The omega-6 arachidonic fatty acid, but not the omega-3 fatty acids, inhibits osteoblastogenesis and induces adipogenesis of human mesenchymal stem cells: potential implication in osteoporosis.

UNLABELLED: Arachidonic fatty acid (AA) induces adipogenesis in human mesenchymal stem cells cultures, and high concentrations inhibit osteoblastogenesis; whereas eicosapentaenoic and docosahexaenoic fatty acids do not induce adipogenesis and do not inhibit osteoblastogenesis. In mesenchymal stem cells, omega-6 arachidonic polyunsaturated fatty acid promotes the differentiation of adipocytes and inhibits the osteoblast differentiation. While omega-3 fatty acids do not affect the adipogenic differentiation their effects on osteoblastogenesis are less relevant. An increased ratio of omega-3/omega-6 fatty acid consumption can prevent bone mass loss. INTRODUCTION: Consumption of omega-3 may protect against osteoporosis since they may inhibit osteoclastogenesis. However, with aging, MSC in bone marrow are increasingly differentiated into adipocytes, reducing the number of osteoblasts. Products derived from omega-6 and omega-3 metabolism may affect MSC differentiation into osteoblasts and adipocytes. METHODS: Human MSC have been differentiated into osteoblasts or adipocytes in the presence of omega-6 (AA), or omega-3 (DHA and EPA), and osteoblastic and adipocytic markers have been analyzed. RESULTS: AA decreases the expression of osteogenic markers and the osteoprotegerin/receptor activator of nuclear factor kappa ß ligand gene expression ratio (opg/rankl). High concentrations of AA inhibit the mineralization and cause the appearance of adipocytes in MSC differentiating into osteoblasts to a higher extent than DHA or EPA. In MSC differentiated into adipocytes, AA increases adipogenesis, while DHA and EPA do not affect it. AA caused the appearance of adipocytes in undifferentiated MSC. The lipoxygenase gene (alox15b) is induced by omega-3 in MSC induced to osteoblasts, and by omega-6 in MSC induced to adipocytes. CONCLUSIONS: An increase in the intake of omega-3 respect to omega-6 may provide protection against the loss of bone mass, since omega-6 favors the osteoclastic activity by diminishing the opg/rankl gene expression in osteoblasts and promotes MSC differentiation into adipocytes, thus diminishing the production of osteoblasts.

Analysis of genetic diversity of southern Spain fig tree (Ficus carica L.) and reference materials as a tool for breeding and conservation.

The common fig tree (Ficus carica L.) is a Mediterranean crop with problematic cultivar identification. The recovery and conservation of possible local varieties for ecological production requires the previous genetic characterization of the available germplasm. In this context, 42 lines corresponding to 12 local varieties and two caprifigs, in addition to 15 reference samples have been fingerprinted using 21 SSR markers. A total of 77 alleles were revealed, detecting a useful level of genetic variability within the local germplasm pools. UPGMA clustering analysis has revealed the genetic structure and relationships among the local and reference germplasm. Eleven of the local varieties could be identified and defined as obtained clusters, showing that SSR analysis is an efficient method to evaluate the Andalusian fig tree diversity for on-farm conservation.

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress-related genes.

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas (Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15-17 days after inoculation) and (iii) when all plants had developed disease symptoms (20-22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier (i.e. GR and SOD) or to higher levels (i.e. DAO) in the incompatible interaction. Expression of apx, cat, sod, lipoxygenase (lox) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier (lox) or higher (actin) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.

Cryopreserved human bone marrow mononuclear cells as a source of mesenchymal stromal cells: application in osteoporosis research.

