An improved method for the enzymatic transformation of nucleosides into 5′-monophosphates

An improved method to transform nucleosides into 5-monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn²⁺. Under optimal conditions, the extent of nucleoside transformations into nucleoside-5-monophosphates were 41–91% (mol).Revisions requested 22 September 2004; Revisions received 11 October 2004     

Degradation of DNA into 5′-Monodeoxyribonucleotides in the Presence of Mn2+ Ions

DNA is known to be aggregated by metal ions including Mn²⁺ ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn²⁺ ions ([Mn]/[P] = 46.3) at 70 °C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol–chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Cd²⁺, as transition element metal ions, were effective as to the degradation into dNMP. Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺, as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl^?, CH₃OO^?, and NO₃ ^? were found to increase the degradation rate. Sixty μg of the 120 μg of the starting DNA in 450 μl was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn²⁺ ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH^? ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5′-NMP, based on the H¹NMR spectra. This prosess should prove to be a new process for the production of 5′-dNMP in addtion to the exonuclease.     

Isolation of the 5′-End of Plant Genes from Genomic DNA by TATA-Box-Based Degenerate Primers

We report a rapid and simple method for isolating the 5′-end of plant genes from genomic DNA by polymerase chain reaction (PCR) with TATA-box-based degenerate primers (TDPs). The TDPs were specially designed according to the TATA box, which is conserved in the promoter region of most plant genes. The unknown 5′-ends of several genes in different plants were isolated by PCR with gene-specific primers of the known core fragment and the TDPs. Our method does not require the arduous RNA manipulations and expensive enzyme treatments of the popular rapid amplification of cDNA ends (RACE) and its variants, and so could be a cheap practical alternative.     

Is the binding of magnesium (II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance

Previous reports on the interaction between calmodulin (CaM) and Mg2+ range from no binding to a binding constant of 10(4) M-1 [for a summary, see Cox, J. A., Comte, M., Malnoe, A., Berger, D., & Stein, E. A. (1984) Met. Ions Biol. Syst. 17, 215-273]. In order to resolve the controversy, we used 25Mg NMR to study the binding of Mg2+ to apo-CaM, CaM.Ca2(2)+ (in which sites III and IV are occupied by Ca2+), CaM.La2(3)+ (in which sites I and II are occupied by La3+), and the two tryptic fragments of calmodulin, TR1C (containing sites I and II of CaM) and TR2C (containing sites III and IV of CaM). In each system, a "titration set" and a "temperature set" were obtained, and the spectral data were analyzed by total band-shape analysis to calculate the association constant (Ka) and off-rate (koff). As in the case of Ca2+ binding, sites I and II and sites III and IV were treated as two sets of equivalent sites, and a Ca2+/Mg2+ competition experiment suggested that Mg2+ competes with Ca2+ for the same sites. For both CaM.Ca2(2)+ and TR1C, moderately large Ka (2000 and 3500 M-1, respectively) and moderate off-rates (koff = 2300 and 3000 s-1, respectively, at 25 degrees C) were observed. For both CaM.La2(3)+ and TR2C, binding of Mg2+ was weaker by a factor of ca. 10 (Ka = 300 and 200 M-1, respectively) while the off-rates were also moderate (koff = 3500 and 2200 s-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Topographical analysis of trypsin active site structure by means of the “inverse substrate” method

Trypsin-specific substrate analogs, “inverse substrates,” carrying the fluorescent dimethylaminonaphthalene group were synthesized. Preparation of acyl trypsins in which the fluorescent group is attached to the catalytic residue through a spacer group of various chain lengths was successfully carried out by use of these inverse substrates. The topographical structure of the trypsin active site vicinity was estimated on the basis of the fluorescence spectra of these acyl trypsins.     

Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA–binding protein

Dda, one of three helicases encoded by bacteriophage T4, has been well-characterized biochemically but its biological role remains unclear. It is thought to be involved in origin dependent DNA replication, recombination-dependent replication, anti-recombination, and recombination repair. The Gp32 protein of bacteriophage T4 plays critical roles in DNA replication, recombination, and repair by coordinating protein components of the replication fork and by stabilizing ssDNA. Previous work demonstrated that stimulation of DNA synthesis by Dda helicase appears to require direct Gp32–Dda protein–protein interactions and that Gp32 and Dda form a tight complex in the absence of ssDNA. Here we characterize the effects of Gp32–Dda physical and functional interactions through changes in the duplex DNA unwinding and ATPase activities of Dda helicase in the presence of different variants of Gp32 and different DNA repair and replication intermediate structures. Results show that Gp32–Dda interactions can be enhancing or inhibitory, depending on the Gp32 domain seen by Dda. Protein–protein interactions with Gp32 stimulate the unwinding activity of Dda, an effect associated with increased turnover of ATP, suggesting a higher rate of ATPase-driven translocation. Dda–Gp32 interactions also promote the unwinding of DNA substrates at higher salt concentrations and in the presence of substrate-bound DNA polymerase. Conversely, the formation of Gp32 clusters on ssDNA can inhibit unwinding, suggesting that Gp32–ssDNA formation sterically regulates which portions of replication and recombination intermediates are accessible for processing by Dda helicase. The data suggest a mechanism of replication fork restart in which Gp32 promotes Dda activity in template switching while preventing premature fork progression.     

