Showing papers in "DNA Research in 1998"

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

Abstract: In this series of projects of sequencing human cDNA clones which correspond to relatively long transcripts, we newly determined the entire sequences of 100 cDNA clones which were screened on the basis of the potentiality of coding for large proteins in vitro. The cDNA libraries used were the fractions with average insert sizes from 5.3 to 7.0 kb of the size-fractionated cDNA libraries from human brain. The randomly sampled clones were single-pass sequenced from both the ends to select clones that are not registered in the public database. Then their protein-coding potentialities were examined by an in vitro transcription/translation system, and the clones that generated proteins larger than 60 kDa were entirely sequenced. Each clone gave a distinct open reading frame (ORF), and the length of the ORF was roughly coincident with the approximate molecular mass of the in vitro product estimated from its mobility on SDS-polyacrylamide gel electrophoresis. The average size of the cDNA clones sequenced was 6.1 kb, and that of the ORFs corresponded to 1200 amino acid residues. By computer-assisted analysis of the sequences with DNA and protein-motif databases (GenBank and PROSITE databases), the functions of at least 73% of the gene products could be anticipated, and 88% of them (the products of 64 clones) were assigned to the functional categories of proteins relating to cell signaling/communication, nucleic acid managing, and cell structure/motility. The expression profiles in a variety of tissues and chromosomal locations of the sequenced clones have been determined. According to the expression spectra, approximately 11 genes appeared to be predominantly expressed in brain. Most of the remaining genes were categorized into one of the following classes: either the expression occurs in a limited number of tissues (31 genes) or the expression occurs ubiquitously in all but a few tissues (47 genes).

Complete Sequence and Gene Organization of the Genome of a Hyper-thermophilic Archaebacterium, Pyrococcus horikoshii OT3

Abstract: The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http://www.nite.go.jp.

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.

Abstract: In this paper, we report the sequences of 100 cDNA clones newly determined from a set of sizefractionated 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.

Complete Nucleotide Sequences of 93-kb and 3.3-kb Plasmids of an Enterohemorrhagic Escherichia coli O157:H7 Derived from Sakai Outbreak

Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived from an outbreak in Sakai city, Japan in 1996, possesses two kinds of plasmids: a 93-kb plasmid termed pO157, found in clinical EHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1, prevalent in EHEC strains isolated in Japan. Complete nucleotide sequences of both plasmids have been determined, and the putative functions of the encoded proteins and the cis-acting DNA sequences have been analyzed. pO157 shares strikingly similar genes and DNA sequences with F-factor and the transmissible drug-resistant plasmid R100 for DNA replication, copy number control, plasmid segregation, conjugative functions and stable maintenance in the host, although it is defective in DNA transfer by conjugation due to the truncation and deletion of the required genes and DNA sequences. In addition, it encodes several proteins implicated in EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and type II secretion system. pOSAK1 possesses a ColE1-like replication system, and the DNA sequence is extremely similar to that of a drug-resistant plasmid, NTP16, derived from Salmonella typhimurium except that it lacks drug resistance transposons.

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

Abstract: 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.

The Dictyostelium Developmental cDNA Project: Generation and Analysis of Expressed Sequence Tags from the First-Finger Stage of Development

Abstract: In an effort to identify and characterize genes expressed during multicellular development in Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, li- brary S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other or- ganisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will pro- vide a useful resource for investigating the genetic networks that regulate multicellular development of this

Structural analysis of Arabidopsis thaliana chromosome 5. V. Sequence features of the regions of 1,381,565 bp covered by twenty one physically assigned P1 and TAC clones.

