Typing of urinary JC virus DNA offers a novel means of tracing human migrations

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

Lactone-ring-cleaving enzyme: Genetic analysis, novel RNA editing, and evolutionary implications

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA–DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of β-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis

Ro09-0198 is a tetracyclic polypeptide of 19 amino acids that recognizes strictly the structure of phosphatidylethanolamine (PE) and forms a tight equimolar complex with PE on biological membranes. Using the cyclic peptide coupled with fluorescence-labeled streptavidin, we have analyzed the cell surface localization of PE in dividing Chinese hamster ovary cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membrane-bound cyclic peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced scrambling of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex treatment had no effect on furrowing, rearrangement of microtubules, and nuclear reconstitution, but specifically inhibited both actin filament disassembly at the cleavage furrow and subsequent membrane fusion. These results suggest that the redistribution of the plasma membrane phospholipids is a crucial step for cytokinesis and the cell surface PE may play a pivotal role in mediating a coordinate movement between the contractile ring and plasma membrane to achieve successful cell division.

Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter.

The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997) Genes Dev. 11, 345–357]. Although cac1Δ cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual cac1Δ cells switch from transcriptionally “off” to transcriptionally “on” more often per cell cycle than wild-type cells. In addition, cac1Δ cells were defective for transcriptional silencing near internal tracts of C1–3A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription–PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C1–3A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the Escherichia coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

Oncogene-dependent apoptosis is mediated by caspase-9

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as reflected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked drug-induced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.

Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ70 factors of bacterial RNA polymerases in Arabidopsis thaliana

Genes for σ-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1, SIG2, and SIG3, for polypeptides possessing amino acid sequences for domains conserved in σ70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription

Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an ≈350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an ≈600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion

We have reported some type II restriction-modification (RM) gene complexes on plasmids resist displacement by an incompatible plasmid through postsegregational host killing. Such selfish behavior may have contributed to the spread and maintenance of RM systems. Here we analyze the role of regulatory genes (C), often found linked to RM gene complexes, in their interaction with the host and the other RM gene complexes. We identified the C gene of EcoRV as a positive regulator of restriction. A C mutation eliminated postsegregational killing by EcoRV. The C system has been proposed to allow establishment of RM systems in new hosts by delaying the appearance of restriction activity. Consistent with this proposal, bacteria preexpressing ecoRVC were transformed at a reduced efficiency by plasmids carrying the EcoRV RM gene complex. Cells carrying the BamHI RM gene complex were transformed at a reduced efficiency by a plasmid carrying a PvuII RM gene complex, which shares the same C specificity. The reduction most likely was caused by chromosome cleavage at unmodified PvuII sites by prematurely expressed PvuII restriction enzyme. Therefore, association of the C genes of the same specificity with RM gene complexes of different sequence specificities can confer on a resident RM gene complex the capacity to abort establishment of a second, incoming RM gene complex. This phenomenon, termed “apoptotic mutual exclusion,” is reminiscent of suicidal defense against virus infection programmed by other selfish elements. pvuIIC and bamHIC genes define one incompatibility group of exclusion whereas ecoRVC gene defines another.

Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice

We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein–coupled receptor that stimulates inositol trisphosphate receptor and Ca2+ influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in Bacillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.