High-voltage Electrophoresis Research Articles

Polysaccharides Analysis of Cell Wall Using Carbohydrate Gel Electrophoresis.

The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting thepolysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated ascompared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.

Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate.

Cress seeds release allelochemicals that over-stimulate the elongation of hypocotyls of neighbouring (potentially competing) seedlings and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here. Low-molecular-weight cress-seed exudate (LCSE) from imbibed Lepidium sativum seeds was fractionated by phase partitioning, paper chromatography, high-voltage electrophoresis and gel-permeation chromatography (on Bio-Gel P-2). Fractions, compared with pure potassium salts, were bioassayed for effects on Amaranthus caudatus seedling growth in the dark for 4 days. The LCSE robustly promoted amaranth hypocotyl elongation and inhibited root growth. The hypocotyl inhibitor was non-volatile, hot acid stable, hydrophilic and resistant to incineration, as expected for K+. The root inhibitor(s) had similar properties but were organic (activity lost on incineration). The root inhibitor(s) remained in the aqueous phase (at pH 2.0, 6.5 and 9.0) when partitioned against butan-1-ol or toluene, and were thus hydrophilic. Activity was diminished after electrophoresis, but the remaining root inhibitors were neutral. They became undetectable after paper chromatography; therefore, they probably comprised multiple compounds, which separated from each other, in part, during fractionation. On gel-permeation chromatography, the root inhibitor co-eluted with hexoses. Cress-seed allelochemicals inhibiting root growth are different from the agent (K+) that over-stimulates hypocotyl elongation and the former probably comprise a mixture of small, non-volatile, hydrophilic, organic substances. Abundant components identified chromatographically and by electrophoresis in cress-seed exudate fitting this description include glucose, fructose, sucrose and galacturonic acid. However, none of these sugars co-chromatographed and co-electrophoresed with the root-inhibitory principle of LCSE, and none of them (in pure form at naturally occurring concentrations) inhibited root growth. We conclude that the root-inhibiting allelochemicals of cress-seed exudate remain unidentified.

Uranium Sorption on “Tezontle” Volcanic Rock

It is described a study that demonstrates that hexavalent uranium ions were sorbed by the naturally occurring mineral using a batch technique. This mineral is found in abundant quantities in Mexico. Our study focused on the separation of UVI from synthetic aqueous systems of both H2O-UO2(NO3)2.6H2O (acid) and H2ONa4[ UO2(CO3)3] (basic). The chemical speciation was performed by using high voltage electrophoresis, and the uranium content was determined by UV-Vis Spectroscopy. The quantified U(VI) sorption by tezontle from acidic and basic systems was 2.72 and 1.68 μmol/g, respectively, and the sorption behavior is discussed considering the surface charge of the tezontle at different pH values based on the point of zero charge characteristic of this material.

Potassium, not lepidimoide, is the principal 'allelochemical' of cress-seed exudate that promotes amaranth hypocotyl elongation.

Background and Aims Imbibed cress (Lepidium sativum L.) seeds exude ‘allelochemicals’ that promote excessive hypocotyl elongation and inhibit root growth in neighbouring competitors, e.g. amaranth (Amaranthus caudatus L.) seedlings. The major hypocotyl promoter has recently been shown not to be the previously suggested acidic disaccharide, lepidimoic acid (LMA), a fragment of the pectic polysaccharide domain rhamnogalacturonan-I. The nature of the hypocotyl promoter has now been re-assessed. Methods Low-molecular weight cress-seed exudate (LCSE) was fractionated by high-voltage electrophoresis, and components with different charge:mass ratios were tested for effects on dark-grown amaranth seedlings. Further samples of LCSE were size-fractionated by gel permeation chromatography, and active fractions were analysed electrophoretically. Key Results The LCSE strongly promoted amaranth hypocotyl elongation. The active principle was hydrophilic and, unlike LMA, stable to hot acid. After electrophoresis at pH 6·5, the only fractions that strongly promoted hypocotyl elongation were those with a very high positive charge:mass ratio, migrating towards the cathode 3–4 times faster than glucosamine. Among numerous naturally occurring cations tested, the only one with such a high mobility was potassium. K+ was present in LCSE at approx. 4 mm, and pure KCl (1–10 mm) strongly promoted amaranth hypocotyl elongation. No other cation tested (including Na+, spermidine and putrescine) had this effect. The peak of bioactivity from a gel permeation chromatography column exactly coincided with the peak of K+. Conclusions The major ‘allelopathic’ substance present in cress-seed exudate that stimulates hypocotyl elongation in neighbouring seedlings is the inorganic cation, K+, not the oligosaccharin LMA.

