Hormona del crecimiento humana recombinante (HGHr) : biosíntesis,purificación y producción de anticuerpos monoclonales

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANLUANLhttp://www.uanl.mx

TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

Background: The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of gene