Atomic spectroscopic evidence for the absence of a low-molecular-weight (486) antigen in RNA extracts shown to transfer delayed-type hypersensitivity in vitro

Ribonucleic acid-rich extracts obtained from the spleens or lymph nodes of guinea pigs skin test sensitive to mono-(p-azobenzenearsonate)-N-chloracetyl-l-tyrosine (ARS-NAT) (MW 486) were able to convert “nonsensitive” peritoneal exudate cells (PEC) to a state of specific immunologic sensitivity, as assessed by the cell-migration-inhibition correlate of delayed hypersensitivity. Specific inhibition of migration of RNA-treated PEC by ARS-NAT antigen was observed while no inhibition of migration occurred with RNA alone or by incubation with unrelated antigens. The RNA used to transfer sensitivity was assessed for arsenic (As) content as a chemical marker for the ARS-NAT antigen utilizing two methods: a Gutzeit As assay, and atomic absorption spectroscopy (AAS). Preliminary chemical analysis utilizing the Gutzeit assay, which detects as little as 1 μg As, failed to detect As in 3200–4800 μg of RNA or in cell suspensions from the spleen, lymph nodes, and liver of immunized guinea pigs. Further attempts to detect As utilizing AAS, where the limit of As sensitivity was 0.1 ng, failed to detect As in 250 μg to 10 mg of “ARS-NAT-sensitive” RNA, suggesting that, if As is associated with the RNA-rich extracts, it could be present in an amount of no more than 5 pg in 500 μg of RNA; this corresponds to less than 0.0000065% ARS-NAT antigen. These results suggest an informational role for the RNA extracts in our delayed hypersensitivity system, paralleling similar evidence for the action of RNA extracts in antibody systems.     

Analysis of spleen focus-forming virus-specific RNA sequences coding for spleen focus-forming virus-specific glycoprotein with a molecular weight of 55,000 (gp55).

The 32S RNA of the Friend strain of spleen focus-forming virus (SFFV) contains two sets of sequences: about half is specific to SFFV, and the other half is in common with the sequence of the helper lymphatic leukemia virus. Fingerprinting analysis of RNase T1 oligonucleotides showed that the SFFV-specific sequences were located in two distinct regions: in the 3' half and near the 5' terminus of the genome. Translation of SFFV RNA in a cell-free system yielded three SFFV-specific polypeptides: two main products with molecular weights of about 47,000 (P47) and 16,000 (P16) and a variable amount of a product with a molecular weight of 40,000 (P40). P47 was translated from polyadenylic acid-containing fragments of 1,500 to 3,000 nucleotides with SFFV-specific sequences from the 3' half of the genome, whereas P16, which contained peptides in common with those of P47, was synthesized by smaller RNA. P47 formed in vitro was found to be structurally related to the protein portion of a glycoprotein, gp55, specifically found in SFFV-infected cells in vitro. It is concluded from the results that a defective env gene containing SFFV-specific sequences in the 3' half of the genome codes for SFFV-specific gp55.     

A novel low molecular weight ribonucleic acid (RNA) related to transforming growth factor alpha messenger RNA

Human transforming growth factor alpha (TGF alpha) is coded for by an mRNA of about 4800 nucleotides. The cDNA sequence demonstrates that the 50 amino acid TGF alpha is embedded in a larger 160 amino acid precursor protein. We report here that in addition to the 4800 nucleotide TGF alpha mRNA, there is a novel second RNA species of about 350 nucleotides that hybridizes to a human TGF alpha cDNA probe. This small RNA species has been found in the RNA of several human tumor cells including HT1080, A549, A431, A2058, and A673. We have demonstrated an inverse relationship between the amounts of the 4800 nucleotide TGF alpha mRNA and the 350 nucleotide novel RNA in these human cells. Restriction enzyme cleavage of a human TGF alpha cDNA probe into three separate domains consisting of a processed coding region and 5'- and 3'-preprocessed coding and untranslated regions showed that only the 3'-untranslated region hybridized to the 350 nucleotide RNA. Using sense and anti-sense single-stranded 3'-untranslated region probes, we determined that the 350 nucleotide RNA band may be composed of multiple species of RNA which are related to the anti-sense DNA strand that is opposite to the strand that codes for the 4800 nucleotide TGF alpha mRNA.     

The association of protein with the polyadenylic acid of HeLa cell messenger RNA: evidence for a "transport" role of a 75,000 molecular weight polypeptide.

