Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization

The complete nucleotide sequence of hepatitis B virus (HBV) DNA from Dane particles of subtype adr was determined. The 3215-bp sequence showed the presence of genes for the surface antigen (226 amino acids) and core antigen (183 amino acids), in addition to two (long and small) open reading frames (ORFs) capable of coding the 843 and 154 amino acids. These ORFs differed from those of the other adr clones so far reported [Ono et al., Nucl. Acids Res. 11 (1983) 1747–1757; Fujiyama et al., Nucl. Acids Res. 11 (1983) 4601–4610]. The gene organization of HBV DNA was found to be well conserved irrespective of subtype. The direct repeat of the undecanucleotide sequence near the 5′ ends of the short (S) and long (L) strands of HBV DNA and the two small direct repeats between both 5′ ends were found to be characteristic structures.     

Possible complex translocation t(9; 14; 13)(q12;pl?;q31) in mother of a child with 9p-trisomy syndrome

Summary A mentally retarded boy with trisomy 9p is described. This trisomy arose through aberrant segregation of translocation chromosome during meiosis in his mother, who has a complex translocation involving chromosomes 9, 13, and 14. Based on both G-, Q-banding, and DNA replication patterns, the patient's karyotype was identified as 47,XY,-13, +(9;13) (9pter9q12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter). We suppose his mother's karyotype to be 46,XX,-9,-13,-14,+t(9;13) (9pterq12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter), +t(9;14) (9qter9q12::14pl?14qter). His phenotypically normal brother and sister are also carriers, having the same translocation chromosome as their mother. Clinical findings of the patient included peculiar face with hypertelorism, prominent nasal bridge and deformed helix, marked delay of osseous development, hypoplastic phalangia in fingers and toes, dysplastic nails and absence of digital triradii.     

Suppression of nucleic acid synthesis in chromatin of Spirogyra during conjugation process

Marked changes in RNA- and DNA-synthesizing activities of thechromatin fraction were found to be connected with the conjugationprocess of Spirogyra sp. RNA-synthesizing activity of the chromatin fraction was muchlower in conjugating and zygote cells than in vegetatively growingcells. No significant differences were observed in the templateactivities of purified DNA prepared from vegetatively growing,conjugating, and zygote cells. The activities of DNA purifiedfrom vegetatively growing and zygote cells and calf thymus werestrongly inhibited at different levels by binding with histonesprepared from vegetatively growing and zygote cells. DNA-synthesizingactivity of the chromatin fraction of conjugating or zygotecells was much lower than that of vegetatively growing cells. (Received December 24, 1971; )     

Molecular clock of silent substitution: At least six-fold preponderance of silent changes in mitochondrial genes over those in nuclear genes

Summary For each of eleven different types of nuclear genes, comparisons of the protein coding sequences were made between human, mouse and rat pairwisely, and the evolutionary rate of silent substitution, v _S ^nucl. , was estimated. It is shown that the v _S ^nucl. is not only very high (=5.37×10^–9/site/yr), but also approximately uniform for different genes regardless of the types, which confirms our previous results (Miyata et al. 1980b). This is in sharp contrast to the rate of protein evolution which differes greatly from protein to protein. Furthermore the v _S ^nucl. is shown to be approximately constant with respect to different divergence times, at least within a short time period (75 Myr). Based on these observations, we propose a new molecular clock which has several advantages over a protein clock. Using this clock, we show that the rate of amino acid replacement in the immunoglobulin Ck gene of b4 rabbit is unexpectedly high, almost comparable to the rate of silent changes. This rate may be the highest one for protein evolution that we know so far. We further examine the rate of silent substitutions in mitochondrial genes comparing mouse and rat. Surprisingly the rate is extremely high (35×10^–9/site/yr), at least 6-times as high as the corresponding rate of nuclear genes. Based on the estimate, we discuss a possible origin of the rapid rate found in mitochondrial DNA.     

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney

BACKGROUND: Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. METHODS: In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. RESULTS: Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70-80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 x 10(5) relative light units (RLU)/g tissue; 50%: 31.3 x 10(5) RLU/g tissue; 100%: 57.9 x 10(5) RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. CONCLUSIONS: Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease.     

Cloning and characterization of two xyloglucanases from Paenibacillus sp. strain KM21

Two xyloglucan-specific endo-beta-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley beta-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.     

Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.     

Genogroup II noroviruses efficiently bind to heparan sulfate proteoglycan associated with the cellular membrane

Norovirus (NV), a member of the family Caliciviridae, is one of the important causative agents of acute gastroenteritis. In the present study, we found that virus-like particles (VLPs) derived from genogroup II (GII) NV were bound to cell surface heparan sulfate proteoglycan. Interestingly, the VLPs derived from GII were more than ten times likelier to bind to cells than were those derived from genogroup I (GI). Heparin, a sulfated glycosaminoglycan, and suramin, a highly sulfated derivative of urea, efficiently blocked VLP binding to mammalian cell surfaces. The reagents known to bind to cell surface heparan sulfate, as well as the enzymes that specifically digest heparan sulfate, markedly reduced VLP binding to the cells. Treatment of the cells with chlorate revealed that sulfation of heparan sulfate plays an important role in the NV-heparan sulfate interaction. The binding efficiency of NV to undifferentiated Caco-2 (U-Caco-2) cells differed largely between GI NV and GII NV, whereas the efficiency of binding to differentiated Caco-2 (D-Caco-2) cells did not differ significantly between the two genogroups, although slight differences between strains were observed. Digestion with heparinase I resulted in a reduction of up to 90% in U-Caco-2 cells and a reduction of up to only 50% in D-Caco-2 cells, indicating that heparan sulfate is the major binding molecule for U-Caco-2 cells, while it contributed to only half of the binding in the case of D-Caco-2 cells. The other half of those VLPs was likely to be associated with H-type blood antigen, suggesting that GII NV has two separate binding sites. The present study is the first to address the possible role of cell surface glycosaminoglycans in the binding of recombinant VLPs of NV.