Aizo Matsushiro

Complementary DNA Cloning and Characterization of Pearlin, a New Class of Matrix Protein in the Nacreous Layer of Oyster Pearls

Abstract: Calcified shell layer is composed of two polymorphs of CaCO3, aragonite or calcite, and an organic matrix. The organic matrix consists of EDTA-soluble and insoluble fractions. These fractions are thought to regulate the formation of the elaborate shell structure. After decalcification of powdered pearl with 0.3 M EDTA, an EDTA-insoluble fraction was extracted with 0.3 M EDTA/8 M urea. This extraction step enabled us to purify a new class of EDTA-insoluble protein, Pearlin, almost homogeneously. Pearlin has a molecular weight of about 15 kDa and contains a sulfated mucopolysaccharide. We cloned the complementary DNA coding for Pearlin and deduced its complete amino acid sequence. Sequence analysis reveals that Pearlin is composed of 129 amino acids with a high proportion of Gly (10.8%), Tyr (10.0%), Cys (8.5%), Asn (7.7%), Asp (7.7%), and Arg (7.7%). Northern blot analysis showed that Pearlin messenger RNA was expressed specifically in mantle epithelium. From the sequencing data, Pearlin was shown to be quite different from the fibrous protein rich in Ala and Gly. The function of this protein in biomineralization is discussed.

Specialized transduction of tryptophan markers in Escherichia coli K12 by bacteriophage ∅80

Abstract A UV-inducible temperate phage ∅80, capable of lysogenizing on E. coli K12, has been isolated. Some of its properties have been investigated. Prophage ∅80 is closely linked to the try marker on the chromosome of strain K12. Specialized transduction of try + genes from tryptophan positive ( try + ) to tryptophan negative cells ( try − ) by ∅80 has been demonstrated. No markers except those in the try region have been transduced by this phage. The frequency of transduction resembles that of other transduction systems such as λ. Some try + transductants have been found to be heterogenotes. Heterogenotic cultures produce high frequency transducing (HFT) lysates, which contain both active phage ∅80 and defective transducing phage ∅80 dt carrying try genes. Each ∅80 dt strain carries the set of genes of the tryptophan operon, replacing a part of the phage genome which includes the immunity-related locus.

Cell cycle-dependent expression of the mouseRad51 gene in proliferating cells

The mouseRad51 gene is a mammalian homologue of theEscherichia coli recA and yeastRAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouseRad51 mRNA was examined in synchronized mouse m5S cells. TheRad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression ofRad51 mRNA. By immunohistochemical analyses, the mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouseRad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Suppressor-sensitive mutants of coliphage ∅80

Abstract About fifty suppressor sensitive (sus) mutants of phage ∅80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in ∅80. Some presumed “early” mutants were also found. Defective lysogens were isolated from a ∅80-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage ∅80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage ∅80 was determined. Clustering of all head genes and also of tail genes, as in phage λ, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of ∅80 was also similar to that of phage λ: namely, the cluster of head genes was found to be located at one end of the vegetative map of ∅80, being followed by that of tail genes, and presumed “early” genes are located at the other end of the map.

Reversible and irreversible stages in the transition of cell surface marker during the differentiation of pluripotent teratocarcinoma cell induced with retinoic acid

Abstract Treatment of a pluripotent teratocarcinoma cell line with retinoic acid (RA) results in the disappearance of peanut agglutinin (PNA) receptors, accompanied with the decrease in F9 antigens and the enhanced secretion of plasminogen activator. However, this type of differentiation was inhibited by feeder cells. Furthermore, the transition of PNA receptor was reversible on the cells treated with RA for 2 days and became irreversible by an additional 2-day treatment with RA. Thus, two stages of teratocarcinoma cell differentiation—reversible and irreversible—were demonstrated.

Characteristics of the transducing elements of bacteriophage φ80

Abstract The density of bacteriophage φ80 was measured by density gradient equilibrium centrifugation in CsCI solutions. This density was the same in phages derived from a lysogenic or a lytic growth cycle. The distribution of low-frequency transducing (LFT) particles of φ80 in a CsCl density gradient was shown to be much broader than that of plaque-forming particles. The transducing phages in the LFT lysates appear to transmit their characteristic densities, as well as definite try markers, to the φ80 dt particles in the high-frequency transducing lysates derived from them. The φ80 dt particles can be divided into two classes according to their requirement for helper phages in transduction. One class transduces more efficiently in the presence of active helper phage, while with the other class the active phage does not enhance or even reduces the transduction frequency. A new transducing phage type, which is not defective since it multiplies without help from the active phage, has been isolated. This phage, called φ80 pt (plaque-forming tryptophan), forms a small turbid plaque. A tryptophan auxotroph lysogenized by φ80 pt becomes try+, although it always segregates out some try− nonlysogenic cells. The buoyant density of φ80 pt in CsCI solution is greater than that of φ80, suggesting that a larger genetic fragment is carried in this phage.

Induction of teratocarcinoma cell differentiation: Effect of the inhibitors of DNA synthesis

Induction of teratocarcinoma cell (F9) differentiation was studied by using inhibitors of DNA synthesis and several agents known to be differentiation inducers. Inhibition of DNA synthesis induced changes in cell surface marker F9 and stimulated the production of plasminogen activator (PA) in a manner that is dependent upon de novo synthesis of RNA and protein. The results thus indicate close association between inhibition of DNA synthesis and induction of cell differentiation. This approach will be useful in investigating the mechanism of teratocarcinoma cell differentiation.

Presence of protein complex is prerequisite for aragonite crystallization in the nacreous layer.

We have isolated a protein complex from the nacreous layer of pearl beads and oyster shells. This complex was mainly composed of pearlin and pearl keratin. Addition of a minute amount of the complex to a calcium-carbonate-saturated solution containing Mg2+ induced aragonite crystallization. The complex was dissociated to individual components in the presence of EDTA and urea. Conversely, the complex was reconstituted from a mixture of components upon incubation with Ca2+ and urea. The mixture of the components was unable to induce aragonite crystallization, but the reconstituted complex recovered this capacity. Thus it is concluded that the complex is the indivisible functional unit required for aragonite crystallization.

Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius.

We cloned and sequenced the dextranase (Dex) (1,6-alpha-glucanhydrolase; EC 3.2.1.11)-encoding gene from Streptococcus salivarius (Ss) strain M-33. Recombinant clones from an Ss genomic library specifying Dex activity were identified as colonies surrounded by transparent halos on blue dextran plates. One of the clones had a 4.3-kb KpnI fragment containing the gene coding for an 826-amino-acid polypeptide with a molecular mass of 87.9 kDa, which corresponds well to that of native Dex from the Ss culture supernatant. There was no sequence homology between the gene encoding Ss Dex and the gene encoding dextran glucosidase of S. mutans, or between their protein products.

Cloning of a cDNA encoding the Tcp-1 (t complex polypeptide 1) homologue of Arabidopsis thaliana.

We isolated a plant homologue of Tcp-1 (t complex polypeptide 1) cDNA from Arabidopsis thaliana. It encodes a 545-amino acid (aa) protein, which has extensive aa and nucleotide sequence homology to the corresponding proteins of mouse, yeast, fruit fly and archaebacteria.