No significant differences were observed in the amounts of DNA strand breaks produced by copper between male and female liver cells of long-evans cinnamon (LEC) strain rats.

The amounts of DNA single strand breaks that are oxidative damage produced by copper were examined by comet assay in the liver cells of an inbred strain of Long-Evans Cinnamon (LEC) rats that spontaneously develops fulminant hepatitis. At 4 weeks of age, copper contents in the liver of LEC rats were approximately 30-fold higher than those of WKAH rats that are control rats used in the present study. Copper accumulated in the liver of LEC rats in an age-dependent manner and no significant differences were observed between copper contents in the livers of males and females at each week of age from 4 to 15 weeks. No significant amounts of DNA strand breaks were found in the liver cells of both male and female WKAH rats from 4 to 15 weeks of age. DNA strand breaks were produced in the substantial population of LEC rat liver cells at 10 weeks of age and induced in an age-dependent manner from 10 to 15 weeks of age. The amounts of DNA strand breaks produced by copper accumulation in the liver cells of female LEC rats are not more abundant than those in the cells of male rats, although it has been reported that hepatitis in female rats is more serious than that in male rats.     

近交系大鼠DNA指纹图与微卫星DNA比较分析

为了建立一种对近交系大鼠遗传物质进行精确可靠、快速简便的监测方法,利用DNA指纹技术对国内6个品系8个近交系大鼠群体进行了DNA多态性的分析,并与PCR扩增微卫星DNA技术进行了比较.其结果显示(1)不同品系之间DNA指纹图差异较大,同一群体不同个体间DNA指纹图带的相似系数和共有带率除SHR(哈)和WKY(哈)小于0.7外,其他均大于0.9.不同地区同一SHR间和WKY间DNA指纹图也存在差异,相同DNA不同次制作的DNA指纹图谱基本一致.(2)不同品系个体间微卫星DNA具有显著多态性;同一群体不同个体之间除SHR(哈)的SMST位点和WKY(哈)的AGT位点出现一定的差异外,其他均没有差异.DNA指纹图能更精确可靠地反映出动物个体间的遗传背景,而微卫星DNA遗传监测方法比较简便快捷.     

Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae

Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.     

Characterization of infectious bronchitis virus isolates by slot blot hybridization

We used slot blot hybridization of the hypervariable regions of the S1 subunit of spike peplomer gene to identify and characterize infectious bronchitis virus (IBV) strains. Template DNA was created from six reference strain IBVs of different serotypes and immobilized on a nitrocellulose membrane. We synthesized digoxigenin-labeled probes from reference and unknown field viruses and hybridized them to template DNA. All reference strains could be distinguished and isolates identified by serotype if they were at least 95% identical to a reference strain. This slot blot hybridization procedure was specific and reproducible, and strain typing was consistent with the S1 sequencing of the IBV genome. This study thus provides a simple and rapid method for typing of IBV.     

Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of mycobacterium paratuberculosis

Reference strains and 31 clinical isolates of M. paratuberculosis, mainly from goats, were analysed for restriction fragment length polymorphism (RFLP). Restriction digests of bacterial DNA were hybridized with a repetitive insertion sequence, IS900, to obtain banding patterns for comparison of strains. Twenty-five of the 31 field-strains hybridized with IS900, and five hybridization patterns were identified. It was not possible to identify specific patterns for goat strains of M. paratuberculosis. Four hybridization patterns were similar, whereas the fifth pattern of a sheep strain diverged considerably in position and number of band. Six goat strains failed to hybridize with IS900, and the absence of IS900 was verified by the polymerase chain reaction and hybridization with an oligonucleotide probe. The six IS900-negative goat strains had diverging phenotypic properties, and the identification of these strains is discussed. The present study shows that M. paratuberculosis strains infecting goats are genetically similar to cattle strains and that IS900 is a specific genetic element for identification of M. paratuberculosis.     

Inhibition of replication induces non-apoptotic cell death in fibroblast cell lines derived from LEC rats

Hydroxyurea (HU), an anticancer drug, inhibits ribonucleoside diphosphate reductase and reduces pool sizes of deoxyribonucleoside triphosphate (dNTP). The reduction of dNTP results in inhibition of DNA replication. The cytotoxic effect of HU was investigated using fibroblast cell lines from LEC rats. LEC rat cells showed significantly higher sensitivity to HU than did cell lines from control WKAH rats. No significant differences were observed between the percentages of apoptotic cells in either LEC or WKAH rat cells that had been treated with HU and those that had not been treated with HU. LEC rat cells also showed significantly higher sensitivity to aphidicolin, which blocks DNA synthesis by inhibiting DNA polymerase alpha, than did WKAH rat cells. In both LEC and WKAH rat cells, intensified bands of p53 protein were observed immediately after treatment with HU. Although the high level of p53 protein persisted in WKAH rat cells until 6 hr post-incubation time after treatment with HU, the level of p53 protein had decreased at 6 hr post-incubation time in LEC rat cells. When the cells were X-irradiated in the absence or presence of HU, the ratio of the surviving fraction without HU to that with HU only slightly increased after X-irradiation in WKAH rat cells. In contrast, the ratio in LEC rat cells significantly increased after X-irradiation in a dose-dependent manner.     

