[The green alga Chlamydomonas reinhardtii as an attractive model system].
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Animals↗
Biomedical subjects
H Fukuzawa
Publications and source records attributed to H Fukuzawa.
A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I. Generation of 3433 non-redundant expressed sequence tags.
To understand genetic information carried in a unicellular green alga, Chlamydomonas reinhardtii, normalized and size-selected cDNA libraries were constructed from cells at photoautotrophic growth, and a total of 11,571 5'-end sequence tags were established. These sequences were grouped into 3433 independent EST species. Similarity search against the public non-redundant protein database indicated that 817 groups showed significant similarity to registered sequences, of which 140 were of previously identified C. reinhardtii genes, but the remaining 2616 species were novel sequences. The coverage of full-length protein coding regions was estimated to be over 60%. These cDNA clones and EST sequence information will provide a powerful source for the future genome-wide functional analysis of uncharacterized genes.
Animals↗
Expressed sequence tags from immature female sexual organ of a liverwort, Marchantia polymorpha.
A total of 970 expressed sequence tag (EST) clones were generated from immature female sexual organ of a liverwort, Marchantia polymorpha. The 376 ESTs resulted in 123 redundant groups, thus the total number of unique sequences in the EST set was 717. Database search by BLAST algorithm showed that 302 of the unique sequences shared significant similarities to known nucleotide or amino acid sequences. Six unique sequences showed significant similarities to genes that are involved in flower development and sexual reproduction, such as cynarase, fimbriata-associated protein and S-receptor kinase genes. The remaining unique 415 sequences have no significant similarity with any database-registered genes or proteins. The redundant 123 ESTs implied the presence of gene families and abundant transcripts of unknown identity. Analyses of the coding sequences of 61 unique sequences, which contained no ambiguous bases in the predicted coding regions, highly homologous to known sequences at the amino acid level with a similarity score greater than 400, and with stop codons at similar positions as their possible orthologues, indicated the presence of biased codon usage and higher GC content within the coding sequences (50.4%) than that within 3' flanking sequences (41.9%).
Amino Acid Sequence↗
Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants.
The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16,103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.
Blotting, Southern↗
Validity of sleep log compared with actigraphic sleep-wake state II.
Twenty-five young people (Y group), three elderly people and seven people with various sleep disorders (SD group) kept a sleep log for 2-7 days, and their wrist-activity was monitored simultaneously. The sensitivity and specificity of the sleep log, and the ratio of agreement between the sleep log and actigraphic sleep-wake state were calculated. The sensitivity and specificity in Y group were 87.93+/-6.49% and 96.51+/-2.37%, respectively. The sensitivity in SD group was significantly lower than in Y group. Even in Y group one-hour agreement ratios dropped during the sleep onset period.
Adult↗
Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing.
In plants, calcium-dependent calmodulin-independent protein kinases (CDPKs) are the predominant calcium-regulated protein kinases and their genes are encoded by a multigene family. A CDPK gene was cloned from a liverwort, Marchantia polymorpha, which showed a high level of sequence similarities to other higher plant CDPK genes. The liverwort CDPK gene consisted of 9 exons and 8 introns. The 6th and 7th exons (Exon 6A and Exon 6B) were almost identical except for 4-amino acid substitutions, both of which coded for EF-hands in the calcium-binding domain. RT-PCR analysis revealed that two species of mature mRNA containing either Exon 6A or Exon 6B were generated from a single CDPK gene by mutually exclusive alternative splicing. Both histidine-tagged fusion proteins derived from cDNAs containing either Exon 6A or Exon 6B exhibited calcium-dependent protein kinase activity in vitro. Preferential accumulation of the mature mRNA with Exon 6A detected in male sexual organ implies possible sexual control of the ratio between the two CDPK isozymes through alternative splicing. Functions and evolution of CDPKs are discussed based on the structure and expression of the liverwort CDPK gene.
Alternative Splicing↗
CO(2)-responsive transcriptional regulation of CAH1 encoding carbonic anhydrase is mediated by enhancer and silencer regions in Chlamydomonas reinhardtii.