BACKGROUND: Mesenchymal stromal cells (MSC) are an invaluable tool for research and therapeutic application regarding degenerative diseases such as osteoporosis. METHODS: Human MSC from cryopreserved mononuclear (c-MSC) cell populations were isolated from bone marrow (BM) and compared with MSC isolated directly from the same BM for immunophenotype, differentiation capacity and Parathormone (PTH) response. RESULTS: c-MSC showed a similar immunophenotype, division and differentiation capacity as standard MSC obtained from the same BM. This capacity was maintained during various culture-growing passages. Treatment with PTH(1-34) from days 6 to 24, after c-MSC induction to osteoblasts and adipocytes, had no significant effect on osteoblastogenesis yet inhibited adipogenesis. This effect was similar in MSC from the same BM. DISCUSSION: We propose cryopreservation of mononuclear cells obtained from BM as a simple and convenient means for routine storage of MSC to be used for therapeutic and research applications.

Tissue factor as an effector of angiogenesis and tumor progression in hematological malignancies.

In the last few years, it has become clear that the processes of tumor angiogenesis, metastasis and invasiveness are highly dependent on components of the blood coagulation cascade. One of the key proteins in coagulation is tissue factor (TF). In addition, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. TF significantly participates in tumor-associated angiogenesis and its expression levels have been correlated with the metastatic potential of many types of hematological malignancies. Signaling pathways initiated by both, tissue factor-activated factor VII (TF-FVIIa) protease activation of protein-activated receptors (PARs), and phosphorylation of the TF-cytoplasmic domain, appear to regulate these tumoral functions. Advances in antiangiogenic therapies and preclinical studies with TF-targeted therapeutics are hopeful in the control of tumor growth and metastasis, but continued studies on the regulation of TF are still needed. In the last few years, the use of approaches of functional genomics and proteomics has allowed the discovery of new proteins involved in the origin of the neoplasia and their participation in the development of the disease. This review attempts to establish a cellular and molecular causal link between cancer coagulopathy, angiogenesis and tumor progression in hematological malignancies.

L-arabinose resistance test with Salmonella typhimurium as a primary tool for carcinogen screening.

The L-arabinose resistance test with Salmonella typhimurium (Ara test) is a forward mutation assay which selects a single phenotypic change (from L-arabinose sensitivity to L-arabinose resistance) in a unique tester strain (an araD mutant). The present study examined the ability of the Ara test to identify as mutagens different classes of chemical carcinogens, including six with detection problems in most bacterial assays. A noncarcinogen related in chemical structure to the selected carcinogen was included whenever possible. A total of 25 organic compounds was assayed by means of a standard mutagenesis procedure: the plate-incorporation test in the presence (if required) of 10% S9 from rat liver induced with Aroclor 1254. Chemicals giving negative or inconclusive results were then retested using other common in vitro mutagenesis conditions. The Ara test detected as mutagens all but one (Urethane) of the 15 established carcinogens and six out of the nine chemicals with questionable or nonassayed carcinogenicity. The compound mutagenic at the lowest dose was ethionine (0.57 nmol), one of the carcinogens undetected by the popular histidine reverse mutation assay (Ames test) or by the SOS chromotest. Actually, only benzo(a)pyrene was found mutagenic at a dose (0.77 nmol) close to that of ethionine. The data reported in this paper suggest that the Ara forward mutation test is less prone than the His reverse mutation test or the SOS chromotest to misclassify as negative carcinogenic compounds. Consequently, it should be considered as an alternative to other bacterial assays in general, massive, and primary screening for genotoxic carcinogens.

Molecular analysis of ultraviolet-induced mutations in a xeroderma pigmentosum cell line.

We have isolated and characterized 47 ultraviolet light-induced hprt mutants from a simian virus 40-transformed excision-repair-deficient xeroderma pigmentosum cell line (complementation group A). Twenty-one independent mutations were found, of which the majority were point mutations. Eleven of these were identified as base changes, nine of which could be attributed to ultraviolet damage on the transcribed DNA strand. Both transitions and transversions were found among the single base changes. A large proportion of the mutations (13/21) resulted in aberrant splicing of the hprt gene, suggesting that the target size for mutations resulting in aberrant splicing must be quite large. A small number of spontaneous mutations were identified, most of which were large deletions. Our data provide a spectrum for the intrinsic mutations resulting from ultraviolet damage in human cells in the absence of repair.

Tissue factor (TF) and urokinase plasminogen activator receptor (uPAR) and bleeding complications in leukemic patients.

