Tamotsu Hashimoto-Gotoh

Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues.

Using the differential hybridization screening method between osteoblastic and fibroblastic cells, a cDNA clone coding for an osteoblast specific protein, named OSF-1, consisting of 168 amino acid residues including a possible 32 amino acid long leader sequence, was isolated from murine osteoblastic cell line MC3T3-E1. The OSF-1 gene was shown by Northern blotting analysis to be expressed in mouse calvarial osteoblast-enriched cells and in mouse brain tissues, but not in thymus, spleen, kidney, liver, lung, testis or heart. The human counterpart was also found in cDNA libraries from human osteosarcoma cell line MG63 and normal brain tissues. DNA sequence analysis revealed four amino acid sequence differences between the mouse and human, of which only one is located in the mature protein. This extremely high sequence conservation suggests that OSF-1 plays a fundamental role in bone and brain functions.

Random replication and random assortment model for plasmid incompatibility in bacteria.

Abstract To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.

Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior

KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimers disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.

Bone mass increase specific to the female in a line of transgenic mice overexpressing human osteoblast stimulating factor-1

We have reported that transgenic mice overexpressing human osteoblast stimulating factor-1 (osf1) under the control of the human osteocalcin promoter have a significantly higher bone mineral content and density than nontransgenic littermates. Consequently, bone mass loss due to estrogen deficiency was compensated for in ovariectomized female mice. Here, we show that in this transgenic line, the bone mass increase was evident in female, but not male, mice, as evaluated using the ash assay, double-emission X-ray analysis, and calcein double-labeling to determine the bone formation rate. To elucidate a possible influence on gene expression, we analyzed genomic structures of the inserted transgene and its flanking regions in mouse chromosomes. The results revealed that the transgene was integrated in the mouse repetitive sequences, 234-bp-long Γ-satellite repeats, as inverted multiple (5 + 8) copies. Twelve copies at most seemed to be functional, but no direct evidence supporting female-specific mRNA synthesis of the transgene was obtained.

Characterization of the spontaneous elimination of streptomycin sensitivity (SmS) on high-copy-number plasmids: SmS-enforcement cloning vectors with a synthetic rpsL gene.

The strAS or rpsL+ gene, encoding a ribosomal protein, S12, expresses its streptomycin-sensitivity (SmS) phenotype dominantly over strAR or rpsL- gene. Therefore, strAR cells that harbor plasmids with strAS alleles are phenotypically SmS. It was found that the SmS phenotype is unstable, and such cells eventually switch to the Sm-resistance (SmR) phenotype, especially when the strAS gene was cloned on high-copy-number (HCN) plasmids. It seemed that the strA gene cloned on HCN plasmids was toxic to Escherichia coli host cells and, during prolonged cultivation, plasmids with an inactivated strAS gene, mostly carrying insertion sequence elements, such as IS1, IS5 and gamma delta, were selected. The instability of the strA gene was particularly enhanced when the Val51 residue in the middle of S12 protein was replaced by Leu, suggesting enhanced toxicity of the altered S12. Since the strAS gene was stably maintained throughout approx. 100 cell doublings when its expression was abolished, most probably it is the gene product rather than the nucleotide sequence itself that is responsible for the instability of strA gene on HCN plasmids. To improve the stability of the SmS phenotype, the previously reported ampicillin-resistance-conferring and SmS-enforcing plasmid vector, pHSG670, was reconstructed. The resulting vector, pHSG683, confers chloramphenicol resistance, enforces SmS on strAR and supE- host bacteria, and has multiple cloning sites within the coding region of synthetic rpsL gene. When pHSG683 DNA was prepared from strAR and sup+ cells grown in tryptophan-rich medium with Cm and Sm, less than 10(-6) plasmids failed to enforce SmS on strAR and supE- cells in tryptophan-less medium with Cm.(ABSTRACT TRUNCATED AT 250 WORDS)

KF-1 Ubiquitin Ligase: An Anxiety Suppressor

Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located on the endoplasmic reticulum (ER), may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior against light or heights. It is surmised that KF-1 degrades some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinsons disease (PD). Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice may be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds.

KF-1 Ubiquitin Ligase: Anxiety Suppressor Model

Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.

Differential expression of presenilin-α and -β (PSα and PSβ) in Xenopus laevis: embryonic phosphorylation of PSα

Abstract Mutations in genes encoding the highly homologous proteins, presenilin-1 and -2 (PS1 and PS2), are linked to the development of early-onset Alzheimers disease. On the other hand, presenilins are known to play a critical role(s) in cell fate decisions during embryonic development in Caenorhabditis elegans . The messenger RNAs (mRNAs) of amphibian presenilin homologues PSα and PSβ are most abundantly synthesized in the brain and the ovary, but are differentially degraded upon oocyte maturation and at the midblastula transition (MBT), respectively. In this study, we examined the spatiotemporal distribution of PSα and PSβ proteins and their post-translational modification. The results were essentially consistent with the mRNA data and revealed moreover that PSα was present exclusively as processed molecules in the early embryos, while PSβ was present mainly as unprocessed molecules (90%). Furthermore, the C-terminal fragment (CTF) of PSα was phosphorylated upon oocyte maturation and dephosphorylated at MBT, while no phosphorylation of the PSβ CTF was detectable. Human PS1 CTF exogenously injected was also phosphorylated in Xenopus oocytes induced to mature in vitro by progesterone treatment. Two phosphorylation loci were mapped at Thr 320 and Ser 334 in the hydrophilic loop region of PSα. Our results suggest that PS1 and PS2 may play different roles under physiological conditions despite their high structural similarity.

Detection of exonuclease activities in restriction endonuclease preparations using an enforcement plasmid for kanamycin-resistance selection

A new enforcement (kyosei-) cloning plasmid vector, designated pKF4, was constructed which confers kanamycin resistance (KmR) and enforces streptomycin sensitivity (SmS). Since it is important to employ restriction endonuclease (ENase) preparations free of exonuclease (Exo) activities for effective use of the kyosei-cloning procedure [Hashimoto-Gotoh et al., Gene 137 (1993) 211-216], ENases such as HpaI and SmaI purchased from four different suppliers were examined for possible contamination by exonucleases using pKF4. The plasmid DNA was digested with either ENase, ligated and transformed into Escherichia coli mutants, rpsL, supE, trpR. With pKF4 intact DNA (approx. 8 ng), 2.3 x 10(5) KmR transformant and four KmRSmR transformant colonies were obtained; the efficiency of transformation plating (ETP) of the intact DNA was approx. 2 x 10(-5). On the other hand, the ETP values were significantly higher by one to three orders of magnitude when cut and re-joined DNAs were used under the same conditions in six out of eight ENase samples examined. The results indicate that even commercially supplied ENases, that should have passed their quality control test, could have been contaminated with Exo sufficient to interfere with effective use of the kyosei-cloning method. Therefore, it is advisable to examine ENase samples for possible contamination with Exo activities, in order to choose the right preparations for this method at the beginning of the experiments.