Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.     

Proliferation and Teratogenicity of Aino Virus in Chick Embryos

Aino virus (AIV; JaNAr 28 strain) 10³ TCID₅₀/0.2 ml was inoculated in the yolk sac of 8-day-old chick embryos. Recovery and titration of the virus from various organs including the central nervous system (CNS) and skeletal muscle were performed at 2, 4, 7, 10 and 13 days after inoculation (PI). AIV was systemically disseminated and proliferated even 2 days PI. The titers of the recovered virus from the CNS and from skeletal muscle was the highest at 4 days PI and declined with time, whereas hydranencephaly, arthrogryposis and cerebellar hypoplasia developed at 7 days PI and gradually progressed until 13 days PI.     

Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens

 We investigated the optimum conditions for the formation of nitrile hydratase (NHase), which acts on indole-3-acetonitrile, in Agrobacterium tumefaciens. Good inducers for enzyme formation have been found to be roughly classified into three representative types of amides such as pivalamide, crotonamide and ɛ-caprolactam. When the strain was cultivated in the optimum culture medium containing ɛ-caprolactam as an inducer, in particular, the specific activity of NHase in the culture was 13 000 times higher than that without addition of amides, nitriles or acids. In this case, NHase formed accounted for 12% of the total cellular soluble protein. The purified NHase did not act on ɛ-caprolactam, and ɛ-caprolactam was not degraded during the cultivation by the strain, suggesting that ɛ-caprolactam seems to keep driving the NHase induction mechanism. Received: 3 March 1995/Received revision: 13 July 1995/Accepted: 7 September 1995     

Genomic imprinting of the human serotonin-receptor (HTR2) gene involved in development of retinoblastoma.

Epidemiological and genetic studies of retinoblastoma (RB) suggested that imprinted genes might be genetically linked to the RB gene. In this study, we found that the human serotonin-receptor, HTR2, gene, which had been mapped nearby the RB gene on chromosome 13, was expressed only in human fibroblasts with a maternal allele and not in cells without a maternal allele. The 5' genomic region of the human HTR2 gene was cloned by PCR-mediated method. Only the 5' region of the gene was methylated in cells with the maternal gene, and it was not methylated in cells without the maternal gene. A polymorphism of PvuII site of the gene was also found and useful for the segregation analysis in a family of a RB patient and for analysis of loss of heterozygosity on chromosome 13 in tumor and its parental origin. These results suggest that the human HTR2 gene might be affected by genomic imprinting and that exclusive expression of the maternal HTR2 gene may be associated with the delayed occurrence of RB, which had lost the maternal chromosome 13.     

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.     

Genetic Mapping of Four Mouse bHLH Genes Related toDrosophilaProneural Geneatonal

It has been shown that mammalian neurogenesis is partly controlled by multiple basic helix–loop–helix (bHLH) genes, as inDrosophila.Recently, mouse homologs ofDrosophila atonal,a proneural gene encoding a bHLH protein required for chordotonal organ and photoreceptor development, have been characterized to obtain further insights into the molecular nature of mammalian neurogenesis. Here, to assess their potential involvement in genetic neural disorders, we have determined genetic map positions for four mouseatonal-related genes,Atoh1, Atoh2, Atoh3,andNdrf,which encode MATH-1, MATH-2, MATH-3, and NDRF, respectively. Interspecific backcross analysis indicated thatAtoh1andAtoh2were located in separate positions of Chr 6 and thatAtoh3andNdrfwere mapped to Chr 10 and Chr 11, respectively. Thus, these structurally related genes are located separately on multiple chromosomes.     

An Integrated Map with Cosmid/PAC Contigs of a 4-Mb Down Syndrome Critical Region

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.     

Spatial distribution of cpDNA and mtDNA haplotypes in a hybrid zone betweenPinus pumila andP. parviflora var.pentaphylla (Pinaceae)

Pinus species exhibit paternal chloroplast inheritance and maternal mitochondrial inheritance. This independent inheritance of two cytoplasmic genomes provides an exceptional environment for discriminating female (seeds) and male (pollen) components of gene flow across hybridizing species. We obtained mitochondrial genetic markers diagnostic toP. parviflora var.pentaphylla andP. pumila by PCR amplification of the intron ofnad1 on mtDNA, and examined the spatial-distribution pattern of the mtDNA haplotypes in a hybrid zone betweenP. parviflora var.pentaphylla andP. pumila in the Tanigawa Mountains of Japan. These data, in conjunction with previous information on cpDNA haplotypes and needle morphology, revealed contrastive patterns of introgression of two cytoplasmic genomes. CpDNA introgression has occurred uni-directionally fromP. parviflora var.pentaphylla toP. pumila. Conversely, mtDNA introgression has occurred in the opposite direction, fromP. pumila toP. parviflora var.pentaphylla. Levels of introgression are roughly equivalent for cpDNA and mtDNA. The contrastive spatial distribution pattern of cpDNA and mtDNA haplotypes could be caused by differential movement of seeds and pollen for interspecific genetic exchange.