Higher sensitivity of LEC strain rat in radiation-induced acute intestinal death.

LEC strain rats (LEC rats), which have been known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation as compared to WKAH strain rats (WKAH rats). Radiation-induced acute intestinal death occurred at doses higher than 6.5 Gy in LEC rats, and at doses higher than 12.8 Gy in WKAH rats, respectively. By the probit analysis of survival data, it was shown that the LD50/7 value of LEC rats was estimated to be 7.03 Gy which was significantly lower than that (12.99 Gy) of WKAH rats. Histopathological examinations of small intestines from LEC rats 2 days after irradiation at the dose of 8.5 Gy showed severe epithelial death together with edema, whereas little or no significant changes were noted in intestinal epithelium of 8.5 Gy-irradiated WKAH rats. These results suggest that the radiosensitivity of LEC rats to ionizing radiation appears to be higher than that of other strains of rats.     

Comparison of different Oncorhynchus masou virus (OMV) strains by DNA restriction endonuclease cleavage analysis.

Seven strains of Oncorhynchus masou virus (OMV) genomes were analyzed with the restriction endonucleases BamHI, EcoRI, HindIII and SmaI. The restriction patterns of OMV strain DNAs were divided into four groups. Restriction profiles of high passage strains (00-7812, 65th passage, and H-83, 60th passage) were different from those of low passage strains (00-7812, 8th passage, and H-83, 6th passage) when digested with BamHI, HindIII and SmaI. However, no difference was observed between the restriction patterns of high and low passage viral DNA with EcoRI. There was no distinct difference observed between the restriction patterns of tumor tissue-derived and coelomic fluid-derived strains. By using 32P-labelled DNA of standard OMV (strain 00-7812) as a probe, most of the fragments of other OMV strain DNAs were hybridized.     

伪狂犬病病毒Ea株UL31基因克隆、序列分析及其在大肠杆菌中的表达

以伪狂犬病病毒Ea株基因组DNA为模板,通过PCR扩增含UL31基因的1 000bp片段,扩增产物克隆于pMD18-T中,双脱氧末端终止法序列测定.通过OM1GA2.0软件包分析发现,Ea株UL31基因编码271个氨基酸,蛋白质分子量为30.38 kD,与Ka株UL31基因核苷酸与氨基酸序列同源性均在98%以上.将α-疱疹病毒亚科9个不同成员UL31同源基因编码的氨基酸序列进行多重比对分析,发现4个保守的功能性结构域.将该片段插入原核表达载体pGEX-KG中GST下游,构建的原核表达质粒pGEX-UL31在大肠杆菌BL32(DE3)中获得了高效表达,SDS-PAGE结果显示,表达的融合蛋白质分子量为56 kD.     

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.     

Restriction endonuclease analysis of various strains of Mycobacterium paratuberculosis isolated from cattle

Deoxyribonucleic acid (DNA) preparations from 3 reference strains of Mycobacterium paratuberculosis and from 23 isolates of M paratuberculosis obtained from cattle in New Zealand were characterized by restriction endonuclease analysis, using the enzymes BstE II, Pvu II, and Bcl I. Patterns of DNA fragments for strain 18 (one of the reference strains) differed markedly from patterns of other strains, indicating genetic differences between strain 18 and the other strains of M paratuberculosis evaluated. The other 2 reference strains (TMC 1613 and Weybridge strain 316) and all but 1 of the isolates from cattle had identical patterns with the 3 enzymes. These 2 reference strains differed from each other in their dependence on exogenous mycobactin, but this was not reflected in their restriction patterns. The single variant isolate from cattle had patterns identical to those of the other isolates, using Pvu II and Bcl I, and had only 1 fragment line difference with BstE II. Although close genetic homogeneity of cattle strains of M paratuberculosis prevented development of a typing system on the basis of restriction endonuclease analysis, the results provided a basis for genomic comparison with other closely related organisms.     

Analysis of the cell cycle of fibroblasts derived from the LEC rat after X-irradiation

The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G1 arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts. Primary-cultured fibroblasts derived from LEC and BN rats were exposed to 4 Gy of X-ray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G1--and G2/M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L7 and W8, which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV40 T-antigen, L7 cells but not W8 cells showed impaired G1 arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after X-irradiation, if they are kept naive as not immortalized with SV40 T-antigen.     

Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization.

Restriction endonuclease analysis and DNA hybridization revealed five ovine strains of Mycobacterium paratuberculosis from South Africa had identical DNA patterns to an ovine strain from Canada. Genetically this strain type has features in common with the two major groups of M. paratuberculosis.     

