Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Screening for glycosylphosphatidylinositol-anchored proteins in the Paracoccidioides brasiliensis transcriptome

Open reading frames in the transcriptome of Paracoccidioides brasiliensis were screened for potential glycosylphosphatidylinositol (GPI)-anchored proteins, which are a functionally and structurally diverse family of post-translationally modified molecules found in a variety of eukaryotic cells. Numerous studies have demonstrated that various GPI anchor sequences can affect the localization of these proteins in the plasma membrane or the cell wall. The GPI anchor core is produced in the endoplasmic reticulum by sequential addition of monosaccharides and phospho-ethanolamine to phosphatidylinositol. The complete GPI anchor is post-translationally attached to the protein carboxyl-terminus by GPI transamidases. Removal of this GPI lipid moiety by phospholipases generates a soluble form of the protein. The identification of putative GPI-attached proteins in the P. brasiliensis transcriptome was based on the following criteria: the presence of an N-terminal signal peptide for secretion and a hydrophobic region in the C-terminus presenting the GPI-attachment site. The proteins that were identified were in several functional categories: i) eight proteins were predicted to be enzymes (Gel1, Gel2, Gel3, alpha-amylase, aspartic proteinase, Cu-Zn SOD, DFG5, PLB); ii) Ag2/PRA, ELI-Ag1 and Gel1 are probably surface antigens; iii) Crh-like and the GPI-anchored cell wall protein have a putative structural role; iv) ECM33 and Gels (1, 2 and 3) are possibly involved in cell wall biosynthesis, and v) extracellular matrix protein is considered to be an adhesion protein. In addition, eight deduced proteins were predicted to localize in the plasma membrane and six in the cell wall. We also identified proteins involved in the synthesis, attachment and cleaving of the GPI anchor in the P. brasiliensis transcriptome.

The cell wall of Paracoccidioides brasiliensis: insights from its transcriptome

The cell wall of a human pathogenic fungus is in contact with the host, serves as a barrier against host defense mechanisms and harbors most fungal antigens. In addition, cell wall biosynthesis pathways have been recognized as essential to viability and as specific drug targets. Paracoccidioides brasiliensis is a dimorphic fungus that presents mycelium morphology in the free environment and causes infection in a yeast form. The morphogenetic conversion is correlated with changes in the cell wall composition, organization and structure. Based on transcriptome analysis, the enzymes involved in the biosynthesis and remodeling of cell wall polysaccharides, as well as several cell wall-associated molecules of P. brasiliensis, were identified and addressed in further detail.

Evoluçäo da atividade e expressäo de enzimas e modificaçäo de polímeros da parede celular de mamöes durante o amadurecimento / Evolution of the activity and enzyme expression and modification of the cellular wall of papayas during the ripening

Foram analisadas expressão dos genes e atividades enzimáticas da pectinametilesterase, beta-galactosidase e poligalacturonase, bem como modificação in situ de polímeros da parede celular de frutos de mamão em diversos estádios de amadurecimento. Após a clonagem e sequenciamento de fragmentos do gene para cada enzima sua expressão foi analisada por Northern blot. A imunolocalização dos polímeros da parede foi feita por microscopia eletrônica. De acordo com os resultados apresentados, é possível evidenciar indução da transcrição do gene para a beta-galactosidase, presença constante de transcrito para a poligalcaturonase e ausência de evidências da expressão do gene para pectinametilesterase...

Cloning and expression of a cell wall associated protein gene of Mycobacterium tuberculosis.

Genomic library of Mycobacterium tuberculosis (SIHV) was constructed into T7 promoter based vector pBluescript II KS (+/-) in Escherichia coil. The expression of mycobacterial antigens from recombinants was detected by immunoscreening using E-coli-preabsorbed hyperimmune rabbit anti-M tuberculosis cell wall associated proteins serum. Among 980 clones tested by immunoscreening, a clone pJJ5 was found to produce mycobacterial antigen. This clone was further characterized by immunoblotting and found to produce a 36 kDa of cell wall associated antigen Detection of antibodies response to this antigen in the serum of the majority of the individuals with tuberculosis indicates that this antigen is capable of stimulating a humoral immune response.