Characterization of Nucleic Acid of Pichinde Virus

The nucleic acid of Pichinde virus was found to be single-stranded ribonucleic acid (RNA) as determined by sensitivity to ribonuclease, by alkaline degradation, by buoyant density in cesium sulfate, and by analysis of the base composition. The RNA of the virion could be separated into five components which had sedimentation coefficients corresponding to 31S, 28S, 22S, 18S and 4 to 6S. The 28S, 18S, and possibly the 4 to 6S RNAs appear to be derived from host cell components incorporated into the virion, whereas the 31S and 22S components appear to represent the genome of the virus.     

Viral RNAs Associated with Ribosomes in Sindbis Virus-Infected HeLa Cells

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.     

Sedimentation characteristics of virion RNA of Machupo virus reproducing in the presence of actinomycin D

Actinomycin D treatment (0.005-05 g/ml) of Vero and BHK-21 cells infected with Machupo virus suppressed the synthesis of ribosomal RNAs but did not affect the production of infectious Machupo virus. Virion RNAs contained 3 high molecular weight RNA species: 28-31 S, 22-24 S and 18 S. In the presence of actinomycin D [3H]-uridine incorporated only in 30-31 S and 22-24 S RNA species. The data are supported by previous results which show that Machupo virus genome contains two RNA species: "large" (30-31 S) and "small" (22-24 S).     

Lymphocytic Choriomeningitis Virus RNAs

IT has been proposed that lymphocytic choriomeningitis (LCM) virus and immunologically related viruses should be placed in a new taxonomic group, with LCM virus as the prototype virus^1,2. They were called arenoviruses because they contain, as a unique feature, electron-dense, sand-like or ribo-some-like granules². The LCM virus contains RNA and we have previously reported that this RNA can be separated into three components by density gradient centrifugation³.     

In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA.

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region 1a-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region 1b synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.     

SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

Cucumber mosaic virus, a model for RNA virus evolution

Taxonomic relationships: Cucumber mosaic virus (CMV) is the type member of the Cucumovirus genus, in the family Bromoviridae . Additional members of the genus are Peanut stunt virus (PSV) and Tomato aspermy virus (TAV). The RNAs 3 of all members of the genus can be exchanged and still yield a viable virus, while the RNAs 1 and 2 can only be exchanged within a species.
Physical properties: The virus particles are about 29 nm in diameter, and are composed of 180 subunits (T = 3 icosahedral symmetry). The particles sediment with an s value of approximately 98. The virions contain 18% RNA, and are highly labile, relying on RNA–protein interactions for their integrity. The three genomic RNAs, designated RNA 1 (3.3 kb in length), RNA 2 (3.0 kb) and RNA 3 (2.2 kb) are packaged in individual particles; a subgenomic RNA, RNA 4 (1.0 kb), is packaged with the genomic RNA 3, making all the particles roughly equivalent in composition. In some strains an additional subgenomic RNA, RNA 4A is also encapsidated at low levels. The genomic RNAs are single stranded, plus sense RNAs with 5' cap structures, and 3' conserved regions that can be folded into tRNA-like structures.
Satellite RNAs: CMV can harbour molecular parasites known as satellite RNAs (satRNAs) that can dramatically alter the symptom phenotype induced by the virus. The CMV satRNAs do not encode any proteins but rely on the RNA for their biological activity.
Hosts: CMV infects over 1000 species of hosts, including members of 85 plant families, making it the broadest host range virus known. The virus is transmitted from host to host by aphid vectors, in a nonpersistent manner.
Useful web sites: http://mmtsb.scripps.edu/viper/1f15.html (structure); http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/10040001.htm (general information)     

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.     

Density Gradient Centrifugation Studies on Lymphocytic Choriomeningitis Virus and on Viral Ribonucleic Acid

Lymphocytic choriomeningitis (LCM) virus, Traub strain, was purified from BHK-21 tissue culture medium. The virus was then analyzed by equilibrium centrifugation and rate zonal centrifugation in sucrose gradients. A buoyant density in sucrose of 1.18 g/ml was found and the S(20, w) value was estimated to be about 470 to 500S. Furthermore, the (3)H-uridine-labeled ribonucleic acid (RNA) from virus was extracted from LCM virus and analyzed by rate zonal centrifugation. Two major and one minor single-stranded RNA components were found with sedimentation coefficients of 28, 22, and 18S.     

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.     

NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF CHIRONOMUS TENTANS SALIVARY GLAND CELLS

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.     

Differences in physical and biological properties of 50S ribosomes and 23S RNAs derived from tight and loose couple 70S ribosomes

Tight couple (TC) 50S ribosomes on treatment with kethoxal lose their capacity to associate with 30S ribosomes whereas loose couple (LC) 50S ribosomes on such treatment fully retain their association capacity. The same is true for 23S RNAs isolated from treated 50S ribosomes or isolated 23S RNAs directly treated with kethoxal, so far as their capacity to associate with 16S RNA is concerned. At certain Mg++ concentrations TC 23S RNA is highly susceptible to the nucleolytic action of single-strand specific enzyme RNase I; LC 23S RNA is quite resistant. The Mg++-dependencies of the two species of 23S RNAs for association with 16S RNA are also quite different. The fluorescence enhancement of ethidium bromide due to binding to TC 23S RNA is slightly less than LC 23S RNA. The hyperchromicity of LC 23S RNA due to thermal denaturation is somewhat more than TC 23S RNA. LC 23S RNA has slightly more elliptic CD spectrum than TC 23S RNA. These results clearly show that 23S RNAs present in TC and LC 50S ribosomes are distinct from each other. It has been recently demonstrated in this laboratory that they can be interconverted by the agents involved in translocation and thus appear to be conformomers.     

激光显微切割分离细胞的微量RNA质量鉴定体系的 建立

摘要: 探索一套激光显微切割(Laser capture microdissection, LCM)分离细胞后获得的微量RNA质量鉴定标准操作流程。选取3个低温保存的胃癌旁组织样本, 冰冻切片进行甲酚紫染色和病理学检查, 利用激光显微切割技术分离非癌上皮细胞, 提取RNA并以Agilent 2100生物分析仪鉴定RNA的纯度和完整性。同时, 选择高、中、低3种不同表达丰度的6个基因(EF1A, ACTB, GAPHD, B2M, MED1, CK20), 在每个基因的5′和3′端设计引物, RT-PCR扩增。以3个培养细胞制备的高质量RNA和3个有降解的胃癌旁组织样本RNA作对照, RT-PCR扩增结果与Agilent 2100生物分析仪的结果高度一致。结果显示冻存组织进行冰冻切片结合病理学检查后, LCM获取细胞提取微量RNA采用RT-PCR进行质量鉴定是一种操作简单的稳定方法, 可以作为肿瘤基因组研究的有效和常规方法。