Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal sister chromatid exchange

An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPC-11 between 3' regions of the C gamma 2a and C gamma 2b heavy chain genes results in duplication of the C gamma 2a heavy chain gene and generation of a novel recombination joint. The USCE occurs between (TC)n tracts adjacent to alternating purine-pyrimidine tracts. We have investigated the capacity of both the donor regions and the recombinant product involved in this event to adopt left-handed Z-DNA and intramolecular triplexes. The results of chemical probing with diethylpyrocarbonate and osmium tetroxide at the base pair level demonstrate that under the influence of negative supercoiling the alternating purine-pyrimidine regions of these plasmids can adopt Z-DNA at neutral pH, and the oligopurine.oligopyrimidine (pur.pyr) regions of these regions can adopt intramolecular triplexes at low pH (less than or equal to pH 6.0). At intermediate pH values, mixtures of both structures are present. Increasing the negative superhelical density of the plasmid does not increase the amount of triplex present at neutral pH indicating that the presence of long Z-DNA segments adjacent to pur.pyr tract prevents intramolecular triplex formation. In summary, we conclude that the sequences involved in the USCE can form either an intramolecular triplex in the (TC)n tract or Z-DNA in the alternating purine-pyrimidine tract and that Z-DNA will predominate under physiological conditions. The presence of segments which adopt Z-DNA at a site of USCE suggests that formation of this structure may enhance recombination between adjacent pur.pyr tracts.     

A Computer Simulation Study of Vntr Population Genetics: Constrained Recombination Rules Out the Infinite Alleles Model

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.     

(dT-dC)n and (dG-dA)n tracts arrest single stranded DNA replication in vitro.

Previous in vivo studies have indicated that (dT-dC)n.(dG-dA)n tracts (referred to here as (TC)n.(GA)n), which are widely dispersed in vertebrate genomes, may serve as pause or arrest signals for DNA replication and amplification. To determine whether these repeat elements act as stop signals for DNA replication in vitro, single stranded DNAs including (TC)n or (GA)n tracts of various lengths, were prepared by cloning such tracts into phage M13 vectors, and were replicated with the Klenow fragment of the E. coli DNA polymerase I, or with the calf thymus DNA polymerase alpha, by extension of an M13 primer. Gel electrophoresis of the reaction products revealed that the replication was specifically arrested around the middle of both (TC)n and (GA)n tracts of n greater than or equal to 16. However, whereas in the (TC)n tracts the arrests were less prominent at pH = 8.0 than at pH = 6.5-7.5, and were completely eliminated at pH = 8.5, the arrests in the (GA)n tracts were stronger at the higher pH values. These results, and previous data, suggest that the arrests were caused by formation of unusual DNA structures, possibly triple helices between partially replicated (TC)n or (GA)n tracts, and unreplicated portions of these sequences.     

Abundance and degree of dispersion of genomic d(GA) n ·d(TC) n sequences

Summary The abundance of d(GA) _n ·d(TC) _n tracts was determined in genomes of rodents and primates. Dot blot hybridization assays revealed that such tracts constitute 0.40%, 0.30%, and 0.40%, respectively, of the rat, hamster, and mouse genomes, but only 0.07% and 0.05% of the human and monkey genomes. A plaque hybridization assay of rat and human genomic libraries showed that 37% and 16%, respectively, of the recombinant phages in these libraries contain d(GA) _n ·d(TC) _n tracts. A survey of sequences stored in the GenBank data bank showed that a significant fraction of the stored rodent genes (about 2.0%) contain long d(GA) _n ·d(TC) _n tracts (n> 30) with <10% mismatching. The primate genes contain only shorter tracts (n<15) with <10% mismatching. In addition, the rodent and the primate genes contain tracts with larger degrees of mismatching. The chicken, which represents an entirely different branch of the evolutionary tree, was found to be as low in d(GA) _n ·d(TC) _n tracts as the primates. It is suggested that a common ancestor of the rodents has acquired the ability to amplify d(GA) _n ·d(TC) _n tracts.     

RecA independent recombination of poly[d(GT)-d(CA)] in pBR322.

Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly[d(GT)-d(CA)] carried in a derivative of pBR322 were recombinogenic in a recA host. Recombination brought about by poly[d(GT)-d(CA)] tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly[d(GT)-d(CA)] tracts, recombination also occurred between sequences bordering the dinucleotide repeats. (ii) recombination was enhanced when two poly[d(GT)-d(CA)] tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs. The mechanism by which poly[d(GT)-d(CA)] stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E. coli may involve formation of Z-DNA.     

Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.     