Abstract: The nucleotide sequences of 21 P1 and TAC clones which have been precisely localized to the fine physical map of the Arabidopsis thaliana chromosome 5, were determined, and their sequence features were analyzed. The total length of the regions sequenced in this study were 1,381,565 bp, bringing the total length of the chromosome 5 sequences determined so far to 6,691,670 bp together with the regions of the 69 clones previously reported. By computer-aided analyses including similarity search against protein and EST databases and gene modeling with computer programs, a total of 337 potential protein-coding genes and/or gene segments were identified on the basis of similarity to the reported gene sequences. An average density of the genes and/or gene segments thus assigned was 1 gene/4,100 bp. Introns were identified in 76.7% of the potential protein genes for which the entire gene structure were predicted, and the average number per gene and the average length of the introns were 3.9 and 176 bp, respectively. These sequence features are essentially identical to those in the previously reported sequences. The numbers of the Arabidopsis ESTs matched to each of the predicted genes have been counted to monitor the transcription level. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http:@www.kazusa.or.jp@arabi

Phosphate Transporter Gene Family of Arabidopsis thaliana

Abstract: Using a high-affinity phosphate transporter gene of Arabidopsis thaliana, PHT1, as a probe, three Arabidopsis homologs were isolated, named PHT4, PHT5 and PHT6, in addition to the previously isolated PHT2 and PHT3. The amino acid sequences deduced from the three nucleotides were 32-42% homologous with microbial phosphate transporters of Saccharomyces cerevisiae (PHO84), Neurospora crassa (PHO-5) and Glomus versiforme (GvPT). PHT1, PHT2, PETS and PHT6 were clustered in a 25-kbp region of chromosome V. PHT1 and PHT4 transcripts were detected in roots. Interestingly, suspension-cultured cells expressed only PHT4- PHT4 and PHT5 located within a genetic distance of 6.4 cM on chromosome II, and were close to a phosphate accumulation mutant pho2. Genomic sequencing revealed no difference in the sequences of the two genes in both pho2 and wild-type. The PHT4 transcript was expressed at similar levels in the mutant and wild-type. These results demonstrate that neither PHT4 nor PHT5 is allelic to PH02.

A Novel Superoxide Dismutase Gene Encoding Membrane-bound and Extracellular Isoforms by Alternative Splicing in Caenorhabditis elegans

Abstract: We have identified a novel Cu/Zn superoxide dismutase gene (termed SOD-4) in Caenorhabditis elegans. Characterization of its complementary DNA revealed that the gene encodes two isoforms by alternative splicing, SOD4-1 and SOD4-2 which differ in their C-terminal exons. Their predicted ammo acid sequences include a consensus signal peptide at their N-termini and are homologous to the extracellular-types of Cu/Zn superoxide dismutase in mammals. In addition, SOD4-2 possesses a putative transmembrane domain at the C-terminal region. When transiently expressed in Chinese hamster ovary cells, both types were found in the membranes and SOD4-1 also in the culture fluid. It is, therefore, indicated that SOD4-1 is an extracellular form and SOD4-2 a membrane-bound form, the latter representing a novel type of SOD. In C. elegans, SOD4-2 mRNA was found to be preferentially expressed in eggs.

Genes from nine genomes are separated into their organisms in the dinucleotide composition space.

Abstract: A set of 16 kinds of dinucleotide compositions was used to analyze the protein-encoding nucleotide sequences in nine complete genomes: Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp., Methanococcus jannaschii, Archaeoglobus fulgidus, and Saccharomyces cerevisiae. The dinucleotide composition was significantly different between the organisms. The distribution of genes from an organism was clustered around its center in the dinucleotide composition space. The genes from closely related organisms such as Gram-negative bacteria, mycoplasma species and eukaryotes showed some overlap in the space. The genes from nine complete genomes together with those from human were discriminated into respective clusters with 80% accuracy using the dinucleotide composition alone. The composition data estimated from a whole genome was close to that obtained from genes, indicating that the characteristic feature of dinucleotides holds not only for protein coding regions but also noncoding regions. When a dendrogram was constructed from the disposition of the clusters in the dinucleotide space, it resembled the real phylogenetic tree. Thus, the distinct feature observed in the dinucleotide composition may reflect the phylogenetic relationship of organisms.