Tissue factor (TF) and urokinase receptor (uPAR) are key cellular receptors triggering, respectively, coagulation and fibrinolysis. Bleeding complications among leukemic patients have been related to an abnormal expression of TF by blast cells and/or to an abnormal fibrinolytic response. In this study the expression of TF and uPAR has been assessed in 18 acute non-lymphoblastic and 8 lymphoblastic leukemic blast cells using several methodological approaches. TF mRNA was evaluated by in situ hybridization and TF and uPAR antigen were evaluated immunologically in cell lysates and on the cell surface by flow cytometry. In addition, TF-procoagulant activity was measured in coagulation-based assays. The reliability of these methods was corroborated in six leukemic cell lines of different lineages and states of maturation. Disseminated intravascular coagulation was detected in two M3 leukemia patients whose blast cells expressed high amounts of TF. Hyperfibrinolysis was detected in one M1 and two M2 patients, whose blast cells displayed a high content of uPAR antigen, but no TF. Furthermore, M5 leukemia blast cells expressed both TF and uPAR, although no hemostatic defects or bleeding complications were detected in these patients. Taken together, although a limited number of patients was included in this study, these data suggest that in leukemia patients exhibiting bleeding, either TF or uPAR are expressed by their blast cells. However, the presence of these receptors does not necessarily imply the existence of a hemostatic disorder.

Allele-frequency determination of BsmI and FokI polymorphisms of the VDR gene by quantitative real-time PCR (QRT-PCR) in pooled genomic DNA samples.

The vdr gene is a candidate for osteoporosis susceptibility, with conflicting results in association studies. We have designed and optimized an individual allele-specific and DNA pooling PCR-based methodology to quantitate BsmI and FokI polymorphisms of the vdr gene and studied single-nucleotide polymorphisms (SNPs) from pooled DNA samples. The allele frequency in DNA pooling experiments has been analyzed by kinetic PCR: quantitative real-time PCR (QRT-PCR). A Spanish cohort of 225 healthy postmenopausal women was studied. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA) and quantitative ultrasound calcaneous densitometry. Allele-specific PCR amplification of BsmI and FokI genes showed full concordance with the PCR-RFLP approach. The prevalence of the three BsmI VDR genotypes was 19.1, 44.9 and 36.0% for BB, Bb and bb, respectively. In the case of the FokI locus, the prevalence of genotypes was 40.4, 48.0 and 11.6% for FF, Ff and ff, respectively. No positive correlation was found between polymorphism and BMD. The DNA pooling procedure was validated. No differences were found in allele frequencies and T-score data obtained using the high throughput DNA pooling approach, as compared to known individual frequencies. In our hands, this is a very useful approach to study quantitative (thus polygenic) traits like osteoporosis susceptibility.

Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts.

Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5-20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or "Nature's pesticides" (the major toxic chemicals ingested by phytoplankton filter-feeders), and/or the result of human activities remains to be determined.

Promutagen activation by fish liver as a biomarker of littoral pollution.

Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1-, 18.6- and 42.8-fold higher capability to activate benzo(a)pyrene, 2-acetyl-aminofluorene and 2-aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose-response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2-aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments.

Study of the causes of direct-acting mutagenicity in coffee and tea using the Ara test in Salmonella typhimurium.

