Sequence diversity of human rotavirus strains investigated by northern blot hybridization analysis.

Rotavirus genomic RNAs, derived from a series of human isolates that exhibit variability in the pattern of migration of the double-stranded RNA on polyacrylamide gels, were transferred to diazobenzyloxymethyl paper, and their sequence diversity was investigated. Hybridization of cDNA probes prepared from the 11 segments of rotavirus RNA indicated that considerable sequence diversity exists among these viruses. Under conditions of both low and high stringency, hybridization analysis of virus collected between 1975 and 1980 suggested that the variation among rotavirus strains may have occurred by a process involving both "drift" and "shift" in the sequence of the rotavirus genomic segments.     

Gel electrophoresis of rotavirus RNA derived from six different animal species.

Rotavirus RNA prepared from calf, pig, mouse, deer, foal and dog-adapted human isolates was compared using polyacrylamide gel electrophoresis. Reproducible differences in the RNA migration patterns were found between all isolates. There were 11 clearly resolved segments in the pig, mouse and foal samples. The calf rotavirus RNA and deer rotavirus RNA separated into 9 bands and 10 bands, respectively. The dog-adapted human virus migrated in 12 bands, and this probably results from the complex passage history of the original human rotavirus isolate.     

长春地区冬季流行的轮状病毒核酸电泳型的比较

轮状病毒是婴幼儿腹泻的主要病原,在我国亦广泛流行,根据病毒基因组的电泳分析,可以研究其分子流行病学的规律。文献报道该病毒在流行区往往以一个电泳型感染为主,我们对长春地区1984～1985年和1985～1986年两个冬季流行的婴幼儿腹泻轮状病毒基因组PAGE分析的结果,也表明两个冬季都是以电泳长型感染为主,其中优势株与本室     

RNA polymerase associated with human rotaviruses in diarrhea stools.

RNA polymerase activity was detected in six stools which were partially purified by high-speed centrifugation from infants with rotavirus gastroenteritis, but was not detected in five stools which were negative for rotavirus by counterimmunoelectrophoresis and radioimmunoassay. The polymerase activity was associated with the 1.38-g/ml rotavirus band after purification in a CsCl gradient.     

Identification of phototrophic sulfur bacteria through the analysis of lmwRNA band patterns

Several phototrophic sulfur bacteria were identified preliminarily through the analysis of the low-molecular-weight RNA fraction (lmwRNA) of bacterial cells. This fraction includes the ribosomal 5S RNA and several transfer RNAs. These molecules were separated by high-resolution electrophoresis in polyacrylamide gels, and the resulting band patterns were used as fingerprints for the identification of the organisms. We examined a large number of well-characterized reference strains together with a broad range of purple sulfur bacterial isolates from freshwater and marine environments. A cluster analysis was run using the similarity matrix calculated from the band patterns. Despite the shortcomings of the method, close relatives were clustered together yielding a number of groups consistent with the phylogenetic arrangement established through the analyses of a few available 16S rRNA gene sequences. Thus, the classification obtained gives further support to rearrangement of the group as the analyses of 16S rRNA gene sequences had previously suggested. We conclude that the analysis of lmwRNA band patterns is a rapid and simple tool for grouping and preliminarily identifying new isolates of phototrophic sulfur bacteria. Received: 5 February 1998 / Accepted: 15 June 1998     

Molecular differentiation of two races of Fusarium oxysporum special form cubense

Total cellular DNA was extracted from 28 isolates of Fusarium oxysporum from vascular wilt of banana. The DNA samples were digested with the restriction endonuclease Hae III, and separated in agarose gels. Two characteristic banding patterns were seen in ethidium bromide stained gels. The two patterns correlated to strains believed to be of pathogenic races 1 and 4. A minor variation to these was detected with isolates of race 1 from Tanzania, which gave a typical race 1 band pattern, with an additional band of approx. 10 kb.     

