Dymron herbicide residues in paddy soil and channel water.
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Agriculture↗
Biomedical subjects
M Hirosawa
Publications and source records attributed to M Hirosawa.
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.
Amino Acid Sequence↗
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.
Brain Chemistry↗
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.
Alternative Splicing↗
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.
Amino Acid Sequence↗
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.
Adult↗
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.
Adult↗
HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.
HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge
Animals↗
Detection of spurious interruptions of protein-coding regions in cloned cDNA sequences by GeneMark analysis.
cDNA is an artificial copy of mRNA and, therefore, no cDNA can be completely free from suspicion of cloning errors. Because overlooking these cloning errors results in serious misinterpretation of cDNA sequences, development of an alerting system targeting spurious sequences in cloned cDNAs is an urgent requirement for massive cDNA sequence analysis. We describe here the application of a modified GeneMark program, originally designed for prokaryotic gene finding, for detection of artifacts in cDNA clones. This program serves to provide a warning when any spurious split of protein-coding regions is detected through statistical analysis of cDNA sequences based on Markov models. In this study, 817 cDNA sequences deposited in public databases by us were subjected to analysis using this alerting system to assess its sensitivity and specificity. The results indicated that any spurious split of protein-coding regions in cloned cDNAs could be sensitively detected and systematically revised by means of this system after the experimental validation of the alerts. Furthermore, this study offered us, for the first time, statistical data regarding the rates and types of errors causing protein-coding splits in cloned cDNAs obtained by conventional cloning methods.
Alternative Splicing↗
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.
5' Untranslated Regions↗
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.
Adult↗
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.
Adult↗
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.
Animals↗
Allogeneic hematopoietic stem cell transplantation for patients with hemophagocytic syndrome (HPS) in Japan.
Seventeen cases (age at onset, 1 month to 18 years; M/F, 9/8) of hemophagocytic syndrome which received allogeneic hematopoietic stem cell transplantation (SCT) in Japan during the period 1988-1998 are reported. The patients consisted of six familial inheritance-proven erythrophagocytic lymphohistiocytosis (FEL), five familial inheritance-unknown and infective agents-unknown HLH (of which two were highly likely to have been FEL with characteristic CNS signs), and six aggressive Epstein-Barr virus (EBV)-related HLH (of which two were natural killer cell-type large granular leukemia/lymphoma-associated hemophagocytic syndrome, EBV-NK-LGLL-HPS). All cases were treated intensively with immuno-chemotherapy, or with chemotherapy before SCT. As sources of SCT, 12 cases received bone marrow cells (sibling six, father one, URD five), two cord blood, two purified CD34-positive cells, and one PBSC. SCTs were successful in all 17 cases, apart from one receiving CD34-positive SCT. Following SCT, four patients relapsed and five died with a median follow-up of 23 months. Among the relapsed cases, the two EBV-NK-LGLL-HPS previously published as successfully transplanted were included. Among the fatal cases, three patients died from relapsed active disease and the remaining two from fatal post-SCT EBV-positive T cell lymphoma and extensive chronic GVHD, respectively. As of the end of September 1998, 10 patients are alive without disease for 3.5 months to 147 months, while two post-SCT patients are still having therapy for residual/recurrent disease. The Kaplan-Meier analysis showed a 2-year event-free survival after SCT as 54.0+/-13.0%.
Adolescent↗
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.
Amino Acid Sequence↗
CyanoBase, a www database containing the complete nucleotide sequence of the genome of Synechocystis sp. strain PCC6803.
CyanoBase (http://www.kazusa.or.jp/cyano/) is a database containing genomic information on the cyanobacterium Synechocystis sp. strain PCC6803. It furnishes an annotation to each of the 3168 protein genes deduced from the entire nucleotide sequence of this genome. Information on the genome can be directly accessed through three different menus: a clickable physical map of the genome, a gene classification list, and a keyword search menu, all of which are accessible from the main page of the database. The entry page for a gene annotation contains the following information: the location of the gene on the genome, the nucleotide and deduced amino acid sequence of the gene, the result of a similarity search, and the classification of the deduced gene product according to its function. This page has reverse-links to the local physical map and gene classification list so that relevant genes can be searched in terms of their location on the genome and their function. In addition, the main page of CyanoBase provides engines for similarity searches between a query sequence and the entire genome sequence and for keyword searches, in addition to numerous links to pages containing related information.
Bacterial Proteins↗
Prediction of translation initiation sites on the genome of Synechocystis sp. strain PCC6803 by Hidden Markov model.
We developed a computer program, GeneHackerTL, which predicts the most probable translation initiation site for a given nucleotide sequence. The program requires that information be extracted from the nucleotide sequence data surrounding the translation initiation sites according to the framework of the Hidden Markov Model. Since the translation initiation sites of 72 highly abundant proteins have already been assigned on the genome of Synechocystis sp. strain PCC6803 by amino-terminal analysis, we extracted necessary information for GeneHackerTL from the nucleotide sequence data. The prediction rate of the GeneHackerTL for these proteins was estimated to be 86.1%. We then used GeneHackerTL for prediction of the translation initiation sites of 24 other proteins, of which the initiation sites were not assigned experimentally, because of the lack of a potential initiation codon at the amino-terminal position. For 20 out of the 24 proteins, the initiation sites were predicted in the upstream of their amino-terminal positions. According to this assignment, the processed regions represent a typical feature of signal peptides. We could also predict multiple translation initiation sites for a particular gene for which at least two initiation sites were experimentally detected. This program would be effective for the prediction of translation initiation sites of other proteins, not only in this species but also in other prokaryotes as well.
Bacterial Proteins↗
Analysis of sequence patterns surrounding the translation initiation sites on Cyanobacterium genome using the hidden Markov model.
Sequence patterns surrounding the translation initiation sites of Cyanobacterium were precisely analyzed by the hidden Markov model (HMM) based on the actual translation initiation sites. In a previous study, 72 actual protein coding regions and their translation initiation sites on the genome of Synechocystis sp. strain PCC6803 were determined by Sazuka et al. using protein two-dimensional electrophoresis and microsequening. In this work, we extracted the sequence patterns surrounding translation initiation sites as HMM using the computer program YEBIS. The constructed HMM could recognize all but one translation initiation site. The HMM contains an AG-rich region (5.7 bp on average), as the Shine-Dalgarno sequence exclusively contains purines, upstream of the translation initiation site (-9.7 position on average) and a CT rich region (4.2 bp on average) just upstream from the translation initiation site. In addition, we found that the second amino acid (-4.5,6) could be classified into two types, one of which had C as their second codon while another of which has a nucleotide distribution relatively similar to the distribution among amino acids in the 72 proteins. This fact corresponds well to our earlier finding that when the second nucleotide of the second amino acid of a translated protein was C, an initial methionine was processed and that otherwise the methionine was intact with high frequency.
Base Sequence↗