Characterization of a Caenorhabditis elegans recA-like Gene Ce-rdh-1 Involved in Meiotic Recombination

Abstract: A recA-like gene was identified in the Caenorhabditis elegans genome project database. The putative product of the gene, termed Ce-rdh-1 (C. elegans RAD51 and DMC1/LIM15 homolog 1), consists of 357 amino acid residues. The predicted amino acid sequence of Ce-rdh-1 showed 46-60% identity to both RAD51 type and DMC1/LIM15 type genes in several eukaryote species. The results of RNAi (RNA-mediated interference) indicated that repression of Ce-rdh-1 blocked chromosome condensation of six bivalents and dissociation of chiasmata in oocytes of F1 progeny. Oogenesis did not proceed to the diakinesis stage. Accordingly, all the eggs produced (F2) died in early stages. These results suggest that Ce-rdh-1 participates in meiotic recombination.

Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.

Abstract: Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.

Intron-exon structure of ubiquitin c-terminal hydrolase-L1.

Abstract: Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by tremor, bradykinesia, postural instability and muscular rigidity. Neuropathologically, it is defined by cell loss in the substantia nigra associated with the presence of intraneuronal inclusions called Lewy bodies (LB) and processes engorged with proteinaceous material, the Lewy neurites. The recent identification of mutations in the a-synuclein gene in some familial forms of PD 3 ' 6 and the demonstration of accumulation of the protein in Lewy bodies and Lewy neurites 7 suggest that abnormal protein aggregation may cause the disease. In addition to asynuclein, LBs contain ubiquitin, proteasomal subunits 2 and ubiquitin C-terminal hydrolase-Ll, 5 pointing to a potential participation of the ubiquitin-proteasome degradation pathway in the onset of the disease. We confirmed this hypothesis by identifying a missense mutation in the ubiquitin C-terminal hydrolase LI (UCH-L1) gene in a German family with an autosomal dominant form of PD. 4 UCH-L1 is highly specific for neurons and cells of the diffuse neuroendocrine system, and represents 1% to 5% of the total brain protein extract. 8 Northern blot analysis reveals a 1.3-kb brain-specific transcript broadly represented in all areas of the brain tested, particularly in the substantia nigra (Fig. 1). Enzymes belonging to the ubiquitin C-terminal hydrolases family can remove small amides and esters at the carboxyl terminus of ubiquitin and have been shown to hydrolyze peptides and small proteins similarly (reviewed in 9). They contribute to the pool of free monomeric ubiquitin molecules by hydrolysing the ubiquitin polymeric proprotein or ubiquitin-ribosomal fusion proteins during the maturation of the ribosome. 9 The Ile93Met mutation observed in 2 PD patients 4 causes a partial loss of catalytic activity of the UCH-L1 enzyme which could induce aberrations in the degradation pathway and lead to aggregation of proteins. To permit the rapid screening for mutations and assess the UCH-L1 role in PD, we have established specific amplification assays for all of its coding exons.

Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana

Abstract: We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.

Complete Genomic Structure, DNA Polymorphisms, and Alternative Splicing of the Human AF-6 Gene

Abstract: In our previous work, detailed deletion mapping of ovarian cancers indicated that a 300-kb region of chromosome 6q27 was likely to contain one or more putative tumor suppressor genes associated with development of this type of cancer. DNA sequencing in the region disclosed the presence of AF-6, a gene that had been identified as the ALL-1 fusion partner involved in acute myeloid leukemias with t(6;ll)(q27;q23) translocations. In the work reported here, we determined the complete genomic sequence of the AF-6 gene, including exon-intron boundaries, and found six DNA polymorphisms. One of them, an insertion/deletion polymorphism, determined the presence or absence of seven amino acids in the AF-6 product. We also identified two alternatively spliced forms of the gene; the two novel transcripts would encode additional C-terminal peptides in comparison to the reported protein. Sequencing of seven cosmid clones that covered the entire gene revealed 32 exons (not including one exon involved in the insertion/deletion polymorphism), spanning approximately 140 kb of genomic DNA. These results may contribute to an understanding of the

A physical map of Arabidopsis thaliana chromosome 3 represented by two contigs of CIC YAC, P1, TAC and BAC clones.

