Novel cross-strand three-purine stack of the highly conserved 5′-GA/AAG-5′ internal loop at the 3′-end termini of Parvovirus Genomes

We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study a 2×3 asymmetric internal loop structure of the highly conserved `5-(GA)/(AAG)-5 bubble' present at the 3-end hairpin of the single-stranded DNA genome of parvoviruses. This motif contains an unpaired adenosine stacked between two bracketed sheared GA pairs. However, the phenomenal cross-strand G-G and A-A stacking in the tandem sheared GA pairs has undergone considerable change. A novel three-purine stacking pattern is observed instead; the inserted A18 base is completely un-stacked from its neighboring G17 and A19 bases, but well stacked with the cross-strand A4 and G3 bases to form a novel A4/A18/G3 stack that is different from the double G/G, A/A or quadruple G/G/G/G stack present in the 5-(GA)/(AG)-5 or 5-(GGA)/(AGG)-5 motifs. Unlike the bulged purine residue that usually causes about 20 degree kink in the helical axis of the parent helix when bracketed by canonical GC or AT base pairs, no significant kink is observed in the present helix containing a bulged-adenine that is bracketed by sheared G A pairs. The phosphodiesters connecting G3-A4 and G17-A18 residues adopt unusual torsional angles close to the trans domain, yet that connecting A18-A19 residues resumes the normal (g <sup>–</sup>) value. The well structured `5-(GAA)/(AG)-5' internal loop in the parvovirus genomes explains its resistance to single-strand specific endonuclease susceptibility.     

Surveying CpG methylation at 5′-CCGG in the genomes of rice cultivars

We investigated the CpG methylation status of the sequence CCGG in the rice genome by using methylation-sensitive AFLP and subsequent Southern analyses with the isolated AFLP fragments as probes. CpGs located in single- or low-copy-sequence regions could be grouped into two classes on the basis of their methylation status: methylation status at the class 1 CpG sites was conserved among genetically diverse rice cultivars, whereas cultivar-specific differential methylation was frequently detected among the cultivars at the class 2 CpG sites. The frequency of occurrence of methylation polymorphism between a pair of cultivars was not related to the genetic distance between the two. Through mapping, five class 2 CpG sites were localized on different chromosomes and were not clustered together in the genome. Segregation analysis of the cultivar-specific methylations with their target sites indicated that the differential methylation was stably inherited in a Mendelian fashion over 6 generations, although alterations in the methylation status at the class 2 CpG sites were observed with a low frequency.     

Catalytic roles of the AMP at the 3′ end of tRNAs

Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3 terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3 terminus of tRNA.     

Remarkable induction of UV-signature mutations at the 3′-cytosine of dipyrimidine sites except at 5′-TCG-3′ in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh^−/−) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh^−/− epidermis and dermis than in the wild-type (Polh^+/+) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh^−/− mice were determined and compared with mutant sequences from irradiated Polh^+/+ mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C → T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5′-TCG-3′ sequence context than at the other dipyrimidine contexts in the Polh^+/+ epidermis, whereas the mutations were induced remarkably and exclusively at the 3′-cytosine of almost all dipyrimidine contexts with no preference for 5′-TCG-3′ in the Polh^−/− epidermis. In addition, in Polh^−/− mice, a small but remarkable fraction of G → T transversions was also observed exclusively at the 3′-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh^+/+ genotype.     

Synthesis of 2′-Deoxy[5′-13C]Ribonucleotides and HMQC-Noesy NMR Study of the Dickerson's Dodecamer with 13C-Labeling at the 5′ Positions

Abstract

In addition to the synthesis of 2′-deoxy[5′-¹³C]ribonucleosides (6) via the D-[5-¹³C]ribose derivative (4), the construction of the corresponding dodecanucleotide with the Dickerson's sequence and its HMQC-NOESY NMR analysis are described.     

Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses

Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-³²P]guanosine 5′-triphosphate or S-adenosyl[methyl-³H]methionine to form “cap” structures of the type m⁷G(5′)pppN^m-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m⁷G(5′)pppA^m, m⁷G(5′)pppA^mpGp, and m⁷G(5′)pppA^mpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution.     

Comparison of the Nucleotide Sequence at the 5′ End of RNAs from Nine Aphthoviruses, Including Representatives of the Seven Serotypes

The sequence of about 70 nucleotides at the 5' end of the RNAs of nine different aphthoviruses (foot-and-mouth disease viruses), including representatives of the seven serotypes of the virus, has been determined by partial enzyme digestion of (32)P-end-labeled S fragment-that part of the RNA lying to the 5' side of the poly(C) tract and including the 5' end of the molecule. The S fragments were prepared from polyadenylated virus-specific RNA extracted from infected cells by digestion with RNase H in the presence of oligo(dG)(12-18). The first 27 nucleotides from the 5' end were highly conserved in all the RNAs. This region was followed by a more variable region of about 15 nucleotides, showing some length and sequence heterogeneity and including potential but probably nonutilized initiation codons. In agreement with previous homology studies, the sequencing results showed that the European serotypes A, O, and C form a group distinct from the SAT serotypes and that the Asia 1 serotype is closely related to the European group. The lengths of the S fragments of two different RNAs were confirmed as containing 360 to 400 nucleotides by gel electrophoresis with reference to nucleotide markers of known size.     