Chlamydomonas reinhardtii adapts to the stress of CO(2)-limiting conditions through the induction of a set of genes including CAH1, which encodes a periplasmic carbonic anhydrase. CAH1 is up-regulated under low-CO(2) conditions (air containing 0.04% [v/v] CO(2)) in the presence of light, whereas it is down-regulated under high-CO(2) conditions (5% [v/v] CO(2)) or in the dark. In an effort to identify cis-elements involved in the transcriptional regulation of CAH1, a series of 5'-nested deletions of the region upstream of CAH1 were fused to a promoterless arylsulfatase reporter gene (ARS). The upstream region from -651 to +41 relative to the transcription start site was sufficient to regulate the expression of ARS with kinetics similar to those of endogenous CAH1. Deletion of the region between -651 and -294 resulted in a significant decrease in the level of arylsulfatase activity expressed under low-CO(2) conditions. The 543-bp upstream region from -651 to -109, without any promoter elements, CAAT-box, or TATA-box, could confer CO(2) and light responsiveness on the beta(2)-tubulin minimal promoter. This 543-bp region was divided into two parts: a 358-bp silencer region from -651 to -294, which represses the minimal promoter activity under high-CO(2) conditions, and a 185-bp enhancer region from -293 to -109, which activates the promoter under low-CO(2) conditions in the presence of light.
5' Untranslated Regions↗
The effects of various GTP analogues on microtubule assembly.
We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.
Adenine Nucleotides↗
[Small heat shock proteins in cyanobacteria].
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Adaptation, Physiological↗
Coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in mitochondria of a liverwort Marchantia polymorpha.
The liverwort Marchantia polymorpha mitochondrial DNA encodes almost all tRNAs required for mitochondrial translation except for the isoleucine (AUU, AUC) and threonine (ACA, ACG) codons, while the missing tRNAs are supplied in part by the nucleus and imported in mitochondria. In this paper, we report a finding of two radically different nuclear tRNAVal(AAC) genes and import of the corresponding tRNA isoacceptors in M.polymorpha mitochondria. This finding is surprising since the mtDNA encodes the gene for tRNAVal(UAC), which alone was considered sufficient for translating all four valine codons GUN by the U/N wobble mechanism. The present results suggest for the first time that the import of ncDNA-encoded tRNAs may result in decoding overlaps in plant mitochondria. The coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in liverwort mitochondria and the significance for the decoding mechanism as well as evolution of tRNA import are discussed.
Biological Transport↗
Validity of sleep log compared with actigraphic sleep-wake state.
Seven women and 11 men, mean age 30.1 years, kept a sleep log for 5-7 days, and their wrist activity was monitored each minute. Sleep-wake state in the sleep log and actigraphic sleep-wake state were compared, and the sensitivity and specificity of the sleep log were calculated for each subject. The ratio of agreement between these two parameters was computed for each subject. The sensitivity and specificity of the sleep log were 72.73-97.56% (mean 86.71%) and 92.85-99.68% (mean 97.04%), respectively. The agreement ratio was 87.30-97.85% (93.48%), but 1-h agreement ratios from midnight dropped during the sleep onset period.
Activity Cycles↗
Failure in detection of Epstein-Barr virus and cytomegalovirus in specimen obtained by fine needle aspiration biopsy of thyroid in patients with subacute thyroiditis.
To study the possible role of viral infection in the etiology of subacute thyroiditis (SAT), we measured serum virus-specific antibodies to measles, rubella, mumps, type I herpes, chicken pox, human parvovirus B19 and cytomegalovirus (CMV) in 10 patients with SAT during the course of illness. In spite of the presence of IgG to each virus in more than 70% of patients, no significant changes in the IgG titers were observed except those to measles, rubella, chicken pox or CMV in only 10% of patients, respectively. Then we examined the presence of virus DNA in specimens of 9 patients with SAT obtained by fine-needle aspiration biopsy (FNAB) of the thyroid. DNA was amplified to detect that of Epstein-Barr virus and CMV by polymerase chain reaction. However, none of them were detected in all the specimens. Whereas previous studies suggest the involvement of viral infection in the pathogenesis of SAT, we failed to demonstrate significant changes in serum antiviral antibody titers or to detect viral DNA in the specimens obtained by FNAB of the thyroid. Thus further studies are clearly required to establish the definite role of viral infection in the pathogenesis of SAT.
