Replicative Synthesis Research Articles

Replacement of murine leukemia virus readthrough mechanism by human immunodeficiency virus frameshift allows synthesis of viral proteins and virus replication.

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model.

DNA replication and RNA synthesis in thymocyte nuclei microinjected into the cytoplasm of artificially activated mouse eggs.

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.

On proteins, DNA and topology

In living cells, genomic DNA is organized into multiple topological domains that restrict the rotation of the double helix and cause DNA supercoiling. DNA topology is important for the intranuclear DNA packaging and plays a role in the regulation of gene expression. Particular cellular proteins are involved in the maintenance and the control of topological DNA organization. In turn, DNA topology can affect the functioning of DNA-processing proteins. That is why various aspects of the topology-mediated interrelationship between DNA and proteins attract a great deal of attention.Three interesting papers touching on this subject from different directions were published in the January and February issues of Nucleic Acids Research. Pavlov et al. report on the finding and cloning of a new DNA-binding protein isolated from hyperthermophilic archaebacterium [1xIdentification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri. Pavlov, N.A et al. Nucleic Acids Res. 2002; 30: 685–694Crossref | PubMedSee all References][1]. This protein, termed 7kMk, bends DNA, leading to the formation of ∼140-bp nucleosome-like loops with negative constrained DNA supercoiling. The change in DNA topology is most likely to be due to a left-handed orientation of the DNA loop, similar to a histone core of eukaryotic or archaeal nucleosomes. The 7kMk–DNA complex adds to the list of nucleoprotein structures with looped DNA configuration that are thought to be involved in DNA packaging and in transcription regulation.Bentin and Nielsen examined whether the rotary constraints within DNA topological domains affect the performance of RNA polymerase [2xIn vitro transcription of a torsionally constrained template. Bentin, T and Nielsen, P.E. Nucleic Acids Res. 2002; 30: 803–809Crossref | PubMedSee all References][2]. To model transcription of a torsionally constrained template in vitro, they immobilized covalently closed, circular DNA to streptavidin-coated beads using a peptide nucleic acid (PNA)–biotin conjugate, stably targeted to a specific DNA site. For synthesis of RNAs up to 900 nt, the rate of constrained transcription elongation was as high as with rotary free DNA. Preliminary data (not shown in the paper) suggested that this could also be the case for even longer RNAs. The authors therefore concluded that genes can be transcribed in vivo by some RNA polymerases with high efficiency, despite the fact that newly synthesized RNA is entangled around the template in the narrow confines imposed by DNA topology.In another study on proteins, DNA and topology, Kuhn et al. addressed the important question of whether the rolling DNA synthesis known as RCA is inhibited by topological constraints [3xRolling-circle amplification under topological constraints. Kuhn, H et al. Nucleic Acids Res. 2002; 30: 574–580Crossref | PubMedSee all References][3]. For this, the PNA-assisted assembly of topologically linked, earring-like DNA constructs has been applied. The RCA efficiency was unaffected when the linked templates were employed. The finding that certain DNA polymerases can carry out replicative synthesis in a topologically constrained setting could have practical implications in the area of DNA diagnostics.These recent studies on the interplay of DNA topology and DNA-binding proteins will stimulate more research on the theme covering its various aspects, with a rewarding impact on molecular biotechnology.

Segment-specific expression of alkaline phosphatase in the Tubifex embryo requires DNA replication and mRNA synthesis.

During embryogenesis of the oligochaete annelid Tubifex, segments VII and VIII specifically express mesodermal alkaline phosphatase (ALP) activity in the ventrolateral region. In this study, using specific inhibitors, we examined whether this segment-specific expression of ALP activity depends on DNA replication and RNA transcription. BrdU-incorporation experiments showed that presumptive ALP-expressing cells undergo the last round of DNA replication at 12-24 hr prior to emergence of ALP activity. When this DNA replication was inhibited by aphidicolin, ALP development was completely abrogated in the ventrolateral mesoderm. Similar inhibition of ALP development was also observed in alpha-amanitin-injected embryos. While injection of alpha-amanitin at 24 hr prior to the emergence of ALP activity exerted inhibitory effects on ALP development, injection at 14 hr was no longer effective. In contrast, ALP activity developed normally in cytochalasin-D-treated embryos in which cytokinesis was prevented from occurring for 36 hs prior to appearance of ALP activity. These results suggest that the segment-specific development of ALP activity in the Tubifex embryo depends on DNA replication and mRNA transcription, both of which occur long before the emergence of ALP activity.

8-(Hydroxymethyl)-3,N(4)-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases.

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.

Rolling-circle amplification under topological constraints.

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics.

