The Kinetochore-Microtubule Coupling Machinery Is Repurposed in Sensory Nervous System Morphogenesis

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Endothelial specific granules in the umbilical veins of the postnatal rabbit

Summary A remarkable increase in number of endothelial specific granules was observed in the rabbit umbilical veins between 2 and 5 days after birth. Electron microscopy indicated that the granules were segregated in the Golgi complex of the endothelial cells and released into the vascular lumen during the postnatal obliteration stage of this vessel.Incubation of the postnatal vessels in Ringer solution containing a histamine releasing compound induced remarkable morphological alterations of these cytoplasmic components; a reduction of their osmiophilia, swelling with a widened space separating the granular matrix from the limiting membrane, fusion to each other and expulsion of their contents into the vascular lumen, as in mast cell degranulation by this drug, were noted.High-performance liquid chromatography of the homogenized vessels demonstrated appreciable concentrations of histamine in the postnatal samples. There was a correlation between the histamine concentration and the quantity of granules in the respective postnatal samples.The present study strongly suggests that the granules are reservoirs of histamine and have an important role in the obliteration of this vessel.This work was supported in part by Grant in Aid for Scientific Research (# 448087) to S. Fujimoto from the Ministry of Education of Japan     

遗传标记与数量性状基因间连锁关系的分析

本文讨论标记基因与数量性状主基因连锁关系的一般分析方法,包括重组值的估计和有关遗传假设的测验。并以我们水稻遗传试验中两个具有互补和重叠作用的卷叶基因和一个矮秆基因试验结果的分析为例作了较详细的示范。     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways

In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.     

RAP80 interacts with the SUMO-conjugating enzyme UBC9 and is a novel target for sumoylation

RAP80, a nuclear protein with two functional ubiquitin-interaction motifs (UIMs) at its N-terminus, plays a critical role in the regulation of estrogen receptor alpha and DNA damage response signaling. A yeast two-hybrid screen identified the SUMO-conjugating enzyme UBC9 as a protein interacting with RAP80. The interaction of RAP80 with UBC9 was confirmed by co-immunoprecipitation and GST pull-down analyses. The region between aa 122-204 was critical for the interaction of RAP80 with UBC9. In addition, we demonstrate that RAP80 is a target for SUMO-1 modification in intact cells. Expression of UBC9 enhanced RAP80 mono-sumoylation and also induced multi-sumoylation of RAP80. In addition to SUMO-1, RAP80 was efficiently conjugated to SUMO-3 but was only a weak substrate for SUMO-2 conjugation. These findings suggest that sumoylation plays a role in the regulation of RAP80 functions.     

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness¹. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)^2,3 and a C-terminal 10xHis tag⁴, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression⁵. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme⁶. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl₂ and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.     

Empirical evidence for key hosts in persistence of a tick-borne disease

An important epidemiological consequence of aggregated host-parasite associations occurs when parasites are vectors of pathogens. Those hosts that attract many vectors will tend to be the focus of transmission. But to what extent, and can we identify characteristics of these key hosts? We investigated these questions with respect to the host-tick relationship of the yellow-necked mouse, Apodemus flavicollis, a critical host in the maintenance of the zoonotic disease, tick-borne encephalitis. Transmission of the virus occurs when ticks feed in a 'co-feeding' aggregation. Thus, the number and frequency of co-feeding groups provides an estimate of the potential rate of virus transmission. We recorded the spatio-temporal variations in co-feeding on a population of rodents in conjunction with recording individual host characteristics. Using Lorenz curves, we revealed conformation of tick-borne encephalitis transmission potential to the 20/80 Rule, where the 20% of hosts most infested with ticks were accountable for 80% of transmission potential. Hosts in the transmission cohort were identified as the sexually mature males of high body mass. Therefore control efforts targeted at this group would substantially reduce transmission potential compared to non-targeted control of the population, which resulted in a linear reduction in transmission potential. Focusing on the 'wrong' functional group would have little impact upon transmission potential until a considerable proportion of the population had been subject to control. However, individuals can change their functional status over time making it difficult to predict the contribution of these individuals to future transmission.     

INO80 participates in the pathogenesis of recurrent miscarriage by epigenetically regulating trophoblast migration and invasion

The INO80 complex, a SWI/SNF family chromatin remodeler, has regulatory effects on ESC self-renewal, somatic cell reprogramming and blastocyst development. However, the role of INO80 in regulating trophoblast cells and recurrent miscarriage (RM) remains elusive. To investigate the in vivo effects of Ino80 in embryo development, we disrupted Ino80 in C57 mice, which resulted in embryonic lethality. Silencing of Ino80 led to decreased survival capacity, migration and invasion of trophoblasts. Furthermore, RNA high-throughput sequencing (RNA-seq) revealed that Ino80 silencing closely resembled the gene expression changes in RM tissues. To investigate the mechanisms for these results, RNA-seq combined with high-throughput sequencing (ChIP-seq) was used in trophoblast cells, and it showed that Ino80 physically occupies promoter regions to affect the expression of invasion-associated genes. Last, Western blotting analyses and immunofluorescence staining revealed that the content of INO80 was reduced in RM patients compared to in healthy controls. This study indicates that INO80 has a specific regulatory effect on the viability, migration and invasion of trophoblast cells. Combined with its regulation of the expression of invasion-associated genes, it has been proposed that epigenetic regulation plays an important role in the occurrence of RM, potentially informing RM therapeutic strategies.