The poly(A) in HeLa cell messenger RNA appears to be associated with proteins in a poly(A)-protein complex that can be isolated after treatment of mRNA-protein complexes with nuclease. The particle survives repeated sedimentation and zonal electrophoresis; [³⁵S]methionine in protein bands together with [³H]adenosine in poly(A). The largest (newest) poly(A)-ribonucleoprotein contains the largest poly(A) and the highest proportion of the most prominent polypeptide, P75 (M_r = 75,000). In addition, treatment of cells with 3′ deoxyadenosine (3′dA, cordycepin) prevents the labeling of new poly(A) as well as the appearance of [³⁵S]methionine-labeled P75 in the larger poly(A)-protein complexes. Furthermore, the pre-existent P75, detected by densitometric scan of polyacrylamide gels containing proteins from the larger poly(A)-ribonucleo-protein, also disappears in 3′dA-treated cells. These data suggest a role for the P75 in the appearance of new mRNA in the cell cytoplasm.     

Characterization of a low molecular weight glycolipid antigen from Cryptosporidium parvum

Cryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG1 subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose3-glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1-3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection.     

Polyadnylic acid sequences in RNA able to transfer specific delayed type hypersensitivity in vitro.

Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with 10^?3M cycloheximide, (Cy)], although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.     

Synthesis of low molecular weight RNA components in cells with a temperature-sensitive polymerase II

AF 8 cells are a mutant cell line of baby hamster kidney cells with a temperature-sensitive polymerase II activity. When these cells grow at the non-permissive temperature (40 degrees C) the syntheseis of low molecular weight RNA components D, C and A is preferentially inhibited, whereas the synthesis of rRNA, tRNA, 5 S RNA and component L is affected only a little or not at all. These results indicate that polymerase II catalyzes the synthesis of components D, C and A.     

Inhibition of peptide initiation by a low molecular weight RNA from rabbit reticulocytes

A heat-stable inhibitor of protein synthesis has been isolated from the postribosomal supernatant of rabbit reticulocytes. Its activity is not susceptible to protease treatment but is destroyed by incubation with alkali. Inhibitory activity can be quantitatively recovered in the aqueous phase after phenol extraction and has the ultraviolet absorption spectrum of a nucleic acid. It is concluded that the inhibitor is RNA. The inhibitory activity sediments in the range of 3 S, but it has not been demonstrated whether the inhibitor RNA is a single molecular species. The inhibitory RNA does not affect peptide elongation but rather blocks a step of peptide initiation. It does not interfere with the formation of the ternary complex between initiation factor 2, GTP, and methionyl-tRNAMetf and does not activate a protein kinase phosphorylating initiation factor 2. The inhibitory RNA appears to be a novel type of RNA that inhibits polypeptide initiation at a step involving ribosomal subunits.     

Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences.

We report the design, synthesis, and characterization of molecular beacons (MB) consisting of three distinct fluorophores, 6-carboxyfluorescein (Fam), N,N,N',N'-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5). The primary light absorber/energy donor (Fam) is located on one terminus of the MB, whereas the primary energy acceptor/secondary donor (Tam) and secondary acceptor (Cy5) are located at the other terminus of the MB. In the absence of target DNA or RNA, the MB exists in the stem-closed form. Excitation of Fam initiates an energy transfer cascade from Fam to Tam and further to Cy5 generating unique fluorescence signatures defined as the ratio of the emission from each of the three fluorophores. This energy transfer cascade was investigated in detail by steady-state and time-resolved fluorescence spectroscopy, as well as fluorescence depolarization studies. In the presence of the complementary target DNA, the MB opened efficiently and hybridized with the target separating Fam and Tam by a large distance, so that energy transfer from Fam to Tam was blocked in the stem-open form. This opening of the MB generates a "bar code" fluorescence signature, which is different from the signature of the stem-closed MB. The fluorescence signature of this combinatorial fluorescence energy transfer MB can be tuned by variation of the spacer length between the individual fluorophores.     

Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms

Low-molecular weight RNA (LMW RNA) analysis using staircase electrophoresis was performed for several species of eukaryotic and prokaryotic microorganisms. According to our results, the LMW RNA profiles of archaea and bacteria contain three zones: 5S RNA, class 1 tRNA and class 2 tRNA. In fungi an additional band is included in the LMW RNA profiles, which correspond to the 5.8S RNA. In archaea and bacteria we found that the 5S rRNA zone is characteristic for each genus and the tRNA profile is characteristic for each species. In eukaryotes the combined 5.8S and 5S rRNA zones are characteristic for each genus and, as in prokaryotes, tRNA profiles are characteristic for each species. Therefore, stable low molecular weight RNA, separated by staircase electrophoresis, can be considered a molecular signature for both prokaryotic and eukaryotic microorganisms. Analysis of the data obtained and construction of the corresponding dendrograms afforded relationships between genera and species; these were essentially the same as those obtained with 16S rRNA sequencing (in prokaryotes) and 18S rRNA sequencing (in eukaryotes).     

A new retinoid-binding protein with a low molecular weight

Cellular retinoid-binding proteins and nuclear receptors may mediate the intracellular transport and the action of retinoids in the control of differentiation and tumorigenesis. We report a new retinoid-binding protein (Ret BP) with a molecular size of 4,000 that binds retinol, retinoic acid, and some of their derivatives. Purification of Ret BP from chick skin cytosol involved DEAE-Sephadex, Sephadex G-100, and Mono Q column chromatography. The Ret BP-retinoid complex eluted at 195 mM NaCl during Mono Q column chromatography using a 0-300 nM NaCl gradient. Superose-12 column chromatography indicated a molecular size of 4,000 for Ret BP. The binding protein showed a pI of 6.8 on electrofocusing in ampholines of pH 3-10. Ret BP may act as an affinant for retinoids in the cell, and may serve to dispense the ligands to their respective functionally active sites.     

Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach

A new chemotaxonomic method is presented for the identification of eubacteria. This method is based on one-dimensinal gel electrophoresis of total RNA extracts from eubacteria. Only low molecular weight (<150 nucleotides) RNA, comprising 5S ribosomal and transfer RNA, was used for the identification. The high resolution of the electrophoresis, better than half a nucleotide, allowed construction of low molecular weight (LMW) RNA profiles that contained 10 to 20 bands per strain. LMW RNA profiles of a set of eubacterial reference strains showed on variation in dependence on culture conditions or physiological state of the cells. Computer-assisted data evaluation, including six molecular weight markers, enabled the calculation of relative nucleotide units (RNU) for every band. The resulting normalized band pattern allowed the identification of identical strains on different gels. The relative position of the single bands from the different groups of RNAs made an identification of bacterial strains to genus and often species level possible.Especially valuable for the identification were the large, class 2 tRNAs that showed certain variation among species of the same genus and varied considerably among different genera. RNA profiles can provide a rapid and inexpensive screening technique for the taxonomic classification of single bacterial strains. Potential fields of application for this technique might be bacterial taxonomy, biotechnology and ecology.     

Specific activation of murine B cells by low molecular weight polyamino-polycarboxylic acids (ampholytes).

Commercial preparations of ampholytes (AM), which consist of mixtures of large numbers of polyamino-polycarboxylic acids of molecular weight less than 1000 and whose chemical composition is otherwise not specified by the manufacturer, were found to induce blastogenesis in spleen cell cultures from various strains of mice. The blastogenic response of the spleen cells to the ampholytes was 2 to 12 times greater than that of the unstimulated cultures, and peak stimulatory activity occurred 2–4 days after stimulation. Preparations consisting of either acidic, neutral, or alkaline ampholytes were all found to be mitogenic, although the alkaline ampholytes generally induced the highest stimulation and were active over the widest concentration range (0.08–80 μg per culture). Studies using spleen cells from nu/nu mice, CBA/N mice, organ distribution studies, and the use of cytotoxic antisera and complement for the depletion of thymus-derived (T) cells or bone marrow-derived (B) cells suggested that ampholytes are mitogenic for murine B cells. These cells arise early in ontogeny, because the ampholytes were found to be as mitogenic for spleen cells from newborn as for adult mice. Further, in concordance with the characteristics of other B-cell mitogens, injection of mice with ampholytes induced polyclonal antibody synthesis. The possibility that blastogenic stimulation was due to a contaminant was ruled out by demonstrating that anti-lipid A-treated, ultrafiltered, and isoelectric-focused ampholytes retained stimulatory activity. The results of these investigations suggest that commercial ampholyte preparations contain an undetermined number of low molecular weight (< 1000) acidic, neutral, and alkaline polyamino-polycarboxylic acids which are specific mitogens for a primitive population of murine B cells.