Cloning and expression of HIV-1 p24 gene in insect cells by using BAC-TO-BAC system

Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection. Supported by the World Bank Boan Program Mallam Nock Joshua: born in 1967, Master of Science To whom correspondence should be addressed (027-7882712-2938)     

Vernalization requires epigenetic silencing of FLC by histone methylation

To ensure flowering in favourable conditions, many plants flower only after an extended period of cold, namely winter. In Arabidopsis, the acceleration of flowering by prolonged cold, a process called vernalization, involves downregulation of the protein FLC, which would otherwise prevent flowering. This lowered FLC expression is maintained through subsequent development by the activity of VERNALIZATION (VRN) genes. VRN1 encodes a DNA-binding protein whereas VRN2 encodes a homologue of one of the Polycomb group proteins, which maintain the silencing of genes during animal development. Here we show that vernalization causes changes in histone methylation in discrete domains within the FLC locus, increasing dimethylation of lysines 9 and 27 on histone H3. Such modifications identify silenced chromatin states in Drosophila and human cells. Dimethylation of H3 K27 was lost only in vrn2 mutants, but dimethylation of H3 K9 was absent from both vrn1 and vrn2, consistent with VRN1 functioning downstream of VRN2. The epigenetic memory of winter is thus mediated by a 'histone code' that specifies a silent chromatin state conserved between animals and plants.