Building the sugarcane genome for biotechnology and identifying evolutionary trends.

BACKGROUND: Sugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome. RESULTS: Three hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences. CONCLUSION: This release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery.

Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.

Efeito da super-expressão do gene RECK na reversão do processo invasivo de glioma humano / RECK gene forced expression effect on the invasiveness in human glioma

Os gliomas são o tipo de tumor primário cerebral mais comum em adultos. A sobrevida média dos pacientes é de cerca de um ano para pacientes de glioblastoma multiforme, o maior grau de malignidade. As terapias disponíveis atualmente, que incluem cirurgia, radioterapia e quimioterapia, não têm sido eficientes devido a vários fatores, em particular a capacidade invasiva das células tumorais. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) é um importante gene supressor de tumor cuja atividade anti-tumoral têm sido associada à sua atividade inibitória sobre algumas MMPs. A perda da função de RECK compromete a integridade tecidual, em parte causada pela atividade aumentada das MMPs. A linhagem celular T98G, derivada de glioblastoma multiforme (GBM), apresenta grande capacidade invasiva e níveis elevados de MMP-2 e -9. A fim de avaliar o efeito de RECK na contenção da invasão no modelo de glioma humano, foi feita a superexpressão de RECK nesta linhagem. A expressão de RECK, MMP-2, MMP-9 a MT1-MMP foram avaliadas por qPCR e por western blotting nas células T98G/RECK+ (células T98G superexpressando RECK após transfecção estável utilizando o vetor pCXN2). O potencial invasivo e migratório das células T98G/RECK+foi inibido, verificado por ensaio transwell. Foram observadas nas células T98G/RECK+ alterações importantes no arranjo do citoesqueleto, mas não nas células controle. Arranjos de actina na forma de "stress fibers" presentes no clone positivo podem ser responsáveis pela alteração observada na migração. A distribuição de FAK foi avaliada por imunocitoquímica e sua expressão por Western blot, mostrando que RECK só altera a distribuição desta proteína no citoesqueleto celular. Quanto ao potencial de inibição de MMPs, observou-se uma diminuição significativa dos níveis gênicos de MMP-9, mas não em termos protéicos ou de atividade de MMPs. Assim, este trabalho contribui para a discussão do papel de RECK na migração de células do modelo de glioma...

Malignant gliomas are the most common type of primary brain tumors in adults. Patient survival is less than one year in average for glioblastoma, the most malignant glioma. Therapies available today, which include surgery, radiotherapy and chemotherapy, have not been successful due to several factors, specially the invasiveness of the tumor. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) is an important tumor suppressor gene whose anti-tumoral activity is associated to its anti-MMPs activity. Lack of functional RECK compromises tissue integrity, in part due to elevated MMPs activity. T98G cells, derived from a human multiform glioblastoma (GBM), were described as a highly invasive glioma cell line, which displays high levels of MMPs 2 and 9. In order to evaluate the effect of RECK in restraining glioma invasion, we overexpressed RECK in these invasive cell line derived from GBM. The expression of RECK, MMP-2, MMP-9 and MT1-MMP was evaluated by qPCR and by western blotting in T98G/RECK+ (T98G cells overexpressing RECK cells, where RECK gene was cloned into the pCXN2 vector generating a stable transfection). The invasion and migration capacity of T98G/RECK+ cells was inhibited in transwell assay. Important cytoskeleton modifications were also observed in T98G/RECK+ cells but not on the control cells. Actin arrangements representing stress fibers on the positive clones may be responsible for motility alteration patterns observed. FAK distribution was assessed through imunocytochemical staining, and its expression evaluated by western blot analyses, showing that RECK forced expression changed the distribution pattern but not FAK expression. Concerning MMPs inhibition, a significant inhibition of MMP-9 gene expression was observed in T98G/RECK+, but neither protein levels nor protein activity were affected. Thus, the present study improves the discussion about RECK role in the migration of glioma cells, an important feature for the failure of...