Dinitropyrenes induce gene mutations in multiple organs of the lambda/lacZ transgenic mouse (Muta Mouse)

Dinitropyrenes (DNPs), 1,3-, 1,6- and 1,8-dinitropyrene, are carcinogenic compounds found in diesel engine exhaust. DNPs are strongly mutagenic in the bacterial mutation assay (Ames test), mainly inducing frameshift type mutations. To assess mutagenicity of DNPs in vivo is important in evaluating their possible involvement in diesel exhaust-induced carcinogenesis in human. For this purpose, we used the lambda/lacZ transgenic mouse (Muta Mouse) to examine induction of mutations in multiple organs. A commercially available mixture of DNPs (1,3-, 1,6-, 1,8-, and unidentified isomer (s) with a content of 20.2, 30.4, 35.2, and 14.2%, respectively) was injected intragastrically at 200 and 400mg/kg once each week for 4 weeks. Seven days after the final treatment, liver, lung, colon, stomach, and bone marrow were collected for mutation analysis. The target transgene was recovered by the lambda packaging method and mutation of lacZ gene was analyzed by a positive selection with galE(-) E. coli. In order to determine the sequence alterations by DNPs, the mutagenicity of the lambda cII gene was also examined by the positive selection with hfl(-) E. coli. Since cII gene (294bp) is much smaller than the lacZ (3024bp), it facilitated the sequence analysis. Strongest increases in mutant frequencies (MFs) were observed in colon for both lacZ (7.5x10(-5) to 43.3x10(-5)) and cII (2.7x10(-5) to 22.5x10(-5)) gene. Three-four-fold increases were observed in stomach for both genes. A statistically significant increase in MFs was also evident in liver and lung for the lacZ gene, and in lung and bone marrow for the cII gene. The sequence alterations of the cII gene recovered from 37 mutants in the colon were compared with 50 mutants from untreated mice. Base substitution mutations predominated for both untreated (91%) and DNP-treated (84%) groups. The DNPs treatment increased the incidence of G:C to T:A transversion (2-43%) and decreased G:C to A:T transitions (70-22%). The G:C to T:A transversions, characteristic to DNPs treatment, is probably caused by the guanine-C8 adduct, which is known as a major DNA-adduct induced by DNPs, through an incorporation of adenine opposite the adduct ("A"-rule). The present study showed a relevant use of the cII gene as an additional target for mutagenesis in the Muta Mouse and revealed a mutagenic specificity of DNPs in vivo.     

Ciona intestinalis cDNA projects: expressed sequence tag analyses and gene expression profiles during embryogenesis

Ascidians are primitive chordates. Their fertilized egg develops quickly into a tadpole-type larva, which consists of a small number but distinct types of cells, including those of epidermis, central nervous system with two sensory organs, endoderm and mesenchyme in the trunk, and notochord and muscle in the tail. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. In addition, the free-swimming and non-feeding larvae metamorphose into sessile and filter-feeding adults. The genome size of Ciona intestinalis is estimated to be about 160 Mb, and the number of genes approximately 15,500. The present Ciona cDNA projects focused on gene expression profiles of fertilized eggs, 32-110-cell stage embryos, tailbud embryos, larvae, and young adults. Expressed sequence tags (ESTs) of the 5'-most end and 3'-most end of more than 3000 clones were determined at each developmental stage, and the clones were categorized into independent clusters using the 3'-end sequences. Nearly 1000 clusters of them were then analyzed in detail of their sequences against a BLASTX search. This analysis demonstrates that, on average, half of the clusters showed proteins with sequence similarities to known proteins and the other half did not show sequence similarities to known proteins. Genes with sequence similarities were further categorized into three major subclasses, depending on their functions. Furthermore, the expression profiles of all of the clusters were analyzed by whole-mount in situ hybridization. This analysis highlights gene expression patterns characteristic to each developmental stage. As a result, the present study provides many new molecular markers for each of the tissues and/or organs that constitutes the Ciona tailbud embryo. This sequence information will be used for further comparative genome studies to explore molecular mechanisms involved in the formation of one of the most primitive chordate body plans. All of the data fully characterized may be viewed at the web site http://ghost.zool.kyoto-u.ac.jp.     

Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 consists of a single chromosome and several plasmids of different sizes, and the nucleotide sequences of the chromosome and three small plasmids (5.2 kb, 2.4 kb, and 2.3 kb) have already been sequenced. We newly determined the nucleotide sequences of four large plasmids, which have been identified in our laboratory (pSYSM:120 kb, pSYSX:106 kb, pSYSA:103 kb, and pSYSG:44 kb). Computer-aided analysis was performed to explore the genetic information carried by these plasmids. A total of 397 potential protein-encoding genes were predicted, but little information was obtained about the functional relationship of plasmids to host cell, as a large portion of the predicted genes (77%) were of unknown function. The occurrence of the potential genes on plasmids was divergent, and parA was the only gene common to all four large plasmids. The distribution data of a Cyanobacterium-specific sequence (HIP1: 5'-GCGATCGC-3') suggested that respective plasmids could have originated from different cyanobacterial strains.     

Change in priming sites for discontinuous DNA synthesis between the monomeric and concatemeric stages of phage T7 replication

Summary We have analyzed the transition sites between primer RNA and DNA in a 589 bp segment of the bacteriophage T7 genome. In the monomeric replication stage, RNA-DNA transition sites are predominantly on the light (L) strand (with, 53 polarity on the genetic map) but rarely on the heavy (H) strand, indicating that replication proceeds semidiscontinuously with the H and L strands corresponding to the leading and lagging strands, respectively. The direction of replication is that expected from the position of the primary origin and also indicates that secondary origins are seldom if ever used. In the concatemeric stage of replication, RNA-DNA transition sites are instead distributed on both strands of the segment with equally high frequency, showing that initiation occurs within the concatemeric molecule per se and by a different mechanism.     

In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain.

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.     

Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches.

The termination of protein synthesis in Escherichia coli requires two codon-specific factors termed RF1 and RF2. RF1 mediates UAA- and UAG-directed termination, while RF2 mediates UAA- and UGA-directed termination. The genes encoding these factors have been isolated and sequenced, and RF2 was found to be encoded in two separate reading frames. The map position of RF1 has been reported as 27 min on the E. coli chromosome, while the RF2 map position has not yet been identified. In this study, two new and independent methods for gene mapping, using pulsed field gel electrophoresis and an ordered bacteriophage library spanning the entire chromosome, were used to localize the map position of the RF2 gene. In addition, the location of the RF1 gene was more precisely defined. The RF2 gene is located at 62.3 min on the chromosome, while the RF1 gene is located at 26.7 min. This approach to mapping cloned genes promises to be a rapid and simple means for determining the gene order of the genome.     

Mycotoxins and fungi in wheat harvested during 1990 in test plots in the state of São Paulo,Brazil

Wheat from two cultivars with contrasting characteristics were harvested in ten experimental plots located in wheat producing areas of the State of São Paulo, Brazil. The samples (10 of each cultivar) were analyzed by a gaschromatographic method for deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), toxins T-2 (T-2) and HT-2, T-2 tetraol, T-2 triol, and by a thin-layer chromatographic method for zearalenone (ZEN), aflatoxins B₁, B₂, G₁, G₂, ochratoxin A and sterigmatocystin. No mycotoxins were detected in 13 samples. DON was found in four samples (0.47–0.59 µg/g), NIV in three samples (0.16–0.40 µg/g), T-2 in two samples (0.40, 0.80 µg/g), DAS in one sample (0.60 µg/g), and ZEN in three samples (0.04–0.21 µg/g). The wheat samples were also examined for the incidence of fungi.Alternaria, Drechslera, Epicoccum andCladosporium were the prevailing genera. Among theFusarium spp.,F. semitectum was present in 19 samples andF. moniliforme in 18 samples. NoF. graminearum was isolated in the samples.Abbreviations DAS diacetoxyscirpenol - DON deoxynivalenol - NIV nivalenol - T-2 T-2 toxin - ZEN zearalenone