Isolation, cloning and characterisation of motifs containing (GA/TC)n repeats isolated from vetch, Vicia bithynica

Microsatellites are widely distributed in plant genomes and comprise unstable regions that undergo mutational changes at rates much greater than that observed for non-repetitive sequences. They demonstrate intrinsic genetic instability, manifested as frequent length changes due to insertions or deletions of repeat units. Detailed analysis of 1600 clones containing genomic sequences of Vicia bithynica revealed the presence of microsatellite repeats in its genome. Based on the screening of a partial DNA library of plasmids, 13 clones harbouring (GA/TC)n tracts of various lengths of repeated motif were identified for further analysis of their internal sequence organization. Sequence analyses revealed the precise length, number of repeats, interruptions within tracts, as well as sequence composition flanking the repeat motifs. Representative plasmids containing different lengths of (GA/TC)n embedded in their original flanking sequence were used to investigate the genetic stability of the repeats. In the study presented herein, we employed a well characterised and tractable bacterial genetic system. Recultivations of Escherichia coli harbouring plasmids containing (GA/TC)n inserts demonstrated that the genetic instability of (GA/TC)n microsatellites depends highly on their length (number of repeats). These observations are in agreement with similar studies performed on repetitive sequences from humans and other organisms.     

Protection of DNA sequences by triplex-bridge formation.

We have demonstrated that the DNA sequence between two triplex-forming polypurine.polypyrimidine (Pu.Py) tracts was protected from DNA modifying enzymes upon formation of triplex DNA structures with an oligodeoxyribonucleotide in which two triplex-forming Pu or Py tracts were placed at the termini (triplex-bridge formation). In model experiments, when two triplex structures were formed between double-stranded DNA with the sequence (AG)17-(N)18-(T)34, and an oligodeoxyribonucleotide, (T)34-(N)18-(GA)17, not only the Pu.Py tracts but also the 18 bp non-Pu.Py sequence in the duplex DNA between the tracts was protected from restriction enzymes, HpaII methylase and DNase I. This protection occurred only when both of the Pu.Py tracts were involved as triplexes. The length of the tracts could be as short as 21 bp, while the difference in length between the non-Pu.Py sequences on the duplex and the oligodeoxyribonucleotide should be within 10 nucleotides. The efficiency of protection was enhanced in the presence of a cationic detergent, cetyltrimethylammonium bromide, during triplex formation. Protection was also observed with another type of the triplex bridge formed between (G)34 and (T)34 tracts with an oligodeoxyribonucleotide, (T)34-(N)20-(G)34. These findings suggest that the protection of specific DNA sequences from enzymes by triplex-bridge formation can be applied to any DNA sequence by placing it between two triplex-forming sequences.     

Phosphatidylinositol metabolism accompanies early activation events in tumor target cell-stimulated human natural killer cells

This study examined the role of phospholipid metabolism in human natural killer (NK) cells upon activation by tumor target cells(TC). The effector cell (EC) population consisted of peripheral blood lymphocytes enriched for NK cells. Upon a 5-min exposure of EC to the NK-sensitive tumor TC K562 and U937, nearly four- and threefold increases in the incorporation of 32P into phosphatidylinositol (PI) occurred, respectively. In contrast, no increase in 32P incorporation into PI was seen when two NK-resistant TC were used. In addition, little or no change in the incorporation of 32P into phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine took place with any of the above TC. Depletion of Leu 11b-positive cells abolished the increase in 32P incorporation into PI when K562 were used in the phospholipid assay. Furthermore, labeling kinetics of this phospholipid turnover showed that it occurred less than 5 min following exposure to NK-sensitive TC and that phosphatidic acid, a breakdown product of phosphoinositides, was produced during this 5-min period. These results indicated that metabolism of a phosphoinositide took place and that it occurred in association with early activation events in NK cells. Quercetin and dibutyryladenosine-cyclic monophosphate (dbcAMP) plus theophylline exerted profound inhibitory effects on both NK activity and PI metabolism, suggesting a linkage between the two events. The inhibitors had no effect on target cell-binding capacity, indicating that the inhibition occurred postbinding. PI metabolism took place in the absence of extracellular calcium even though NK activity was completely abolished under the same conditions. Thus, we have shown PI metabolism, but not other phospholipids, to occur in human NK cells upon exposure to NK-sensitive TC, in association with early activation events. This event was independent of extracellular calcium and could be inhibited by quercetin or dbcAMP plus theophylline.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Acetylcholinesterase-positive intrapineal neuronal system in the palm squirrel Funambulus pennanti (Wroughton)

A well-developed acetylcholinesterase (AChE)-positive neuronal system could be demonstrated in the pineal organ of the palm squirrel. There are two longitudinal nerve tracts which run all along the margin of the pineal organ from its distal to proximal regions. These nerve tracts are confluent distally. Another short, but deep tract was seen in the middle part of the pineal organ which joins one of these tracts. A large number of AChE-positive neurons whose processes actually form the tracts are present all along the pineal organ. They are distinguished into multipolar and pseudounipolar/unipolar neurons. A few neurons seen outside the nerve tract form a network of nerve fibres among the pinealocytes and also link the main tracts. The nerve tracts appear wavy, irregular and tortuous. A large number of round ring-like bodies seen in close association with neuronal perikarya and nerves may represent the axo-somatic and axo-dendritic contacts.     

