Gotowa bibliografia na temat „Northern hybridisation analysis”

RNA northern blots provide robust measurements of gene expression. The simple northern blot technique described in this report has been optimised to provide rapid, reproducible detection and analysis of mature and precursor forms of microRNAs. This protocol economises on the use of commercially available components and secondly reduces the hybridisation step to 2 hours.

Molecular, cytological, and morphological data support the existence of a hybrid population between Schoenus nigricans and Schoenus ferrugineus. This population was found in northeastern Italy, where S. nigricans is central with respect to its natural range and S. ferrugineus is marginal, being most common in the Alps and in central and northern Europe. Molecular marker data show that the putative hybrid population is genetically intermediate between nearby populations of the parent species. Cytological evidence confirmed the hybrid nature of this population, as does the almost complete sterility of plants within the population. Although no seeds were produced by the hybrid population, some possibly fertile pollen grains were produced; this suggests that the possibility of introgression between the two species through the hybrids cannot completely be excluded.Key words: Schoenus, AFLP markers, chromosome behaviour, introgression.

An inhibin alpha-subunit cDNA sequence from the Australian brushtail possum (Trichosurus vulpecula) has been identified and analysed. The cDNA includes an open reading frame encoding a predicted precursor protein of 361 amino acids. The predicted protein sequence includes four possible proteolytic cleavage sites, 12 evolutionarily conserved cysteine residues and three potential N-linked glycosylation sites. The mature alpha-subunit is the carboxyl terminal fragment (alphaC) consisting of 131 amino acids. The full-length precursor protein shows a mean identity with eutherian homologues of 69.8%. The homology is not evenly distributed, with the putative alphaC fragment showing the highest level (79.7%). Using Northern hybridisation, an alpha-subunit transcript of approximately 1.6 kb was detected in adult possum ovary. Using in situ hybridisation and immunocytochemistry, inhibin alpha-subunit was localised exclusively to the granulosa cell layers of follicles. Hybridisation and immunostaining for the inhibin alpha-subunit were first observed in granulosa cells of primary follicles and the expression continued throughout all stages of follicular growth. Inhibin alpha-subunit mRNA and protein were also detected in cells of the corpus luteum. In summary, results indicate considerable conservation of the structure and possible function of the inhibin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages. The expression data suggest that, in the adult possum, inhibin may have a role in ovarian follicular growth from the primary stage of development.

The effect of cucumber mosaic virus (CMV) infection on the phloem sap mRNA population was investigated in pumpkin Cucurbita maxima Duch. cv. Big Max, through analysis of a suppressive subtractive hybridisation (SSH) library. Analysis of the infected phloem library identified 91 highly diverse mRNA species, including enzymes involved in general metabolism, transcription factors and signalling agents. Our analysis indicated that, quantitatively, the effect of CMV infection on the composition of the phloem sap transcriptome was minor in nature. Virtual northern analysis was used to confirm the specific upregulation of these transcripts in the phloem of CMV-infected plants. In silico northern analysis also confirmed that none of the transcripts identified in the SSH library was contained in the population of mRNA species present in the phloem sap of healthy plants. Induction levels ranged from low to high and in situ hybridisation studies showed that transcripts displayed a range of accumulation patterns. Collectively, our findings suggest that plants have evolved a highly robust mechanism for the exchange of information macromolecules between the companion cell (CC) and the sieve tube system. Production of viral movement protein (MP) in the CC is not sufficient for the indiscriminate transport of mRNA into the sieve element. Our findings are discussed in the context of symptom development and likely strong selection pressure, on the viral genome, to encode for a MP that does not adversely interfere with the phloem long-distance trafficking system.