The mutagenic activities of 6 of the chemicals identified in coffee solutions were assayed with the Salmonella Ara test, under experimental conditions optimized for coffee mutagenicity. Caffeine was the only non-mutagenic compound. Among the other 5 chemicals, hydrogen peroxide was the strongest mutagen and chlorogenic acid the weakest; methylglyoxal, glyoxal and caffeic acid exhibited intermediate mutagenicities. The minimal mutagenic doses of these components correlated negatively with their relative concentrations in coffee. It was concluded that chlorogenic acid, caffeic acid, glyoxal and methylglyoxal cannot contribute alone to the mutagenicity of coffee in the Ara test, since their minimal mutagenic concentrations were much higher than their respective levels in the coffee samples assayed. By contrast, 40-60% of the mutagenic activity in coffee and also in tea could be attributed to their H2O2 contents. Catalase abolished more than 95% of the mutagenic activity of coffee, as detected by the Ara test. A similar sensitivity to catalase has been reported by other authors in relation to the coffee mutagenicity identified by the Salmonella His test. Nevertheless, the results presented in this paper suggest that the Ara forward and the His reverse mutation tests are sensitive to the mutagenicity of different constituents in coffee solutions. We propose that the His test, sensitive at high coffee doses, mainly recognizes the mutagenicity of methylglyoxal, whilst the Ara test, sensitive at low coffee doses, mainly detects the mutagenic activity of hydrogen peroxide. The data reported also suggest that the direct-acting mutagenicity(ies) detected by the Ara test in tea solutions is (are) based on similar, if not identical, mechanisms.

Development of new molecular procedures for the detection of genetic alterations in man.

The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus. Mutations are identified as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE). Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR. We have developed this procedure using both bacterial and mammalian cells. With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10(-6) at an EcoRI site in the AraA locus of Salmonella typhimurium. The detection limit with an RsaI site in the lacI gene of Escherichia coli was 10(-5), and we were able to detect DNA damage and repair after treatment with N-methyl-N-nitrosourea (MNU). With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells. In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasI and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions. We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.

New methods to use fish cytochrome P4501A to assess marine organic pollutants.

A new methodology has been developed to assess cytochrome P4501A expression in two South Atlantic Spanish fish, guilthead seabream (Sparus aurata) and grey mullet (Liza aurata), used as pollution bioindicators. Degenerate oligos were used to amplify by reverse transcription and PCR (RT-PCR) specific cyp1A cDNA sequences, used subsequently to design specific primers to get the full cDNA by rapid amplification of cDNA ends. A new assay has been developed to quantitate cyp1A expression by RT-PCR in an automated DNA sequencer. The effect of beta-naphthoflavone inducing biotransformation has been used to compare three distinct pollution biomarkers: EROD activity, ELISA determination of CYP1A, and 2-aminoanthracene (2-AA) activation. Immunodetection by ELISA or Western blot was inconsistent in S. aurata and L. aurata. EROD activity yielded satisfactory results; the higher induction was observed by bioactivation of 2-AA to mutagens detected with strain BA149 of Salmonella typhimurium, in agreement with the high sensitivity previously described for this biomarker. The present paper summarizes the current status of our research.

Biochemical and genetic indices of marine pollution in Spanish littoral.

Increased activities of several detoxifying and antioxidant enzymes were detected in mollusc and fish from Spanish littoral areas with high metal contents. Ethanolic extracts from molluscs contained direct-acting and polar genotoxins of oxidative type, which were detected by strain TA102 of S. typhimurium and catalase-deficient strains of E. coli. Animals from contaminated sites contained less genotoxins than those from control areas. Polluted fishes displayed highly induced cytochrome P-450 activity and increased promutagen activation capabilities. In addition, specific forms of glutathione transferase and superoxide dismutase were induced, particularly highly acidic forms.

The BLAST algorithms: practical application in molecular cloning, marker-assisted selection (MAS) and introgression of wheat.

BLAST (Basic Local Alignment Search) analyses of Hordeum chilense RAPD markers have shown DNA sequence similarities with several plant genes. Biologically significant hits were scored for: plastocianin (Hordeum vulgare), alpha-gliadin (Triticum aestivum), Grandel-6 retrotransposon (Zea diploperennis), retrofit (gag/pol) copia-like, transposon-like element (Oryza longistaminata), copia-like retrotransposon Hopscotch polyprotein (gag/pol) (Zea mays) and several retrotransposons/retroelements from other species (Arabidopsis thaliana, Oryza sativa, Pisum sativum and Zea mays). Statistically significant hits also included endochitinase (Brassica napius), ribulose-1,5-bisphosphate carboxylase (Euglena gracilis), piruvate, orthophosphate dikinase (Flaveria trinervia), and an ORF flanked by ARS sequences (Triticum aestivum). Interestingly, it was also found that the currently recommended BLAST algorithm (version 2.0.11; gapped) at did not produce any biologically significant hits, whereas the previous version (1.4.11; ungapped) did generate biologically significant results. Thus, we suggest using the new BLAST 2.0.x when strong homologies are expected in short sequence stretches between closely related species. In other instances (and particularly when searching for lower similarities, yet spanning significantly larger stretches between more distant species), the BLAST 1.4.x could yield additional results. BLAST searches are valuable tools to identify and clone DNA sequences of interest, with applications in Marker-Assisted Selection (MAS) and wheat introgression.

