Drosophila melanogaster has different ribosomal RNA sequences on S and Y chromosomes.

The primary structures of ribosomal RNAs transcribed from the nucleolus organizers on X and Y chromosomes of Drosophila melanogaster were compared by RNase T₁ fingerprints made with two different systems; i.e. homochromatography on DEAE-cellulose, and polyethyleneimine-cellulose thin-layer chromatography.Ribosomal RNA derived from the X-linked nucleolus organizer was obtained from a strain producing only female larvae and ribosomal RNA derived from the Y-linked nucleolus organizer was isolated from a mutant lacking the X-linked nucleolus organizer.No difference was detected between the fingerprints of 28 S RNA from these animals.In 18 S RNA, however, one oligonucleotide showed a remarkable difference in mobility. The structure of the X-linked organizer-specific oligonucleotide was 5′ U-C-U-U-U-U-U-U-C-C-U-A-U-G 3′, and that of the Y-linked organizer-specific oligonucleotide was 5′ U-C-U-C-U-U-U-U-C-C-U-A-U-G 3′, indicating one base substitution (U á3 C) between them.The absence of 5′-temninal phosphate in this oligonucleotide and available sequence data also suggest that these oligonucleotides did not come from either the 5′ or 3′ terminus of 18 S RNA.D. simulans, whose Y chromosome has no nucleolus organizer (Ritossa &; Atwood, 1966), showed an 18 S RNA fingerprint having only the X-linked organizer-specific oligonucleotide.We conclude from these results that in Drosophila the ribosomal RNA gene sequences are different for the two nucleolus organizers located on the X and Y chromosomes. The implications of those findings concerning the parallel evolution of these genes are discussed.     

Sequence analysis of T1 ribonuclease fragments of 18S ribosomal RNA by 5''-terminal labeling, partial digestion, and homochromatography fingerprinting.

The method employed to determine the sequence of a T1 RNase fragment, A-A-A-A-A-U-A-A-C-A-A-U-A-C-A-Gp, from Novikoff rat hepatoma 18S ribosomal RNA is described. This method is applicable to any oligoribonucleotide produced by specific endonucleases that leave the newly cleaved 5'-end free for labeling with polynucleotide kinase and gamma-(32p)-ATP. The (32p)-labeled oligoribonucleotide is subjected to partial endonucleolytic digestion and fractionated by two-dimensional homochromatography fingerprinting. The nucleotide sequence is determined by following mobility shifts of the labeled and partially digested oligoribonucleotides in homochromatography fingerprinting.     

The use of nuclease P1 in sequence analysis of end group labeled RNA.

A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.     

Fingerprinting studies of the maturation of ribosomal RNA in mammalian cells.

32P-labelled ribosomal RNA of L 5178 Y cells (a mouse cell line) was digested with T1 ribonuclease and fingerprinted by electrophoresis at pH 3.5 on cellulose acetate and homochromatography on DEAE-cellulose thin-layer plate. From this, it can be concluded that 18-S and 28-S RNA have different and characteristic fingerprints and that the number, the size and the frequency of the large T1 oligonucleotides demonstrate that the guanylic residues are randomly interspaced along the molecule. Using a double-labelling technique with 32P-labelled 45-S RNA and 14C-labelled 18-S RNA or 28-S RNA, long T1 oligonucleotides of the 45-S RNA can be divided into three classes: (a) those which are lost during the transition, (b) those which are present in the 18-S RNA and (c) those which are present in the 28-S RNA. These results provide direct evidence for the existence of one common precursor for the two mature ribosomal RNAs. The comparison of the fingerprints of T1-ribonuclease-digested 47-S, 45-S and 41-S RNA shows that the 47-S and 41-S RNA have a characteristic ribosomal pattern. Finally the size, the number and the mobility of the oligonucleotides present in the different RNA precursors but absent from the mature RNA demonstrate that the non-conversed RNA pieces do not have a monotonous and repetitive sequence.     

Rapid cataloging of ribonuclease T1 resistant oligonucleotides from ribosomal RNAs for phylogenetic studies

A rapid and simple method of oligonucleotide cataloging for phylogenetic studies is presented. It involves in vitro 5'-32P-labelling of RNase T1 - resistant oligonucleotides of ribosomal 16S RNA and finger-printing by high voltage electrophoresis and gradient thinlayer chromatography. Oligonucleotide sequences are established by the mobility shift method, using controlled alkali cleavage, high voltage electrophoresis and homochromatography. These procedures facilitate in particular the analysis of long RNase T1 - resistant oligonucleotides. Oligonucleotide catalogs are established fo three Actinomycetes, namely Oerskovia turbata, Actinoplanes philippinensis and Ampullariella regularis. These catalogs are equivalent to those obtained by methods which were described by Sanger and Woese.     

