Disintegration of nascent replication bubbles during thymine starvation triggers RecA- and RecBCD-dependent replication origin destruction.

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA(+) cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction.

Stalled replication fork repair and misrepair during thymineless death in Escherichia coli.

Starvation for DNA precursor dTTP, known as 'thymineless death' (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing.

Hydroxymethanesulfonate from Volcanic Sulfur Dioxide: A "Mineral" Reservoir for Formaldehyde and Other Simple Carbohydrates in Prebiotic Chemistry.

While formaldehyde (HCHO) was likely generated in Earth's prebiotic atmosphere by ultraviolet light, electrical discharge, and/or volcano-created lightning, HCHO could not have accumulated in substantial amounts in prebiotic environments, including those needed for prebiotic processes that generate nucleosidic carbohydrates. HCHO at high concentrations in alkaline solutions self-reacts in the Cannizzaro reaction to give methanol and formate, neither having prebiotic value. Here, we explore the possibility that volcanic sulfur dioxide (SO2) might have generated a reservoir for Hadean HCHO by a reversible reaction with HCHO to give hydroxymethanesulfonate (HMS). We show that salts of HMS are stable as solids at 90°C and do not react with themselves in solution, even at high (>8 M) concentrations. This makes them effective stores of HCHO, since the reverse reaction slowly delivers HCHO back into an environment where it can participate in prebiotically useful reactions. Specifically, we show that in alkaline borate solutions, HCHO derived from HMS allows formation of borate-stabilized carbohydrates as effectively as free HCHO, without losing material to Cannizzaro products. Further, we show that SO2 can perform similar roles for glycolaldehyde and glyceraldehyde, two intrinsically unstable carbohydrates that are needed by various models as precursors for RNA building blocks. Zircons from the Hadean show that the Hadean mantle likely provided volcanic SO2 at rates at least as great as the rates of atmospheric HCHO generation, making the formation of Hadean HMS essentially unavoidable. Thus, hydroxymethylsulfonate adducts of formaldehyde, glycolaldehyde, and glyceraldehyde, including the less soluble barium, strontium, and calcium salts, are likely candidates for prebiotically useful organic minerals on early Earth.

Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.

Identification of a novel member of the snail/Gfi-1 repressor family, mlt 1, which is methylated and silenced in liver tumors of SV40 T antigen transgenic mice.

DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.

Reduction in antiviral activity of human interferon-gamma in acidic media with reference to structural change.

The fluorescence intensity of a unique tryptophan 36 in human interferon-gamma was drastically decreased below pH 4 with a concomitant decrease of antiviral activity. The region of residues 32-42 of human interferon-gamma was found by calculation to have a low hydrophobicity together with a high helical hydrophobic moment, and the net electric charge of this region having an amphiphilic helical structure changed significantly near pH 4. These results suggest that the region of residues 32-42 plays an important role in exhibiting antiviral activity.

Improved restriction landmark cDNA scanning and its application to global analysis of genes regulated by nerve growth factor in PC12 cells.

Restriction landmark cDNA scanning (RLCS) is a novel method by which more than 1000 genes can be simultaneously and quantitatively displayed as two-dimensional gel spots. Here we present an adaptation that allows an individual spot to correspond to a unique gene species without redundancy in more than two gel patterns. Using this improved RLCS, we examined global changes on the gene expression of PC12 cells before and after treatment with nerve growth factor. Among a total of 3000 spots, 21 (0.70%) and 91 (3.03%) spots newly appeared and became more intense with treatment. On the other hand, 15 (0.50%) and 44 (1.47%) spots disappeared, becoming less intense with treatment. These observations suggest that approx. 6% of the detected PC12 genes are up-(3.73%) or down-(1.97%) regulated when the cells differentiate to neuronal cells. In comparison with the results obtained using the expressed-sequence-tag approach, previously reported by Lee et al. (Proc. Natl. Acad. Sci. USA 92 (1995) 8303-8307), RLCS should be useful for quantitatively examining the global change of differentially expressed genes of various expression levels.

Cloning of a novel gene specifically expressed in clonal mouse chondroprogenitor-like EC cells, ATDC5.

We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.

A genetic linkage map of the mouse using restriction landmark genomic scanning (RLGS).