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.).

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70-80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.

Coffee is highly mutagenic in the L-arabinose resistance test in Salmonella typhimurium.

The present study shows that the L-arabinose resistance test in Salmonella typhimurium detects coffee as a strong mutagen in the absence of mammalian microsomal activation. The response of the Ara forward mutation assay was 8.5 times higher than that of TA104, which is the most sensitive to coffee of the tester strains of the Ames test. Both the mutagenesis protocol (preincubation test) and the additional genetic characteristics of the bacterial tester strain (excision repair deficiency, normal lipopolysaccharide barrier, and the presence of plasmid pKM101) were critical factors in the optimal induction by coffee of forward mutations to L-arabinose resistance. All ten samples of roasted coffee analyzed with the Ara assay were highly mutagenic: one cup of coffee (150 ml) was calculated to induce 3-4 X 10(6) AraR mutants. In contrast, coffee prepared from unroasted beans (green coffee) had no mutagenic activity. Regular- and sugar-roasted coffees showed similar mutagenicities, but the specific mutagenic activity of instant coffees (1559 AraR mutants/mg) was almost 2 times that of noninstant ones (834 AraR mutants/mg). The Ara assay allowed the direct testing of coffee, although it was demonstrated that lyophilization has no effect on the mutagenicity of this beverage. Like roasted coffee, roasted barley induced a large number of AraR mutants per mg (227), though its specific mutagenic activity was approximately 4 and 7 times lower than that of noninstant and instant coffees, respectively.

Cross-species amplification of the Hordeum chilense genome using barley sequence-tagged-sites (STSs).

A selection of 51 barley Sequence-Tagged Sites (STSs) were studied for their utility in Hordeum chilense. They included four primer sets from wheat origin and six primer sets from oat origin. Forty-four primer pairs amplified H. chilense products consistently. Five primer pairs were suitable for studying the introgression of H. chilense in wheat because they amplified H. chilense products of distinct size. Six of the STSs showed polymorphism between different H. chilense accessions. The results showed that barley STSs could be useful for the genetic characterization of H. chilense, tritordeums and derived introgression lines.

Automated laser-induced fluorescence DNA sequencing: equalizing signal-to-noise ratios significantly enhances overall performance.

We have significantly optimized the performance of the Perkin-Elmer/Applied Biosystems (PE/ABI) 373Stretch DNA sequencer. Best results were obtained using 19% less ddNTPs and 75% more dNTPs relative to the manufacturer's recommended reaction mixture. Other changes included 4.25% (instead of 4%) acrylamide gels run at 38 W (instead of 40 W). These variations produced a significant equalization of the signal-to-noise profile, resulting in longer reads. The overall accuracy improvement for unedited pGEM 1000-base sequences analyzed by the ABI100 basecaller was 8.2%. High accuracy gains were observed in the 501- to 850-base region (6.0%) and, most significantly, from that point to the end of the sequence (41.6%). A decrease in accuracy was also found for the first 30 bases, which usually correspond to vector sequence. Our study has focused primarily on double-stranded DNA (dsDNA) using the DyeDeoxy Terminator chemistry kit and 48-cm well-to-read (WTR) gels since, in our hands, such a combination represents the quicker, most accurate, versatile, and productive choice for large-scale DNA sequencing. Nevertheless, our findings could be also exploited in other sequencing strategies using ssDNA templates, the DyePrimer chemistry, alternative enzyme preparations, or different WTR lengths on the 373Stretch or other sequencing machines.