A fluorimetric assay for available lysine in proteins

A two-dimensional fingerprinting gel system that provides sensitive analyses with high resolution of T1-resistant oligonucleotides of large RNA molecules is described. Unique oligonucleotides less than 30 bases in length are recovered quantitatively while longer oligonucleotides are recovered in very large (～90%) yields by active transfer of the fingerprint to DEAE paper. After elution of the oligonucleotides from DEAE paper, secondary analysis is performed by digestion of oligonucleotides with pancreatic RNase and separation of the products by high-voltage electrophoresis on polyethyleneimine cellulose. The complete analysis of up to 40 oligonucleotides can be accomplished within 4 days.     

Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase

A method is described for the determination of nucleotide sequences in DNA by using specific oligonucleotides as primers for copying specific regions by DNA polymerase. The method was applied to bacteriophage f1 DNA using the synthetic octanucleotide A-C-C-A-T-C-C-A as primer and a sequence (sequence A) of 81 nueleotides was determined. Synthesis was carried out in the presence of manganese and with one of the deoxyribotriphosphates (dCTP or dGTP) replaced by the corresponding ribotriphosphate so that mixed oligonucleotides were found which could be specifically split at the ribonucleotide residues by the appropriate ribonuclease or by alkali. The relative order of the digestion products was determined by fractionating the undigested oligonucleotides according to size on a two-dimensional system and digesting the isolated products. In the presence of rGTP the octanucleotide appeared to prime at a second site giving rise to a second sequence (B) besides sequence A. The complementary sequence to sequence A, which corresponds to the plus strand of f1 DNA and to the messenger RNA, contains five nonsense codons, four of which are in the same phase, and two possible initiation codons. It also contains a repetitive sequence which suggests its evolutionary origin by duplication.     

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.     

Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA

Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton &; Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.     

A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA.

A method for the isolation of RNA fragments originating from defined regions of bacteriophage Qbeta RNA minus strands is described. Large RNase T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA. The nucleotide sequences (13 to 26 nucleotides) and map positions of these oligonucleotides were known from previous work (Billeter, M. A. (1978) J. Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the average) using terminal adenylate transferase, these pure oligonucleotides were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in vitro. Fragments in the size range of 100 to 500 nucleotides were then generated by partial digestion with RNase T1. Fragments hybridized to such oligonucleotides were recovered by chromatography on poly(U)-Sephadex and then resolved according to their size by polyacrylamide gel electrophoresis. The specificity and reproducibility of the method as well as its suitability for the sequence analysis of Qbeta RNA was verified by using in particular a linker oligonucleotide derived from a Qbeta RNA region near the 3' end. The sequence catalogues of the RNase T1 and RNase A oligonucleotides of two fragments isolated in this way, 202 and 310 nucleotides in length, were established and all fragments isolated were shown to contain a sequence complementary to the linker oligonucleotide.     

Properties of 2'-fluorothymidine-containing oligonucleotides: interaction with restriction endonuclease EcoRV

2'-Fluorothymidine (Tf) was synthesized via an improved procedure with (diethylamino)sulfur trifluoride. The compatibility of the analogue with DNA synthesis via the phosphoramidite method was demonstrated after complete enzymatic digestion of the oligonucleotides d(Tf11T) and d(Tf3T), the sole products of which were 2'-fluorothymidine and thymidine in the expected ratio. The 2'-fluorothymidine was also incorporated into the EcoRV recognition sequence (underlined), within the complementary oligonucleotides d(CAAACCGATATCGTTGTG) and d(CACAACGATATCGGTTTG). Thermal melting characteristics of these duplexes showed a significant decrease in stability only when both of the thymidine residues in one of the strands were replaced. In contrast, when all of one strand of a duplex contained 2'-fluorothymidine, as in d(Tf11T).d(A12), a substantially higher Tm and cooperativity of melting was observed relative to the unmodified structure. EcoRV cleaved a duplex that contained a 2'-fluorothymidine at the scissile linkage in each strand at two-thirds of the rate obtained for the unmodified structure. A duplex containing two 2'-fluorothymidine residues in one strand and none in the other was cleaved at one-third of the rate in its unsubstituted strand, whereas the cleavage rate was reduced to 22% in its modified strand.     

Analysis of the pancreatic-ribonuclease-digestion products of alfalfa-mosaic-virus ribonucleic acid. Sequence homologies between the different RNAs.