Abstract: We have constructed a physical map of Arabidopsis thaliana chromosome 3 by ordering the clones from CIC YAC, P1, TAC and BAC libraries using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 112 DNA markers, 145 YAC end sequences, and 156 end sequences of P1, TAC and BAC clones. The entire genome of chromosome 3, except for the centromeric and telomeric regions, was covered by two large contigs, 13.6 Mb and 9.2 Mb long. This physical map will facilitate map-based cloning experiments as well as genome sequencing of chromosome 3. The map and end sequence information are available on the KAOS (Kazusa Arabidopsis data Opening Site) web site at http://www.kazusa.or.jp/arabi/.

Molecular Characterization of Mouse Pneumocystis carinii Surface Glycoprotein A

Abstract: Since the mouse offers an easily manipulated experimental animal model for the study of the im- munopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and character- ized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P. carinii. A cDNA library was constructed in bacteriophage Agtll from P. carinii -infected mouse lung poly(A+) RNA. Using a nucleic acid probe derived from a conserved region of the mouse P. carinii gpA structural gene, cDNAs encoding gpA were identified. A composite full-length gpA coding sequence was assembled from two overlapping cDNA clones. A DNA element homologous to the rat P. carinii upstream conserved sequence (UCS) was identified at the 5' end of several of the mouse P. carinii gpA cDNA clones, just upstream of the sequences encoding gpA structural gene isoforms. Using primer extension analysis, two neighboring putative transcriptional start sites were located on UCS-gpA mRNAs approximately 25 and 30 nt, respectively, upstream of the most 5' gpA cDNA clone isolated, suggesting a 5' UCS of 489 or 494 nucleotides in mouse P. carinii gpA. A comparative alignment of the composite mouse P. carinii gpA deduced amino acid sequence with gpA homologs from rat, human and ferret P. carinii demonstrated 156 identical residues, including 46 cysteines, further supporting the hypothesis for conserved secondary structure, as well as function, for gpA from all P. carinii.

Absence of Mutation in the β- and γ-Synuclein Genes in Familial Autosomal Dominant Parkinson's Disease

Abstract: Communicated by Mituru Takanami* To whom correspondenc bee addressed shoul Christiad N nLavedan, PhD National Institute of Healths , National Centerfor Human Genome Research, Laborator of Genetic Diseasy eResearch, Building: 49, Roo 4m9 4B67 Convent Driv, e MSC4470, Bethesda, Maryland 20892, USA, Tel +1-301-435-1196,Fax +1-301-402-2170, E-mail: lavedan@nhgrinihgovf Present address: Novartis 93 Pharmaceutical8 s Corporation,Clopper Road, Gaithersburg MD, 20878, USA

The Polyphosphate Kinase Gene of Pseudomonas aeruginosa

Abstract: We have cloned and sequenced a gene encoding polyphosphate kinase (PPK) from Pseudomonas aeruginosa PAO1. The gene immediately follows the hemB gene encoding porphobilinogen synthase responsible for heme synthesis. The predicted amino acid sequence of P. aeruginosa PPK is similar to those of PPKs previously characterized except that it possesses an extra stretch of 46 amino acids at its N-terminus, which has significant similarity to the Ras-related protein ARA5 of Arabidopsis thaliana. When P. aeruginosa PPK was overproduced in Escherichia coli, ATP-dependent polyphosphate-synthesizing activity was drastically enhanced, confirming that the protein is a PPK.

A Novel Plasmid Recombination Mechanism of the Marine Cyanobacterium Synechococcus sp. PCC7002

Abstract: We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The specific recombination sites on the smallest plasmid pAQl were localized by studying the properties of pAQl-derived shuttle-vectors. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination. Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements. Interestingly, this element is over-represented not only in pAQl and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp. PCC6301, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these cyanobacteria.

