Structural analysis of a novel class of R-M controller proteins: C.Csp231I from Citrobacter sp. RFL231

Controller proteins play a key role in the temporal regulation of gene expression in bacterial restriction-modification (R-M) systems and are important mediators of horizontal gene transfer. They form the basis of a highly cooperative, concentration-dependent genetic switch involved in both activation and repression of R-M genes. Here we present biophysical, biochemical, and high-resolution structural analysis of a novel class of controller proteins, exemplified by C.Csp231I. In contrast to all previously solved C-protein structures, each protein subunit has two extra helices at the C-terminus, which play a large part in maintaining the dimer interface. The DNA binding site of the protein is also novel, having largely AAAA tracts between the palindromic recognition half-sites, suggesting tight bending of the DNA. The protein structure shows an unusual positively charged surface that could form the basis for wrapping the DNA completely around the C-protein dimer.     

Meiosis-specific yeast Hop1 protein promotes pairing of double-stranded DNA helices via G/C isochores

In eukaryotes, genetic exchange between homologs is facilitated by a tripartite proteinaceous structure called the synaptonemal complex (SC). Several lines of evidence indicate that the genes that encode components of SC are essential for meiotic chromosome pairing and recombination. However, the molecular mechanism by which SC proteins promote these processes is obscure. Here, we report that Saccharomyces cerevisiae Hop1 protein, a component of SC, promotes pairing between two double-stranded DNA helices containing a centrally located G/C isochore. Significantly, pairing was rapid and robust, and required four contiguous G/C base pairs. Using a series of truncated DNA double helices we show that 20 bp on either side of 8 bp target G/C sequence is essential for pairing. To our knowledge, Hop1 is the first protein shown to do so from yeast or any other organism. These results indicate that Hop1 protein is likely to play a direct role in meiotic chromosome pairing and recombination.     

Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry

In the double-stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid-bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N-terminal helix-loop-helix (H-L-H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid-bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.     

A Full-Length Dof1 Transcription Factor of Finger Millet and its Response to a Circadian Cycle

DOF1 (DNA binding with one finger) plays an important role in regulating C/N metabolism in cereals. In order to validate its role in the regulation of nitrogen use efficiency (NUE) and photosynthetic efficiency in finger millet, 5′–3′ RACE PCR was performed to obtain and characterize full-length Dof1 genes of high and low grain protein finger millet genotypes. The full-length DOF1 ORFs were both 1,284 nt long and were 98.8 % similar over 427 amino acids containing the characteristic Dof domain. Comparison of both the EcDof1 protein sequences with the Dof1 of other cereals revealed high sequence similarity to the Dof1 of rice. Southern hybridization carried out using the probe developed from the region encoding the highly variable C-terminal region of EcDof1 showed the presence of four copies of the DOF1 gene in finger millet, which might explain the high NUE and photosynthetic performance of finger millet. Since the genes involved in C/N metabolism are regulated diurnally and play crucial roles in determining grain protein content during grain filling, the diurnal expression of EcDOF1 was assessed in two finger millet genotypes (GE 3885 and GE 1437) with differing grain protein content (13.8 % and 6.15 % respectively). It was found that EcDOF1 exhibited diurnal regulation and peak differential pattern expression with early phasing in GE3885 and late phasing in GE1437. Differential expression of DOF1 might alter the regulation of genes involved in C/N metabolism affecting grain protein composition of finger millet genotypes.     

茶树DELLA基因家族的鉴定及表达分析

利用生物信息学方法,从茶树（Camellia sinensis（L.）O.Ktze.）全基因组数据库中分析获得DELLA蛋白的家族成员,并对它们的系统进化关系、蛋白序列特征、基因表达特异性及其与茶树次生代谢物的相关性进行分析。结果显示：茶树基因组中共有5个DELLA基因,分别为：TEA009882（CsGAI）、TEA022818（CsRGA1）、TEA010112（CsRGL1）、TEA008736（CsRGL2）和TEA020933（CsRGL3）;其编码的氨基酸数量在525~594之间,均定位于细胞核。该蛋白的二级和三级结构分析结果表明,茶树DELLA蛋白结构中含有大量的α螺旋及少量β转角结构。蛋白保守结构域分析结果显示该蛋白与拟南芥（Arabidopsis thaliana（L.）Heynh.）具有高度的同源性,均具有GRAS、DELLA等保守结构域。基因的表达特异性分析结果表明,在茶树不同组织部位中,TEA009882、TEA022818和TEA010112基因的表达量均较高,而TEA020933和TEA008736的表达量在各组织中均较低;茶树DELLA基因的表达受到干旱、NaCl、低温及茉莉酸甲酯等非生物逆境胁迫的调控,且其表达量与茶树次生代谢物的积累间存在相关性。推测茶树DELLA基因广泛参与了茶树生长发育及非生物逆境胁迫的响应,以及对次生代谢物生物合成过程的调控。     

