Kazunori Kondo

hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1.

Proteins of the tolloid/bone morphogenetic protein (BMP)‐1 family play important roles in the differentiation of cell fates. Among those proteins are BMP‐1, which plays a role in cartilage and bone formation in mammals, the TOLLOID protein, which is required for the establishment of the dorsoventral axis of Drosophila embryos and BP10/SpAN, which are thought to act in the morphogenesis of sea urchins. These proteins have some properties in common. First, they contain the astacin metalloprotease domain, the CUB domain and the epidermal growth factor‐like domain. Second, they are expressed in embryos at stages expected for their role in cell differentiation. Third, at least BMP‐1 and TOLLOID are thought to interact with proteins of the transforming growth factor‐beta family. We report that the hch‐1 gene of the nematode Caenorhabditis elegans encodes a tolloid/BMP‐1 family protein. The protein has the characteristic domains common to the tolloid/ BMP‐1 family. Like other members of the family, it is expressed in embryos. However, the phenotype of hch‐1 mutants shows that it is required for normal hatching and normal migration of a post‐embryonic neuroblast. Furthermore, in spite of its expression in embryogenesis, it is not required for the viability of embryos. These results show new functions of the tolloid/BMP‐1 family proteins and give insight into their evolution.

Molecular cloning and characterization of avian N-methyl-D-aspartate receptor type1 (NMDA-R1) gene

Birds have several advantages in the study of memory formation, as imprinting and passive avoidance behaviors in chick are often used as model systems. However, the primary structure of the bird N-methyl-d-aspartate (NMDA) responsive glutamate receptor, which is assumed to play a critical role in memory formation, has not been determined. In this report we describe the cDNA cloning of a subunit of NMDA receptors (NMDA-R1) from duck and analysis of its structure and distribution in the brain. The N-terminal 898 amino acids of the NMDA-R1 were well conserved between duck and mammals, but the homology was completely lost in the C-terminus. In situ hybridization showed that the duck NMDA-R1 gene was expressed throughout the brain as it is in mammals.

Characterization of a novel rice kinesin O12 with a calponin homology domain

Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.

Molecular cloning of the kainate-binding protein and calmodulin genes which are induced by an imprinting stimulus in ducklings

For the formation of imprinting in birds, protein synthesis is known to be essential in the medial hyperstriatum ventrale (MHV) of the forebrain after presentation of an imprinting stimulus. We have searched for the genes whose expressions are increased in ducklings MHV during formation of imprinting, and identified kainate-binding protein and calmodulin genes. This may reflect the formation of glutamatergic pathways in MHV.

DIFFERENTIAL EXPRESSION OF INDIVIDUAL SUPPRESSOR TRNATRP GENE FAMILY MEMBERS IN VITRO AND IN VIVO IN THE NEMATODE CAENORHABDITIS ELEGANS

ABSTRACT Eight different amber suppressor tRNA (suptRNA) mutations in the nematode Caenorhabditis elegans have been isolated; all are derived from members of the tRNATrp gene family (K. Kondo, B. Makovec, R. H. Waterston, and J. Hodgkin, J. Mol. Biol. 215:7–19, 1990). Genetic assays of suppressor activity suggested that individual tRNA genes were differentially expressed, probably in a tissue- or developmental stage-specific manner. We have now examined the expression of representative members of this gene family both in vitro, using transcription in embryonic cell extracts, and in vivo, by assaying suppression of an amber-mutated lacZ reporter gene in animals carrying different suptRNA mutations. Individual wild-type tRNATrp genes and their amber-suppressing counterparts appear to be transcribed and processed identically in vitro, suggesting that the behavior of suptRNAs should reflect wild-type tRNA expression. The levels of transcription of different suptRNA genes closely parallel the extent of genetic suppression in vivo. The results suggest that differential expression of tRNA genes is most likely at the transcriptional rather than the posttranscriptional level and that 5′ flanking sequences play a role in vitro, and probably in vivo as well. Using suppression of a lacZ(Am) reporter gene as a more direct assay of suptRNA activity in individual cell types, we have again observed differential expression which correlates with genetic and in vitro transcription results. This provides a model system to more extensively study the basis for differential expression of this tRNA gene family.

Photocontrol of Calmodulin Interaction with Target Peptides using Azobenzene Derivative

Calmodulin (CaM), a physiologically important Ca(2+)-binding protein, participates in numerous cellular regulatory processes. It is dumbbell shaped and contains two globular domains connected by a short alpha-helix. Each of the globular domains has two Ca(2+)-binding sites, the EF hands. CaM undergoes a conformational change upon binding to Ca(2+), which enables it to bind to specific proteins for specific responses. Here, we successfully photocontrolled CaM binding to its target peptide using the photochromic compound N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis-trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. In order to specifically incorporate PAM, CaM mutants having reactive cysteine residues in the functional region were prepared; PAM was stoichiometrically incorporated into the cysteine residues in these mutants. Further, we prepared the target peptide, M13, fused with yellow fluorescent protein (YFP) to monitor the CaM-M13 peptide interaction. The binding of the PAM-CaM mutants, N60C, D64C and M124C, to M13-YFP was reversibly photocontrolled upon UV-VIS light irradiation at appropriate Ca(2+) concentrations.

Characterization of the Plant-Specific Rice Kinesins

Kinesin is a motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics and signal transduction. Kinesin converts chemical energy from ATP into mechanical force. Kinesin family is classified into some subfamilies. Some species of kinesin derived from vertebrate have been well studied. However, not so many studies for kinesins of plants have been done yet. Recently, the genome sequences of rice were completed. Bioinformatical analyses revealed that at least 41 kinesin-related proteins were encoded on the rice genome. In this study, we focused on the two rice kinesins; 1. O12 that has a calponin homology domain, 2. K23 that belongs to At1 subfamily in kinesin-7. The cDNAs of the kinesin motor domain was subcloned into expression vector pET and transformed into E. coli BL21 (DE3). kinesin motor domains were expressed and purified by Co-NTA column. The biochemical characterizations of the two rice kinesins were studied. The microtubule-dependent ATPase activity of the two rice kinesins motor domains were 30∼60-fold lower than that of conventional kinesin. Kinetic analyses using stopped-flow demonstrated that ATP binding to O12 in the absence of microtubule was extremely slow compared with that of conventional kinesin. While, ATP binding to K23 was not accelerated by microtubule. Furthermore, interestingly ATPase activity of O12 in the absence microtubule regulated by actin. The O12-tail fused with GFP was observed to localize in the actin filament in the onion cell. The two plant specific rice kinesin O12 and K23 were shown to have unique enzymatic properties.