Kiyozo Asada

The expression profile of microRNAs in mouse embryos

MicroRNAs (miRNAs), which are non-coding RNAs 18–25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering ∼80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

cDNA Cloning of Human Calpastatin: Sequence Homology Among Human, Pig, and Rabbit Calpastatins

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.

Amplification of the 16S-23S spacer region in rRNA operons of mycoplasmas by the polymerase chain reaction

Summary Nucleotide sequences of the spacer region between 16S and 23S DNA in the ribosomal RNA operons of mycoplasmas were identified by analysis of products of the polymerase chain reaction (PCR) amplified from the corresponding regions of 12 species of this family. Three common PCR primers, F1, F2, and R1, were designed by analysis of similarity between these sequences. Primers F1 and R1 produced fragments of 340 to 660 bp when the DNA of each species was used as the template. Specific amplification of the spacer region was confirmed by a second round of PCR in which the amplified products were used as the templates and F2 and R1 were used as the primers. No discrete band was observed in electrophoresis when human or mouse DNA served as the template with use of primers F1 and R2, which suggests that many mycoplasmal species that sometimes contaminate a culture of eukaryotic cells can be detected by the PCR.

Detection of Mycoplasma hyopneumoniae DNA by the polymerase chain reaction.

DNA amplification by the polymerase chain reaction (PCR) was examined to detect DNA of Mycoplasma hyopneumoniae, an etiological agent of porcine pneumonia. A pair of synthetic primers was selected that specify the amplification of a 520-basepair DNA fragment in a reiterative sequence of M. hyopneumoniae genome. The PCR product was detected by direct gel electrophoresis or by blot hybridization to a synthetic oligonucleotide probe. The specificity of PCR for M. hyopneumoniae was confirmed by lack of cross-reactivity to DNA from other porcine mycoplasmas.

Characterization of a functional domain of human calpastatin

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Nucleotide Sequence of the Arginine Deiminase Gene of Mycoplasma hominis

The arginine deiminase gene of Mycoplasma hominis was amplified by the polymerase chain reaction, and its entire nucleotide sequence was determined. This gene consists of 1227 base pairs encoding 409 amino acids, and has 35.2% guanine plus cytosine content. Nucleotide sequence homologies of the arginine deiminase gene between M. hominis and M. arginini, and between M. hominis and M. orale were 82.1 and 80.8%, respectively, suggesting that this gene is highly conserved among arginine‐utilizing Mycoplasma species.

The Polymerase Chain Reaction for Mycoplasma pulmonis

in vitro DNA amplification by polymerase chain reaction was examined to detect Mycoplasma pulmonis. A pair of synthetic oligonucleotide primers was constructed, and used to amplify a unique sequence of M. pulmonis DNA. Amplified products were detected by agarose gel electrophoresis and verified by blot hybridization with a synthetic oligonucleotide probe. This system detected cellular DNA of M. pulmonis but not M. arthritidis or M. neurolyticum, and thus appears to be useful for M. pulmonis diagnosis.

cDNA cloning of an aspartic proteinase secreted by Candida albicans.

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1α (EF-1α), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1α. An intron is located in the 5′ untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1αtef-c (97%) than to the P. purpurea EF-1αtef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.

486. Optimized Conditions for Retroviral Gene Transfer by RetroNectin|[reg]| Bound Virus Infection Method

In retroviral gene transduction, the efficiency of viral infection is suppressed by the proteoglycans and some other materials secreted by the producer cell lines. In order to remove these inhibitors, we previously developed the rFN-CH-296 (RetroNectin®)-bound virus infection method. In the present study, we further optimized the conditions of gene transfer efficiency into human leukemia cell lines, K562, TF-1, HL-60, and CEM cells utilizing RetroNectin® plates with galv-pseudo-typed or amphotropic retrovirus vector, and applied the optimized protocol to the CD34+ BMCs in a closed system with gas permeable culture bag.