiae Bristol N2 GTPase-activating protein mRNA, partial cds. -Caenorhtibdins tlegatis phospholipase C PLC210 mRNA, completi cds. -C degans transport-associated protein Tap2A -Atlartic salmon. deltex mRNA, complete cds. -human hyperpolarization-activated cation channel HAC3 -mouse probable G protein-coupled receptor form "-cells precursor -mouse protein phosphatase PP2A, 55 kD regulatory subunit. alpha isoform -rat giant protein p619 -human zinc finger protein 85 -human zinc finger protein 184 -human zinc finger protein 51 -mouse probable RNA helicase -Arabidopsis thaliaim SNF2 protein homolog -human zinc finger protein 132. -human putative ATP-dependent RNA helicase -C elegans CTD-binding SR-like protein rA9 mRNA, :omplete cds. -rat kinesin-like protein KIF1B -mouse osteomodulin, complete cds. -human actin binding protein MAYVEN mRNA. complete cds. -human ptasmacytoma-associated neuronal glycoprotein PANG • mouse outer arm dynein beta heavy chain gene, complete cds. -Paramecium leiraurelia collagen alpha l(XIV) chain precursor -ch cken myosin heavy chain, cardiac muscle beta isoform -human exrensin -Volvox carteri hematopoieric-specific IL-2 deubiquitinatirg enzyme gene, complete cds -mouse dipeptidyl peptidase IV like protein -human oxysterol-binding protein, complete cds. -mouse oxysterol-binding protein, complete cds -mouse a) Homology search was performed by Smith-Waterman algorithm, using BioView Toolkit and GeneMatcher (revision 3.3, Paracel Inc. USA) against OWL database (release 31.4). The homologous protein with the highest score was listed, when it satisfied the following conditions, i) the protein was functionally annotated, ii) the aligned region exceeded 200 amino acid residues, and iii) percent identity in the algined region was 30% or greater, b) Function was classified based on the annotation of the entry of the homologous protein in the database, c) The values mean the ratio of the length of aligned region to the original length of the query sequence, in percentage. the size-fractionated human fetal brain cDNA libraries Nos. 4 and 6 (insert sizes ranging from 4 to 7 kb) previously constructed. 3 ' 4 In this report, 20 cDNA clones (KIAA1497-KIAA1508, KIAA1528-KIAA1531, KIAA1538 and KIAA1541-KIAA1543) were selected from the adult brain libraries and the remaining 80 cDNA clones were obtained from the fetal brain cDNA libraries. According to our selection system for unidentified genes, the clones with unidentified sequences at both ends were chosen by single-pass sequencing and homology search against the GenBank database (release 116.0) excluding expressed sequence tags and genomic sequences. 4 For selection of cDNA clones to be entirely sequenced, we used an in vitro transcription/translation system to examine their protein-coding capacities and/or a computerbased method based on GeneMark analysis to predict protein-coding potentialities on their 5'-end sequences in this study. 4 ' 5 Eighty-eight cDNA clones were selected by the in vitro expression system, while 12 cDNA clones (KIAA1532-KIAA1543) were chosen by the computerassisted method. Entire sequencing of these clones was performed according to the methods previously described in detail. 4 Thirty clones (KIAA1509-KIAA1538) seemed to carry spurious coding interruption caused by errors of the reverse transcriptase or by retained intron sequences. For these cases, the sequences of the regions causing interruption of open reading frame (ORF) were reexamined by direct sequencing of the major products of reverse transcription-coupled polymerase chain reaction (RT-PCR) to predict authentic protein-coding sequences in brain. 6 As the results of these confirmations, spurious interruptions were found in the following cDNA clones: ORFs in 23 clones (KIAA1509-KIAA1512, KIAA1514-KIAA1517,  KIAA1519-KIAA1521,  KIAA1523,  KIAA1524, KIAA1526-KIAA1528, KIAA1530 and KIAA1532-KIAA1537) were found to carry single or multiple insertions, most of which probably corresponded to retained intronic sequences; ORFs in 10 clones (KIAA1513, KIAA1515, KIAA1520-KIAA1522, KIAA1524, KIAA1525, KIAA1529, KIAA1531 and KIAA1538) were found to carry single or multiple deletions; ORFs in 2 clones (KIAA1523 and KIAA1524) were frame-shifted by a single nucleotide deletion. For those genes, the revised sequences by the RT-PCR experiments, not the actual cloned cDNA sequences, were deposited to GenBank/EMBL/DDBJ  databases and used for prediction of their proteincoding sequences unless otherwise stated. The results of the comparison between the cloned DNA and the revised DNA sequences are available through the World Wide Web site at http://www.kazusa.or.jp/huge. Notably, clones for seven genes (KIAA1444, KIAA1447, KIAA1455, KIAA1471, KIAA1498, KIAA1502 and KIAA1506) seemed to lack regions encoding C-terminal portions due to the presence of a Not I site in their coding regions because cDNAs were digested with Not I before ligation to a vector. In contrast, clones for two genes (KIAA1445 andKIAA1450) were found to lack the 5'portions of the sequences due to the presence of an internal Not I site. For these genes, the nucleotide sequences of only the region between two Not I sites were determined, since their original clones were most likely to harbor intermolecularly ligated two independent cDNAs. 7 After these revisions, the average size of the cDNA sequences reached 4.4 kb and that of the predicted coding region was approximately 856 amino acid residues. Physical maps of the 100 cDNA sequences analyzed are shown in Fig. 1, where the ORFs and the first ATG codons in respective ORFs are indicated by solid boxes and triangles, respectively. Repeat sequences are also shown in Fig. 1. Eleven genes had 5'-untranslated regions longer than 1 kb. We could not completely rule out the possibility that these clones retained a 5'-intron upstream of the predicted protein coding region, since the RNAs used to construct the cDNA libraries contained heterogeneous nuclear RNAs besides the cytoplasmic mRNAs. Chromosomal loci of 54 newly identified genes were determined by using human-rodent hybrid panels, GeneBridge 4 (Research Genetics Inc., USA) 8 or CCR (Coriell Cell Repositories, USA) since their mapping data were not available. The chromosomal locations of the 42 genes, which are highlighted by asterisks in Table 1, were fetched from the UniGene database (http://www.ncbi.nlm.nih.gov/UniGene). The chromosomal locations of the remaining four genes, which are highlighted by sharp (#) in Table 1, were taken from the description of the GenBank database. a) The definition of homologues used here was the proteins found in the databases satisfying the following conditions: i) the length ranged from 80% to 125% of the query sequence; ii) the ratio of the length of aligned region to that of the original sequence of the query was 80% or greater; iii) percent identity was 30% or greater. The method of homology search was the same to that explained in

