Biomedical subjects

T Ohsumi

Publications and source records attributed to T Ohsumi.

At least 19 recordsLinked to original sources

PubMed2001

Immunohistopathologic demonstration of pleuropneumonia associated with Morganella morganii in a piglet.

Serofibrinous pleuropneumonia in a piglet was examined microbiologically and immunohistopathologically. Large numbers of Morganella morganii were isolated from the pneumonic lesion, but no other pathogens were identified. A large amount of M. morganii antigen was demonstrated, and its distribution was closely associated with the histologic lesion. This finding suggests that pleuropneumonia in piglets might be caused by M. morganii.

Animals↗

PubMed1999

Prevalence of Sarcocystis spp. cysts in Japanese and imported beef (Loin: Musculus longissimus).

A survey was carried out to investigate the occurrence of Sarcocystis infection in the loin (Musculus longissimus) of Japanese and imported beef. In all, the muscle tissue of 482 samples were examined by histological method. The prevalence of Sarcocystis unspecified species cysts was lower in Japanese beef (total 6.31%: 0% in Holstein castrated, 12.96% in Holstein milk cow, 3.33% in Japanese shorthorn and 11.58% in Japanese black cattle) than in beef imported from America (36.78%) or Australia (29.49%). The infection density of imported beef, especially in American, was higher than in Japanese beef. All detected cysts except one were identified as Sarcocystis cruzi. One thick walled cyst was found in Australian beef but it could not be distinguished as either Sarcocystis hirsuta or Sarcocystis hominis.

Animals↗

PubMed1998

Evaluation of cytotoxicity of calcium phosphate cement consisting of alpha-tricalcium phosphate and dicalcium phosphate dihydrate.

A newly developed calcium phosphate cement, MM, was evaluated for tissue irritability by means of cell cultures. In this study, MM showed extensive mild cytotoxicity compared with other cements before setting. Inhibition of adhesion of L-929 cells was not observed after contact with MM for 24 hours. The influence of MM on colony formation was approximately the same as that of another calcium phosphate cement and less than that of a glass ionomer cement. Toxicity of MM after setting was compared with four cements; another calcium phosphate cement, glass ionomer cement, silicate cement and zinc oxide eugenol cement, but MM showed the least influence on cell morphology. Judging from these results, MM appears to be less cytotoxic than the cements in current use.

Animals↗

PubMed1997

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted. The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.

Animals↗

PubMed1997

High efficiency selection of full-length cDNA by improved biotinylated cap trapper.

We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.

Animals↗

PubMed1997

Linkage map of Syrian hamster with restriction landmark genomic scanning.

We have constructed the linkage map with precise genetic analysis of the Syrian hamster, Mesocricetus auratus, according to the restriction landmark genomic scanning (RLGS) spot mapping method. Although only 3.2-6.6% of the total RLGS spots between the two strains, ACN and BIO 14.6, showed genetic variance, 572 loci were found to be polymorphic. Out of 569 RLGS loci and 3 other loci, 531 were mapped with the backcross (ACN x BIO 14.6) F1 x BIO 14.6. The cumulative map was 1111.6 cM, indicating that the spots/loci are located throughout the genome at 1.94 cM intervals on average. Thus, RLGS provides us with a rapid tool to construct the genetic map of any species, even if it has less genetic variation.

Animals↗

PubMed1996

High-efficiency full-length cDNA cloning by biotinylated CAP trapper.

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

Animals↗

PubMed1996

Five candidate genes for hamster cardiomyopathy did not map to the cardiomyopathy locus by FISH analysis.

The Syrian cardiomyopathic hamster (BIO14.6), that develops both muscular dystrophy and progressive cardiomyopathy, is widely used as an animal model of autosomal recessive cardiomyopathy mimicking human hypertrophic cardiomyopathy, and five genes have been proposed as strong candidates for the cause of cardiomyopathy. We recently mapped the cardiomyopathy locus of the hamster to the centromeric region of chromosome 9qa2.1-b1 by construction of a genetic linkage map of the Syrian hamster. Thus, we analyzed the loci of the five candidate genes, alpha tropomyosin, cardiac troponin T, adhalin, calpain 3 and cardiac myosin binding protein-C, by the FISH method, and found that these genes were mapped on the distal portion of chromosome 12qa5 and 4pa2 and the proximal portion of chromosomes 9qb7, 1qc1.1 and 1qb3, respectively. These results provide strong evidence that the five candidate genes previously proposed are not related to the hamster cardiomyopathy.

