Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA.

A cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene (AAF) induced rat hepatocellular carcinoma was screened by synthetic DNA probes deduced from a partial amino acid sequence of glutathione S-transferase P subunit that had been isolated from the tumor by two-dimensional gel electrophoresis. One of the four clones analyzed contained an mRNA region encoding the total amino acid sequence of this enzyme subunit and the complete 3'-noncoding region. The nucleotide sequence indicates that this enzyme subunit has 209 amino acids (calculated Mr=23,307) distinct from other glutathione S-transferase subunits such as Ya and Yc. Comparison of the amino acid sequences between these proteins indicates that glutathione S-transferase P subunit gene has been evolved from the ancestral gene at an earlier stage than the separation of Ya and Yc and that there are at least three domains having a considerable homology with each other in these enzymes. The very large increase of this mRNA in chemically induced hepatocellular carcinoma suggests a characteristic derepression of this gene during hepatocarcinogenesis.     

Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a.

By random transposon Tn5 insertions, we previously identified six virulence-associated SalI fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigella flexneri 2a. In this study, we analyzed the sites of 134 independent Tn5 insertions on four contiguous SalI fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichia coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for full expression of the virulence phenotype of shigellae.     

Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene.

Inactivation of the retinoblastoma gene appears to have a fundamental role in the genesis of retinoblastoma, osteosarcoma, and other malignant tumors. The gene is generally inactivated because of loss-of-function mutations, although epigenetic phenomena, such as hypermethylation of the promoter region, could possibly have the same effect. We investigated the methylation pattern at the 5' end of the retinoblastoma gene, including its promoter region and exon 1, in DNA purified from 56 primary retinoblastomas. We found five tumors with evidence for hypermethylation, all from unilateral, simplex patients. No methylation abnormalities were detected in DNA purified from the leukocytes from these patients. It is interesting that in one of these tumors the hypermethylation was confined to one allele. There were no mutations in a 1,306-bp sequence including the hypermethylated region that might account for the allele-specific hypermethylation. We believe that the hypermethylation of the retinoblastoma gene that we found in these tumors corresponds to the allelic inactivation of the gene, and we speculate that erroneous hypermethylation without alteration of nucleotide sequence occasionally plays a role in the genesis of this cancer. If this is true, then retinoblastomas with hypermethylation might be treatable with chemotherapeutic agents that interfere with methylation of DNA.     

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.     

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

Integration is essential for efficient gene expression of human immunodeficiency virus type 1.

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.     

Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey.

We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivation of gene expression controlled by a long terminal repeat, and viral RNA and protein syntheses, were examined by transfection and infection experiments. Three structural (gag, pol, and env) and two regulatory (tat and rev) gene mutants were not infectious, whereas vif, vpx, and nef were dispensable for infectivity and mutant viruses were highly cytopathic. In transient transfection assays, a rev mutant produced mainly small mRNA species and no detectable virus protein and particles. The transactivation potential of a tat mutant was about 10-fold less than that of wild-type DNA, generating small amounts of virus.