Functional Classification of Predicted Gene Products
The gene products predicted from the cDNA sequences were classified by homology and/or motif search against the following public databases: protein sequence database, OWL (release 31.4), 9 databases of predicted protein sequences from yeast 10 Table 2, the functions of 53 gene products were classified into five functional categories. Among them, 41 gene products indicated significant sequence similarity to functionally annotated proteins (

KIAA 1444
(fg/ng of poly (A)* RNA) Figure 2. Expression profiles of 100 newly identified genes examined by RT-PCR ELISA. The tissue expression levels of the 100 human genes were analyzed by using the RT-PCR ELISA according to the methods previously described in detail. 14 Gene names are given as KIAA numbers at the left side of each set of color codes. Tissue and brain region names are indicated above the top sets of color codes. A color conversion panel shown at the bottom was used for displaying mRNA levels as color codes. The mRNA levels are expressed in equivalent amounts (fg) of the authentic cDNA plasmids in 1 ng of starting poly(A)+ RNAs. Besides 10 tissues, 9 regions of the adult central nervous system (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substantia nigra, subthalamic nucleus, thalamus, and spinal cord) and fetal brain were included in the expression profiling. As a control, mRNA levels in fetal liver were also examined.
Prediction of Unidentified Human Genes [Vol. 7, of functional motifs/domains, since they did not show sequence similarity to functionally annotated proteins ( Table 2-2). In total, 48 gene products (91% of genes functionally annotated here) were suggested to have functions relating to cell signaling/communication, nucleic acid management or cell structure/motility. To find the genes structurally conserved in others, we tentatively defined "homologues" as genes with at least 30% amino acid identity spanning almost the entire region (more than 80% coverage against the query protein sequence). As shown in Table 3, 32 KIAA gene products were found to have "homologues" in the databases.

Expression Profiles of Predicted Genes
The expression profiles of the genes newly identified in this study are represented in Fig. 2 by color codes. 14 The expression levels of six genes (KIAA1444, KIAA1446, KIAA1472, KIAA1479, KIAA1484 and KIAA1497) were relatively higher in brain than in other tissues. Among them, KIAA1446 and KIAA1497 were homologues of rat brain-enriched guanylate kinase-associated protein 15 and mouse neuronal leucine-rich repeat protein I, 16 respectively. The gene product of KIAA 1472 exhibited similarity in sequence through almost the entire regions to KIAA0374 protein, whose expression was relatively high in brain although it's function was unknown. KIAA 1535 was a human counterpart of mouse hyperpolarization-activated cation channel HAC3, and its expression pattern suggests that this gene might play an important role in development of the human brain since the expression of KIAA 1535 was higher in fetal brain than in adult brain. These expression profiles provide us important information for identifying biologically important genes characterized in this project.