Animals↗

PubMed1996

A genetic linkage map of the Syrian hamster and localization of cardiomyopathy locus on chromosome 9qa2.1-b1 using RLGS spot-mapping.

The Syrian cardiomyopathic hamster (BIO14.6) has an inherited form of progressive myocardial necrosis and congestive heart failure. Although widely studied as an animal model for human hypertrophic cardiomyopathy, further genetic analysis has been limited by a scarcity of DNA markers. Until now, only six autosomal linkage groups have been described and the number of polymorphic loci was extremely limited. In this study, we applied the restriction landmark genome scanning (RLGS) spot-mapping method to construct a genetic map of the Syrian hamster (Mesocricetus auratus) using 72 back-cross progeny. Although the polymorphic rate is very low (3-7%) between the strains, 531 polymorphic spots/loci were mapped, showing the power of this approach and reasonable applicability to other organisms lacking a well-defined genetic map. Further, the spot markers which flank the cardiomyopathy (cm) locus were cloned to determine the chromosomal location of cm by fluorescent in situ hybridization (FISH) analysis, resulting in the assignment of the locus to the centromeric region of hamster chromosome 9qa2.1-b1. Several candidate genes responsible for hypertrophic cardiomyopathy in humans have been excluded.

Animals↗

PubMed1996

Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exon.

We examined the genomic structure of the reeler gene in Orleans reeler mouse mutant. Exon skipping of the reeler gene caused a 220 bp deletion in the transcript, resulting in a frame shift of the reeler gene which disrupts the 8th EGF-like motif of the reeler product. Surprisingly, the skipped exon was inserted by the 7104 bp L1 element which carried the full-length stretch of the mouse L1 sequence, consisting of a 212 bp F-type tandem repeat, open reading frame 1 (ORF1), ORF2, the polyadenylation signal and a poly A stretch. The transposed L1 sequence was flanked by 13 bp of the target sequence at both ends. ORF1 and ORF2 of this L1 repeat element are thought to encode a component of the RNP particle and the reverse transcriptase, respectively. Orleans reeler was originally established by spontaneous mutation caused by L1 insertion, and this L1 sequence is considered to be potentially active for transposition in mouse genome.

Amino Acid Sequence↗

PubMed1995

Loss of heterozygosity in chromosomes 1, 5, 7 and 13 in mouse hepatoma detected by systematic genome-wide scanning using RLGS genetic map.

We have developed an RLGS-based scanning system to detect DNA alteration in tumor tissues, using 575 mapped spots/loci in a single gel. This system is very powerful for screening and identifying not only loss of heterozygosity (LOH) but also DNA methylation change. In this study, we applied this system to search for the LOH of hepatoma from an interspecific F1 hybrid between Mus spretus and C57BL/6 with SV40 early T antigen transgene connected to a mouse major urinary protein enhancer/promoter. Comparing the RLGS profiles of each tumor to that of the normal tissue showed significant LOH in chromosomes 1, 5, 7 and 13.

Animals↗

PubMed1995

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.

Animals↗

PubMed1995

An expanded system of restriction landmark genomic scanning (RLGS Ver. 1.8).

The restriction landmark genomic scanning (RLGS) method is a high-speed genome scanning system which is based on the concept that restriction enzyme sites can be used as landmarks throughout the genome. It employs direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme, followed by high-resolutional two-dimensional electrophoresis. Recently, this system was further developed to lower cost and to simplify the procedure. This paper reviews the RLGS principle and the breakthroughs enabling its further development. Also presented is the precise protocol of the newest version (RLGS Ver. 1.8) that offers cost effectiveness and an expanded production system. Finally, the advantages of this new RLGS method and prospects for its widespread application are discussed.

Animals↗

PubMed1995

A spot cloning method for restriction landmark genomic scanning.

We introduce two new methods for target cloning of DNA fragments corresponding to spots on the two-dimensional profile of restriction landmark genomic scanning (RLGS). One is a restriction trapper-based method and the other is a polymerase chain reaction (PCR) mediated method. Both are designed to select the target DNA fragments from a large amount of unlabeled background DNA fragments in the RLGS gel which produce background clones. The restriction trapper method is simple, with a cloning efficiency that is not biased by the length of the target DNA nor by its GC content. On the other hand, the PCR-mediated method is efficient for cloning DNA fragments from a small amount of starting materials. These methods provide us with powerful tools for isolating DNA clones identified by the RLGS system as interesting spots. This paper reports the precise protocols of these methods and discusses their application and usefulness.

