Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd.

Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis. A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII. Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development. Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region. The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data. Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus. KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis. Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome. Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation. Further analysis of the klp-3 gene in C. elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development.

Prediction of the coding sequences of unidentified human genes. XXI. The complete sequences of 60 new cDNA clones from brain which code for large proteins.

As an extension of a sequencing project of human cDNA clones which encode large proteins of unidentified genes, we herein present the entire sequences of 60 cDNA clones for the genes named KIAA1879-KIAA1938. The cDNA clones were isolated from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain and amygdala, and their protein-coding sequences were predicted. Thirty-seven cDNA clones entirely sequenced in this study were selected as cDNAs which have coding potentiality by in vitro transcription/translation experiments, and the remaining 23 cDNA clones were chosen by computer-assisted analysis of terminal sequences of cDNAs. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.5 kb and 2.2 kb (733 amino acid residues), respectively. Sequence analyses against the public databases enabled us to annotate the functions of the predicted products of the 25 genes; 84% of these predicted gene products (21 gene products) were classified into proteins related to cell signaling/communication, nucleic acid management, and cell structure/motility. In addition to the sequence information about these 60 genes, their expression profiles were also studied in some human tissues including brain regions by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Prediction of the coding sequences of unidentified human genes. XXII. The complete sequences of 50 new cDNA clones which code for large proteins.

As an extension of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 50 cDNA clones, named KIAA1939-KIAA1988. cDNA clones to be entirely sequenced were selected by two approaches based on their protein-coding potentialities prior to sequencing: 10 cDNA clones were chosen because their encoding proteins had a molecular mass larger than 50 kDa in an in vitro transcription/translation system; the remaining 40 cDNA clones were selected because their putative proteins-as determined by analysis of the genomic sequences flanked by both the terminal sequences of cDNAs using the GENSCAN gene prediction program-were larger than 400 amino acid residues. According to the sequence data, the average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.6 kb and 1.9 kb (643 amino acid residues), respectively. From the results of homology and motif searches against the public databases, the functional categories of the 31 predicted gene products could be assigned; 25 of these predicted gene products (81%) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility. The expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, the products of which were quantified by enzyme-linked immunosorbent assay.

Prediction of the coding sequences of unidentified human genes. XVI. The complete sequences of 150 new cDNA clones from brain which code for large proteins in vitro.

We have carried out a human cDNA sequencing project to accumulate information regarding the coding sequences of unidentified human genes. As an extension of the preceding reports, we herein present the entire sequences of 150 cDNA clones of unknown human genes, named KIAA1294 to KIAA1443, 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 reached 4.8 kb and 2.7 kb (910 amino acid residues), respectively. From sequence similarities and protein motifs, 73 predicted gene products were functionally annotated and 97% of them were classified into the following four functional categories: cell signaling/communication, nucleic acid management, cell structure/motility and protein management. Additionally, the chromosomal loci of the genes were assigned by using human-rodent hybrid panels for those genes whose mapping data were not available in the public databases. The expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

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.

Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

To accumulate information on the coding sequences of unidentified genes, we have carried out a sequencing project of human cDNA clones which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unidentified human genes, named KIAA1776 and KIAA1780-KIAA1878, from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain, hippocampus and amygdala. Most of the cDNA clones to be entirely sequenced were selected as cDNAs which were shown to have coding potentiality by in vitro transcription/translation experiments, and some clones were chosen by using computer-assisted analysis of terminal sequences of cDNAs. Three of these clones (fibrillin2/KIAA1776, MEGF10/KIAA1780 and MEGF11/KIAA1781) were isolated as genes encoding proteins with multiple EGF-like domains by motif-trap screening. The average sizes of the inserts and corresponding open reading frames of eDNA clones analyzed here reached 4.7 kb and 2.4 kb (785 amino acid residues), respectively. From the results of homology and motif searches against the public databases, the functional categories of the predicted gene products of 54 genes were determined; 93% of these predicted gene products (50 gene products) were classified as proteins related to cell signaling/communication, nucleic acid management, or cell structure/motility. To collect additional information on these genes, their expression profiles were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Identification of novel transcribed sequences on human chromosome 22 by expressed sequence tag mapping.