PRV闽A株Bam HI片段克隆及其第7片段的鉴定

用鸟枪法将ＰＲＶ闽Ａ株的ＢａｍＨＩ酶切片断克隆到ｐＢＲ３２２质粒中，再经抗性筛选、酶切鉴定和菌落原位杂交，证实已克隆了ＰＲＶ闽Ａ株１４个ＢａｍＨＩ酶切片段中的１２个，从而构建了其基因文库，通过Ｓｏｕｔｈｅｒｎ转印杂交和酶切图谱分析鉴定了重组质粒ｐＰＲ１２８，其插入片段含量包含了ＰＲＶ闽Ａ株糖蛋白ｇｐ５０基因在内的ＢａｍＨＩ－７片段，核酸长约６．８ｋｂ。     

鹅细小病毒VP1-VP3非重叠序列地高辛探针的制备和应用

根据GPV H1株核苷酸序列，设计了扩增VP1-VP3基因非重叠序列的1对引物，对其结构蛋白VP1与VP3非重叠核苷酸序列进行PCR扩增，将PCR产物纯化、回收后制备出GPV VP1-VP3基因DIG标记核酸探针，其标记效率达到0．1pg／μl。特异性检测结果表明，该探针能与GPV不同毒株核酸发生特异性杂交，而与对照的DPV、GPMV等病毒的核酸杂交反应均为阴性；敏感性检测结果表明该探针对GPV的最低检出量为0．032ng。上述试验结果表明该探针可以用于GPV感染临床病料的检测。     

猪瘟病毒cF114株E2基因在家蚕杆状病毒表达系统中的融合表达

为进一步利用家蚕杆状病毒表达系统研制猪瘟病毒(CSFV)新型的亚单位疫苗,将猪瘟病毒cF114株囊膜糖蛋白E2基因克隆至带有GST标签的分泌表达杆状病毒转移载体pAcSecG2T的EcoRⅠ和BamHⅡ位点之间,获得了带有杆状病毒囊膜蛋白gp67信号肽-GST-E2元件的重组杆状病毒转移载体pAcSecG2T-E2,与线性化家蚕杆状病毒Bm-BacPAK6 DNA共转染家蚕BmN细胞,筛选重组病毒(Bm-BacPAK6-E2),PCR证实所分离的重组病毒含有目的片段E2基因。SDS-PAGE图谱显示,感染重组病毒后,在细胞培养液上清和家蚕幼虫、蛹的血淋巴中均可检测到一条分子量为63 kD左右的特异性条带;感染Bm-BacPAK6-E2家蚕蛹的血淋巴可检测到GST活性,接种病毒148 h后重组蛋白的表达水平达到17.11μg/mL。     

马立克氏病病毒６４８株囊膜糖蛋白gI基因的克隆和表达的研究

根据马立克氏病病毒（ＭＤＶ）强毒株ＧＡ的基因序列，设计和合成一对引物，以特超强毒６４８株基因组ＤＮＡ为模板，通过ＰＣＲ技术，扩增其囊膜糖蛋白gI基因阅读框（ＯＲＦ）中，除去其Ｎ－端编码疏水区的１６５个碱基对（bp)以外的蓁部分；将ＰＣＲ产物按正确的阅读框架定向克隆到表性载体pGEX-6P-1中谷胱甘肽转移酶（ＧＳＴ）基因的下游，将重组质粒转化进大肠杆菌ＢＬ２１株，在１．０mMIPTG浓度和３０℃的条件下诱导，gI-GST基因融合蛋白获得了理想的表达；经聚丙烯酰胺凝脉电泳，West-ern-blot试验，通信班下其表达的融合蛋白产物大小为预期的６３ＫＤ。将表达产物回收后免疫小鼠，所得抗血清可与ＭＤＶ感染的鸡胚成纤维细胞（ＣＥＦ）在免疫荧光试验（ＦＡ）中呈细胞膜阳性染色。试验结果表明，在大肠杆菌中表达的６４８株ＭＤＶgI基因的融合蛋白产物保留了天然蛋白的某些抗原性。     

DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

Identification of a locus for susceptibility to renal cell carcinoma in the Long-Evans Cinnamon rat

The Long-Evans Cinnamon (LEC) mutant rat shows higher incidence of renal cell carcinomas induced by a treatment with the chemical carcinogen N-diethylnitrosamine, as compared to the normal control rat. We performed the first genome-wide scan for genes responsible for susceptibility to chemically induced renal cell carcinoma in an F2 intercross obtained by mating the LEC and Fischer-344 (F344) rats. The genotype of 71 (F344 x LEC) F2 progenies was determined with the use of 338 simple sequence length polymorphisms (SSLPs) spread over the genome. The F2 rats which carried renal cell carcinoma were shown to possess the incidence of homozygosity of the LEC allele which is higher than that of the other genotypes at SSLP markers on chromosome 5 (chi2 = 17.5 for D5Rat21). Our linkage analysis has led to the revelation of a novel gene that influences susceptibility to renal cell carcinoma on rat chromosome 5.