5′-Halogenated Formycins as Inhibitors of 5′-Deoxy-5′-methylthioadenosine Phosphorylase: Protection of Cells Against the Growth-Inhibitory Activity of 5′-Halogenated Adenosines

Abstract

A series of 5′-halogenated formycins, including the chloro-, bromo- and iodo- derivatives, were synthesized. These compounds are competitive inhibitors of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAPase) with K_i values in the range of 10^?7 M, making them the most potent inhibitors of MTAPase reported to date. These compounds protect cells from the growth-inhibitory action of 5′-halogenated adenosines, which must be activated by MTAPase. The syntheses of 5′-halogenated formycin B derivatives, which inhibit purine nucleoside phosphorylase, are also described.     

The absence of the CpNpG methylation at the 5′-terminal region of the human calcitonin gene in norm and leukemias

The inner cytosine methylation was analyzed in the CCWGG sequences of the 5′-terminal region of the human calcitonin gene from peripheral blood and bone marrow cells in various forms of leukemia. Since these sequences remain nonmethylated both in the norm and in various leukemia forms, the CpG dinucleotide hypermethylation of the 5′-end of the human calcitonin gene, characteristic for the development of leukemias, does not spread to adjacent CpNpG sequences.     

Synthesis and SAR of geminal substitutions at the C5′ carbosugar position of pyrimidine-derived HCV inhibitors

The installation of geminal substitution at the C5′ position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5′ position led to the installation of the dimethyl group as the optimal functionality. An improved route was subsequently designed to access these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These compounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in rodent as well as in higher species.     

New ionic targets of 3,3′,5′-triiodothyronine at the plasma membrane of rat Sertoli cells

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT₃ on calcium (⁴⁵Ca²⁺) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT₃. Our results indicate that rT₃ induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT₃ occurred in <5 min in Sertoli cells. In addition, the rT₃ metabolite, T₂, also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT₃ also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na⁺/K⁺ ATPase pump. The rapid response of rT₃ is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells.     

Distribution of 5′-nucleotidase in the renal interstitium of the rat

Summary The hydrolysis of 5-AMP by 5-nucleotidase is the main source of adenosine. In various tissues adenosine is a local mediator adjusting the organ work to the available energy. In the kidney it regulates renal hemodynamics, glomerular filtration rate and renin release via specific receptors of the arteriolar walls. By immunocytochemistry we identified interstitial and tubular sites of 5-nucleotidase in the rat kidney. In the interstitium the enzyme was detected only in the cortical labyrinth, the compartment that comprises all arteriolar vessels besides other putative targets of adenosine. The 5-nucleotidase-positive cells of the interstitium were identified as fibroblasts. The fibroblasts are in close contact with the tubules as well as with the vessels. Thus, any 5-AMP released by the tubules into the interstitial space would be converted to adenosine in the direct vicinity of its assumed targets. Adenosine produced by tubular cells would hardly have access to its known targets, since 5-nucleotidase is restricted to the luminal cell surface. Pathological events affecting the fibroblasts might influence renal function by modifying the interstitial adenosine production.     

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Abstract

Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of “complementary addressed modification titration” (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylaminojbenzylamino- group (C1RCH₂NH)- at 5′-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E₁, E₂ and their derivatives E₁ ^phn, E₂ ^phn bearing N- (2-hydroxyethyl)-phenazinium residues Phn- both at 5′- and 3′- ends covalently linked via ethylenediamine linker were used as effectors. Effectors E₁ and E₂ (E₁ ^Phn and E₂ ^Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8^m) reagents were used for the target modification. The reagent X8^m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8^m and effectors E₁, E₂, E₁ ^phn, E₂ ^Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group CIRCH₂NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either CIRCH₂NH- group or TT-mismatch at the junction.     

Differential methylation at the 5′ and the 3′ CCGG sites flanking the X chromosomal hypervariable DXS255 locus

Summary The degree of methylation at the 5 and 3 CCGG sequences flanking the variable number of tandem repeat (VNTR) region of the DXS255 locus at Xp11.22 was analysed separately in several haematopoietic cell lineages. The 5 CCGG site on active chromosomes was found to be completely methylated in B and T lymphocytes and granulocytes. Methylation of the 5 site on inactive X chromosomes differed between females (0%–60%), but was consistent in different cell lineages obtained from individual females. In contrast, methylation at the 3 CCGG site on active chromosomes was found to vary in B lymphocytes (40%–100%), whereas complete methylation was found in T lymphocytes and granulocytes. The extent of methylation on inactive X chromosomes was found to differ significantly between B lymphocytes (17%), T lymphocytes (54%) and granulocytes (82%). Thus, methylation at the 5 CCGG site seems to be primarily related to the status of X chromosome inactivation, whereas methylation at the 3 CCGG site is mainly subject to cell-lineage-specific influences.