Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.     

Isolation and characterization of a novel reassortant between avian Ty-1 and simian RRV rotaviruses.

A reassortant, TyRh, was isolated after coinfection of MA104 cells with avian Ty-1 and simian RRV rotaviruses. Hybridization and serological studies showed that the reassortant's 4th gene, which encodes Vp4, was derived from the simian RRV rotavirus parent, whereas the remaining 10 genes were derived from the avian Ty-1 rotavirus parent.     

Glycosaminoglycans and proteoglycans synthesized by rat limb buds during prechondrogenic and chondrogenic stages

The sulfated glycosaminoglycans synthesized in the forelimb plates of rats on days 12, 13, 14, and 15 of gestation were characterized by their susceptibility to various glycosaminoglycan lyases. On days 12 and 13, heparan sulfate accounted for approximately 65% of the newly synthesized sulfated glycosaminoglycans. Small amounts of dermatan sulfate and chondroitin sulfates were also observed. On day 14, the relative amount of chondroitin 4-sulfate began to increase, there being a compensatory decrease in the amount of heparan sulfate. 35S-Sulfate-labeled material was extracted from day-13 forelimb plates with 4 M guanidine/HCl without proteolysis. Using ultracentrifugation on a sucrose density gradient, the extract was separated into two peaks: a light peak (L) mainly composed of heparan sulfate, and a faster-sedimenting peak (M) mainly composed of chondroitin sulfate. The cartilage-type proteoglycan (H) was first detectable on day 14 of gestation, indicating that chondrogenesis in rat forelimb plates starts on day 14 of gestation. In addition to these previously identified glycosaminoglycans or proteoglycans, we isolated an unknown component in the glycosaminoglycan preparations obtained from limb plates during these developmental stages. This component was not found in glycosaminoglycan preparations obtained either from the brain or tail of rat fetuses at the same stages.     

A direct evidence of the localization of mitochondrial calmodulin

The presence and localization of mitochondrial calmodulin was directly proved immuno-electron microscopically by the protein A-gold technique. In the ultra-pure mitochondria the complexes of anti-calmodulin antibody and protein A-gold clearly showed the localization of mitochondrial calmodulin on the inner membrane and in the matrix space.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

Effect of thioacetamide,growth hormone or partial hepatectomy on spermidine acetylase activity of rat liver cytosol

Rat liver cytosol extracts catalyzed the formation of monoacetylspermidine when incubated with acetyl-CoA and spermidine.This activity was enhanced 15-fold by administration of thioacetamide (150 mg/kg). The peak of activity occurred 18–24 h after treatment with the drug and then declined reaching control levels by 76 h. Previous studies have shown that ornithine decarboxylase activity was also greatly increased over this time period. Putrescine content in the liver was increased 80–90-fold at 18–24 h and then declined. Spermidine levels were decreased significantly over the period 12–24 h after thioacetamide treatment and then increased substantially at later times. These results are consistent with the hypothesis that, at early times after administration of thioacetamide, the increase in putrescine content is brought about both by decarboxylation of ornithine and by degradation of monoacetylspermidine.Spermidine acetylase activity was also measured in liver extracts prepared after two other physiological stimuli known to enhance ornithine decarboxylase activity were used. Both growth hormone treatment and partial hepatectomy produced an early 2–3-fold increase in the cytosolic spermidine acetylase activity.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.