To identify sequences on the human genome that are actually transcribed, we mapped expressed sequence tags (ESTs) of long cDNAs ranging from 4 kb to 7 kb along a 33.4-Mb sequence of human chromosome 22, the first human chromosome entirely sequenced. By the EST mapping of 30,683 long cDNAs in silico, 603 cDNA sequences were found to locate on chromosome 22 and classified into 169 clusters. Comparison of the genomic loci of these cDNA sequences with 679 genes already annotated on chromosome 22q revealed that 46 clusters represented newly identified transcribed sequences. To further characterize these sequences, we sequenced 12 cDNAs in their entirety out of 46 clusters. Of these 12 cDNAs, 6 were predicted to include a protein-coding region while the remaining 6 were unlikely to encode proteins. Interestingly, 3 out of the 12 cDNAs had the nucleotide sequences of the opposite strands of the genes previously annotated, which suggested that these genomic regions were transcribed bi-directionally. In addition to these newly identified 12 cDNAs, another 12 cDNAs were entirely sequenced since these cDNAs were likely to contain new information about the predicted protein-coding sequences previously annotated. In the cases of KIAA1670 and KIAA1672, these single cDNA sequences covered two separately annotated transcribed regions. For example, the sequence of a clone for KIAA1670 indicated that the CHKL and CPT1B genes were co-transcribed as a contiguous transcript without making both the protein-coding regions fused. In conclusion, the mapping of ESTs derived from long cDNAs followed by sequencing of the entire cDNAs provided indispensable information for the precise annotation of genes on the genome together with ESTs derived from short cDNAs.

Prediction of the coding sequences of unidentified human genes. XVIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In our series of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 100 cDNA clones of unidentified genes, named KIAA1544 to KIAA1643, 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 reached 4.6 kb and 2.8 kb (930 amino acid residues), respectively. By computer-assisted database search of the deduced amino acid sequences, 48 predicted gene products were classified into the five functional categories of proteins relating to cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. Homology search against the databases for proteins deduced from yeast, nematode and fly full genome sequences revealed only one gene (KIAA1630) was entirely conserved among human and these three organisms in the 100 genes reported here. Additionally, their chromosomal loci were determined by using human-rodent hybrid panels unless they were already assigned in the public databases. Furthermore, the expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Characterization of cDNA clones selected by the GeneMark analysis from size-fractionated cDNA libraries from human brain.

We have conducted a sequencing project of human cDNAs which encode large proteins in brain. For selection of cDNA clones to be sequenced in this project, cDNA clones have been experimentally examined by in vitro transcription/translation prior to sequencing. In this study, we tested an alternative approach for picking up cDNA clones having a high probability of carrying protein coding region. This approach exploited 5'-end single-pass sequence data and the GeneMark program for assessing protein-coding potential, and allowed us to select 74 clones out of 14,804 redundant cDNA clones. The complete sequence data of these 74 clones revealed that 45% of them encoded proteins consisting of more than 500 amino acid residues while all the clones thus selected carried possible protein coding sequences as expected. The results indicated that the GeneMark analysis of 5'-end sequences of cDNAs offered us a simple and effective means to select cDNA clones with protein-coding potential although the sizes of the encoded proteins could not be predicted.