Purification strategies for Drosophila mtDNA replication proteins in native and recombinant form. Mitochondrial single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs) serve critical roles in DNA replication, repair, and recombination (). Mitochondrial SSBs (mtSSBs) share similar physical and biochemical properties with Escherichia coli SSB (, , , , , ), with which they exhibit a high degree of amino acid sequence conservation (, , ). Considering the roles served by E. coli SSB in bacterial replication in helix destabilization () and in enhancing DNA polymerase processivity (, ) and fidelity (, ), we purified Drosophila mtSSB and studied its effects on in vitro DNA synthesis by pol γ, in an assay that mimics lagging DNA strand synthesis in mitochondrial replication (, , , , , , , , , ). Our biochemical data are consistent with an important role for mtSSB in initiation and elongation of DNA strands in mitochondrial DNA (mtDNA) replication, that has been documented genetically by the fact that a null mutation in the gene for the yeast homolog (RIM1) results in complete loss of mtDNA in vivo (). More recently, we have demonstrated that an insertion in the third intron of the Drosophila gene (lopo) results in developmental lethality, concomitant with the loss of mtDNA and respiratory capacity ().

Retinoic acid inhibits fibronectin and laminin synthesis and cell migration of human pleural mesothelioma in vitro

The effects of retinoic acid (RA) on cell replication, fibronectin and laminin synthesis, integrin expression and haptotactic migration of three mesothelioma cell cultures of different histotype, one epithelioid, one fibromatous and one biphasic, were evaluated. Cell growth was not affected by RA, while RA treatment decreased the synthesis of fibronectin and laminin and inhibited the migration of all three mesotheliomas on substrates of fibronectin and laminin; on the contrary, the expression of some integrins was not significantly modified by RA. These data indicate that RA may lead to a decrease of mesothelioma cell local invasion; this can correlate with a modification induced by RA on mesothelioma tumor progression in vivo.

Conditional Coupling of Leading-strand and Lagging-strand DNA Synthesis at Bacteriophage T4 Replication Forks

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.

The effects of complexity, of simplicity and of scaling in thermal-hydraulics

This lecture has a twofold purpose. First, we will assess the state of the art and the trends in thermal-hydraulics (T-H) technology, within the context of replicating and non-replicating information systems. Four T-H examples are used to illustrate that an ever-increasing complexity in formulating and analyzing problems leads to inefficiency, obsolescence and evolutionary failure. By contrast, simplicity, which allows for parsimony, synthesis and clarity of information, ensures efficiency, survival and replication. This comparison (complexity versus simplicity) also provides the requirements and guidance for a success path in T-H development. The second objective of this paper is to demonstrate that scaling provides the means to process information in an efficient manner, as required by competitive (and, thereby, replicating) systems. To this end, the lecture summarizes the essential features of the Fractional Change, Scaling and Analysis approach, which offers a general paradigm for quantifying the effects that an agent of change has on a given information system. The paper will further demonstrate that a single concept and a single method may be used to scale and analyze all transport processes in a given field of interest (fluid mechanics, heat transfer, etc.) and/or across fields and disciplines (mechanics, biology, etc.) Therefore, the paradigm: (1) ensures economy and efficiency in addressing and resolving technical or scientific problems; and (2) enables a ‘cultural cross-pollination’ between different information systems (disciplines). By means of a simple example in the Appendix, we shall: (1) demonstrate the efficiency to be gained through scaling; and (2) illustrate the inefficiency and wastefulness of computer-based safety studies as presently conducted.

Nidovirus genome replication and subgenomic mRNA synthesis. Pathways followed and cis-acting elements required.

Nidoviruses, specifically coronaviruses and arteriviruses, are recognized as unique among RNA viruses for utilizating a discontinuous transcription step during sgmRNA synthesis. Details of this pathway and of genome replication remain enigmatic, and identification of cis-acting elements and trans-acting factors involved is far from complete. The apparent absence of a common leader in toroviruses and differences in genome size among the viruses hint that fundamental differences in pathways of RNA synthesis might still be revealed. What are the current views?

Werner’s syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage

Werner’s syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Elongation of tandem repetitive DNA by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis at a hairpin-coil transitional state: a model of amplification of a primordial simple DNA sequence.

DNA is replicated by DNA polymerase semiconservatively in many organisms. Accordingly, the replicated DNA does not become larger than the original DNA (template DNA), implying that replicative synthesis by DNA polymerase alone cannot explain the diversification of primordial simple DNA. We demonstrate that a single-stranded tandem repetitive oligodeoxyribonucleic acid (oligoDNA) composed of a palindromic or quasi-palindromic motif sequence and 25-50% GC content is elongated in vitro to more than 20,000 bases at 70-74 degrees C by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis without a bimolecular primer-template complex. The efficiency of elongation decreased when the palindromic structure of the oligoDNA was destroyed or when the GC content of the oligoDNA was outside the range of 25-50%. The thermal melting transition profile of the oligoDNA, as observed by ultraviolet spectroscopy, exhibited a biphasic curve, reflecting a duplex-hairpin transition at 31-40 degrees C and a hairpin-coil transition at 70-77 degrees C. The optimal reaction temperature for the elongation, for instance, of oligoDNA (AGATATCT)(6) (72 degrees C) was very close to its hairpin-coil transition melting temperature (70.4 degrees C), but was markedly higher than the temperature at which duplex oligoDNA can exist stably (<35.9 degrees C). These results suggest that a hairpin-based "intramolecular primer-template structure" is formed transiently in the oligoDNA, and it is elongated by the DNA polymerase to long DNA through repeated cycles of folding and melting of the hairpin structure. We discuss the implication of this phenomenon, "hairpin elongation", from the standpoint of potential amplification of simple DNA sequences during the evolution of the genome.

Poveikis ir atsakas biologiniuose vyksmuose

Kiekvienas biologinis vyksmas susideda iš dviejų dalių: poveikio ir atsako. Atsako santykis su poveikiu gali būti tiesinis ar netiesinis. Tačiau kada poveikis kinta plačiose ribose, atsakas kinta taip pat ir grafiškai atvaizduotas primena kiek palinkusią „S“ raidę. Kai poveikis laipsniškai stiprėja, atsakas pradžioje yra gana silpnas. Tik kada poveikis pasiekia tam tikro stiprumo, atsakas tampa tolygus poveikiui. Jei poveikis toliau stiprėja, atsakas tampa vėl netolygus arba visai nekinta. Biologinių vyksmų poveikio – atsako priklausomybė gali būti vertinama dviem lygiais: pirmas – molekulių sąveikų, atomų ir jungčių mainų lygis, antras – ląstelės-individo-organizmo lygis. Pirmojo lygio poveikio-atsako santykis yra ribojamas sąlygų ir reaguojančių medžiagų kiekio; reakcija paklūsta netiesinės-tiesinės-netiesinės priklausomybės dėsniui. Antrojo lygio – ląstelių ir organizmų – visi vyksmai remiasi cheminiu-fizikiniu pagrindu; ląstelės-individo atsakas į poveikį gali būti vertinamas kaip tikimybė, bet populiacijos atsakas artėja prie tiesioginės priklausomybės. Tokia būsena ir sąlygotumų reikšmė aiškinama trimis pavyzdžiais: DNR dvigubėjimu, DNR nurašymu ir baltymų sinteze. Iš sąlygotumų svarbiausia yra genetinė reguliacija. Žmogaus organizme, be kitų, prisideda aukštesnio lygio sąlygotumai: jausmai, valia, sąmonė. Kaip sąlygotumai turėtų būti vertinami tikėjimas, stebuklai, aiškiaregystė ir kiti panašūs reiškiniai. Neigimas nieko neįrodo, ir tai yra silpnoji mokslo pusė.

Precise Mapping of the Replication and Transcription Promoters of Human Parainfluenza Virus Type 3

The terminal RNA regions of the genomic and antigenomic RNAs of the paramyxoviruses and rhabdoviruses are known to contain sequences essential for directing RNA replication and transcription. The 3′ terminus (leader region) of the negative-sense, genomic RNA of the rhabdoviruses and paramyxoviruses is known as the leader (Le) promoter and directs synthesis of positive-sense replication and transcription products. The 3′ terminus of the antigenome is termed the trailer complementary (TrC) promoter and directs the synthesis of genomic RNA. By creating mutations in the corresponding regions of an HPIV3 minireplicon in which the viral protein coding sequences were replaced by the luciferase gene, we were able to precisely define the elements of the leader promoter involved in directing positive-strand replication of HPIV3. Nucleotides 1 through 12 (from the terminus) formed a domain critical for replication. The region from nucleotides 13 through 55 was important but not crucial for replication, while G residues at positions 79, 85, and 91 comprised another domain critical for replication. It was also shown that the TrC promoter is similar, though not identical, to the Le promoter. Nucleotides 1 through 12 of the TrC promoter were critical for synthesis of genomic RNA, though specific positions behaved differently from the corresponding positions of the Le promoter. While many of these mutations could not be analyzed for transcription because they completely abrogated genomic RNA synthesis (the template for transcription), we were surprised to find that no mutations in the leader promoter which decreased replication had any significant effect on transcription. However, mutations in the intergenic sequence and gene start signal following the leader and preceding the luciferase message severely decreased transcription, but not replication.