We have developed a multiplex method of genome analysis, restriction landmark genomic scanning (RLGS) that has been used to construct genetic maps in mice. Restriction landmarks are end-labeled restriction fragments of genomic DNA that are separated by using high resolution, two-dimensional gel electrophoresis identifying as many as two thousand landmark loci in a single gel. Variation for several hundred of these loci has been identified between laboratory strains and between these strains and Mus spretus. The segregation of more than 1100 RLGS loci has been analyzed in recombinant inbred (RI) strains and in two separate interspecific genetic crosses. Genetic maps have been derived that link 1045 RLGS loci to reference loci on all of the autosomes and the X chromosome of the mouse genome. The RLGS method can be applied to genome analysis in many different organisms to identify genomic loci because it uses end-labeling of restriction landmarks rather than probe hybridization. Different combinations of restriction enzymes yield different sets of RLGS loci providing expanded power for genetic mapping.

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5.

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.

A PCR-mediated method for cloning spot DNA on restriction landmark genomic scanning (RLGS) gel.

A PCR-mediated direct cloning for target spot DNA from RLGS gel has been established. The method consists of PCR amplification of adaptor-ligated spot DNA fragments without excluding similar-sized DNA fragments co-localized on RLGS gel, and following selective ligation with the NotI-dT vector. Applying this method, we have successfully cloned several DNA fragments derived from target spots whose intensities change developmentally due to DNA methylation in the telencephalon of C3H/HeN mice. Since only a few micrograms of total DNA is sufficient for our spot cloning, our method may be highly useful when the total DNA sample prepared for cloning is limited.

Comparative analysis of mouse NotI linking clones with mouse and human genomic sequences and transcripts.

NotI cleavage sites are frequently associated with CpG islands that identify the 5' regulatory sites of functional genes in the genome. Therefore we analyzed a sample of 22 NotI linking clones prepared from mouse brain DNA, to determine whether these mouse NotI site associated clones could be used for comparative analysis of mouse and human genomes by cross-reaction with both mouse and human genomic DNA and RNA in Southern and Northern hybridization. We further examined whether we could establish the identity of these clones with known genes by comparing the nucleotide sequences surrounding the NotI site with the GenBank database. We observed that 70% of the clones cross-hybridized with human DNA and that 4 of 11 tested clones (36%) detected a transcript in human HeLa cells RNA whereas 73% clones (8/11) detected transcripts in mouse RNAs from one or more organs. Single pass sequence analysis was successful on 16 of 19 clones. The GC content in these sequence was very high (48.8% to 73.8%) suggesting that 12 of 16 sequenced clones contained a CpG island. Three out of 19 clones showed significant similarity with previously analyzed mouse gene sequences in GenBank, including the mouse rRNA gene family, cathepsin and the scip POU-domain genes. In addition, two sequences showed significant similarity to the human and rabbit protein phosphatase 2A-beta subunit and the human transforming growth factor-beta. Thus, 5 of 16 clones showed homology with identified genes. These results and the recent work of using RLGS methods for genetic mapping indicate that NotI linking clones can be used to efficiently cross reference a comparative analysis of the mouse and human genomic maps.

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes.

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.

Comprehensive sequence analysis of translation termination sites in various eukaryotes.

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.

A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration.

We detected a missense mutation in exon 10 of tau that causes a substitution at codon 279 (N279K) in a Japanese patient with a familial background of parkinsonism and dementia originally described as pallido-nigro-luysian degeneration. This mutation is the same as one seen in a Caucasian family with pallido-ponto-nigral degeneration. The similarities between these two families suggest a common genetic mechanism that may account for the peculiar distribution of neuroglial degeneration with tauopathy.

Effect of hypertension on lysosomal enzyme activities in aortic endothelial cells.

In order to obtain information about the changes in lysosomal enzyme activities in arterial endothelial cells under hypertensive conditions, a biochemical study was performed on 5 lysosomal enzymes, acid phosphatase, N-acetyl-beta-glucosaminidase (NAGase), cathepsin B, cathepsin D and beta-glucuronidase, in endothelial cells isolated by an enzymatic technique from the aorta of spontaneously and renal hypertensive rats, and normotensive control rats. The aortic endothelial cells in the old spontaneously and the renal hypertensive rats showed increased activities of enzymes examined in comparison with those in the age-matched control rats. Endothelial cells in young spontaneously hypertensive rats did not show any elevated enzyme activities compared with those in the controls, and the enzyme activities tended to increase with aging. From this, it is deduced that hypertension activates lysosomal enzyme activities in aortic endothelial cells. The differences in the activities of NAGase, cathepsin B and cathepsin D between hypertensive and control animals increased markedly with advancing age. These activated lysosomal enzymes seem to be involved in the developmental mechanism of arterial endothelial cell injury in hypertension and in further development of hypertensive vascular changes.

