Denaturing Sucrose Gradients Research Articles

An encapsidated, subgenomic messenger RNA encodes the coat protein of carnation mottle virus

The translation strategy of carnation mottle virus (CarMV) in vitro has been generally assumed to involve internal initiation events on full-length, genomic RNA (4.3 kb). We suggest that this is, at least in part, incorrect. Encapsidated RNA, fractionated on denaturing sucrose gradients, or total RNA from CarMV-infected leaves, fractionated under non-denaturing conditions, was translated in an mRNA-dependent rabbit reticulocyte cell-free system. Evidence for subgenomic RNAs which encode a polypeptide of Mr 38 000 was found. This product was shown to be related to authentic CarMV coat protein by partial proteolysis with alpha-chymotrypsin and SDS/polyacrylamide-gel electrophoresis.

An Efficient Procedure for Purification of an Isolate of Peanut Mottle Virus from Wild Peanut and Determination of Molecular Weights of the Viral Components1

Abstract An efficient procedure was developed for purification of peanut mottle virus (PMV) from pea (Pisum sativum cv. Little Marvel) that yielded 10–19 mg virus/ kg infected tissue. Virus was extracted from frozen infected tissue in 0.01 M potassium phosphate buffer, pH 8.0, with 0.001 M dithioerythritol, followed by clarification with chloroform (15%, v/v) and precipitation by KCl and polyethylene glycol. Virus was resuspended in 0.01 M borate-phosphate buffer, pH 8.3, with 0.2 M urea prior to density gradient centrifugation. Purified virus sedimented as a single component with a sedimentation coefficient of 149 S. The molecular weight of the single coat protein was estimated as 36,100 daltons in 12% polyacrylamide gels. The single nucleic acid isolated from PMV on sucrose gradients was degraded by RNase, but not DNase. The molecular weight of the RNA was estimated as 3.1 × 106 daltons on nondenaturing and denaturing sucrose gradients.

Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B.

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.

Analysis of vaccinia virus transcriptional complexity in vitro and in vivo: characterization of RNase T1-resistant 5'-terminal oligonucleotides

Vaccinia virus mRNAs synthesized in vitro and in vivo, polyadenylated leader sequences synthesized in vitro in the absence of added GTP, CTP, or UTP or in the presence of 20 micrograms of actinomycin D per ml, and high-molecular-weight RNA synthesized in vitro under limiting ATP concentrations were labeled specifically in the cap structure using [alpha-32P]GTP and vaccinia-soluble enzyme extracts. The complexity of RNase T1-resistant 5'-terminal oligonucleotides was analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 190 unique T1-resistant 5'-terminal oligonucleotides were observed from vaccinia virus 8 to 12S RNA synthesized in vitro. A somewhat greater complexity was observed with polyadenylated leader sequences and actinomycin D RNAs where unique T1-resistant oligonucleotides ranged from approximately 210 to 280 5'-terminal fragments. On a composite fingerprint of the above RNAs, more than 300 identifiable unique T1-resistant 5'-terminal oligonucleotides were observed. Significantly, close to 300 T1-resistant fragments were derived from RNA sedimenting faster than 18S on denaturing sucrose gradients. Analysis of vaccinia RNAs synthesized in vivo in the absence of either de novo protein synthesis or DNA replication or in the presence of actinomycin D gave essentially similar profiles of 5'-terminal T1-resistant oligonucleotide fingerprints consisting of approximately 200 fragments. Analysis of the 5'-terminal T1-resistant oligonucleotides of vaccinia RNAs present after DNA replication showed essentially the same pattern of early T1-fragments albeit in reduced amounts but in addition revealed a complex pattern of T1-resistant oligonucleotides unique to this class of vaccinia RNA.

Human interferon gamma is encoded by a single class of mRNA.