Novel insights into ascorbate retention and degradation during the washing and post-harvest storage of spinach and other salad leaves

Post-harvest treatments of pre-packaged salad leaves potentially cause l-ascorbate loss, but the mechanisms of ascorbate degradation remain incompletely understood, especially in planta. We explored the extent and pathways of ascorbate loss in variously washed and stored salad leaves. Ascorbate was assayed by 2,6-dichlorophenolindophenol titration, and pathways were monitored by 14C-radiolabelling followed by high-voltage electrophoresis. All leaves tested showed ascorbate loss during storage: lettuce showed the greatest percentage loss, wild rocket the least. Spinach leaves were particularly prone to losing ascorbate during washing, especially with simultaneous mechanical agitation; however, washing in the presence of hypochlorite did not significantly increase ascorbate loss. In spinach, [14C]oxalate was the major product of [14C]ascorbate degradation, suggesting that commercial washing causes oxidative stress. This study highlights that ascorbate/dehydroascorbic acid are lost via the oxidative pathway during washing and post-harvest storage of salad leaves. Thus changes to washing procedures could potentially increase the post-harvest retention of ascorbate.

Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method.

Background and aims Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals (•OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to ‘fingerprint’ •OH-attacked polysaccharides.Methods We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during •OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography.Key Results Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA–pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently.Conclusions GalA–pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents (•OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by •OH. The evidence shows that •OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). •OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.

BF*F allotype of the alternative pathway of complement: A marker of protection against the development of antiphospholipid antibodies in patients with systemic lupus erythematosus.

B factor (BF) from the alternative complement pathway seems to participate in the pathophysiology of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To study the allotypic variability of BF in SLE and their associations with clinical and autoantibodies profile. BF allotypes were determined by high-voltage agarose gel electrophoresis, under constant cooling, followed by immunofixation with anti-human BF antibody, in 188 SLE patients and 103 controls. Clinical and serological data were obtained from medical examination and records. No significant differences of BF variants between patients and controls were found, neither in relation to epidemiologic or clinical manifestations. Associations of phenotype BF SS07 and allotype BF*S07 were found with anticardiolipin IgM (aCl-IgM) antibodies (p = 0.014 and p = 0.009 respectively), but not with aCl-IgG, lupus anticoagulant (LA), anti β2GPI or clinical APS. A significant decrease in BF*F allotype (p = 0.043) and BF SF phenotype (p = 0.018) was detected in patients with anti-phospholipid antibodies as a whole (aCl-IgG, aCl-IgM, LA and anti β2GPI). There is a link between phenotype BF SS07 and allotype BF*S07 with aCl-IgM in SLE patients; BF*F allotype could be considered a marker of protection against the development of antiphospholipid antibodies in these patients.

Separation and purification of Escherichia coli-expressed human thymosin-α1 using affinity chromatography and high-performance liquid chromatography

In this study, a human thymosin-α1 (hTα1) fusion protein was overexpressed in Escherichia coli ( E. coli). The hexahistidine-tagged hTα1 fusion protein was obtained in soluble form in cells of the engineered E. coli strain BL21 (DE3)/pET-28a-hTα1 that had been induced with isopropyl -D-1-thiogalactopyranoside (IPTG). The recombinant protein accounted for approximately 50–60% of the total protein. We then developed and validated a separation method for hTα1 from E. coli cells based on thermal denaturation, nickel-resin affinity chromatography and high-performance liquid chromatography. The purification method showed good reproducibility and was easy to operate. Purified recombinant hTα1 of high homogeneity was characterized and found to be of high purity (over 99%), as determined by high-voltage electrophoresis and high-performance liquid chromatography analysis. Isoelectric focusing analysis indicated a pI of approximately 4.0, and full wavelength screening showed an optimal absorbance wavelength at around 214 nm.