Promotion of mitosis by activated protein kinase B after DNA damage involves polo-like kinase 1 and checkpoint protein CHFR

The role of the protein kinase B (PKB/Akt) in the regulation of cell survival and proliferation is well established. PKB is a key effector in the phosphatidylinositol 3-kinase pathway and plays a role in the initiation of S phase and in the G(2)-M transition. I report here that activated PKB shortens the G(2) arrest induced by DNA damage and promotes early entry into mitosis. Activated PKB supports high levels of expression and activity of the polo-like kinase 1 (Plk1) after DNA damage as cells accumulate in G(2). The checkpoint protein CHFR implicated in degradation of Plk1 is involved in the regulation of Plk1 by PKB. PKB phosphorylates CHFR in vitro and in vivo. Expression of a mutant form of CHFR that cannot be phosphorylated by PKB results in reduction of levels of Plk1 and inhibition of mitotic entry under normal conditions and after DNA damage. Results of this study support a model in which PKB facilitates mitotic resolution of DNA damage-induced G(2) arrest by inhibiting the checkpoint function of CHFR. The deregulated activation of PKB that occurs frequently in tumors might inhibit CHFR activity after DNA damage and therefore promote Plk1 accumulation leading to the disruption of the DNA damage checkpoint.     

GADD45A protein plays an essential role in active DNA demethylation during terminal osteogenic differentiation of adipose-derived mesenchymal stem cells

Methylation and demethylation of DNA are the complementary processes of epigenetic regulation. These two types of regulation influence a diverse array of cellular activities, including the maintenance of pluripotency and self-renewal in embryonic stem cells. It was generally believed that DNA demethylation occurs passively over several cycles of DNA replication and that active DNA demethylation is rare. Recently, evidence for active DNA demethylation has been obtained in several cancer, neuronal, and embryonic stem cell lines. Studies in embryonic stem cell models, however, suggested that active DNA demethylation might be restricted to the early development of progenitor cells. Whether active demethylation is involved in terminal differentiation of adult stem cells is poorly understood. We provide evidence that active DNA demethylation does occur during terminal specification of stem cells in an adipose-derived mesenchymal stem cell-derived osteogenic differentiation model. The medium CpG regions in promoters of the Dlx5, Runx2, Bglap, and Osterix osteogenic lineage-specific genes were demethylated during the increase in gene expression associated with osteogenic differentiation. The growth arrest and DNA damage-inducible protein GADD45A was up-regulated in these processes. Knockdown of GADD45A led to hypermethylation of Dlx5, Runx2, Bglap, and Osterix promoters, followed by suppression of the expression of these genes and interruption of osteogenic differentiation. These results reveal that GADD45A plays an essential role in gene-specific active DNA demethylation during adult stem cell differentiation. They enhance the current knowledge of osteogenic specification and may also lead to a better understanding of the common mechanisms underlying epigenetic regulation in adult stem cell differentiation.     

Reproductive barrier and genomic imprinting in the endosperm of flowering plants

In flowering plants, success or failure of seed development is determined by various genetic mechanisms. During sexual reproduction, double fertilization produces the embryo and endosperm, which both contain maternally and paternally derived genomes. In endosperm, a reproductive barrier is often observed in inter-specific crosses. Endosperm is a tissue that provides nourishment for the embryo within the seed, in a similar fashion to the placenta of mammals, and for the young seedling after germination. This review considers the relationship between the reproductive barrier in endosperm and genomic imprinting. Genomic imprinting is an epigenetic mechanism that results in mono-allelic gene expression that is parent-of-origin dependent. In Arabidopsis, recent studies of several imprinted gene loci have identified the epigenetic mechanisms that determine genomic imprinting. A crucial feature of genomic imprinting is that the maternally and paternally derived imprinted genes must carry some form of differential mark, usually DNA methylation and/or histone modification. Although the epigenetic marks should be complementary on maternally and paternally imprinted genes within a single species, it is possible that neither the patterns of epigenetic marks nor expression of imprinted genes are the same in different species. Moreover, in hybrid endosperm, the regulation of expression of imprinted genes can be affected by upstream regulatory mechanisms in the male and female gametophytes. Species-specific variations in epigenetic marks, the copy number of imprinted genes, and the epigenetic regulation of imprinted genes in hybrids might all play a role in the reproductive barriers observed in the endosperm of interspecific and interploidy crosses. These predicted molecular mechanisms might be related to earlier models such as the "endosperm balance number" (EBN) and "polar nuclei activation" (PNA) hypotheses.