Callus culture has been an inevitable step in genetic transformation of monocotyledonous (monocot) species. The induction and maintenance of embryogenic calluses is time-consuming, laborious and also requires experience. A straightforward and callus-free transformation procedure was developed and demonstrated for two monocot species, bermudagrass (Cynodon spp.) and creeping bentgrass (Agrostis stolonifera). Stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA or pTOK233 binary vectors. Green shoots were directly produced from infected stolon nodes 4–5 weeks after hygromycin selection. Without callus formation and with minimum tissue culture, this procedure allowed us to obtain well-rooted transgenic plantlets in only 7 weeks and greenhouse-grown plants in only 9 weeks. The established plants were screened by PCR; the transgenic nature of the plants was demonstrated by Southern hybridisation analysis. Expression of the transgenes was confirmed by northern hybridisation analysis and GUS staining. Based on the number of transgenic plants obtained and the number of stolon nodes inoculated, transformation frequencies of 4.8%–6.1% and 6.3%–11.3% were achieved for bermudagrass and creeping bentgrass, respectively. The rapid and efficient production of transgenic plants without callus induction is a significant improvement for genetic transformation of monocot species.

Objective:To highlight the potential role of molecular biological studies in examining the expression of genes of interest in brain tissue to elucidate the pathophysiological basis of the major psychoses. Method:To review the principles underlying the available techniques for expression studies. Results:Detection of messenger RNA by in situ hybridisation and quantitation by Northern analysis are powerful tools to detect abnormalities in gene expression in brain tissue. Conclusion:The availability of simple techniques to examine the expression of RNA and protein products of individual genes, including examination at the level of individual cells, offers a clear opportunity to define the molecular basis of the major psychoses.

Phylogenetic analyses of DNA-sequence data have revealed that the southern hemisphere species of Veronica are derived from within the northern hemisphere Veronica clade. Previous analyses focussed on the species in New Zealand and included at maximum 7 of 23 species of section Labiatoides from Australia. In the present study, we used nuclear ribosomal-ITS and plastid ndhF–rpl32-spacer sequence data of all species currently recognised in Australia to analyse phylogenetic patterns. Most importantly, herbaceous species from coastal calcareous sands or limestone habitats do not form a clade with those from shady, moist forest habitats, as formerly believed, but seem to be independently derived from woody species. Incongruence between results from nuclear- and plastid-DNA markers suggest hybridisation to be an important factor in the evolution of the group. Our sample of V. parnkalliana included alleles similar to V. decorosa and V. novae-hollandiae at both loci, which suggests a hybrid origin.

Background—bcl-2and bax belong to thebcl-2-related gene family, which marks a new class of genes that influence apoptosis. Thebcl-2 oncogene acts as a broad antiapoptotic factor and extends both normal and tumour cell survival. In contrast, the bax gene is a promoter of apoptosis.Aims—To analyse the expression of bcl-2 andbax in pancreatic cancer and correlate the results with clinical parameters.Patients—Pancreatic cancer tissue samples were obtained from 28 female and 32 male patients (median age 63, range 43–79 years) having surgery for pancreatic cancer. Normal pancreatic tissues obtained from 18 previously healthy organ donors served as controls.Methods—The levels ofbcl-2 and baxmRNA expression were analysed by northern blot and the exact site of mRNA transcription was determined by in situ hybridisation. The presence of the corresponding proteins was determined by immunohistochemistry.Results—Northern blot analysis indicated that, in comparison with the normal pancreas,bcl-2 mRNA was overexpressed in 30% andbax mRNA in 61% of the pancreatic cancer samples. Concomitant overexpression ofbcl-2 and bax was present in 26% of the cancer samples. Pancreatic adenocarcinomas exhibited 3.7-fold and 5.4-fold increases (p<0.001) inbcl-2 and baxmRNA levels respectively. In situ hybridisation showed that bothbcl-2 and baxmRNA were expressed in the cancer cells. Immunohistochemical analysis showed positive Bcl-2 and Bax immunostaining in 28 and 83% of the cancer samples respectively. In multivariate analysis (Cox regression model), bax expression was found to be a strong indicator of survival (p<0.001). Patients whose tumours exhibited Bax immunostaining lived significantly longer (12 months) than those whose tumours were Bax negative (five months) (p<0.039). In contrast, no relation was found between Bcl-2 and survival time.Conclusions—The data indicate that genes that are involved in the regulation of apoptosis are upregulated in human pancreatic cancer cells. Prolonged survival times in patients in whom apoptosis promoting factors are upregulated indicate that apoptotic pathways are of biological significance in pancreatic cancer.