Estimation of Coupling Strength in Regenerated Lamprey Spinal Cords Based on a Stochastic Phase Model

We present a simple stochastic model of two coupled phase oscillators and a method of fitting the model to experimental spike-train data or to sequences of burst times. We apply the method to data from lesioned isolated lamprey spinal cords. The remaining tracts at the lesion site are either regenerated medial tracts, regenerated lateral tracts, control medial tracts, or control lateral tracts. We show that regenerated tracts on average provide significantly weaker coupling than control tracts. We compare our model-dependent estimate of coupling strength to a measure of coordination based on the size of deflections in the spike-train cross-correlation histogram (CCH). Using simulated data, we show that our estimates are able to detect changes in coupling strength that do not change the size of deflections in the CCH. Our estimates are also more resistant to changes in the level of dynamic noise and to changes in relative oscillator frequency than is the CCH. In simulations with high levels of dynamic noise and in one experimental preparation, we are able detect significant coupling strength although there are no significant deflections in the CCH.     

Expanded HOXA13 polyalanine tracts in a monotreme

SUMMARY The N-terminal region of human HOXA13 has seven discrete polyalanine tracts. Our previous analysis of these tracts in multiple major vertebrate clades suggested that three are mammal-specific. We now report the N-terminal HOXA13 repetitive tract structures in the monotreme Tachyglossus aculeatus (echidna). Contrary to our expectations, echidna HOXA13 possesses a unique set of polyalanine tracts and an unprecedented polyglycine tract. The data support the conclusion that the emergence of expanded polyalanine tracts in proteins occurred very early in the stem lineage that gave rise to mammals, between 162 and 315 Ma.     

Evidence for an early calcium-independent event in the activation of the human natural killer cell cytolytic mechanism

In this study, we examined the functional status of human natural killer (NK) cells after their direct interaction with the NK-sensitive tumor target cell (TC), K562. Human peripheral blood lymphocytes depleted of adherent cells were incubated for 4 hr with unlabeled K562 cells at an effector cell (EC) to TC ratio of 2:1. After incubation, the EC were separated from the TC via centrifugation over a single-step Percoll gradient. K562-treated and separated EC were subsequently shown to be unable to lyse fresh K562 TC when retested in the standard chromium-release assay. Kinetic studies revealed that greater than 90% inactivation of NK cell-mediated cytotoxicity (CMC) could be achieved within 2 hr. Inactivation of NK-CMC by K562 was not caused by a specific loss of NK cells, as detected by changes in the expression of two NK cell-associated markers, Leu-7 and Leu-11, or to alterations in EC viability and target binding cell capacity. Interestingly, NK inactivation also occurred in medium devoid of extracellular calcium, although parallel testing of NK-CMC in the same medium resulted in no chromium release. NK inactivation, however, was significantly prevented when the EC and TC were co-incubated at 4 degrees C, or in medium without magnesium. Additional studies revealed that inactivation of NK-CMC could be achieved with another NK-sensitive, but not with an NK-resistant TC. Overall, we demonstrated that NK cells rapidly lost their lytic potential after direct interaction with a sensitive TC, although the cells remained viable, expressed the same percentage of Leu-7 and Leu-11, and could still bind the TC; and NK inactivation occurred in the absence of extracellular calcium, but not when EC and TC were incubated in medium without magnesium. These latter results provide evidence for an early event in the activation of human NK cells that is binding dependent, temperature sensitive, and independent of extracellular calcium.     

Analysis of Antiretroviral Nucleosides by Electrospray Ionization Mass Spectrometry and Collision Induced Dissociation

Abstract

Antiretroviral nucleoside drugs used against the human immunodeficiency virus (HIV) infection have been analyzed using negative ion electrospray ionization (ESI) mass spectrometry and collision-induced dissociation (CID-MS/MS). Mass fragmentation of azidothymidine (AZT), didanosine (ddI), dideoxycytidine (ddC) and dideoxythiacytidine (3TC) were obtained at different cone voltages and collision energies. Fragmentation of purines and pyrimidines occurred by different pathways. For purines (ddI), the fragmentation was similar to those found in endogenous nucleosides; mainly the pseudo molecular ion is present (M-H) and a cleavage through the glycosidic bond forming (B) was observed. For pyrimidines (AZT, ddC, 3TC), the fragmentation pathways were different from endogenous nucleosides; for AZT, the fragmentation occurred primarily through the elimination of the azido group in the 3′-position (M-H₂-N₃), whereas ddC and 3TC presented more complex fragmentation patterns. For ddC, fragmentation appeared to be dominated by a retro Diels-Alder mechanism (M-CONH). For 3TC, the sulfur atom in the sugar moiety provided greater stability to the charge, producing fragments where the charge resided initially in the dideoxyribose (M-C₂O₂H₆).     

Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin

Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes. These studies show that (a) two of the three polar residues, S174, T177, or D176 and two of the three conserved alanine residues, A179 and A184 present in the 15-residue evolutionary conserved region are essential for Cbl-binding by human TC, and (b) TC gene is transferred in a syntenic manner to different chromosomes, at least before the divergence of mouse/rat and human.     

87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants

Transgenic tomato plants (Lycopersicon esculentum) overexpressing the prosystemin transgene have been shown previously to accumulate a soluble 87 kDa cystatin constitutively. We report here that this protein can be found in a crystalline form which can be purified using a glycerol/sucrose gradient. Midgut homogenate of third-instar larvae of two coleopteran pest insects, Callosobruchus maculatus and Zabrotes subfasciatus, had their proteolytic activity content significantly inhibited by tomato cystatin (TC). In leaves of wild-type tomato plants, cystatin mRNA accumulated systemically in response to wounding, treatment with methyl jasmonate (MJ) and when supplied with systemin, corroborating the anti-herbivorous activity. Accumulation of cystatin mRNA occurred when plants were supplied with chitosan and oligogalacturonic acid fragments (OGA), suggesting an effect of TC against pathogens. Moreover, this protein reduced the growth of two fungi, Fusarium solani and Trichoderma viride in vitro. Taken together, the data reinforce a role for TC in defense response against pests or pathogens.     

Feeding habits of bearded seals (Erignathus barbatus) from the Svalbard area, Norway

The objective of the present study was to assess the feeding habits of bearded seals (Erignathus barbatus) from the Svalbard area, Norway. Stomach and/or intestine contents were collected from 47 individuals shot between May and September (1989–1996). Prey specimens were identified from whole specimens or hard items such as fish otoliths, crustacean exoskeletons, cephalopod beaks, polychaete jaws, gastropod operculae and mollusc shell remains. To show the dietary contribution of the different prey items found in the gastrointestinal tracts, the frequency of occurrence and the numerical frequency methods were used. Fifty-nine percent or more of the gastrointestinal tracts contained five or more prey organisms. The most frequent fish species recorded was polar cod (Boreogadus saida), which was found in 49% of the gastrointestinal tracts, followed by sculpins (Cottidae spp.) (44%), long rough dab (Hippoglossoides platessoides) (28%) and the stout eelblenny (Lumpenus medius) (18%). The most frequent crustaceans were the spider crab (Hyas araneus) (59%), Sabinea septemcarinatus (54%), Sclerocrangon boreas (46%) and Lebbeus polaris (18%). Of the molluscs, the whelk Buccinum spp. occurred in 18% of the gastrointestinal tracts. Other species such as cephalopods, polychaete worms, amphipods and echiuran worms occurred in small amounts. There were no significant differences in foraging behaviour between males and females. This study also shows that in the Svalbard area bearded seals are benthic feeders and utilise a wide variety of organisms. Accepted: 26 August 1998     

Activity of digestive proteinases and carbohydrases in the alimentary tract of the fig leaf roller,Choreutis nemorana Hübner (Lepidoptera: Choreutidae)

.The fig leaf roller or Fig-tree Skeletoniser, Choreutis nemorana (Lep.: Choreutidae), is a destructive pest of fig trees found in some fig-growing areas of Iran. The larvae feed on the upper level of leaves, near the main vein. In this study, digestive carbohydrases including α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and proteinases including trypsin, chymotrypsin and elastase were investigated. The results showed that the carbohydrases were present in the alimentary tracts of the pest. Optimum pH for α-glucosidase and β-glucosidase activity was at pH 6.0 and 7.0, respectively. Maximum activity of α-galactosidase and β-galactosidase occurred at pH 6.0. Total proteolitic activity against the substrate azocasein was optimally occurred at pH 10.0. The greatest activity of trypsin, chymotrypsin and elastase was determined at pH 10.0, 11.0 and 11.0, respectively. Zymogram analyses using nitrocellulose membrane revealed two trypsin isoforms in which one of them was completely inhibited by Soybean Kunitz inhibitor and the other was notably inhibited.