Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different-colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were samplcd. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating inde- pendently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once.

Better prediction of protein contact number using a support vector regression analysis of amino acid sequence

Background: Protein tertiary structure can be partly characterized via each amino acid's contact number measuring how residues are spatially arranged. The contact number of a residue in a folded protein is a measure of its exposure to the local environment, and is defined as the number of C-beta atoms in other residues within a sphere around the C-beta atom of the residue of interest. Contact number is partly conserved between protein folds and thus is useful for protein fold and structure prediction. In turn, each residue's contact number can be partially predicted from primary amino acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more accurate contact number prediction method from protein primary sequence. Results: We predict contact number from protein sequence using a novel support vector regression algorithm. Using protein local sequences with multiple sequence alignments (PSI-BLAST profiles), we demonstrate a correlation coefficient between predicted and observed contact numbers of 0.70, which outperforms previously achieved accuracies. Including additional information about sequence weight and amino acid composition further improves prediction accuracies significantly with the correlation coefficient reaching 0.73. If residues are classified as being either contacted or non-contacted, the prediction accuracies are all greater than 77%, regardless of the choice of classification thresholds. Conclusion: The successful application of support vector regression to the prediction of protein contact number reported here, together with previous applications of this approach to the prediction of protein accessible surface area and B-factor profile, suggests that a support vector regression approach may be very useful for determining the structure-function relation between primary sequence and higher order consecutive protein structural and functional properties.

The use and implications of ribosomal DNA sequencing for the discrimination of digenean species

In just over a decade, the use of molecular approaches for the recognition of parasites has become commonplace. For trematodes, the internal transcribed spacer region of ribosomal DNA (ITS rDNA) has become the default region of choice. Here, we review the findings of 63 studies that report ITS rDNA sequence data for about 155 digenean species from 19 families, and then review the levels of variation that have been reported and how the variation has been interpreted. Overall, complete ITS sequences (or ITS1 or ITS2 regions alone) usually distinguish trematode species clearly, including combinations for which morphology gives ambiguous results. Closely related species may have few base differences and in at least one convincing case the ITS2 sequences of two good species are identical. In some cases, the ITS1 region gives greater resolution than the ITS2 because of the presence of variable repeat units that are generally lacking in the ITS2. Intraspecific variation is usually low and frequently apparently absent. Information on geographical variation of digeneans is limited but at least some of the reported variation probably reflects the presence of multiple species. Despite the accepted dogma that concerted evolution makes the individual representative of the entire species, a significant number of studies have reported at least some intraspecific variation. The significance of such variation is difficult to assess a posteriori, but it seems likely that identification and sequencing errors account for some of it and failure to recognise separate species may also be significant. Some reported variation clearly requires further analysis. The use of a yardstick to determine when separate species should be recognised is flawed. Instead, we argue that consistent genetic differences that are associated with consistent morphological or biological traits should be considered the marker for separate species. We propose a generalised approach to the use of rDNA to distinguish trematode species.

XSophe-Sophe-XeprView (R). A computer simulation software suite (v. 1.1.3) for the analysis of continuous wave EPR spectra

The XSophe-Sophe-XeprView((R)) computer simulation software suite enables scientists to easily determine spin Hamiltonian parameters from isotropic, randomly oriented and single crystal continuous wave electron paramagnetic resonance (CW EPR) spectra from radicals and isolated paramagnetic metal ion centers or clusters found in metalloproteins, chemical systems and materials science. XSophe provides an X-windows graphical user interface to the Sophe programme and allows: creation of multiple input files, local and remote execution of Sophe, the display of sophelog (output from Sophe) and input parameters/files. Sophe is a sophisticated computer simulation software programme employing a number of innovative technologies including; the Sydney OPera HousE (SOPHE) partition and interpolation schemes, a field segmentation algorithm, the mosaic misorientation linewidth model, parallelization and spectral optimisation. In conjunction with the SOPHE partition scheme and the field segmentation algorithm, the SOPHE interpolation scheme and the mosaic misorientation linewidth model greatly increase the speed of simulations for most spin systems. Employing brute force matrix diagonalization in the simulation of an EPR spectrum from a high spin Cr(III) complex with the spin Hamiltonian parameters g(e) = 2.00, D = 0.10 cm(-1), E/D = 0.25, A(x) = 120.0, A(y) = 120.0, A(z) = 240.0 x 10(-4) cm(-1) requires a SOPHE grid size of N = 400 (to produce a good signal to noise ratio) and takes 229.47 s. In contrast the use of either the SOPHE interpolation scheme or the mosaic misorientation linewidth model requires a SOPHE grid size of only N = 18 and takes 44.08 and 0.79 s, respectively. Results from Sophe are transferred via the Common Object Request Broker Architecture (CORBA) to XSophe and subsequently to XeprView((R)) where the simulated CW EPR spectra (1D and 2D) can be compared to the experimental spectra. Energy level diagrams, transition roadmaps and transition surfaces aid the interpretation of complicated randomly oriented CW EPR spectra and can be viewed with a web browser and an OpenInventor scene graph viewer.