Prediction of the coding sequences of unidentified human genes. XV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In order to obtain information on the coding sequences of unidentified human genes, we newly determined the sequences of 100 cDNA clones of unknown human genes, which we named KIAA1193 to KIAA1292, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The results of our particular strategy to select cDNA clones which have the potentiality of coding for large proteins in vitro revealed that the average sizes of the inserts and the corresponding open reading frames reached 5.2 kb and 2.8 kb (933 amino acid residues), respectively. By the computational analysis of the predicted amino acid sequences against the OWL and Pfam databases, 58 predicted gene products were classified into the following five functional categories: cell signaling/communication, cell structure/motility, nucleic acid management, protein management and metabolism. It was also found that 30 gene products had homologues in the public databases which were similar in sequence throughout almost their entire regions to the newly identified genes. The chromosomal loci of the genes were assigned by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of the genes were studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Prediction of the coding sequences of unidentified human genes. XIX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

As an extension of our human cDNA project for accumulating sequence information on the coding sequences of unidentified genes, we here present the entire sequences of 100 cDNA clones of unidentified genes, named KIAA1673-KIAA1772, from three sets of size-fractionated cDNA libraries derived from human adult whole brain, hippocampus, and fetal whole brain. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.9 kb and 2.7 kb (corresponding to 895 amino acid residues), respectively. By computer-assisted analysis of the deduced amino acid sequences, 44 predicted gene products were classified into five functional categories of proteins relating to cell signaling/communication, nucleic acid management, cell structure/motility, protein management, and metabolism. Furthermore, the expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse-transcription-coupled polymerase chain reaction, the products of which were quantified by enzyme-linked immunosorbent assay.

Characterization of long cDNA clones from human adult spleen.

As an extension of our analysis of long cDNAs, we here report the characterization of cDNA clones from human adult spleen. From 2000 cDNA clones randomly sampled from a size-fractionated human spleen cDNA library (average size 4.5 kb), 97 clones were selected for sequencing according to their ability to code for protein at the 5'-end sequences and the novelty of their end sequences. The sequence data of these clones demonstrated that 87 out of 97 cDNA clones were derived from independent human genes. The average sizes of the inserts and corresponding open reading frames of these 87 cDNAs reached 4.5 kb and 1.4 kb (corresponding to 468 amino acid residues), respectively. In addition to these sequence analyses in silico, the expression profiles of the genes were also studied in ten human adult tissues by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay. The results indicated that spleen could be used as an additional source of human long cDNAs to complement the list of human genes.

Prediction of the coding sequences of unidentified human genes. XIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

As a part of our cDNA project for deducing the coding sequence of unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0919 to KIAA1018. The sequencing of these clones revealed that the average sizes of the inserts and corresponding open reading frames were 4.9 kb and 2.6 kb (882 amino acid residues), respectively. A computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes contained sequences similar to known genes, 53% of which (46 genes) were categorized as proteins relating to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Prediction of the coding sequences of unidentified human genes. XI. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In our series of projects for accumulating sequence information on the coding sequences of unidentified human genes, we have newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0711 to KIAA0810. These cDNA clones were selected according to their coding potentials of large proteins (50 kDa and more) in vitro. The average sizes of the inserts and corresponding open reading frames were 4.3 kb and 2.6 kb (869 amino acid residues), respectively. Sequence analyses against the public databases indicated that the predicted coding sequences of 78 genes were similar to those of known genes, 64% of which (50 genes) were categorized as proteins functionally related to cell signaling/communication, cell structure/motility and nucleic acid management. As additional information concerning genes characterized in this study, the chromosomal locations of the clones were determined by using human-rodent hybrid panels and the expression profiles among 10 human tissues were examined by reverse transcription-coupled polymerase chain reaction which was substantially improved by enzyme-linked immunosorbent assay.

Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In this paper, we report the sequences of 100 cDNA clones newly determined from a set of size-fractionated human brain cDNA libraries and predict the coding sequences of the corresponding genes, named KIAA0819 to KIAA0918. These cDNA clones were selected on the basis of their coding potentials of large proteins (50 kDa and more) by using in vitro transcription/translation assays. The sequence data showed that the average sizes of the inserts and corresponding open reading frames are 4.4 kb and 2.5 kb (831 amino acid residues), respectively. Homology and motif/domain searches against the public databases indicated that the predicted coding sequences of 83 genes were similar to those of known genes, 59% of which (49 genes) were categorized as coding for proteins functionally related to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations and the expression profiles of all the genes were also examined. For 54 clones including brain-specific ones, the mRNA levels were further examined among 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substantia nigra, subthalamic nucleus, and thalamus), spinal cord, and fetal brain.