Does hypertension confer a hypercoagulable state in stroke-prone spontaneously hypertensive rats?

OBJECTIVE: To verify whether hypertension confers a hypercoagulable state in a hypertensive animal model. DESIGN: The parameters of blood coagulation were compared between stroke-prone spontaneously hypertensive rats (SHR-SP) and Wistar-Kyoto (WKY) rats. Each rat group consisted of a younger subgroup at 8-12 weeks old (n = 12) and an older subgroup at 16-20 weeks old (n = 12). METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fluorogenic PT, fibrinogen, fibrin/fibrinogen degradation products (FDP), thrombin-anti-thrombin III complex (TAT), factor Xa activity, anti-thrombin III (AT-III), tissue factor pathway inhibitor (TFPI), protein C and C1 inhibitor were measured in both rat groups. RESULTS: There was no significant difference in FDP and TAT levels between SHR-SP and WKY rats even at 16-20 weeks when SHR-SP developed severe hypertensive vascular lesions. Contrary to expectations, fluorogenic PT and factor Xa activity were significantly lower in SHR-SP than in WKY rats. While there was no significant difference in AT-III, TFPI and protein C activities between SHR-SP and WKY rats, C1 inhibitor activity was significantly higher in SHR-SP than in WKY rats. The elevated C1 inhibitor activity was inversely correlated with the reduced factor Xa activity. Gel-filtered fractionated plasma with C1 inhibitor activity had an inhibitory effect on the purified rat factor Xa, and immunodepletion of C1 inhibitor from the fractionated plasma attenuated the inhibitory effect CONCLUSION: These results suggest that SHR-SP get into a hypocoagulable state rather than a hypercoagulable state, and that the reduction of factor Xa activity in SHR-SP may be related to the elevation of C1 inhibitor activity.

Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing.

We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

Orientation of HLA-DNA gene and identification of a CpG island-associated gene adjacent to DNA in human major histocompatibility complex class II region.

We have constructed the detailed physical map of the HLA class II gene region by the pulsed field gel electrophoresis (PFGE) and cosmid walking technique. In this process, the DNA gene was found to be located telomeric to DPA1 with the 5'----3' orientation which is the same as the DPA1 and DPA2 genes, but opposite to the DQA1, DQA2 and DRA genes. This orientation is reverse to that of the counterpart gene in the rabbit major histocompatibility complex region. About 30 kb downstream from the DNA gene towards DOB, a CpG island characterized by clustered sites for rare cutting restriction enzymes and frequently associated with the 5' end of housekeeping genes was identified by PFGE and cosmid walking. From a complementary DNA (cDNA) library constructed from a Epstein-Barr virus-transformed B-cell line, a cDNA clone was isolated using the genetic probe from this CpG island. Its nucleotide sequences suggested that it represented a new non-HLA gene with a single copy which was of little genetic polymorphism and named NAT (DNA-associated transcript). Northern blot analysis showed that the NAT gene was expressed with a 4-kb transcript in all of tissues examined so far.

No difference in the nucleotide sequence of the DQ beta beta 1 domain between narcoleptic and healthy individuals with DR2,Dw2.

Narcolepsy is a sleep disorder completely associated with HLA-DR2,Dw2. We demonstrated the 100% presence of three DQ beta fragments (EcoRI 2.4-kb, BamHI 2.9-kb, and PstI 12-kb) in narcoleptic patients that were detected in healthy DR2 controls only at decreased frequencies. In this paper, we have cloned the DQ beta gene from three Japanese narcoleptic patients and sequenced their beta 1 domain in order to study the sequence polymorphisms that might exist in the DQ beta genes of patients, but no difference in sequence could be found between narcoleptic and healthy individuals, suggesting that narcolepsy is not due to mutation in the DQ beta gene. In this context, a possible role of the HLA class II antigens in narcolepsy is discussed.