Polyadenylated RNA from human peripheral blood lymphocytes and spleen cells, treated with different inducers for IFN-gamma production, was fractionated on denaturing sucrose gradients. Two IFN-gamma mRNA peaks at 12S and 16S were consistently observed. Nucleotide sequence analysis of cDNA clones showed that the 12S IFN-gamma mRNA from the different sources is identical to the gel fractionated 18S IFN-gamma mRNA which gave rise to the IFN-gamma cDNA clone p69 (1). Nucleotide sequence analysis of several IFN-gamma cDNA clones showed the presence of a CGA (Arg) codon at position 140 of mature IFN-gamma in contrast with the CAA (Gln) codon, which is found in p69 (1). Specifically primed cDNA extension on total induced polyadenylated RNA revealed the presence of a single mRNA species having a 5' untranslated region of 125-130 nucleotides. The nucleotide sequence of this region has been obtained. These data suggest that a single human IFN-gamma gene, which has very little polymorphism, gives rise to a single size class of mRNA.

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)(+) RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I-IV. Polypeptides synthesized in vitro were labeled with ((35)S)-cysteine and ((3)H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I-IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.

Purification and characterization of the messenger RNA for the heavy chain of rat immunoglobulin E.

The isolation and translational properties of rat immunoglobulin E (IgE) heavy chain mRNA are described. The mRNA has a sedimentation coefficient of approximately 18S, a chain length of about 2000 nucleotides and directs the synthesis in vitro of a polypeptide of 65000 molecular weight in an mRNA-dependent rabbit reticulocyte lysate. Inclusion of dog pancreatic microsomes in the cell-free translation system resulted in a heavy chain product of about 75000 molecular weight, presumably as a consequence of glycosylation in vitro. This species co-migrated in an SDS polyacrylamide gel with mature IgE heavy chain. Substantial purification of heavy chain mRNA was achieved by denaturing sucrose gradient centrifugation and agarose gel electrophoresis.

Transcription of adenoviral genetic information in isolated nuclei. Characterization of viral RNA sequences synthesized in vitro.

The virus-specific RNA sequences synthesized in nuclei isolated from adenovirus type 2-infected HeLa cells comprise a fraction of the total RNA similar to that observed with RNA made in vivo. By 16 h after infection, for example, some 25% of the RNA made in isolated nuclei is transcribed from adenoviral DNA. Only 10 to 15% of the adenoviral RNA sequences synthesized in nuclei isolated during the late phase of infection are transcribed by form III RNA polymerase. This RNA, whose synthesis is resistant to 0.5 microgram/ml, but sensitive to 200 microgram/ml of alpha-amanitin, sediments at about 5 S in denaturing sucrose gradients and must, therefore, represent the virus-associated RNAs. The remainder of the sequences are transcribed by form II RNA polymerase and sediment as several RNA species in denaturing sucrose gradients. The largest of these exhibits the size, 55 to 60 S, expected of a complete transcript of the major, adenoviral transcriptional unit expressed during the late phase. Hybridization of RNA 32P-labeled in nuclei isolated during the late phase of infection to restriction endonuclease fragments of adenoviral DNA immobilized on nitrocellulose filters suggests that sequences of this transcriptional unit are indeed transcribed in vitro. To make a detailed assessment of the fidelity of transcription in isolated nuclei, transcription reactions were performed in the presence of 5-mercuricytidine 5'-triphosphate and the RNA mercurated in vitro separated from endogenous RNA by chromatography on sulfhydryl-agarose columns by a stringent procedure. After demercuration, RNA made in nuclei isolated 20 h following adenovirus type 2 infection was hybridized to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus type 2 DNA. Such RNA is complementary to the r strand of adenoviral DNA from 16.6 units to a point to the right of 98.3 units. These sequences comprise the major transcriptional unit expressed during the late phase (Fig. 1). It is therefore clear that the fidelity of transcription of adenoviral DNA by form II RNA polymerase is preserved in isolated nuclei. Two 1-strand transcriptional units, those of the IVa2 and ts36 genes (see Fig. 1) are also active at 20 h after infection. The results of similar analysis of RNA made in nuclei isolated 4 and 12 h after infection are also presented and discussed in terms of the mapping of individual transcriptional units within the type 2 adenoviral genome and the temporal regulation of adenoviral gene expression during productive infection.

Partial characterization of procollagen messenger ribonucleic acid in a murine chondrosarcoma.