Prediction of the coding sequences of unidentified human genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

To extend our cDNA project for accumulating basic information on unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human adult and fetal brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA1019 to KIAA1118. The sequencing of these clones revealed that the average size of the inserts and corresponding open reading frames were 5.0 kb and 2.6 kb (880 amino acid residues), respectively. Database search of the predicted amino acid sequences classified 58 predicted gene products into the five functional categories, such as cell signaling/communication, cell structure/motility, nucleic acid management, protein management and cell division. It was also found that, for 34 gene products, homologues were detected in the databases, which were similar in sequence through almost the entire regions. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Sequence homologies among mitochondrial DNA-coded URF2, URF4 and URF5.

Amino acid sequences coded for by 13 different genes of mammalian mitochondrial DNA (mtDNA) including 8 unassigned open reading frames (URFs) were compared in pairs. It was found that significant homologies exist among the amino acid sequences of the three URFs (URF2, URF4 and URF5) with a probability of occurrence of less than 10(-5). This result strongly suggests that the 3 URFs evolved from a single ancestral gene by a series of gene duplications. A phylogenetic tree based on the alignment of the URF sequences from mammals, an insect, a fungus and protozoa revealed a very remote divergence of the 3 URFs, going back to a time before separations of animal/protozoa and animal/fungus.

Expression profile of mRNAs from human pancreatic islet tumors.

In order to understand the tIssue specificity of the endocrine pancreas, it is important to clarify the expression profile of mRNAs in various states of the tIssue. A total of approximately 9000 non-redundant expressed genes from human pancreatic islets and insulinoma have so far been determined as expressed sequence tags (ESTs) and deposited in public databases. In the present study towards the identification of a complete set of genes expressed in human pancreatic islets, we have determined 3'-ESTs of 21267 clones randomly selected from a cDNA library of human pancreatic islet tumors. Clustering analysis generated 6157 non-redundant sequences comprising 2323 groups and 3834 singletons. Nucleotide and peptide database searches show that 3103 of them represent known human sequences or homologs of genes identified in other species and 58 are new members of structurally related families. The sequences were classified on the basis of the putative protein functions encoded, and were assigned to the respective chromosome by database analysis. The sequences were also compared with the EST databases (dbEST and EPConDB) including ESTs from normal pancreatic islet, insulinoma, and fetal pancreas. Since 3384 genes were newly found to be expressed in human pancreatic islets and 587 of them were unique to the islets, this study has considerably expanded the catalog of genes expressed in the endocrine pancreas. The larger collection of pancreatic islet-related ESTs should provide a better genome source for molecular studies of differentiation, tIssue-specific functions, and tumorigenesis of the endocrine pancreas as well as for genetic studies of diabetes mellitus.

Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs.

The terminal sequences of long cDNAs from human brains were subjected to an improved method of motif-trap screening. This process resulted in the identification of three novel genes that encode proteins with 27, 27, and six cadherin domains that we denoted as KIAA1773, KIAA1774 and KIAA1775, respectively. Sequence analysis indicated that the products of these genes were non-classical cadherins. KIAA1773 was found to be a mammalian homologue of the Drosophila dachsous gene but the remaining two genes did not have any likely homologues in public databases. Assessment of their expression in rat tissues indicated that these genes are expressed in highly distinct and tissue-specific patterns. Notably, KIAA1775 is expressed almost exclusively in the olfactory bulb in the rat brain. In situ hybridization further showed that KIAA1775 is strongly expressed by the mitral and tufted cells in the main and accessory olfactory bulbs, suggesting that KIAA1775 may be important in the formation and maintenance of neuronal networks, particularly those in the olfactory bulb. This study clearly shows the importance and usefulness of our cDNA project in search for genes encoding large proteins, as this project has allowed us to identify several novel non-classical cadherin genes that have thus far not been detected by conventional methods.

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence.

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.