The ground beetle Scarites laevigatus Fabricius, 1792 is a specialized predator occurring in the Mediterranean sandy shores, currently threatened with disappearance due to the widespread modification of beach ecosystems. The present study purposes a morphological analysis of the northern Adriatic form, described by Puel in 1938 as a subspecies with the name venetianus, in comparison with the typical form of this taxon and S. terricola. The examination of pronotum, elytra, body shape, male genitalia and wing development, suggests that the studied population is different from the nominotypical form and might originate from the introgressive hybridisation of S. terricola with the north Adriatic populations of S. laevigatus. This hypothesis is discussed in the light of current knowledge of the systematics and ecology of the putative parent species.

Cardiac hypertrophy defines an adaptive process brought about in response to sustained increases in haemodynamic work. Cardiomyocytes undergo an initial compensatory phase in which enlargement and contractility alterations normalise wall stress and maintain adequate perfusion of organs. In pathological hypertrophy, this deteriorates to a decompensated state characterised by ventricular dysfunction and predisposition to heart failure. In contrast, physiological hypertrophy and associated enhanced cardiac functioning arising from chronic exercise training does not progress to heart failure. Determination of the molecular pathways underlying myocardial hypertrophy remains a challenge for cardiovascular research. The objective of the work presented in this thesis was to identify genes differentially expressed during pathological and physiological hypertrophy in order to enhance our knowledge of the mechanistic processes involved. A reverse Northern hybridisation method was applied to profile the expression of specifically selected genes in the hypertrophic models examined. Functional categories represented in the gene panel assembled included cardiac contractile and cytoskeletal markers, matrix metalloproteinases, vasoactive pathway factors, calcium handling genes, ion channels, cardiac regulatory factors, signalling pathway intermediates, apoptotic factors and histone deacetylases. In order to investigate pathological hypertrophy, a deoxycorticosterone acetate-salt (DOCA-salt) rat model was utilised. DOCA-salt treated rats used in this study demonstrated a 1.4-fold increase in heart weight to body weight ratio compared to controls. Impaired cardiac function indicative of a decompensated pathological phenotype in the DOCA-salt treated group was demonstrated by way of decreased chamber size, impaired myocardial compliance and significantly reduced cardiac output. Reverse Northern hybridisation analysis of 95 selected genes identified a number of candidates with differential expression in hearts of DOCA-salt treated rats. Increased gene expression was demonstrated for the collagenase MMP1 and stress-activated signal transduction factor Sin1. In contrast, the sarcoplasmic reticulum calcium ATPase SERCA-2 and anti-apoptotic factor BCL2l-10 genes exhibited decreased expression. To investigate changes in gene expression associated with physiological hypertrophy, use was made of an endurance run-trained rat model. The run-trained rats used in this study demonstrated a 24.1% increase in heart weight to body weight ratio and improvements in performance consistent with physiological cardiac adaptation. These performance indicators included improvements in systolic volume, cardiac output, myocardial compliance and bio-energetic function. Reverse Northern hybridisation expression analysis of 56 genes identified a number of differentially expressed mRNA transcripts in run-trained hypertrophied hearts. Four genes shown to demonstrate reduced expression in the run-trained rat model were interleukin-1 receptor associated kinase (IRAK1) and the developmentally expressed transcription factors Nkx-2.3, dHAND, and IRX-2. Based upon the reverse Northern hybridisation results, four genes were selected for Western blotting analysis of rat cardiac tissue. Of these, MMP1 and a putative isoform of Sin1 exhibited increased levels in DOCA-salt treated hypertrophic left ventricular tissue, results that correlate with the findings of increased mRNA expression for these two genes. Therefore, this study identified MMP1 and Sin1 as candidates involved in pathological but not physiological hypertrophy. This finding is in accord with other recent investigations demonstrating that pathological hypertrophy and physiological hypertrophy are associated with distinct molecular phenotypes. An aside to the major objective of identifying genes differentially regulated in left ventricular hypertrophy involved the application of the P19CL6 cell in vitro model of cardiomyogenesis to compare protein expression during hypertrophy and development. The Sin1 isoform, found to be up-regulated during DOCA-salt induced hypertrophy, was also shown to be more abundant in differentiating, than non-differentiating, P19CL6 cells. This result is consistent with the developing paradigm that implicates 'fetal' genes in the hypertrophic remodelling process.

