Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.     

Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of ∼ 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.     

Does life originate from a single molecule?

Life did not emerge in a single step. In chemical evolution, the first formation of a self-replicating molecule was probably one of the most critical bottlenecks, which was overcome only with a very low probability. If only one such event was successful, present-day life originates from a single molecule. In this case, homochirality in DNA and RNA is explained almost without further assumptions. By contrast, the enantiomer excess, produced by the deterministic mechanisms suggested so far, is smaller than the statistical standard deviation, unless the postulated initial number of molecules is very--in some mechanisms unreasonably--large. A certain chiral nonuniformity of natural monosaccharides other than (deoxy)ribose supports the idea that homochirality originates not from such small molecules but from an early RNA-like oligomer. This nonuniformity seems also hard to explain by any deterministic mechanism.     

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.     

C. elegans MCM-4 is a general DNA replication and checkpoint component with an epidermis-specific requirement for growth and viability

DNA replication and its connection to M phase restraint are studied extensively at the level of single cells but rarely in the context of a developing animal. C. elegans lin-6 mutants lack DNA synthesis in postembryonic somatic cell lineages, while entry into mitosis continues. These mutants grow slowly and either die during larval development or develop into sterile adults. We found that lin-6 corresponds to mcm-4 and encodes an evolutionarily conserved component of the MCM2-7 pre-RC and replicative helicase complex. The MCM-4 protein is expressed in all dividing cells during embryonic and postembryonic development and associates with chromatin in late anaphase. Induction of cell cycle entry and differentiation continues in developing mcm-4 larvae, even in cells that went through abortive division. In contrast to somatic cells in mcm-4 mutants, the gonad continues DNA replication and cell division until late larval development. Expression of MCM-4 in the epidermis (also known as hypodermis) is sufficient to rescue the growth retardation and lethality of mcm-4 mutants. While the somatic gonad and germline show substantial ability to cope with lack of zygotic mcm-4 function, mcm-4 is specifically required in the epidermis for growth and survival of the whole organism. Thus, C. elegans mcm-4 has conserved functions in DNA replication and replication checkpoint control but also shows unexpected tissue-specific requirements.     

Biochemical significance of enhanced activity of fluorinated 1,25-dihydroxyvitamin D3 in human cultured cell lines

Several human cancer cells possess receptors for 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]. In these cells 1,25-(OH)2D3 has a biphasic concentration-dependent regulatory effect on cell replication and specifically induces its own metabolism. We have studied the effects on these parameters of the native hormone together with those of two analogues fluorinated at the 24-carbon and of 1,24R,25-trihydroxyvitamin D3[1,24R,25-(OH)3D3]. The difluorinated analogue 24,24-difluoro-1,25-(OH)2D3[24,24-F2-1,25-(OH)2D3] is an approximately fivefold more potent inhibitor of cellular replication than the native hormone, while 1,24R,25-(OH)3D3 is about fivefold less potent. This enhanced potency of the fluorinated analogue parallels its enhanced potency in in vivo studies of its effects on calcium and mineral metabolism. However, although the analogue retains replication stimulatory activity, it is clearly no more potent than the native hormone in this activity: 1,24R,25-(OH)3D3 has no significant stimulatory activity. Exposure of the cells to 1,25-(OH)2D3 at 0.05 nM for 6 h increases the subsequent conversion of labelled hormone to aqueous phase soluble compounds by 6.7-fold. None of the other compounds had a similar effect at this concentration. At 10 nM all 1-hydroxylated compounds increased aqueous phase radioactivity about equally (13 to 17-fold); this effect is still specific since 25-OH D3 had no such effect even at 10 nM. Studies on the effects of the fluorinated analogues upon receptor binding of hormone in cell cytosols and uptake of hormone by intact cells clearly demonstrate that the enhanced activity of these analogues is not due to higher receptor affinity or more rapid access to intracellular receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.