Reactive oxygen species‐induced release of intracellular ascorbate in plant cell‐suspension cultures and evidence for pulsing of net release rate

*Apoplastic ascorbate has been proposed to confer resistance to oxidative stresses, e.g. ozone. We investigated reactive oxygen species (ROS)-induced secretion and catabolism of ascorbate. *Late-growth-phase cultured cells of rose and Arabidopsis were preloaded with [(14)C]ascorbate. Radiolabelled metabolites and secretion products were analysed by high-voltage electrophoresis. *In both species, exogenous 1 mM hydrogen peroxide (H(2)O(2)) rapidly stimulated [(14)C]ascorbate and [(14)C]dehydroascorbate accumulation in the medium (apoplast). Net (14)C export was most rapid within 100 s of washing, and often showed superimposed pulses, of c. 10-s duration, whose amplitude was greater after H(2)O(2) treatment. Oxidative stress did not cause indiscriminate metabolite leakage from the cells. H(2)O(2) caused c. 20-40% of the intracellular [(14)C]ascorbate to be irreversibly catabolized to [(14)C]oxalyl-threonate and related products; however, the great majority of the extracellular radioactivity remained as [(14)C]ascorbate and [(14)C]dehydroascorbate. Much of the apoplastic dehydroascorbate was probably reabsorbed by the cells and reduced back to ascorbate. *The data show that exported ascorbate can serve an apoplastic antioxidant role in these late-growth-phase cells without being irreversibly lost, whereas in early-growth-phase cells most extracellular ascorbate is irreversibly degraded. In conclusion, cultured plant cells can respond actively to oxidative stress by reversibly exporting ascorbate into the apoplast.

Electrodischarge treatment of alcohols to produce carbonic nanomaterials

The method and products of the electrodischarge treatment of alcohols have been studied. The method of separation of the CNM produced from inferior alcohols by the high-voltage electrophoresis method has been proposed and tested. As the X-ray diffraction analysis results show, the application of only the electrophoresis method is not enough for the complete separation of the crystallites into the different allotropic forms of carbon. It is necessary to use complex methods of the CNM enrichment.

Charge equilibria and p K values of 5-carboxypyranomalvidin-3-glucoside (vitisin A) by electrophoresis and absorption spectroscopy

Apparent p K values of the wine pigment, 5-carboxypyranomalvidin-3-glucoside (vitisin A), were determined using UV–vis spectroscopy, viz. p K a1=0.98 (±0.10), p K H1=4.51 (±0.03) and p K H2=7.57 (±0.02). An additional ionisation constant at high pH (p K a4=8.84±0.06) was established by high-voltage paper electrophoresis. These data in conjunction with previously published p K a values determined by high-voltage electrophoresis suggest that in wine (pH 3.2–3.8), 5-carboxypyranomalvidin-3-glucoside exists as a complex mixture of hydrated and non-hydrated, partially ionised species with the predominant species being the quinonoidal base ( λ max 498 nm).

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization

Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.

Phosphorylation of Amphiphysin I by Minibrain Kinase/Dual-specificity Tyrosine Phosphorylation-regulated Kinase, a Kinase Implicated in Down Syndrome

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

Urinary polyamine levels in patients with gastrointestinal malignancy.

Polyamine levels (putrescine, spermidine, and spermine) were determined in 24-hour urine samples by a high voltage electrophoresis technique. Eleven of 14 patients with widespread metastatic gastrointestinal cancer had two or more elevated polyamine values. Two patients with cancer confined to the colon and two with cancer spread within the pelvis had two or more elevated polyamine values. Three patients who had all known disease removed by surgery, two patients with gastric lymphoma, and one patient with a vilous adenoma of the colon had normal values. Individual values of spermidine and spermine appeared to be elevated more frequently than putrescine values in these patients.