Selectivity of RNA chain initiation in vitro. 1. Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides.

A method for the rapid and quantitative analysis of 5'-terminal oligonucleotides of RNAs made in vitro is described. The method involves synthesis of RNA in the presence of [gamma-32P]ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic ribonucleases to release labeled 5'-triphosphate termanated oligonucleotides. The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer system using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the first dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second. RNAs made with E. coli RNA polymerase and lambdacb2, T7, T4, and adenovirus 2 DNA yield characteristic fingerprint patterns. The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed.     

Evolutionary trends in 18S ribosomal RNA nucleotide sequences of rat, mouse, hamster and man.

The large T1 ribonuclease fragments of 18S ribosomal RNA from four mammalian species, rat, mouse, hamster and man, were compared by two-dimensional homochromatography fingerprinting. The nucleotide sequences of the large T1 ribonuclease fragments, polypyrimidines and polypurines which were different among the four mammalian species were determined and compared. The method used for determining nucleotide sequences utilizes 32p-labeling of oligonucleotides at their 5'-termini by polynucleotide kinase, partial digestion by ribonucleases and analysis of labeled spots by homochromatography-fingerprinting. Several examples of point mutations were detected. It was of interest that the 18S rRNA of Chinese hamster has more oligonucleotide sequences in common with those of man that rat or mouse.     

A two-dimensional gel electrophoretic procedure for the separation of complex mixtures of 4–12S RNAs

A two-dimensional gel electrophoretic method suitable for the separation of complex mixtures of RNA species in the size range of 4 to 12 S is described. A 3.6–11% polyacrylamide gradient gel containing a gradient of 0–7 m urea was used in the first dimension, and a transverse 3.6–22.6% polyacrylamide gradient gel containing 5 m urea was used in the second dimension. The method was applied to the separation of total cytoplasmic RNAs from a cellular slime mold. In this method reproducible fingerprints were obtained by the use of visible-marker RNA.     

A method for the isolation of segments from the 5′ ends of retrovirus RNA

A method for the isolation of segments of any desired length from the 5′ end of retrovirus RNA has been tested. The method is based on selection of 5′-specific segments by hybridizing suitably fragmented genomic (35 S) RNA to mercurated strong stop cDNA followed by chromatography on sulfhydryl-agarose. The method has been shown to be effective for Akv viral RNA by observing the T1 oligonucleotide fingerprints of a 5′-enriched fraction. This fingerprint pattern is of lower complexity than that of total 35 S RNA, contains oligonucleotide spots that have previously been assigned as 5′ specific by conventional fingerprinting methods, and does not overlap with the pattern from 3′-specific RNA.     

CTAB–Urea Method Purifies RNA from Melanin for cDNA Microarray Analysis

Melanin represents a major problem for the study of melanoma by microarrays since it is retained during RNA extraction and inhibits the enzymatic reactions used for probe preparation. Here we report a new method for cleaning RNA from melanin, based on the use of the cationic detergent cetyl‐trimethylammonium bromide (CTAB)–urea for RNA precipitation. This method is easy to perform and has a low cost. Purified RNA is recovered with high quality and good yield. CTAB–urea treated RNA from highly pigmented melanoma cells can be successfully reverse transcribed and labeled to obtain probes which can be subsequently used in cDNA microarray experiments, giving consistent and reproducible results.     

Comparison of gas chromatography mass spectrometry methods for the determination of delta9-tetrahydrocannabinol in plasma

A method for the identification of delta9-tetrahydrocannabinol by gas chromatography mass spectrometry has been developed, and this method has been compared with other techniques, such as detection via thin-layer chromatography using tritium labeled delta9-tetrahydrocannibinol and a dual gas chromatographic method. The gas chromatographic mass spectrometric method was found to be equal or superior to other techniques and has the added advantage of being highly specific for the compound analyzed. An alternate approach using chemical ionization is also described; however, this procedure does not show significant advantages over the electron impact method. These methods show a practical lower detection limit of 500 pg ml-1 of plasma in clinical practice.     

