Fingerprinting Procedure Research Articles

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA UCG Arg, tRNA GUC Asp, and tRNA GAU Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA UCG Arg and tRNA GUC Asp determined similarly, and a bovine mitochondrial tRNA GAU Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.

A new method to characterize the translation initiation sites of messenger ribonucleic acids

RNA can be radiolabeled in vitro with 125I in uridine and cytidine residues by initial mercuration of these pyrimidine residues by the method of Dale et al. [Dale, R. M. K., Martin, E., Livingston, D. C., & Ward, D. C. (1975) Biochemistry 14, 2447-2457] and subsequent electrophilic displacement of the mercury with 125I+ generated by the method of Commerford [Commerford, S. L. (1971) Biochemistry 10, 1993-2000]. These two reactions can be manipulated to produce intact high specific activity 125I-labeled mRNA containing approximately equal specific activity in their 5-[125I]iodouridine and 5-[125I]iodocytidine residues. In vitro radiolabeling of satellite tobacco necrosis virus RNA (STNV RNA) by this procedure yields a 125I-labeled mRNA that is biologically active in ribosome protection analyses in that one obtains the correct translation initiation fragments of the mRNA. Exhaustive digestion of a specific 125I-labeled 32 nucleotide long initiation fragment of STNV RNA using four separate nucleotide-specific digestion (hydrolysis) reactions yields four different populations of 125I-labeled digestion products that can be resolved by two-dimensional fingerprint procedures. Characterization of all these 125I-labeled digestion products followed by overlap nucleotide sequence comparisons of these specific digestion products allows a nucleotide sequence determination of the original 32 nucleotide long translation initiation fragment of this mRNA. This suggests that this overall in vitro procedure can be used to determine the nucleotide sequence of the translation initiation site(s) of any mRNA.

Microfingerprints of proteins through labeling acetylation of their proteolytic peptides

Microgram amounts of proteins applied to polyacrylamide gel electrophoresis were subjected to a fingerprinting procedure using a combined proteolysis-acetylation method with the aid of 14C-labeled acetic anhydride of high specific activity. After staining, gel slices were partially dried and were resoaked in a solution of a protease. After elution and acetylation, the resulting peptides were resolved in fingerprints on cellulose thin-layer chromatography plates and subjected to autoradiography with or without sensitization. Yields, completeness of fingerprinting, and possible artefacts were investigated.

Die Isopolymolybdationen in wäßrigen Medien im Bereich Z ≦ 1,14 / Concerning Isopolymolybdate Ions in Aqueous Media in the Range Z ≦ 1.14

The system H+/MoO4 2- was investigated by Raman spectroscopy in the range Z = 0 to 1.14 at initial concentrations CMoO₄ 2- = 0.02 to 2M and concentrations of the ionic medium CMe+ = 2CMoO₄ 2- to 3M Me(Cl,NO3) (Me=Li, Na, K,NH4 , Mg/2). The mole-ratio method and intensity difference diagrams do not indicate any species between MoO4 2- and Mo7O24 6- contrary to propositions in recent papers. A detailed evaluation of the extensive data shows the possible portion of such species to be < 3 % , this value having a statistical certainty of 95%. Comparing the solutions having Z = 1.1 by fingerprint procedures, in all cases Mo7O24 6- is the first main product (detectable by static methods) irrespective of the nature and concentration of the ionic medium. Thus, Mg2+ ions do not influence the course of aggregation of molybdate ions as has been proposed in the literature. This is in accordance with theoretical investigations since the Mo7O24 6- ion has the distinction of a number of favourable structural parameters in the system that become operative in case of scarcity of H+ ions.

5′-Terminal structures of poly(A) + cytoplasmic messenger RNA and of poly(A) + and poly(A) − heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. 32P-labeled RNA was digested with ribonucleases T 2, T 1 and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes. Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m 7G 5′ppp 5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N 6,2′- O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base. Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.

Isolation and identification of the messenger ribonucleic acid for a structural lipoprotein of the Escherichia coli outer membrane.

