The Mechanism of Oocyte Activation Influences the Cell Cycle-Related Genes Expression During Bovine Preimplantation Development

The first cleavage divisions and preimplantation embryonic development are supported by mRNA and proteins synthesized and stored during oogenesis. Thus, mRNA molecules of maternal origin decrease and embryonic development becomes gradually dependent on expression of genetic information derived from the embryonic genome. However, it is still unclear what the role of the sperm cell is during this phase and whether the absence of the sperm cell during the artificial oocyte activation affects subsequent embryonic development. The objective of this study was to determine, in bovine embryos, changes in cell cycle-associated transcript levels (cyclin A, cyclin B, cyclin E, CDC2, CDK2, and CDK4) after oocyte activation in the presence or absence of the sperm cell. To evaluate that, in vitro-produced (IVP) and parthenogenetically activated (PA) embryos (2-4 cells (2-4C), 8-16 cells (8-16C) and blastocysts) were evaluated by real-time PCR. There was no difference in cleavage and blastocyst rates between IVP and PA groups. Transcript level was higher in oocytes than in IVP and PA embryos. Cleaved PA embryos showed higher expression of cyclin A, cyclin B, cyclin E, and CDK2 and lower expression of CDC2 when compared with that from the IVP group. At the time of activation, all transcripts were expressed less in PA than in IVP embryos, whereas at the blastocyst stage, almost all genes were expressed at a higher level in the PA group. These results suggest that in both groups there is an initial consumption of these transcripts in the early stages of embryonic development. Furthermore, 8-16C embryos seem to synthesize more cell cycle-related genes than 2-4C embryos. However, in PA embryos, activation of the cell cycle genes seems to occur after the 8- to 16-cell stage, suggesting a failure in the activation process.

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous gamma H2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the alpha-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-alpha primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.

Characterization of anti-silencing factor 1 in Leishmania major

Anti-silencing factor 1 (ASF1) is a histone chaperone that contributes to the histone deposition during nucleosome assembly in newly replicated DNA. It is involved in chromatin disassembly, transcription activation and in the cellular response to DNA damage. In Leishmania major the ASF1 gene (LmASF1) is located in chromosome 20 and codes for a protein showing 67% of identity with the Trypanosoma brucei TbASF1a. Compared to orthologous proteins, LmASF1 conserves the main residues relevant for its various biological functions. To study ASF1 in Leishmania we generated a mutant overexpressing LmASF1 in L. major. We observed that the excess of LmASF1 impaired promastigotes growth rates and had no impact on cell cycle progress. Differently from yeast, ASF1 overproduction in Leishmania did not affect expression levels of genes located on telomeres, but led to an upregulation of proteins involved in chromatin remodelling and physiological stress, such as heat shock proteins, oxidoreductase activity and proteolysis. In addition, we observed that LmASF1 mutant is more susceptible to the DNA damaging agent, methyl methane sulphonate, than the control line. Therefore, our study suggests that ASF1 from Leishmania pertains to the chromatin remodelling machinery of the parasite and acts on its response to DNA damage.

Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes

mitochondrial genomes are generally thought to be under selection for compactness, due to their small size, consistent gene content, and a lack of introns or intergenic spacers. As more animal mitochondrial genomes are fully sequenced, rearrangements and partial duplications are being identified with increasing frequency, particularly in birds (Class Ayes). In this study, we investigate the evolutionary history of mitochondrial control region states within the avian order Psittaciformes (parrots and cockatoos). To this aim, we reconstructed a comprehensive multi-locus phylogeny of parrots, used PCR of three diagnostic fragments to classify the mitochondrial control region state as single or duplicated, and mapped these states onto the phylogeny. We further sequenced 44 selected species to validate these inferences of control region state. Ancestral state reconstruction using a range of weighting schemes identified six independent origins of mitochondrial control region duplications within Psittaciformes. Analysis of sequence data showed that varying levels of mitochondrial gene and tRNA homology and degradation were present within a given clade exhibiting duplications. Levels of divergence between control regions within an individual varied from 0-10.9% with the differences occurring mainly between 51 and 225 nucleotides 3' of the goose hairpin in domain I. Further investigations into the fates of duplicated mitochondrial genes, the potential costs and benefits of having a second control region, and the complex relationship between evolutionary rates, selection, and time since duplication are needed to fully explain these patterns in the mitochondrial genome. (C) 2012 Elsevier Inc. All rights reserved.

Modularity and evolutionary constraints in a baculovirus gene regulatory network

Abstract Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates that modularity may be a general feature of biological gene regulatory networks.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart. Oncogene (2012) 31, 4245-4254; doi:10.1038/onc.2011.586; published online 9 January 2012

Broad and Cross-Clade CD4(+) T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-gamma secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-gamma and TNF-alpha, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology

We have prepared a DNA-mimicry of nucleosides in which the anti-HIV drug lamivudine (beta-L-2',3'-dideoxy-3'-thiacytidine, 3TC) self-assembles into a base-paired and helically base-stacked hexagonal structure. Face-to-face and face-to-tail stacked 3TC=3TC dimers base-paired through two hydrogen bonds between neutral cytosines by either N-H center dot center dot center dot O or N-H center dot center dot center dot N atoms give rise to a right-handed DNA-mimicry of lamivudine with an unusual highly symmetric hexagonal lattice and topology. In addition, a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate was also obtained as a result of our crystal screenings. This structure is formed through partially face-to-face stacked lamivudine pairs held together by protonated and neutral fragments. However, no helical stacking occurs in this structure in which lamivudine also adopts unusual conformations as the C1'-endo and C1'-exo sugar puckers and cytosine orientations intermediate between the anti and syn conformations. As a conclusion drawn from the nucleoside duplex, the hexagonal DNA-mimicry of lamivudine reveals that such double-stranded helices can be assembled without counterions and organic solvents but with higher crystallographic symmetry instead, because only water crystallizes together with lamivudine in this structure.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

HIV-1 Proviral DNA Loads (as Determined by Quantitative PCR) in Patients Subjected to Structured Treatment Interruption after Antiretroviral Therapy Failure

The impact of Structured Treatment Interruption (STI) in peripheral blood mononuclear cell (PBMC) proviral reservoirs in 41 highly active antiretroviral therapy (HAART)-treated viremic individuals at baseline and 12 weeks after STI was determined using quantitative PCR (qPCR). Viral load increased 0.7 log(10) and CD4 decreased 97.5 cells/mm(3) after 12 weeks. A total of 28 of the 41 individuals showed an increased proviral load, 19 with a statistically significant increase above 10%. An increase in active viral replication is an important factor in the replenishment of the proviral reservoir even for short time periods.