Cells from a rat chondrosarcoma in primary suspension culture were used for labeling studies and isolation of RNA. With extended labeling, a distinct fraction of poly(A+)RNA sedimented in the region of 26S on denaturing sucrose gradients. Similar profiles were obtained with labeled poly(A+)RNA isolated from normal cartilage tissues, as well as from chondrocytes in culture. This mRNA coded for collagenous protein when translated in a cell-free system derived from wheat germ. Among the polypeptides synthesized in vitro, one distinctive component of approximately 150,000 daltons was detected by gel electrophoresis. Polypeptides of identical size, sensitive to bacterial collagenase, were also synthesized in vitro by polysomes isolated from the chondrosarcoma.

The synthesis of presumptive procollagen messenger ribonucleic acid in the calvaria of the developing chick embryo

The presumptive messenger RNAs for type I procollagen were isolated from chick embryo calvaria at various stages of development. Poly(A)-containing RNA fractions from denaturing sucrose gradients directed protein synthesis in a cell-free system derived from wheat germ. Procollagen mRNA activity was detected in a region of about 26 S. Approx. 80% of the labeled proline incorporated into cell-free product was susceptible to digestion by purified bacterial collagenase. The synthesis of procollagen mRNAs was followed during development. Comparison of the in vitro labeled mRNAs from calvaria of day 12–16 embryos indicated that the 26 S component was most pronounced at day 13 and decreased progressively towards day 16. In addition, incubation of calvaria with tritiated nucleosides for 1.5–25 h revealed that 26 S mRNA was significantly labeled only after prolonged periods. The results suggest that procollagen mRNA is a relatively stable species with a prolonged half-life compared to the majority of mRNAs in this tissue.

Photochemical crosslinking of transcription complexes with psoralen. I. Covalent attachment of in vitro SV4O nascent RNA to its double-stranded DNA template

14C-labeled SV40 DNA has been transcribed with E. coli RNA polymerase using 3H-labeled ribonucleotide triphosphates as precursors. The resulting transcription complexes were then photochemically crosslinked with the psoralen derivative, 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT), at 37 degrees C and analyzed in SDS-sucrose gradients. It was found that the photochemical crosslinking procedure caused the nascent RNA chains to co-sediment with their double-stranded (helical) SV40 templates in the denaturing sucrose gradient. This result and several control experiments suggest that covalent linkages have formed between nascent RNA and helical DNA after the photochemical reaction. The crosslinking phenomenon was observed to be independent of the superhelical state of the DNA used as the template. Prior addition of EDTA to stop the transcription is not required for successful crosslinkage.

5'-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.

Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of gag- and env-gene products.

Polyadenylylated mRNA isolated from cells infected with Rauscher murine leukemia virus was fractionated by centrifugation in in a denaturing sucrose gradient into different sizes. Each RNA fraction was injected into oocytes of Xenopus laevis and the virus-specific products were analyzed by immunoprecipitation with polyvalent and monospecific antisera against polypeptides of Rauscher murine leukemia virus, and then by gel electrophoresis and scintillation autoradiography. It was shown that a 35S mRNA species directs the synthesis of a precursor of the internal or group-specific antigens of the virion (the gag-gene products). A 22S mRNA species directs the synthesis of two viral envelope polypeptides and their precursor polypeptide (env-gene products). The results indicate that the gag- and env-related polypeptides of Rauscher murine leukemia virus are synthesized uncoordinately and provide evidence for open and closed cistrons on the virus-specific mRNAs.

Molecular Weight Distribution of RNA Fractionated on Aqueous and 70% Formamide Sucrose Gradients

This chapter discusses that sucrose gradient centrifugation has been used for many years to fractionate RNA according to size. It has also been known for many years that, under nondenaturing conditions, there is no simple relation between RNA sedimentation constants and molecular weights. This observation has been confirmed more recently in studies of eukaryotic mRNAs, which exhibit anomalous sedimentation rates relative to ribosomal RNA on aqueous sucrose gradients. The chapter reviews that aqueous sucrose gradients are still being used to analyze the size distribution of nuclear RNA. The MW distribution of pulse-labeled RNA is also analyzed and found that RNA with MW's equal to and greater than 2 × l0 6 sediment as low as 20S on both aqueous and 70% formamide sucrose gradients. Thus, it is quite contrary to the observation that large nuclear RNA sediments too slowly relative to rRNA on fully denaturing Me 2 SO gradients, the former actually sediments too slowly on nondenaturing or partially denaturing sucrose gradients.