The cells of Escherichia coli strain CP 78 were labeled with [32P]orthophosphate and the total radioactive RNA was prepared from the cells. The mRNA that codes for a structural lipoprotein in the outer membrane was purified from the total RNA by three successive electrophoreses on polyacrylamide slab gels, twice at pH 8.3 and once at pH 3.5 in 7 M urea. Approximately 0.002% of the total radioactive phosphate used was incorporated into the fraction containing the most purified mRNA. The two-dimensional fingerprint of the T1 ribonuclease digest of the 32P-labeled mRNA showed that the purity of the mRNA was as high as 90%. A preliminary sequence analysis was carried out on the T1 ribonuclease oligonucleotides which had been separated by the fingerprinting procedure. By using the established amino acid sequence of the lipoprotein and the genetic code, three relatively long oligonucleotides were assigned to code for three different parts of the lipoprotein. From these data, the present RNA fraction was identified as the lipoprotein mRNA. From the analysis of the T1 ribonuclease oligonucleotides, the mRNA was estimated to be 360 +/- 10 nucleotides in length. Although the length of the mRNA was enough to code for 2 lipoprotein molecules, T1 ribonuclease digestion of the mRNA yielded only 1 mol/mol of mRNA of the individual oligonucleotides assigned to parts of the amino acid sequence of the lipoprotein. This suggests that the mRNA codes for only 1 molecule of the lipoprotein. It was also found that the mRNA has no polyadenylate sequence at the 3' end.

The modified 5′-terinal sequences in messenger RNA of mouse myeloma cells

A fingerprinting procedure has been used to investigate modified nucleotides and 5′-end groups in polyadenylated mRNA of mouse myeloma cells. MPC-11-66.2 cells were labelled in culture with [32P]orthophosphate, total RNA extracted from cytoplasmic fractions, and polyadenylated RNA selected by two cycles of oligo(dT)-cellulose chromatography. The RNA was digested with a mixture of pancreatic, T1 and T2 ribonucleases, which hydrolyses all phosphodiester bonds except those following 2′-O-methyl nucleosides, and the digests were fractionated by electrophoresis on cellulose acetate at pH 3·5, followed by electrophoresis on DEAE-paper at pH 3·5. The fingerprints revealed N6-methyl adenylic acid and a set of ribonuclease-resistant oligonucleotides, about 20 of which were resolved by this technique. The modified nucleotides were present in both microsomal mRNA and mRNA of post-microsomal supernatants. The analysis of the oligonucleotides has indicated that they have structures of the two forms: m7G5′ppp5′NmpNp (type I) and m7G5′ppp5′NmpNmpNp (type II), where m7G is 7-methyl guanosine, Nm are 2′-O-methyl nucleosides and N are standard nucleosides. These structures can only be derived from the 5′-termini of the molecules. The evidence for the structures is as follows. The oligonucleotides were resistant to alkaline hydrolysis but contained an alkali-labile component (7-methyl guanosine). Treating them with alkaline phosphatase released only the 3′-phosphate, and the amount of phosphate resistant to digestion indicated that there were about four “internal” phosphates. All the oligonucleotides were resistant to the 5′-exonuclease spleen phosphodiesterase. However, both venom phosphodiesterase and nucleotide pyrophosphatase cleaved pm7G from the (3′-phosphorylated) oligonucleotides. This cleavage exposed two 5′-phosphates on the other product, indicating that the pm7G was derived from the 5′-terminus by the scissions m7G5′p/ppNmpNp and m7G5′p/ppNmpNmpNp. Digestion of dephosphorylated oligonucleotides with venom phosphodiesterase gave pm7G, pN, either one or two pNm, Pi and some ppNm. These are the products expected from the scissions m7G5′p/ppNm/pN and m7G5′p/ppNm/pNm/pN, with partial hydrolysis of the ppNm. The mRNA contains a minimum of 27 5′-terminal sequences of the two types above. The derivation of these sequences, which represent a catalogue of the 5′-termini of a very large fraction of the myeloma mRNAs, is described. No sequences with a diphosphate linkage have been detected, nor any with a different 5′-terminal nucleoside. The first Nm in the sequences can be any of the standard four or A*m, an unidentified nucleoside which appears to be a 2′-O-methyl adenosine with a base modification, and the second Nm can be any of the standard four. No intermediate, partially modified structures have been detected. The different 5′-terminal sequences in myeloma mRNA vary considerably in frequency.

Transcription by Escherichia coli RNA polymerase of a single-stranded fragment by bacteriophage φX174 DNA 48 residues in length

A single-stranded fragment of bacteriophage φX174 DNA, 48 residues in length and of known sequence, has been transcribed by Escherichia coli DNA-dependent RNA polymerase. After transcription for eight hours at 37 °C the molar yield of RNA synthesized was 20 to 40 times the molar amount of template DNA present. The products of transcription were very heterogeneous in size, indicating multiple points of initiation and termination along the template. Sequence analysis of the heterogeneous transcripts included a two-dimensional fingerprinting procedure designed to separate oligonucleotides containing initiation and termination points from internal, complete oligonucleotides produced by digestion with T 1 or pancreatic ribonuclease. Overlapping sequences were obtained by partial digestion with T 1 ribonuclease and it was shown that the sequence of all but the first five 5′-terminal residues of the template could be determined by this method.