人轮状病毒的细胞培养分离

用CV-1细胞从急性腹泻病儿7份粪便标本中直接分离出4株人轮状病毒(HRV),并适应传代14代。感染细胞出现特征性细胞病变,经电镜、ELISA、免疫荧光染色及病毒RNA电泳等试验证实HRV毒株在CV-1细胞中的繁殖及抗原特异性。病毒滴度为10~(6.0)TCID_(60)。分离的4株HRV毒株均为RNA电泳型长型,经CV-1细胞传7～14代后,RNA图型与原粪样相比未见变异。     

Isolation of an atypical rotavirus causing diarrhea in neonatal ferrets

A rotavirus was isolated from neonatal ferrets (Mustela putorius furo) with diarrhea at a large commercial farm. This virus is classified as an atypical rotavirus, probably belonging to the group C rotaviruses. This classification is based on the lack of the rotavirus group A common antigen and on its distinct dsRNA electropherotype pattern in polyacrylamide gels. The diarrheal disease was reproduced experimentally in neonatal ferrets.     

Gene Mapping of Rotavirus Double-Stranded RNA Segments by Northern Blot Hybridization: Application to Segments 7, 8, and 9

Cloned DNA copies of double-stranded RNA segments 7, 8, and 9 of UK bovine rotavirus were nick-translated with [alpha-(32)P]ATP and hybridized to double-stranded RNA of various rotavirus strains which had been separated on long polyacrylamide gels and then transferred to o-aminophenylthioether paper. Specific hybridization of the UK calf clones to the separated RNA segments allowed the corresponding genes of four different rotaviruses to be rapidly determined.     

Newcastle Disease Virus-Specific RNA: Polyacrylamide Gel Analysis of Single-Stranded RNA and Hybrid Duplexes

Newcastle disease virus-specific [(3)H]uridine-labeled 18S RNA was resolved by polyacrylamide gel electrophoresis into several components with molecular weights from 450,000 to 840,000. The analysis of 35 and 24S virus-specific RNA also revealed several components in each sedimentational class. The conversion of 18S RNA into double-stranded form by hybridization with an excess of unlabeled virion RNA improved the resolution in polyacrylamide gels and revealed at least six distinct components. The same six classes of hybrid duplexes were revealed when (32)P-labeled 50S virion RNA was hybridized with an excess of 18S RNA. The applicability of polyacrylamide gel electrophoresis of hybrid duplexes to the analysis of viral genome structure is discussed.     

Hammondia heydorni: evidence of genetic diversity among isolates from dogs

Canine isolates of Hammondia heydorni from Argentina, Brazil, and the United States were analysed for genetic diversity. A total of 14 isolates were tested for their ability to produce amplification using three PCR assays, one targeting the common toxoplasmatiid ITS-1 region and 2 amplifying novel, H. heydorni-specific loci, HhAP7 and HhAP10. While the ITS-1 fragments could be amplified from all isolates, only six isolates were capable of amplifying the fragments from the novel loci. The PCR products were further investigated for genetic diversity using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Polymorphism in the digestion pattern was evident only at the HhAP10 locus, differentiating two of the Argentinean isolates from the remainder. Mobility shifts on SSCP gels revealed that the two Argentinean isolates were not only different from the other four isolates, but also differed from each other, both at the HhAP7 and HhAP10 loci. The ITS-1 fragments of all isolates were identical by RFLP. However, two distinct mobility patterns resulted when the products were electrophoresed on SSCP gels. Based on the sequence data from the ITS-1 and the two random loci, the isolates could be broadly classified into two distinct groups, within which minor polymorphisms were evident. In contrast, very little heterogeneity occurred in the sequences of corresponding ITS-1 regions of Neospora caninum and Toxoplasma gondii isolates. Thus, it is concluded that there is a considerable degree of microheterogeneity among isolates of H. heydorni. This diversity should be taken into consideration while attempting to elucidate the systematics, diagnostics, and biology of H. heydorni in relation to N. caninum.     

Spectrin band 1 and 2 are antigenically distinct and are not composed of subunits.