Antibodies, Viral↗
Accumulation of nuclear-encoded tRNA(Thr) (AGU) in mitochondria of the liverwort Marchantia polymorpha.
The mitochondrial genome of the liverwort Marchantia polymorpha does not encode the full complement of tRNAs for the threonine and isoleucine codon boxes. To find the missing tRNA genes specifically for tRNA(Thr) in mitochondria, we have searched the genomic library and identified two clones (pTT1 and pTT2), encoding the identical tRNA(Thr) (AGU) gene copy with different 5'- and 3'-flanking sequences. By northern analysis, we demonstrate considerable accumulation of the nuclear encoded tRNA(Thr) and moderate expression of native tRNA(Thr) (GGU) in mitochondria. Nonetheless, the imported and native tRNA(Thr) species together are not sufficient to translate all four threonine codons used in liverwort mitochondria, implicating mitochondrial import of at least one additional threonine isoacceptor tRNA.
Anticodon↗
Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha.
The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes.
Amino Acid Sequence↗
Synthesis of guanosine 5'-triphosphate analogues and their effect on microtubule assembly.
More than 20 base-modified analogues of guanosine 5'-triphosphate including 2'-deoxyguanosine derivatives were synthesized and examined their effect on tubulin polymerization into microtubules (Mts). Among those, 2,6-diamino-8-oxopurineriboside 5'-triphosphate (1d), 2,6-diamino-2'-deoxypurineriboside 5'-tri phosphate (1b) and 8-bromoguanosine 5'-tri phosphate (1g) were shown to have remarkable effect to promote microtubule assembly.
Guanosine Triphosphate↗
Occurrence of nuclear-encoded tRNAIle in mitochondria of the liverwort Marchantia polymorpha.
Although 29 tRNA genes have been deduced from the complete nucleotide sequence of the mitochondrial genome from the liverwort Marchantia polymorpha, a tRNAIle gene decoding AUU and AUC codons is conspicuously absent. In order to address the question of the possible involvement of nuclear-encoded tRNA, we isolated and identified three variant copies of the nuclear-encoded tRNAIle(AAU) gene from the liverwort. Northern analysis showed the presence of nuclear-encoded tRNAIle both in the mitochondrion and the cytosol, while both chloroplast DNA-encoded tRNAIle and nuclear-encoded tRNATyr were absent in liverwort mitochondria. These results unequivocally establish that import of nuclear tRNAIle into mitochondria indeed occurs in one of the most primitive plants, M. polymorpha.
Base Sequence↗
cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC6803.
We have isolated mutants of Synechocystis PCC6803 that grew very slowly in a low-sodium medium, which is unfavorable for HCO3(-) transport, and examined two of these mutants (SC1 and SC2) for their ability to take up CO2 and HCO3(-) in the light. The CO2 transport activity of SC1 and SC2 was much lower than that of the wild type (WT), whereas there was no difference between the mutants and the WT in their activity of HCO3(-) transport. A clone containing a 3.9-kilobase-pair insert DNA that transforms both mutants to the WT phenotype was isolated from a genomic library of WT Synechocystis. Sequencing of the insert DNA in the region of mutations in SC1 and SC2 revealed an open reading frame (designated cotA), which showed significant amino-acid sequence homology to cemA encoding a protein found in the inner envelope membrane of chloroplasts. The cotA gene is present in a single copy and was not cotranscribed with any other gene(s). cotA encodes a protein of 247 amino acids containing four transmembrane domains. There was substitution of a single base in SC1 and two bases in SC2 in their cotA genes. A possible role of the cotA gene product in CO2 transport is discussed.
Amino Acid Sequence↗
[Drug-induced purpura in a patient with chronic hepatitis C due to interferon therapy].
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Chronic Disease↗