An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3′-hydroxylase gene

The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3′H, the gene for flavonoid 3′-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

Isolation of oligonucleotide fragments containing the 5′-ends of nuclear pre-mRNA of Ehrlich carcinoma cells

The isolation of pre-mRNA fragments containing triphosphorylated 5′-ends nucleosides is described. The isolation is based on hydroxyapatite chromatography.     

Acceleration of the template-directed reactions of nucleoside 5′-phosphorimidazolides by acylation

Summary Nucleoside-5-phosphorimidazolides react readily with acylating agents to give N-substituted products that are highly activated. In most cases these acylated derivatives undergo rapid hydrolysis to give nucleoside 5-phosphates, whether or not a complementary template is present. However, guanosine 5-phosphorimidazolide reacts with diethyl pyrocarbonate to give a derivative that oligomerizes rapidly and efficiently in the presence of polycytidylic acid and Pb²⁺. The reaction is complete in about 1 h, whereas the corresponding reaction in the absence of an acylating agent takes several days. However, the final yield of long oligomers is lower when diethyl pyrocarbonate is present.     

DNA polymorphisms in the 5′-flanking region of the HLA-DQA1 gene

The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5 flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5 flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5 end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M72411. Address correspondence and offprint requests to: J. Guardiola.     

Determinants of laminin polymerization revealed by the structure of the α5 chain amino-terminal region

The polymerization of laminin into a cell-associated network--a key step in basement membrane assembly--is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN-LE1-2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network.     

Recruitment of divalent metal ions by incorporation of 4-thio-2′-deoxythymidine or 4-thio-2′-deoxyuridine into DNA

The modified nucleosides 4-thio-2-deoxyuridine (s⁴dU) and 4-thio-2-deoxythymidine (s⁴dT) are incorporated into dinucleosides, and s⁴dT is incorporated into a DNA hairpin loop to provide divalent metal ion binding sites. Binding of two different metal ions to these sites is studied, including Cd(II) as an NMR spectroscopy probe and Cu(II) as a reactive metal ion for DNA cleavage. Binding of Cd(II) to 4-thiouridine (s⁴U) and s⁴dT nucleosides, s⁴dU- and s⁴dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s⁴dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 °C in solutions containing 20.0–40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl₂. Cd(II) binds to the N(3) deprotonated form of s⁴dT with a binding constant (K=1.1×10⁴ M^–1) that is similar to that for Cd(II) binding to d(Tps⁴T) (K=9.2×10³ M^–1). Apparent binding constants (K_app) at pH 7.7 of Cd(II) to dinucleosides d(Gps⁴T), d(s⁴TpG), and d(Gps⁴U) are similar to those of their respective nucleosides s⁴U and s⁴dT, suggesting that neither the phosphate diester nor the second nucleoside has a major effect on Cd(II) binding. Binding of Cd(II) to s⁴U and d(Gps⁴U) is studied by use of ¹¹³Cd NMR and ¹H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps⁴U) as studied by ¹H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s⁴dT in the loop portion of a DNA hairpin loop (K_app=2.7×10³ M^–1 at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in T_m of 14 °C in the presence of 10.0 mM Cd(II) as determined by optical melting experiments. Cu(II) oxidizes s⁴dT to form the disulfide of s⁴dT, limiting the usefulness of further studies with Cu(II).Electronic Supplementary Material Supplementary material is available in the online version of this article at .Abbreviations s⁴dU 4-thio-2-deoxyuridine - s⁴dT 4-thio-2-deoxythymidine - s⁴U 4-thiouridine     

An Improved Procedure for the Synthesis of 2′-0-Methylnucleoside 5′-Diphosphate

2′-0-Methyladenosine 5′-diphosphate has been chemically synthesized with an overall yield of 48% by adopting the direct phosphorylation of the nucleoside by phosphoryl chloride in step 1 and the morpholidate procedure in step 2 of the following scheme: Am¹ - pAm¹ (step 1) - ppAm (step 2). The method has been successfully used in the synthesis of 2′-O-methyluridine 5′-diphosphate and 2′-O-methylcytidine 5′-diphosphate and is applicable to the synthesis of nucleoside 5′-diphosphates in general.     

An EcoRI RFLP in the 5′ region of the human NF1 gene

Von Recklinghausen neurofibromatosis or type l neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.