Alfalfa mosaic virus (AMV) genome consists of three pieces of RNA (24-S, 20-S and 17-s RNA). For infectivity these three RNAs and the coat protein are required. In the absence of coat protein, infectivity is obtained by adding the 12-S RNA also normally present in the virus. This 12-S RNA represents the message for coat protein. Thus a redundancy of the gene for coat protein exists between 12-S RNA and one of the other RNAs. Sequence analysis of the oligonucleotides resulting from pancreatic ribonuclease digestion of the AMV RNAs indicates that the nucleotide sequence of 12-S RNA occurs in 17-S RNA. Analysis of the pancreatic ribonuclease digestion products of the two larger alfalfa mosaic virus RNAs (20-S and 24-S RNA) shows some oligonucleotides containing seven, eight and nine nucleotides with the same structure present in both RNAs. The possibility of a limited nucleotide sequence homology between these two RNAs is discussed. The comparison of the RNase digestion products of 20-S and 24-S RNA with those of 12-S or 17-S RNA revealed no homologous oligonucleotides, thus the origin of 12-S RNA appears to be 17-S RNA.     

Demonstration of the "5-8 S" ribosomal sequence in HeLa cell ribosomal precursor RNA

HeLa cell “5.8 S” ribosomal RNA was digested with T₁ ribonuclease and the digestion products were characterized. In particular several hexa-, or larger, oligonucleotides were well fractionated by T₁ ribonuclease plus alkaline phosphatase fingerprints. The sequences of these large products were determined. The same large products were identified in fingerprints of “native” 28 S RNA, that is, 28 S RNA to which 5.8 S RNA is attached. The products were demonstrably absent in fingerprints of heat-denatured 28 S RNA, which lacks the 5.8 S fragment. The oligonucleotides were present in fingerprints of 32 S RNA, whether previously heated or not. One of the largest 5.8 S oligonucleotides contains an alkali-stable (2′-O-methylated) dinucleotide, Gm-C. This product was identified in fingerprints of methyl-labelled 45 S RNA. These findings prove that the 5.8 S ribosomal sequence is present within HeLa cell ribosomal precursor RNA. In addition to the methylated nucleotide, two pseudouridylate residues were discovered in HeLa cell 5.8 S RNA.     

Amino acid sequence of a neurotoxin from the anemone Radianthus macrodactylus

Amino acid sequence of neurotoxin I isolated from the anemone Radianthus macrodactylus, and consisted of 48 amino acid residues, including six cysteines, was determined by analysis of products of its trypsin and chymotrypsin digestion.     

Avocado sunblotch viroid: primary sequence and proposed secondary structure.

The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). A secondary structure model for ASBV is proposed and contains 67% of its residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity.     

Mode of Action and Specificity of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

A nuclease from N. crassa mycelia was found to attack both heat-denatured and native DNA in endonucleolytic manner. The products of exhaustive degradation of heat-denatured DNA were mainly di- to pentanucleotides bearing 5′-phosphoryl groups. 5′-Mononucleotides amounted to 4.4% of the total products and the base distribution was in the following order: dTMP > dCMP > dGMP > dAMP. Analysis of the residues at 5′- and 3′-termini of the oligonucleotides showed that thymidine was predominant at both termini, especially at 3′- termini. Also the analysis of terminal residues produced by limited digestion (27% and 55.5 % of the substrate were rendered acid soluble, respectively) gave the same results as above. Therefore, it was suggested that N. crassa nuclease has some preference for thymidine residue to hydrolyze the sequence of ?T ↓ pT? or ?T ↓ pX-predominantly. The activity toward synthetic polymers was in the following order; poly d(A-T) ? poly dA poly dT > poly d(G-C) > poly dGpoly dC. The correlation between GC-contents and the activity was also investigated.     

Sequence studies on tRNAPhe from placenta: comparison with known sequences of tRNAPhe from other normal mammalian tissues.

Phenylalanine-specific tRNA was isolated from human placenta and degraded to mixtures of oligonucleotides. Tritium sequence analysis of the digestion products indicates that the sequence of human placenta tRNA^Phe is identical to that of calf liver tRNA^Phe and differs only slightly from that of rabbit liver tRNA^Phe.     

Improved oligonucleotide sequencing by alkaline phosphatase and exonuclease digestions with mass spectrometry

The combination of exonuclease digestion and mass spectrometry has been widely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, especially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late digestion products due to artifact peaks from nucleotide cluster ions. Here we report a procedure that eliminates the effect of the cluster ions. In this method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectrum of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 5'-terminus of the oligonucleotide. With this approach, the signal/noise ratio of the reconstructed molecular weight spectrum is greatly improved for relatively large oligonucleotides, and only a single digestion is needed for sequencing.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

Catalytic hydrogenolysis of poly-iodinated recombinant human insulin-like growth factor II (IGF-II): a potentially useful method for the tritiation of IGF-II.

A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Mono- and di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the iodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.     

Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli.

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.     

Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS).

We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.