Animals↗

PubMed1995

Genetic mapping of restriction landmark genomic scanning loci in the mouse.

Restriction landmark genomic scanning (RLGS) was originally proposed as a high-speed method for surveying a large number of restriction landmarks in genomic DNA. The effort to apply this method to genetic analysis has been made, resulting in developing the new approach for the rapid construction of the genetic map of complex mammalian genomes (RLGS spot mapping). Especially, the use of NotI as the restriction landmark for genetic studies suggests that there is a high probability that a significant number of these RLGS loci will be associated with CpG islands of functional genes. Moreover, it is possible to use the RLGS spot mapping to analyze genetic map-poor species very rapidly for linkage of recessive mutations or segregating traits, because it does not rely upon cloned probes or sequences. In this paper, we summarize the progress that has been made in the practical application of the RLGS method to genetic analysis using congenic strains, recombinant inbred (RI) strains, and in interspecific backcrosses of mice.

Animals↗

PubMed1995

A single gel analysis of 575 dominant and codominant restriction landmark genomic scanning loci in mice interspecific backcross progeny.

We identified 575 new NotI landmarks of C57BL/6(B)- and M. spretus (S)-specific, dominant and codominant loci which were segregated in B x S interspecific backcrosses (BSS), using the restriction landmark genomic scanning (RLGS) spot mapping method. All of these loci were visualized on a single RLGS profile which was produced with NotI-PvuII-PstI. These landmarks include 250 newly identified S-specific spots in addition to the previously reported 325 B-specific spots. The S-specific spots were identified by reading full or half intensity, based on the property that the spot intensity of the autoradiographic signal reflected the copy number of an end-labeled restriction landmark. The cumulative map is 1341 cM and it is based upon 985 meiotic events in 72 backcross progeny. This map covers 90% of the total estimated length of the mouse genetic map. This map provides a good tool for the high-speed genome scanning assay in the mouse genome by a single RLGS gel analysis.

Animals↗

PubMed1994

A genetic linkage map of the mouse using an expanded production system of restriction landmark genomic scanning (RLGS Ver.1.8).

We have developed an expanded system (RLGS Ver.1.8) for producing RLGS patterns that result in a 16-fold increase in the number of gels produced and a 10-fold reduction in the total cost per gel. The major modifications include: 1) performing the blocking and labeling step without phenol extraction or ethanol precipitation; 2) minimizing the reaction volume and the enzyme units in each step; 3) developing a long vertical agarose disc gel electrophoresis for the 1st-dimension; and 4) developing a new apparatus for multiplex vertical 2nd-dimensional electrophoresis. RLGS Ver.1.8 was used with a new combination of restriction enzymes to identify variation for 209 loci between C57BL/6J and DBA/2J. Twenty-six BXD RI strains were analyzed and 195/209 loci were genetically mapped. These loci were mapped in one week of laboratory work by two people. This system provides an important tool for the genetic analysis of new loci in similar genetic resources.

Animals↗

PubMed1993

Ribosome-binding protein p34 is a member of the leucine-rich-repeat-protein superfamily.

Protein p34 is a non-glycosylated membrane protein characteristic of rough microsomes and is believed to play a role in the ribosome-membrane association. In the present study we isolated cDNA encoding p34 from a rat liver cDNA library and determined its complete amino acid sequence. p34 mRNA is 3.2 kb long and encodes a polypeptide of 307 amino acids with a molecular mass of about 34.9 kDa. Primary sequence analysis, coupled with biochemical studies on the topology, suggested that p34 is a type II signal-anchor protein; it is composed of a large cytoplasmic domain, a membrane-spanning segment and a 38-amino-acid-long luminally disposed C-terminus. The cytoplasmic domain of p34 has several noteworthy structural features, including a region of 4.5 tandem repeats of 23-24 amino acids. The repeated motif shows structural similarity to the leucine-rich repeat which is found in a variety of proteins widely distributed among eukaryotic cells and which potentially functions in mediating protein-protein interactions. The cytoplasmic domain also contains a characteristic hydrophilic region with abundant charged amino acids. These structural regions may be important for the observed ribosome-binding activity of the p34 protein.

Amino Acid Sequence↗