Human erythrocyte membranes and freshly isolated spectrin were separated into their constituent peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptides were electrophoresed from slices of such gels into agarose gels containing anti-spectrin antibodies and Triton X-100. In fresh preparations, precipitin arcs were observed only against peptides migrating as bands 1 and 2. It was found that bands 1 and 2 did not cross-react. There were two major arcs from band 1 and one principal arc from band 2, plus minor splitting of these arcs. None of the band 1 arcs fused with band 2 arcs. In fresh erythrocyte ghosts only bands 1 and 2 reacted with anti-spectrin; bands 2.1, 3, and 5, in particular, showed no precipitin arcs. However, in aged ghosts, arcs appeared in the band 3 region; in aged isolated spectrin, arcs appeared in the band 2.1 region; and in trypsin-degraded spectrin, reactive species occurred in all molecular weight classes. It is concluded that spectrin has no subunits smaller than 220,000 molecular weight and that bands 1 and 2 are immunochemically distinct.     

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.     

轮状病毒RNA基因图谱变异分析

用聚丙烯酰胺凝胶电泳法对轮状病毒(RV)标准株进行RNA基因图谱分析,发现人轮状病毒(HRV)Wa株在MA104细胞上连续传5代后,其RNA基因图谱由原来的4:2:3:2模式变为4:3:3:2模式。继续分别在CV-1、MA104细胞上传代后,基因图谱进一步演变为4:3:4:2模式。经比较电泳、病毒空斑纯化,初步证实RVWa有变异林存在。目前尚未见RV标准株变异的报告,有待进一步研究。同时对多个RV标准株及1939年南京市区婴幼儿秋季腹泻的RV流行株进行基因图谱分析,发现不同的人RV及牛RV标准株有相同的基因图谱;不同实验室来源的同一标准株有不同的基因图谱。此外,尚证实HRV是1989年南京市区婴幼儿秋季腹泻的主要病源,总检出率45.1％,电泳长型为流行优势株89.2％。本文尚对RV RNA基因图谱变异的原因及意义进行了讨论。     

Characterization of pre-messenger-RNA-containing nuclear ribonucleoprotein particles from avian erythroblasts.

Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule.     

Isoenzyme patterns and phylogenetic relationships in Acanthamoeba spp. isolated from contact lens containers in Korea

In order to refer to the basic information regarding the identification of isolates obtained from a contact lens container in Korea, the isoelectric focusing gel electrophoresis was employed to compare the isoenzyme band patterns among Acanthamoeba spp. including eight isolates and the simple pairwise dissimilarity analysis was carried out. For an alkaline phosphate development, isolate 7 and Acanthamoeba polyphaga showed homologous band patterns, and isolates 1, 2, and 3 showed the same patterns. For lactate dehydrogenase, similar patterns were observed in isolates 2 and 3. Isolates 3 and 5 showed homologous band patterns for malate dehydrogenase and glucose phosphate isomerase. For hexokinase, isolates 4, 7, and A, hatchetti showed the same band patterns. In others, a considerable number of interstrain polymorphisms was observed in nine isoenzyme band patterns. In Acanthamoeba group II, genetic distances among isolates 1, 2, 3, 4, and 5 ranged from 0.104 to 0.200. In comparison to A. castellanii, A. hatchetti, and A. polyphaga, genetic distances of isolates 7 and 8 were 0.254 and 0.219, respectively. In Acanthamoeba group III, including A. culbertsoni, A. healyi, and A. royreba, isolate 6 had genetic distances which ranged from 0.314 to 0.336. Finally, when comparing to the six reference Acanthamoeba, it was possible to classify isolates 1, 2, 3, 4, and 5, as genetically close-related species and as independent species group. Furthermore, isolates 6, 7 and 8 were identified as independent species as well.     

Two distinct conformations of rat liver ribosomal 5S RNA.

Three different conformers of rat liver 5S ribosomal RNA were investigated by partial nuclease cleavage technique using S1 nuclease and cobra venom endoribonuclease (CVE) as conformational probes. Urea-treated and renatured 5S RNA co-migrate on non-denaturing gels, but exhibit distinct differences in their nuclease cleavage patterns. The most prominent differences in S1 nuclease and CVE accessibility of these conformers are located in region 30-50 and around nucleotides 70 and 90. The third form of 5S RNA with higher electrophoretic mobility was generated by EDTA treatment. The cleavage patterns of this 5S RNA conformer are similar to that characteristic for the renatured 5S RNA. The results demonstrate the difference in secondary structure and possibly different tertiary base-pairing interactions of 5S RNA conformers.