Masashi Takao

Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms

Many biochemical, physiological and behavioural processes show circadian rhythms which are generated by an internal time-keeping mechanism referred to as the biological clock. According to rapidly developing models, the core oscillator driving this clockis composed of an autoregulatory transcription–(post) translation-based feedback loop involving a set of ‘clock’ genes,. Molecular clocks do not oscillate with an exact 24-hour rhythmicity but are entrained to solar day/night rhythms by light. The mammalian proteins Cry1 and Cry2, which are members of the family of plant blue-light receptors (cryptochromes) and photolyases, have been proposed as candidate light receptors for photoentrainment of the biological clock. Here we show that mice lacking the Cry1 or Cry2 protein display accelerated and delayed free-running periodicity of locomotor activity, respectively. Strikingly, in the absence of both proteins, an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, in addition to a possible photoreceptor and antagonistic clock-adjusting function, both proteins are essential for the maintenance of circadian rhythmicity.

A Back-up Glycosylase in Nth1 Knock-out Mice Is a Functional Nei (Endonuclease VIII) Homologue

Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (β-δ) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression.

A new class of DNA photolyases present in various organisms including aplacental mammals.

DNA photolyase specifically repairs UV light‐induced cyclobutane‐type pyrimidine dimers in DNA through a light‐dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S‐transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light‐absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution.

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild‐type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X‐ray irradiation disappeared with time, though more slowly than in the wild‐type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild‐type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV‐sensitive Escherichia coli cells. The gene is altered in a N.crassa mus‐18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild‐type mus‐18 gene complements not only the mus‐18 DNA repair defect of N.crassa, but also confers UV‐resistance on various DNA repair‐deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus‐18 protein was purified from E.coli and found to be an endonuclease for UV‐irradiated DNA. Both cyclobutane pyrimidine dimers and (6‐4)photoproducts are cleaved at the sites immediately 5′ to the damaged dipyrimidines in a magnesium‐dependent, ATP‐independent reaction. This mechanism, requiring a single polypeptide designated UV‐induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.

Isolation and sequence determination of the chicken rhodopsin gene

A chicken genomic library was screened with a bovine opsin cDNA probe. A clone isolated under high stringency hybridization conditions contained DNA sequences highly homologous to all of the five exons of bovine and human opsin genes. Sequence comparison of the putative open reading frame in the chicken DNA fragment of 4.3 kb with bovine opsin cDNA revealed 82% identity for the nucleotide and 87% for the deduced amino acid sequence, indicating that this DNA fragment contains the complete chicken opsin gene. The position of four introns and amino acid sequences at all putative cytoplasmic loops are exactly conserved in chicken and mammals.

Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination-free flow cytometry.

Circulating tumor cells (CTC) are an important biomarker for several solid cancers. Most of the commercially available systems for enumeration of CTC are based on immunomagnetic enrichment of epithelial cell adhesion molecule (EpCAM/CD326)‐expressing CTC before microscopic cell imaging or reverse‐transcription PCR (RT‐PCR). The aim of this study was to establish a practical method for enumeration of CTC using a novel flow cytometer that has a disposable microfluidic chip, which is designed to realize absolute cross contamination‐free measurements and to collect the analyzed cell sample. Although the process of enumeration and labeling of CTC was optimized for this device, the simplified protocol described here could be applied to other flow cytometers. Cultured cancer cells spiked into normal blood were enriched using MACS® EpCAM‐MicroBeads following cell labeling with an allophycocyanin (APC)‐conjugated EpCAM mAb, instead of by intracellular staining of cytokeratins (CK). The EpCAM double‐positive selection/labeling method allows enumeration of intact CTC, maintenance of cellular integrity, and the concomitant performance of a CTC viability test. The combination of the fine‐tuned CTC enrichment process and the cytometric multicolor analysis resulted in a linear relationship between the output cell count and the input cell number from zero to hundreds of cells. In particular, a satisfactory signal/noise ratio was obtained by gate‐exclusion of leukocyte signals using an anti‐CD45 mAb. The entire process had little influence on the viability of the spiked lung cancer cell PC‐9. Measured PC‐9 and breast cancer MCF‐7 cells bearing EpCAM‐MicroBeads, APC‐conjugated EpCAM mAb, and the DNA staining dye SYTO9 grew normally, demonstrating the potential usefulness of the collected samples for further studies. This intact CTC enumeration and analysis procedure (iCeap) would be of great benefit to clinicians by providing them with rapid stratification of antitumor therapy, and to basic researchers by permitting further molecular and cellular characterization of CTC.

Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant

Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei‐like proteins, NEIL1 and NEIL2. A third Nei‐like protein, NEIL3, is composed of an amino‐terminal Nei‐like domain and an unknown carboxy‐terminal domain. In contrast to the other well‐described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1–290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1–290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single‐stranded (ss) DNA but double‐stranded (ds) DNA. The activity is abrogated completely in mutants with an amino‐terminal deletion and at the zinc‐finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.

Demonstration of rod and cone photoreceptors in the lamprey retina by freeze-replication and immunofluorescence

SummaryIn common with other cyclostomata, the Japanese river lamprey (Lampetra japonica) has a retina consisting of distinct types of photoreceptor cells called long and short photoreceptor cells. After freeze-fracture, disc membranes of these photoreceptor cells were characterized in common by a homogeneous distribution of intramembrane particles on the protoplasmic fracture faces, in contrast to those of the myeloid bodies bearing scattering particles.Immunofluorescent examination was applied to the retina with monoclonal antibodies raised against bovine and chicken rhodopsins. Positive immunoreactivity was found to be limited to outer segments of the short cell, leaving the entire body of the long cell and all other components of the retina negative. The results suggest that the short cell is more closely related to a rod-type photoreceptor cell characterized by rhodopsin as its visual pigment.

A Putative Blue-Light Receptor From Drosophila melanogaster

Abstract— A gene encoding a 62.5 kDa homolog of Drosophila melanogaster photolyase was isolated. Purified recombinant protein contained a flavin adenine dinucleotide chromophore. The recombinant protein did not show photolyase activity for either cyclobutane pyrimidine dimers or 6–4 photoproducts in vitro as well as in vivo in Escherichia coli host cells, suggesting that the protein is not a DNA repair enzyme but a blue‐light photoreceptor. Reverse transcription polymerase chain reaction analysis showed that the gene is more expressed in head than in body and that it is more expressed in antennae than in legs, wings and mouth appendages. In a phylogenetic tree of the photolyase family, the Drosophila photolyase homolog is located in a cluster containing 6–4 photolyases and mammalian photolyase homologs, which is only distantly related to the clade of higher plant blue‐light photoreceptors. The mammalian photolyase homologs are more closely related to Drosophila 6–4 photolyase than to the Drosophila photolyase homolog, suggesting different roles of the photolyase homologs.