Detection of femtomolar quantities of the ganglioside GM1 on thin-layer chromatography plates by native choleratoxin and labeled antisera

After separation of gangliosides by thin-layer chromatography, femtomolar quantities of G_M1 were detected by incubating the plate with native choleratoxin, followed by anticholeratoxin and species-specific labeled antiserum. Only stable reagents were involved when antiserum labeled with horseradish peroxidase was used. Native choleratoxin rather than iodinelabeled toxin ensured good reproducibility of the method.     

Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.     

Adenovirus type 2 late mRNA's: structural evidence for 3'-coterminal species.

Adenovirus type 2-infected HeLa cells were labeled with 32PO4 during the period 14 to 17 h postinfection. Viral mRNA's with polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fractionated according to size by electrophoresis through an acrylamide-agarose slab gel. Messenger bands were eluted and partially degraded with alkali. RNA fragments from each band that contain polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fingerprinted two-dimensionally after T1 RNase digestion. Three bands, with mobilities of approximately 26S, 21S, and 18S, shared two large characteristic T1 oligonucleotides in common in the fingerprints of their 3'-terminal sequences. These oligonucleotides were mapped with a Hpa II restriction fragment of adenovirus type 2 DNA with coordinates 49-50.2. We conclude that the three mRNA's are coterminal in sequence at their 3' ends and overlap at internal positions. Implications for the protein-coding potential of these mRNA's and the mechanisms of adenovirus tyep 2 late RNA processing are discussed.     

Use of lysed human platelets to study prostaglandin E2 production

The conversion of 1-¹⁴C-arachidonic acid into prostaglandin E₂ was studied in lysed human platelets. Optimum production of the labeled reaction product was obtained when reduced glutathione and hydroquinone were included in the incubations. The labeled product was characterized by silicic acid column chromatography, thin-layer chromatography, and gas-liquid chromatography and was found to behave as standard prostaglandin E₂. The results indicate that the prostaglandin synthetase in the human blood platelet is similar to prostaglandin synthetases found in other tissues.     

3'-terminal processing of ribosomal RNA precursors in mammalian cells.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.     

Two zinc finger proteins from Xenopus laevis bind the same region of 5S RNA but with different nuclease protection patterns.

Immature oocytes from Xenopus laevis contain a 42S ribonucleoprotein particle (RNP) containing 5S RNA, tRNA, a 43 kDa protein, and a 48 kDa protein. A particle containing 5S RNA and the 43 kDa protein (p43-5S) liberated from the 42S particle upon brief treatment with urea can be purified by anion exchange chromatography. The purified p43-5S RNA migrates as a distinct species during electrophoresis on native polyacrylamide gels. Radiolabeled 5S RNA can be incorporated into the p43-5S complex by an RNA exchange reaction. The resulting complexes containing labeled 5S RNA have a mobility on polyacrylamide gels identical to that of purified p43-5S RNPs. RNP complexes containing 5S RNA labeled at either the 5' or 3' end were probed with a variety of nucleases in order to identify residues protected by p43. Nuclease protection assays performed with alpha-sarcin indicate that p43 binds primarily helices I, II, IV, and V of 5S RNA. This is the same general binding site observed for TFIIIA on 5S RNA. Direct comparison of the binding sites of p43 and TFIIIA with T1 and cobra venom nucleases reveals striking differences in the protection patterns of these two proteins.     

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Triacylglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity of the biosynthesis of triacylglycerols by rat intestinal mucosa was examined by means of radioactive and mass tracers, and thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the triacylglycerols isolated by solvent extraction and thin-layer chromatography. Analyses of the molecular species of the triacylglycerols labeled from monoacylglycerols showed that the 2-monoacylglycerol pathway was responsible for the biosynthesis of a maximum of 90% and the X-1-monoacylglycerol pathway for about 10% of the total radioactive triacylglycerols. Detailed analyses of the molecular species of triacylglycerols labeled fro free fatty acids showed that the phosphatidic acid pathway contributed a minimum of 20-30% of the total labeled triacylglycerol formed. There was a preferential utilization in triacylglycerol biosynthesis of the more unsaturated diacylglycerols arising from the monoacylglycerol pathway and of the more saturated diacylglycerols originating from the phosphatidic acid pathway. The above experiments do not allow a demonstration of the utilization of the sn-2,3-diacylglycerols in triacylglycerol biosynthesis but are not inconsistent with it.