Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

Two genes controlling acute phase responses by the antitumor polysaccharide,lentinan

Lentinan, a -1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) andMus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence lenght polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 andltn1.2) responsible for DT-APR.ltn1.1 is closely linked toD3Mit11 on chromosome 3 andltn1.2 toD11Nds9 on chromosome 11 (P<0.001). The linkage analysis also suggested thatltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that theltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.     

Amphiphilic block copolymer with a molecular recognition site: induction of a novel binding characteristic of amylose by self-assembly of poly(ethylene oxide)-block-amylose in chloroform

Solution properties of amphiphilic methoxy poly(ethylene oxide)-block-amyloses (MPEO-amyloses) in chloroform were investigated by SLS and DLS. The results indicated that MPEO-amyloses dissolved in chloroform containing 2 wt % DMSO by their self-associations. The complexation of MPEO-amylose with methyl orange (MO) was significantly enhanced in the amylose domain of the associate in chloroform. The blue shift of the maximum absorption and strong induced circular dichroism with exciton coupling were observed in the MPEO-amylose MO complex in chloroform. The self-assembly of MPEO-amylose in chloroform shows a unique feature for binding with MO. MPEO-block-amylose is a novel amphiphilic polymer with amylose as a molecular recognition site.     

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.     

Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Removal characteristics of high load ammonia gas by a biofilter seeded with a marine bacterium, Vibrio alginolyticus

When the cells of the newly isolated marine bacterium, Vibrio alginolyticus, were inoculated on to an inorganic packing material in biofilter, and a load of ammonia of 2.4–22.5 g-N kg^–1dry packing material was introduced continuously under non-sterile conditions, the average amount of NH₃removed exceeded 85% over 61-d operation. The maximum removal capacity and the complete removal capacity were 22.8 g-N kg^–1dry packing material dand 18.6 g-N kg^–1dry packing material d, respectively, which were about four times larger than those obtained in autotrophic nitrifying sludge inoculated on the same packing material.     

EST analysis of gene expression in testis of the ascidian Ciona intestinalis

To explore the gene expression underlying spermatogenesis, a large-scale analysis has been done on the cDNAs from testis of the ascidian, Ciona intestinalis. A set of 5,461 expressed sequence tags was analyzed and grouped into 2,806 independent clusters. Approximately 30% of the clusters showed significant sequence matches to the proteins reported in DDBJ/GenBank/EMBL database including a set of proteins closely related to the gene regulation during spermatogenesis, functional and morphological changes of spermatogenic cells during spermiogenesis, and physiological functions of sperm, as well as those with housekeeping functions commonly expressed in other cells. Some clones show similarities to the proteins present in vertebrate lymphocytes, suggesting a primitive immune system in ascidians. We have also found some genes that are known to participate in hormonal regulation of spermatogenesis in vertebrates. The large majority of the genes expressed in Ciona testis show no significant matches to known proteins and the further analysis of these genes may shed new light on the molecular mechanism of spermatogenesis and sperm functions.     

Update of MAGEST: Maboya Gene Expression patterns and Sequence Tags

MAGEST is a database for maternal gene expression information for an ascidian, Halocynthia roretzi. The ascidian has become an animal model in developmental biological research because it shows a simple developmental process, and belongs to one of the chordate groups. Various data are deposited into the MAGEST database, e.g. the 3′- and 5′-tag sequences from the fertilized egg cDNA library, the results of similarity searches against GenBank and the expression data from whole mount in situ hybridization. Over the last 2 years, the data retrieval systems have been improved in several aspects, and the tag sequence entries have increased to over 20 000 clones. Additionally, we constructed a database, translated MAGEST, for the amino acid fragment sequences predicted from the EST data sets. Using this information comprehensively, we should obtain new information on gene functions. The MAGEST database is accessible via the Internet at http://www.genome.ad.jp/magest/.     

Effect of volume expansion on hemodynamic variables in nephrectomized dogs

We have previously demonstrated that blood pressure elevation by acute blood volume expansion is volume-dependent during the infusion period and resistance-dependent in the post-infusion period in normal anesthetized dogs, and that such an increase in blood pressure is associated with a potentiation of the pressor response to norepinephrine. To evaluate the possible renal contribution to these hemodynamic changes, blood volume expansion was performed for 1 h with dextran dissolved in lactated Ringer's solution (20 ml/kg) in 15 nephrectomized dogs. The mean blood pressure, cardiac output and total peripheral resistance at the end of infusion were 126%, 225% and 60%, respectively; 3 h after volume expansion they were 126%, 151%, and 92% respectively. However, in 4 dogs, there was an increase in mean blood pressure (138%) 3 h after volume expansion. This was thought to result from an increase in the total peripheral resistance (133%) associated with the recovery of cardiac output (106%). The pressor response to norepinephrine (0.5 microgram/kg) was potentiated after volume expansion. These results indicate that the handling of volume by the kidney contributed to the maintenance of an elevated level of cardiac output. However, nephrectomy did not seem to interfere with the hemodynamic switching of the causative factor for blood pressure elevation from increased cardiac output to increased total peripheral resistance. Neither was the potentiation of pressor response to norepinephrine affected.