Isolation and Mapping of a Family of Putative Zinc-finger Protein cDNAs from Rice

Abstract: To understand the functions of rice homologues of the Arabidopsis flowering-time gene CONSTANS (CO) and salt-tolerance gene STO, we performed a similarity search of the single-run sequence data of cDNA clones accumulated by the Rice Genome Research Program, and isolated seven rice cDNA clones (S3574, C60910, S12569, R2931, R1479, R1577, and E10707) coding for proteins containing one or two zinc-finger-like motifs. Comparison of the deduced amino acid sequences between these cDNAs and the CO gene revealed significant similarities (46%-61%) in the region of zinc-finger motifs. A domain having a high content of basic amino acids at the C-terminus of the CO protein was found in the corresponding region of proteins predicted from cDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL" and "FCV(L)EDRA," which were present inside each zinc-finger in the Arabidopsis regulatory protein STO, were also found in each of the two zinc-finger regions of proteins predicted from cDNAs R2931, R1479, R1577, and E10707. Using restriction fragment length polymorphism (RFLP) linkage analysis, we determined the chromosomal location of the seven cDNA clones. The position of R2931 on the RFLP linkage map was closely linked to Hd-3, one of the putative quantitative trait loci (QTL) controlling heading date in rice.

Nucleotide Sequence of Plasmid pAQ1 of Marine Cyanobacterium Synechococcus sp. PCC7002

Abstract: We have determined the complete nucleotide sequence of pAQ1, the smallest plasmid of the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The plasmid consists of 4,809 bp and has at least four open reading frames that potentially encode polypeptides of 50 or more amino acids. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, is over-represented not only in plasmid pAQ1 but also in the accumulated cyanobacterial genomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. It suggests that this sequence might mediate gene rearrangement, thus increasing genetic diversity, since recombination events are frequent in cyanobacteria.

The psbO Gene for 33-kDa Precursor Polypeptide of the Oxygen-Evolving Complex in Arabidopsis thaliana — Nucleotide Sequence and Control of its Expression

Abstract: The 33-kDa polypeptide of the oxygen-evolving complex of photosystem II is nuclear-encoded. The single psbO gene of Arabidopsis thaliana, as suggested by Southern hybridization, has been isolated from the genomic library and sequenced. The sequence analysis has revealed that the psbO gene harbors two introns and encodes a precursor polypeptide of 332 amino acid residues; the first 85 amino acid residues represent the transit peptide and the following 247 amino acids constitute the mature polypeptide. The hydrophilic nature of the 33-kDa protein is confirmed by the presence of 27% charged residues. Northern analysis of the total RNA from Arabidopsis indicates that a 1.2-kb transcript represents the psbO gene. It is expressed in a tissue-specific manner -- the steady-state transcript levels being highest in the leaves and virtually undetectable in the roots. Also, expression of the psbO gene is development-dependent and regulated by light in young Arabidopsis seedlings. In a constitutively photomorphogenic mutant of Arabidopsis, pho2 (plumular hook open 2), the psbO gene is de-repressed in young, dark-grown seedlings, resulting in increased transcript abundance compared to the wild-type. These studies, thus, define the influence of at least one regulatory component for psbO expression.

Genomic organization and localization of the human CRMP-1 gene.

Abstract: The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein considered to be involved in the collapsin-induced growth cone collapse during neural development. CRMP-1 belongs to the Unc-33 gene family. Here we report the genomic structure and the localization of the human CRMP-1 gene to chromosome 4pl6.1. Sequence analysis revealed that the human CRMP-1 gene consists of 14 exons. We have also established sequencing assays for all its coding exons. This should permit the rapid screening for mutations to assess CRMP-1 role in genetic disorders mapped in the 4pl6.1 region.

cDNA Cloning, Tissue Distribution and Chromosomal Localization of the Human ID4 Gene

Abstract: A cDNA encoding the human Id4 protein has been isolated from an astrocytoma library. The predicted protein product shares 98% identity with the mouse Id4 protein and is markedly different from that already reported. By FISH analysis, the human ID4 gene was more precisely mapped to chromosome 6p22.3-p23. Northern blot analysis showed that ID4 is mainly expressed in thyroid, brain and fetal tissue and in some nervous system tumor cell lines.