Experimental Analysis of Froude Number Effect on Air Entrainment in the Hydraulic Jump

A hydraulic jump is characterized by strong energy dissipation and mixing, large-scale turbulence, air entrainment, waves and spray. Despite recent pertinent studies, the interaction between air bubbles diffusion and momentum transfer is not completely understood. The objective of this paper is to present experimental results from new measurements performed in rectangular horizontal flume with partially-developed inflow conditions. The vertical distributions of void fraction and air bubbles count rate were recorded for inflow Froude number Fr1 in the range from 5.2 to 14.3. Rapid detrainment process was observed near the jump toe, whereas the structure of the air diffusion layer was clearly observed over longer distances. These new data were compared with previous data generally collected at lower Froude numbers. The comparison demonstrated that, at a fixed distance from the jump toe, the maximum void fraction Cmax increases with the increasing Fr1. The vertical locations of the maximum void fraction and bubble count rate were consistent with previous studies. Finally, an empirical correlation between the upper boundary of the air diffusion layer and the distance from the impingement point was provided.

Analysis of multiple gas solid reactions

Multiple gas solid reactions involving one solid and N gaseous reactants are investigated in this study by using a matched asymptotic expansion technique. Two cases are particularly studied. In the first case all N chemical reaction rates are faster than the diffusion rate. While in the second case only M (M < N) chemical reaction rates are faster than the diffusion rate and the rates of the remaining (N-M) chemical reactions are comparable to that of diffusion. For these two cases the solid concentration profile behaves like a travelling wave. In the first case the wave front velocity is contributed linearly by all gaseous reactants (additive law) while in the second case this law does not hold.

Data analysis in multiple-frequency bioelectrical impedance analysis

The performance of three analytical methods for multiple-frequency bioelectrical impedance analysis (MFBIA) data was assessed. The methods were the established method of Cole and Cole, the newly proposed method of Siconolfi and co-workers and a modification of this procedure. Method performance was assessed from the adequacy of the curve fitting techniques, as judged by the correlation coefficient and standard error of the estimate, and the accuracy of the different methods in determining the theoretical values of impedance parameters describing a set of model electrical circuits. The experimental data were well fitted by all curve-fitting procedures (r = 0.9 with SEE 0.3 to 3.5% or better for most circuit-procedure combinations). Cole-Cole modelling provided the most accurate estimates of circuit impedance values, generally within 1-2% of the theoretical values, followed by the Siconolfi procedure using a sixth-order polynomial regression (1-6% variation). None of the methods, however, accurately estimated circuit parameters when the measured impedances were low (<20 Omega) reflecting the electronic limits of the impedance meter used. These data suggest that Cole-Cole modelling remains the preferred method for the analysis of MFBIA data.

Sensitivity of multiple frequency bioelectrical impedance analysis to changes in ion status

Bioelectrical impedance analysis has found extensive application as a simple noninvasive method for the assessment of body fluid volumes, The measured impedance is, however, not only related to the volume of fluid but also to its inherent resistivity. The primary determinant of the resistivities of body fluids is the concentration of ions. The aim of this study was to investigate the sensitivity of bioelectrical impedance analysis to bodily ion status. Whole body impedance over a range of frequencies (4-1012 kHz) of rats was measured during infusion of various concentrations of saline into rats concomitant with measurement of total body and intracellular water by tracer dilution techniques. Extracellular resistance (R-o), intracellular resistance (R-i) and impedance at the characteristic frequency (Z(c)) were calculated. R-o and Z(c) were used to predict extracellular and total body water respectively using previously published formulae. The results showed that whilst R-o and Z(c) decreased proportionately to the amount of NaCl infused, R-i increased only slightly. Impedances at the end of infusion predicted increases iu TBW and ECW of approximately 4-6% despite a volume increase of less than 0.5% in TBW due to the volume of fluid infused. These data are discussed in relation to the assumption of constant resistivity in the prediction of fluid volumes from impedance data.

