Caracterização funcional de dois cDNAs de cana-de- açúcar : PKCI e SHAGGY

Flowering is controlled by several environmental and endogenous factors, usually associated with a complex network of metabolic mechanisms. The gene characterization in Arabidopsis model has provided much information about the genetic and molecular mechanisms that control flowering process. Some of these genes had been found in rice and maize. However, in sugarcane this processe is not well known. It is known that early flowering may reduce its production up to 60% at northeast conditions. Considering the impact of early flowering in sugarcane production, the aim of this work was to make the gene characterization of two cDNAs previously identified in subtractive cDNA libraries: scPKCI and scSHAGGY. The in silico analysis showed that these two cDNAs presented both their sequence and functional catalytic domains conserved. The results of transgenic plants containing the overexpression of the gene cassette scPKCI in sense orientation showed that this construction had a negative influence on the plant development as it was observed a decrease in plant height and leaf size. For the scPKCI overexpression in antisense orientation it was observed change in the number of branches from T1 transgenic plants, whereas transgenic T2 plants showed slow development during germination and initial stages of development. The other cDNA analyzed had homology to SHAGGY protein. The overexpression construct in sense orientation did not shown any effect on development. The only difference observed it was an increase in stigma structure. These results allowed us to propose a model how these two genes may be interact and affect floweringdevelopment.

Cloning and characterisation of the hint homologue of the Thermophile Thermus thermophilus

Screening of a genomic library of the thermophile Thermus thermophilus revealed a novel thermophilic hint gene, homologues of which are highly conserved in genera from archaea to mammals. Hint belongs to the HIT protein super-family, which contains two broad groups, Fhit, associated with tumour suppression in eukaryotes and Hint with putatitive protein kinase C inhibitory activity. In T. thermophilus the 321bp gene has a GC content of 67% overall and 94.4% in the third nucleotide position, with unusually no thymine as a wobble base. The gene product, a small highly conserved 11996Da predicted soluble cytoplasmic protein, offers an ideal opportunity to investigate thermostabilising amino acid substitutions. Here we report on the characterisation of the novel hint sequence.

Pkc1 acts through Zds1 and Gic1 to suppress growth and cell polarity defects of a yeast eIF5A mutant

eIF5A is a highly conserved putative eukaryotic translation initiation factor that has been implicated in translation initiation, nucleocytoplasmic transport, mRNA decay, and cell proliferation, but with no precise function assigned so far. We have previously shown that high-copy PKCI suppresses the phenotype of tif51A-1, a temperature-sensitive mutant of eIF5A in S. cerevisiae. Here, in an attempt to further understand how Pkc1 functionally interacts with eIF-5A, it was determined that PKCI suppression of tif51A-1 is independent of the cell integrity MAP kinase cascade. Furthermore, two new suppressor genes, ZDS1 and GIC1, were identified. We demonstrated that ZDS1 and ZDS2 are necessary for PKC1, but not for GIC1 suppression. Moreover, high-copy GIC1 also suppresses the growth defect of a PKCI mutant (stt1), suggesting the existence of a Pkc1-Zds1-Gic1 pathway. Consistent with the function of Gic1 in actin organization, the tif51A-1 strain shows an actin polarity defect that is partially recovered by overexpression of Pkc1 and Zds1 as well as Gic1. Additionally, PCL1 and BNI1, important regulators of yeast cell polarity, also suppress tif51A-1 temperature sensitiviiy Taken together, these data strongly Support the correlated involvement of Pkc1 and eIF5A in establishing actin polarity, which is essential for bud formation and G1/S transition in S. cerevisiae.

Floração precoce em cana-de-açúcar - um estudo utilizando ferramentas de análise in silico e proteômica

Sugarcane is one of the most important products of the world and Brazil is responsible for 25 % of the world production. One problem of this culture at northeast of Brazil is the early flowering. In our laboratory, it has been made before four subtractive libraries using early and late flowering genotypes in order to identify messages related to the flowering process. In this work, two cDNAs were chosen to make in silico analysis and overexpression constructs. Another approach to understand the flowering process in sugarcane was to use proteomic tools. First, the protocol for protein extraction using apical meristem was set up. After that, these proteins were separated on two bidimensional gels. It was possible to observe some difference for some regions of these gels as well as some proteins that can be found in all conditions. The next step, spots will be isolated and sequence on MS spectrometry in order to understand this physiological process in sugarcane

Differential expression of HINT1 in schizophrenia brain tissue

Recent findings in the literature suggest a relation between histidine triad nucleotide-binding protein-1 (HINT1) and psychiatric disorders such as major depression, anxiety, and schizophrenia, although its physiological roles are not completely comprehended. Using Western blot, we compared HINT1 protein expression in the postmortem dorsolateral prefrontal cortex and thalamus of schizophrenia patients and healthy controls for contributing to elucidate the role of HINT1 in schizophrenia pathophysiology. HINT1 was found to be downregulated in the dorsolateral prefrontal cortex and upregulated in the thalamus. Our results combined to previous studies in human samples and preclinical models support the notion that HINT1 must be more explored as a potential target for psychiatric disorders.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.