Abasic Sites Induce Triplet-repeat Expansion during DNA Replication in Vitro

The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.

The Extracellular Signal-Regulated Kinase Pathway Contributes to Mitogenic and Antiapoptotic Effects of Peroxisome Proliferators in Vitro

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens thought to induce tumors by altering the balance between mitosis and apoptosis. Previous studies suggest mitogenic growth factors that act through the extracellular signal-regulated kinase (ERK) pathway, including insulin and epidermal growth factor (EGF), modulate peroxisome proliferator-activated receptor α activation as well as the mitogenic activity of peroxisome proliferators. We have investigated whether the ERK pathway plays a role in regulating the growth and survival altering properties of peroxisome proliferators in primary mouse hepatocytes. Exposure of hepatocytes to Wy-14,643 and trichloroacetate resulted in a dose-dependent phosphorylation and activation of ERK. Peroxisome proliferator-induced ERK phosphorylation was blocked when cells were pretreated with the MEK (ERK kinase) inhibitor, PD098059, or the phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and apigenin, suggesting that both MEK and PI3K are involved in the initial response. The pathway leading to peroxisome proliferator-induced ERK activation is different than that induced by phorbol ester or EGF, since the PI3K inhibitors had no effect on ERK phosphorylation induced by these agents. Under defined culture conditions, Wy-14,643 increased the level of BrdU incorporation in primary hepatocytes and suppressed the incidence of apoptosis induced by transforming growth factor β1. In contrast, concentrations of PD098059 that block Wy-14,643-induced ERK phosphorylation also blocked the stimulation of DNA replicative synthesis and suppression of apoptosis by Wy-14,643. These studies indicate that activation of the ERK pathway through a PI3K-dependent mechanism may play a significant role in the tumor-promoting properties of peroxisome proliferators.

In vivo addition of poly(A) tail and AU-rich sequences to the 3' terminus of the Sindbis virus RNA genome: a novel 3'-end repair pathway.

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

Molecular and physiological markers during seed development of peppers (Capsicum annuum L.): DNA replication and β-tubulin synthesis

AbstractThe correlation between physiological markers for seed maturity (acquisition of germinability, definitive morphological traits and desiccation tolerance) and timing of the shutdown of nuclear replication activity and β-tubulin synthesis has been investigated in developing pepper seeds. Nuclear replication activity was analysed by flow cytometry. Expression of β-tubulin, a cell-cycle related structural protein, was detected in western blots of total protein extracts. In pepper embryos, DNA synthesis stopped 42–49 days after anthesis (DAA), at which time seeds started to acquire desiccation tolerance. A clear β-tubulin signal was detected up to 63 DAA, only 1 week before the acquisition of complete desiccation tolerance. Both nuclear replication and β-tubulin synthesis can therefore be used as markers for the physiological stage reached by developing pepper seeds. Detection of β-tubulin signals in embryonic tissues can also be helpful in discriminating immature seeds within a commercial seed lot.

Replication block by an enediyne drug-DNA deoxyribose adduct.

Under anaerobic conditions neocarzinostatin chromophore, an enediyne antibiotic, forms a covalent drug-DNA adduct on the 5' carbon of deoxyribose at a specific single site in a 2-nucleotide bulge, rather than strand cleavage, by a mechanism involving general base-catalyzed intramolecular drug activation to a reactive radical species. We have taken advantage of the selectivity of this reaction to prepare a single-stranded oligonucleotide containing a single drug adduct at a T residue and to study its effect on the template properties of the oligonucleotide in replicative synthesis, as followed by 5'-32P-labeled primer extension by several DNA polymerases. With the Klenow fragment of Escherichia coli DNA polymerase I, synthesis stops at the base immediately 3' to the adduct. The same enzyme, but lacking 3' to 5' exonuclease activity, permits synthesis to proceed by one additional nucleotide. This effect is enhanced when Mn2+ is substituted for Mg2+. T4, herpes simplex virus, and cytomegalovirus DNA polymerases all act like Klenow polymerase. Sequenase (exo-minus T7 DNA polymerase) is qualitatively similar to exo-minus Klenow polymerase but is more efficient in inserting a nucleotide opposite the lesion. With the small-gap-filling human DNA polymerase beta, which lacks intrinsic exonucleolytic activity, primer extension proceeds to the nucleotide opposite the lesion. However, when a gap was created opposite the lesion, polymerase beta adds as many as two additional nucleotides 5' to the adduct site. The fidelity of base incorporation opposite the lesion was not impaired, in contrast with adducts on DNA bases.