Comparative analysis of dendritic cell density and total number in commonly transplanted organs: morphometric estimation in normal mice

Dendritic cells (DC) are considered to be the major cell type responsible for induction of primary immune responses. While they have been shown to play a critical role in eliciting allosensitization via the direct pathway, there is evidence that maturational and/or activational heterogeneity between DC in different donor organs may be crucial to allograft outcome. Despite such an important perceived role for DC, no accurate estimates of their number in commonly transplanted organs have been reported. Therefore, leukocytes and DC were visualized and enumerated in cryostat sections of normal mouse (C57BL/10, B10.BR, C3H) liver, heart, kidney and pancreas by immunohistochemistry (CD45 and MHC class II staining, respectively). Total immunopositive cell number and MHC class II+ cell density (C57BL/10 mice only) were estimated using established morphometric techniques - the fractionator and disector principles, respectively. Liver contained considerably more leukocytes (similar to 5-20 x 10(6)) and DC (similar to 1-3 x 10(6)) than the other organs examined (pancreas: similar to 0.6 x 10(6) and similar to 0.35 x 10(6): heart: similar to 0.8 x 10(6) and similar to 0.4 x 10(6); kidney similar to 1.2 x 10(6) and 0.65 x 10(6), respectively). In liver, DC comprised a lower proportion of all leukocytes (similar to 15-25%) than in the other parenchymal organs examined (similar to 40-60%). Comparatively, DC density in C57BL/10 mice was heart > kidney > pancreas much greater than liver (similar to 6.6 x 10(6), 5 x 10(6), 4.5 x 10(6) and 1.1 x 10(6) cells/cm(3), respectively). When compared to previously published data on allograft survival, the results indicate that the absolute number of MHC class II+ DC present in a donor organ is a poor predictor of graft outcome. Survival of solid organ allografts is more closely related to the density of the donor DC network within the graft. (C) 2000 Elsevier Science B.V. All rights reserved.

Coordinated plant defense responses in Arabidopsis revealed by microarray analysis

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2.375 selected genes were examined simultaneously by cDNA microarray analysis in Arabidopsis thaliana after inoculation with an incompatible fungal pathogen Alternaria brassicicola or treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene, Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology, In leaf tissue inoculated with A. brassicicola, the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Folate-sensitive fragile site FRA10A is due to an expansion of a CGG repeat in a novel gene, FRA10AC1, encoding a nuclear protein

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/ CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5'UTR of a novel gene named FRA10ACl. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10ACl gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of similar to 1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10ACl, of unknown function. Several splice variants leading to alternative 3' ends were identified (particularly in testis). These give rise to FRA10ACl proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10ACl protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10ACl gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site. (C) 2004 Elsevier Inc. All rights reserved.

Familial partial epilepsy with variable foci: Clinical features and linkage to chromosome 22q12

Background: Familial partial epilepsy with variable foci (FPEVF) is an autosomal dominant syndrome characterized by partial seizures originating from different brain regions in different family members in the absence of detectable structural abnormalities. A gene for FPEVF was mapped to chromosome 22q12 in two distantly related French-Canadian families. Methods: We describe the clinical features and performed a linkage analysis in a Spanish kindred and in a third French-Canadian family distantly related to the original pedigrees. Results: Onset of seizures was typically in middle childhood, and attacks were usually easy to control. Seizure semiology varied among family members but was constant for each individual. In some, a pattern of nocturnal frontal lobe seizures led to consideration of the diagnosis of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The Spanish family was mapped to chromosome 22q (multipoint lod score, 3.4), and the new French-Canadian family had a multipoint lod score of 2.97 and shared the haplotype of the original French-Canadian families. Conclusions: Identification of the various forms of familial partial epilepsy is challenging, particularly in small families, in which insufficient individuals exist to identify a specific pattern. We provide clinical guidelines for this task, which will eventually be supplanted by specific molecular diagnosis. We confirmed linkage of FPEVF to chromosome 22q 12 and redefined the region to a 5.2-Mb segment of DNA.