A Novel Gene, ITM, Located between p57KIP2 and IPL, Is Imprinted in Mice

Abstract: We searched for new imprinted genes using a positional cloning method in a region of human chromosome Ilpl5.5, which contains several imprinted genes including p57 KIP2 and IPL, and found a novel ITM gene located between p57 KIP2 and IPL. We also obtained the mouse homologue Itm in its syntenic region of mouse chromosome 7. In humans, its location is 17 kb centromeric to p57 KIP2 and 3 kb telomeric to IPL, and in mice, 15 kb and 2.5 kb, respectively. They are expressed in most tissue, but especially in the kidney and liver, and moderately in the heart, lung and testis. Mice exhibit a functional imprinting resulting in higher expression of maternal alleles in fetal, newborn and most adult tissues, but it is biallelically expressed in the adult kidney and liver where expression is the highest. In addition to the discrepancy between the level of expression and the strength of the imprint, Itm has several unusual features for an imprinted gene, including large introns, moderate GC content and the absence of direct repeats. Our results will be helpful in understanding the intricate regulatory mechanism of imprinted genes.

Structural Characterization of the virB Operon on the Hairy-root-inducing Plasmid A4

Abstract: The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferring tumorigenic symptoms on plants upon infection by its host bacterium, Agrobacterium rhizogenes. The virB operon on pRiA4 has been sequenced and found to be composed of 11 genes, virB1 to virB11, whose products mostly appear to be associated with the cell membrane. A novel structural characteristic is frequent overlappings between the translation termination and initiation codons of adjacent genes. This is indicative of fine tuning of relative translation frequencies for each VirB protein. These results support the view that VirB multisubunit complexes provide facilities for T-DNA transfer at the bacterial cell membrane. The structural organization of the pRiA4 virB operon was essentially identical to that of the previously reported virB operons of tumor-inducing plasmids, pTiC58 and pTiA6, and the corresponding VirB proteins of the three plasmids were extremely homologous to one another. On the basis of the structural similarity of each VirB protein, the phylogenetic relationship among pRiA4, pTiC58, and pTiA6 is discussed.

Contig Map of the Parkinson's Disease Region on 4q21-q23

Abstract: We have constructed a yeast artificial chromosome contig (YAC) map of human chromosome 4q21q23 across the Parkinson's disease region by combining molecular and fluorescence in situ hybridization techniques. This map contains 55 YACs and 51 molecular markers, including 23 polymorphic markers. We have also isolated one PI and 33 bacterial artificial chromosomes located within this contig. Plasmid libraries were generated from 11 of these BAC and PI clones, and 614 random plasmid clones were sequenced for a total of about 200 kb. This contig allowed us to precisely determine the location of 18 transcripts within the D4S2460-D4S2986 interval, including the alpha-synuclein gene found to be mutated in some families with Parkinson's disease.

High-Resolution Mapping of Ribosomal Protein Genes to Human Chromosome 19

Abstract: In a systematic effort for mapping of all the human ribosomal protein (rp) genes, we have found that an unusually large number (12) of rp genes are present on chromosome 19 and subsequently determined their locations on the chromosome by a radiation-hybrid procedure. For this, we isolated cosmid clones corresponding to each gene and placed nine of them on a metric physical map of chromosome 19. Although most genes are scattered over the chromosome, we found three genes are clustered in a 0.6-Mb region at 19ql3.3 and two of them, RPL13A and RPSll, within a single cosmid only 4.3 kb apart. To explore a possible relationship between rp gene defects and human disease, we compared map positions of the rp genes and disease loci on chromosome 19, which led us to find RPS9 gene in the same interval as the gene for retinitis pigmentosa 11. The disease locus has previously been mapped to the 6-cM interval at 19ql3.4 between markers D19S572 and D19S926, which corresponds to less than 2-Mb region on the metric physical map. We mapped RPS9 about 800 kb distal to D19S572.

Mapping of Rice Rf Gene by Bulked Line Analysis

Abstract: A method, bulked line analysis (BLA), was developed for identification of the RFLP markers associated with a target gene. Instead of segregating progenies, conventional lines sharing the same trait were bulked by the BLA method. This method is an alternative approach to the identification of DNA markers linked with a target gene. A major advantage of this method is time-saving for genetic stock development. The advantage is very significant for organisms having a long generation period. This method has been tested by using fertility restoration of rice cytoplasmic male sterility of wild abortive type as a target trait. A fertility-restoring gene was successfully identified by linkage with RFLP markers. This gene was mapped in the middle of the long arm of chromosome 10 of the rice genome.