Cardiac hypertrophy defines an adaptive process brought about in response to sustained increases in haemodynamic work. Cardiomyocytes undergo an initial compensatory phase in which enlargement and contractility alterations normalise wall stress and maintain adequate perfusion of organs. In pathological hypertrophy, this deteriorates to a decompensated state characterised by ventricular dysfunction and predisposition to heart failure. In contrast, physiological hypertrophy and associated enhanced cardiac functioning arising from chronic exercise training does not progress to heart failure. Determination of the molecular pathways underlying myocardial hypertrophy remains a challenge for cardiovascular research. The objective of the work presented in this thesis was to identify genes differentially expressed during pathological and physiological hypertrophy in order to enhance our knowledge of the mechanistic processes involved. A reverse Northern hybridisation method was applied to profile the expression of specifically selected genes in the hypertrophic models examined. Functional categories represented in the gene panel assembled included cardiac contractile and cytoskeletal markers, matrix metalloproteinases, vasoactive pathway factors, calcium handling genes, ion channels, cardiac regulatory factors, signalling pathway intermediates, apoptotic factors and histone deacetylases. In order to investigate pathological hypertrophy, a deoxycorticosterone acetate-salt (DOCA-salt) rat model was utilised. DOCA-salt treated rats used in this study demonstrated a 1.4-fold increase in heart weight to body weight ratio compared to controls. Impaired cardiac function indicative of a decompensated pathological phenotype in the DOCA-salt treated group was demonstrated by way of decreased chamber size, impaired myocardial compliance and significantly reduced cardiac output. Reverse Northern hybridisation analysis of 95 selected genes identified a number of candidates with differential expression in hearts of DOCA-salt treated rats. Increased gene expression was demonstrated for the collagenase MMP1 and stress-activated signal transduction factor Sin1. In contrast, the sarcoplasmic reticulum calcium ATPase SERCA-2 and anti-apoptotic factor BCL2l-10 genes exhibited decreased expression. To investigate changes in gene expression associated with physiological hypertrophy, use was made of an endurance run-trained rat model. The run-trained rats used in this study demonstrated a 24.1% increase in heart weight to body weight ratio and improvements in performance consistent with physiological cardiac adaptation. These performance indicators included improvements in systolic volume, cardiac output, myocardial compliance and bio-energetic function. Reverse Northern hybridisation expression analysis of 56 genes identified a number of differentially expressed mRNA transcripts in run-trained hypertrophied hearts. Four genes shown to demonstrate reduced expression in the run-trained rat model were interleukin-1 receptor associated kinase (IRAK1) and the developmentally expressed transcription factors Nkx-2.3, dHAND, and IRX-2. Based upon the reverse Northern hybridisation results, four genes were selected for Western blotting analysis of rat cardiac tissue. Of these, MMP1 and a putative isoform of Sin1 exhibited increased levels in DOCA-salt treated hypertrophic left ventricular tissue, results that correlate with the findings of increased mRNA expression for these two genes. Therefore, this study identified MMP1 and Sin1 as candidates involved in pathological but not physiological hypertrophy. This finding is in accord with other recent investigations demonstrating that pathological hypertrophy and physiological hypertrophy are associated with distinct molecular phenotypes. An aside to the major objective of identifying genes differentially regulated in left ventricular hypertrophy involved the application of the P19CL6 cell in vitro model of cardiomyogenesis to compare protein expression during hypertrophy and development. The Sin1 isoform, found to be up-regulated during DOCA-salt induced hypertrophy, was also shown to be more abundant in differentiating, than non-differentiating, P19CL6 cells. This result is consistent with the developing paradigm that implicates 'fetal' genes in the hypertrophic remodelling process.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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