火鸡疱疹病毒细菌人工染色体的构建

火鸡疱疹病毒(HVT)为一种-疱疹病毒,因其与马立克氏病病毒(MDV)抗原相关性而被广泛用作预防马立克氏病(MD)的活疫苗.[目的]本研究的目的是构建HVT全基因组感染性细菌人工染色体(BAC).[方法]利用Eco-gpt(黄嘌呤鸟嘌呤磷酸核糖转移酶)基因和BAC载体pBeloBAC11的基本功能序列,构建重组病毒转移载体Pgab-gpt-BAC11.通过将Pgab-gpt-BAC11与HVT感染细胞总DNA共转染原代鸡胚成纤维细胞(CEF),待出现病毒噬斑后,利用霉酚酸(MPA)阻断核酸代谢途径,经过筛选获得纯化的重组病毒purified-Rhvt.提取purified-Rhvt感染细胞总DNA电转化大肠杆菌DH10B感受态细胞,在氯霉素抗性平板上筛选阳性克隆,并用酶切和PCR方法对其进行鉴定.随机选取BAC克隆提取BAC DNA转染次代CEF,完成HVT重组病毒的拯救.[结果]经过6轮筛选后获得纯化的重组病毒,并筛选到25个BAC分子克隆化病毒.其中BAC6、BAC8和BAC10再次启动病毒感染,产生与野生型HVT感染CEF相似的病毒噬斑形态,说明已经获得拯救出的HVT重组病毒.[结论]本研究构建了HVT全基因组感染性细菌人工染色体,建立了HVT反向遗传操作技术平台.     

Electron microscopic studies on the uptake of colloidal thorium dioxide particles by isolated fetal guinea-pig chondrocytes and the distribution of labeled lysosomes in cartilage formed by transplanted chondrocytes

Summary Chondrocytes isolated from fetal, guinea-pig epiphyses were grown in monolayer culture, exposed to thorium dioxide particles, and studied ultrastructurally after varying intervals. The exogenous marker was ingested by endocytosis and subsequently accumulated in lysosomes. After intramuscular injection into young guinea pigs, the thorium dioxidelabeled chondrocytes formed a typical hyaline cartilage. This consisted mainly of rounded or polygonal cells with large, eccentrically located nuclei. The cytoplasm showed an extensive granular endoplasmic reticulum and a well-developed Golgi complex, suggesting active synthesis and secretion of matrix components. Among the other cytoplasmic organelles, lysosomes containing variable amounts of marker particles were observed. After 2–3 weeks the transplants showed signs of cellular degeneration and disintegration. During these processes, lysosomes remained structurally intact and, furthermore, retained the incorporated marker. Thus, thorium dioxide-labeled bodies were found in former chondrocyte lacunae and in the intercellular substance proper. In the latter location labeled bodies could be observed in close proximity to early mineral deposits. These results are discussed with special reference to the cellular origin and lysosomal nature of matrix vesicles in calcifying cartilages.The skilled technical assistance of Mrs. Eva Lundberg and Miss Karin Askfors and the secretarial assistance of Mrs. Ingrid Wäälma are gratefully acknowledged.Financial support was obtained from the Swedish Medical Research Council (proj. no. 12X-3355), the Swedish Cancer Society (proj. no. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Harald and Greta Jeansson Foundations, the C. B. Nathhorst Foundation, the A. O. Swärd Foundation, and from the Funds of Karolinska Institutet.     

Ultrastructural reactions of spinal ganglia to tri-ortho-cresyl-phosphate: Effects of neurotoxicity

Summary The ultrastructure of neurons in spinal ganglia of the domestic fowl poisoned with tri-ortho-cresyl-phosphate (TOCP) shows characteristic changes. The light neurons react to TOCP by a marked increase in the number of neurofilaments. These neurons also contain mitochondria in various degenerative stages. Several of the altered mitochondria show an increasing osmiophilia. Some of the darker neurons display a hypertrophy of the endoplasmic reticulum or a relative increase of neurofilaments. The mitochondria in some of these cells show early stages of degeneration. These changes appear 13 days after TOCP ingestion.     

刺槐根瘤菌与纤维素分解菌对上海青和高粱的促生效应

通过盆栽试验,用刺槐根瘤菌(Rhizobium of Robinia pseudoacacia)与纤维素分解菌(Cellulose-decomposing Bacteria)对高粱和上海青进行单独接种和混合接种,采用针刺、浸种和涂叶3种接种方法,测量其各种生长指标。初步探究刺槐根瘤菌与纤维素分解菌联合对禾本科作物高粱和双子叶作物上海青两种非豆科植物的促生效应。结果表明,在非针刺条件下,两种菌混接组(B组)比单独接种根瘤菌组(D组)的促生效应更显著,上海青B组在灭菌条件下的根长促生率比D组高出48.97%,高粱B组在非灭菌条件下干重的促生率比D组高出30.76%;而且除了高粱的B组干重以外,盆栽试验条件下,B、D两组的其他生长指标在灭菌情况下的促生率比非灭菌条件下的促生率高,差别最大的为上海青B组鲜重,灭菌条件下比非灭菌条件下高出47.13%。根瘤菌与纤维素分解菌混合接种非针刺组,对高粱和上海青的各项生长指标促生效应明显,可为今后进一步开发非豆科作物菌肥提供试验依据。     

Localization of calcitonin gene-related peptide in the small intestine of various vertebrate species

Summary Calcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.     

A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.     

The DNA between Rz and cosR in bacteriophage lambda is nonessential

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.