60Co aqueous speciation and pH effect on the adsorption behavior on inorganic materials

The adsorption of radioactive cobalt from aqueous solution on MgO, MnO 2, TiO 2, SnO, activated carbon and calcined hydrotalcite was studied under static conditions as a function of pH (1, 3, 5, 7, and 10) of the 60Co solution. It was found that under the experimental conditions, 60Co was significantly adsorbed on MnO 2, TiO 2 and activated carbon and in smaller quantities on MgO, SnO and calcined hydrotalcite. The adsorption of 60Co species on MgO, MnO 2, TiO 2 and SnO can be attributed to the direct exchange of ions with a specific group on the adsorbing surface, in accordance with the concept of zero point charge (zpc). The radionuclide content was determined by gamma-spectrometry. The 60Co chemical species present in aqueous solutions with different pH values were surveyed by using high-voltage electrophoresis. It was also found that 60Co was present only as a cationic species at pH 1, 3, 5 and 7 and a neutral species at alkaline pH.

Enfermedad de jarabe de arce: una entidad rara que debemos recordar. A propósito de su manejo dietético

Maple syrup disease is secondary to a deficiency of deshydrogenase complex of a cetoacid of branched-chain. This disease has a recessive autosomic inheritance, with an incidence of 1/200,000 newborns, without differences between male and female. Due to clinical presentation and biochemical response to tiamin, these patients can be classified in five clinical entities: classic, intermediate, intermittent, positive response to tiamin and deficiency of dihydrolipoyl deshydrogenase (E3). In these patients, an increase of seric branched-chain aminoacids is detected, it could be detected by (chromatography) during neonatal period. Valine, isoleucine, and aloisoleucine are increased in serum, orine, and cephaloraquideum liquid by ionic changed chromatography, chromatography of high resolution or high voltage electrophoresis. Patients have two phases in this disease (acute phase and maintaining phase). Objectives in acute phase are based in three topics: to eliminate toxic metabolites, nutritional support and to get anabolism. Utilization of hemodialysis/peritoneal dialysis/blood exchange is one of the first treatments. Dietetic support is the second treatment, with a minimum energy intake and controlling blood levels of aminoacids. Modified dietetic formulas is a main device to treat these patients.

Cyclic GMP-dependent Protein Kinase Regulates CCAAT Enhancer-binding Protein β Functions through Inhibition of Glycogen Synthase Kinase-3

The CCAAT enhancer-binding protein (C/EBPbeta) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPbeta participates in cGMP-regulated transcription of c-fos in osteoblasts (Chen, Y., Zhuang, S., Cassenaer, S., Casteel, D. E., Gudi, T., Boss, G. R., and Pilz, R. B. (2003) Mol. Cell. Biol. 23, 4066-4082). In the present work, we show that cGMP/cGMP-dependent protein kinase (PKG) induced dephosphorylation and activation of C/EBPbeta by inhibiting glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta on Ser9 negatively regulates the enzyme activity, and we found that PKG phosphorylated this site both in vitro and in vivo; the in vivo phosphorylation occurred rapidly and preceded C/EBPbeta dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3beta is a C/EBPbeta kinase in resting cells. We determined that GSK-3beta phosphorylated C/EBPbeta in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPbeta was phosphorylated on these same sites in intact, unstimulated osteoblasts, and phosphorylation was decreased in cGMP-treated cells. Mutation of the GSK-3 phosphorylation sites in C/EBPbeta prevented C/EBPbeta phosphorylation in resting cells, enhanced C/EBPbeta DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPbeta. cGMP regulation of C/EBPbeta was disrupted by a mutant GSK-3beta(Ala9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPbeta by preventing the negative effects of GSK-3 phosphorylation.

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Investigating HIV-1 Polypurine Tract Geometry via Targeted Insertion of Abasic Lesions in the (–)-DNA Template and (+)-RNA Primer

A variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (-)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO(4) footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (-)-DNA template between positions -15 and -13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (-11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile -1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis.