Research note: Identification of a cDNA homologous to the cell-cycle-controlling cdc2 gene in Acroslphonia duriuscula (Acrosiphoniales, Chlorophyta)

Cyclin‐dependent kinases, the most notable of which is cdc2, are key regulators of the cell cycle and are highly conserved in evolution. We have cloned and analyzed one cDNA containing an open reading frame of 337 amino acids from a multinucleate, multicellular green alga, Acrosiphonia duriuscula (Ruprecht) Collins. The deduced protein, named Adcdc2‐1, showed 51% and 54% amino acid sequence identity to yeast cdc2/cdc28 and human cdc2, respectively. Several domains that characterize the cdc2‐related kinases were identified from this sequence, although the PSTAIRE motif was replaced with PPTTIRE. Furthermore, this protein has conserved Tyr and Thr residues that are sites of phosphorylation in cdc2‐related kinases and are important for regulating kinase activity. The present results suggest that the universal cdc2 is conserved in algae with unique structural characteristics.     

Growth of Ecklonia cava (Laminariales, Phaeophyta) sporophytes transplanted to a locality with different temperature conditions

Transplanting experiments were carried out to determine whether the small type sporophytes with short stipe of Ecklonia cava Kjellman (Laminariales, Phaeophyta) growing in a locality with warm temperatures, change into larger type with a long stipe when transplanted to a locality with cooler temperatures. Juvenile E. cava sporophytes, having a stipe shorter than 5 cm long were collected from Tei in Tosa Bay (southern Japan) (seawater temperature 15–29°C) and transplanted to Nabeta Bay (central Japan) (seawater temperature 13–25°C), where larger type E. cava sporophytes characterized by long stipe (ca 1 m) grow. They were attached to artificial reefs at the sea bottom (9 m depth) in Nabeta Bay to monitor their growth. For comparison, juvenile E. cava sporophytes of almost similar size growing in Nabeta Bay were also transplanted in the same way to the same experimental site. Observations of growth of sporophytes from Tei and Nabeta were carried out monthly for 2 years from November 1995 to October 1997. The transplanted Tei and Nabeta sporophytes showed an increase in stipe length and diameter from winter to spring, whereas almost no increase was observed during summer and autumn. At the end of the study period, the stipe of Nabeta sporophytes reached 25.6 cm in length and 17.0 mm in diameter, whereas that of Tei sporophytes reached 11.1 cm in length and 11.2 mm in diameter. The primary blade length was 16.0 cm in Nabeta sporophytes, whereas it was 5.5 cm in Tei sporophytes. Thus, Tei sporophytes still remained smaller than Nabeta sporophytes even under the same environmental conditions.     

Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.     

Impaired auditory-vestibular functions and behavioral abnormalities of Slitrk6-deficient mice

A recent study revealed that Slitrk6, a transmembrane protein containing a leucine-rich repeat domain, has a critical role in the development of the inner ear neural circuit. However, it is still unknown how the absence of Slitrk6 affects auditory and vestibular functions. In addition, the role of Slitrk6 in regions of the central nervous system, including the dorsal thalamus, has not been addressed. To understand the physiological role of Slitrk6, Slitrk6-knockout (KO) mice were subjected to systematic behavioral analyses including auditory and vestibular function tests. Compared to wild-type mice, the auditory brainstem response (ABR) of Slitrk6-KO mice indicated a mid-frequency range (8-16 kHz) hearing loss and reduction of the first ABR wave. The auditory startle response was also reduced. A vestibulo-ocular reflex (VOR) test showed decreased vertical (head movement-induced) VOR gains and normal horizontal VOR. In an open field test, locomotor activity was reduced; the tendency to be in the center region was increased, but only in the first 5 min of the test, indicating altered adaptive responses to a novel environment. Altered adaptive responses were also found in a hole-board test in which head-dip behavior was increased and advanced. Aside from these abnormalities, no clear abnormalities were noted in the mood, anxiety, learning, spatial memory, or fear memory-related behavioral tests. These results indicate that the Slitrk6-KO mouse can serve as a model of hereditary sensorineural deafness. Furthermore, the altered responses of Slitrk6-KO mice to the novel environment suggest a role of Slitrk6 in some cognitive functions.     

CRYOPRESERVATION OF THE CONCHOCELIS OF PORPHYRA (RHODOPHYTA) BY APPLYING A SIMPLE PREFREEZING SYSTEM1

The conchocelis cells of four strains of Porphyra yezoensis Udea and four other Porphyra species were cryopreserved in liquid nitrogen (LN) using a programmable freezer or a simple prefreezing system, which consisted of a styrofoam box and a deep-freezer at ?40° C. The cells differed in their freezing tolerance but survived maximally when prefrozen to ?40° C in a cryoprotective solution composed of 10% dimethylsulfoxide and 0.5 M sorbitol in 50% seawater. The cryopreservation was successfully performed by applying the simple prefreezing system as well as by a programmable freezer. Conchocelis cells thawed from the LN temperature formed colonies and retained the ability to form conchospores that grew into gametophytic thalli. This technique using a simple prefreezing system will accelerate the spread of Porphyra cryopreservation.     

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE ENDANGERED SPECIES PORPHYRA TENERA (BANGIALES,RHODOPHYTA)1

Porphyra tenera Kjellman, widely cultivated in nori farms before the development of artificial seeding, is currently listed as an endangered species in Japan. To confirm whether a wild‐collected gametophytic blade was P. tenera or the closely related species P. yezoensis Ueda, morphological observations and molecular analyses were made on the pure line HGT‐1 isolated from a wild blade. This pure line was identified as P. tenera based on detailed morphological features. Sequences of the nuclear internal transcribed spacer region 1 and the plastid RUBISCO spacer revealed that P. tenera HGT‐1 was clearly different from P. yezoensis f. narawaensis Miura, the main species cultivated in Japan. PCR‐RFLP analysis of the internal transcribed spacer region was found to be a convenient method for rapid discrimination between P. tenera and cultivated P. yezoensis. The restriction patterns generated by the endonucleases Dra I and Hae III were useful for differentiating between both gametophytic and conchocelis stages of P. tenera HGT‐1 and P. yezoensis f. narawaensis strains. Thus, PCR‐RFLP analysis will serve as a valuable tool for rapid species identification of cultivated Porphyra strains, culture collections of Porphyra strains for breeding material and conservation of biodiversity, and, as codominant cleaved amplified polymorphic sequence markers for interspecific hybridization products between P. tenera and P. yezoensis f. narawaensis. Under the same culture conditions, rate of blade length increase and the blade length‐to‐width ratio were lower in P. tenera HGT‐1 than in P. yezoensis f. narawaensis HG‐4. The HGT‐1 became mature more rapidly than HG‐4 and had thinner blades.     

Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity.

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.     

Micronucleus induction in mouse bone marrow by phenacetin administered intraperitoneally or orally

The extent and time course of induction of micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow were examined after administration of phenacetin as an insoluble suspension in olive oil by intraperitoneal injection (i.p.) or gastric intubation (p.o.) to 2 strains of mice, MS/Ae and CD-1, at doses up to 1200 mg/kg. The toxicity of phenacetin and the sensitivity of micronucleus induction differed in the 2 strains, but there was little difference in the extent of MNPCEs induced by the 2 administration routes.