The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Fibrinogen and fibrin in strong magnetic fields. Complementary results and discussion

When fibrin polymerizes in a strong magnetic field, it can be highly oriented. The structural diffraction study of the oriented polymer becomes thus possible. The magnetic birefringence can also be used to study the development of the polymer Fibrinogen in solution is weakly oriented in high magnetic fields. In this work we present complementary results and discussion. The validity of the comparison of the orientation parameters of fibrinogen and fibrin with those of other orientable known biological structures is discussed. The orientation of fibrin formed from fibrin monomer solution is compared to that of fibrin formed by the action of thrombin on fibrinogen. The conditions to obtain highly oriented fibrin gels suitable for three dimensional structure studies are also briefly discussed.     

PCR primers for polymorphic microsatellite loci in the desert locust,Schistocerca gregaria (Orthoptera: Acrididae)

Nine pairs of polymerase chain reaction (PCR) primers that amplify polymorphic microsatellite loci in the desert locust, Schistocerca gregaria (Forskal), were developed using a magnetic bead‐based enrichment protocol. A sample of 48 locusts collected during the 1993 and 1995 upsurge periods in Eritrea, East Africa, were genotyped. The number of alleles per locus ranged from six to 20; the average was 12.67. Allelic distributions were significantly different between samples from different localities.     

Development of microsatellite markers in polyploid persimmon (Diospyros kaki Lf) from an enriched genomic library

The oriental persimmon (Diospyros kaki Lf) is believed to have originated in China with subsequent introduction into Japan and Korea in ancient times. The species was then brought to Europe, Brazil and the USA from Japan in the 19th century. Recent studies highlighted the poor state of identification of cultivars in these countries due to incorrect labelling and presence of synonyms among local varieties. Thus, molecular marker characterization of germplasm resources is of great value for genetic resource preservation and plant breeding of persimmon. Therefore, to identify accessions for further plant breeding and germplasm management, 37 microsatellite loci were developed from a CT/AG‐enriched persimmon genomic library.     

Characterization of microsatellite loci in White‐chinned Petrel (Procellaria aequinoctialis) and cross‐amplification in six other procellariiform species

Six polymorphic dinucleotide microsatellite loci were isolated and characterized from the White‐chinned Petrel Procellaria aequinoctialis, using a degenerate primer and PCR‐based technique to construct and screen an enriched genomic library. Preliminary data on three populations show heterozygosity levels ranging from 0.22 to 0.67 and allele numbers from three to nine. Preliminary data also suggest genetic distance between these three populations (F_ST 0.088). Cross‐species amplification of these six microsatellite loci and one further locus were tested in six other procellariiform species of the genus Procellaria, Macronectes, Thalassarche and Diomedea.     

Combined effect of X‐ray radiation and static magnetic fields on reactive oxygen species in rat lymphocytes in vitro

The aim of this study was to investigate the effect of static magnetic fields (SMF) on reactive oxygen species induced by X‐ray radiation. The experiments were performed on lymphocytes from male albino Wistar rats. After exposure to 3 Gy X‐ray radiation (with a dose rate of 560 mGy/min) the measurement of intracellular reactive oxygen species in lymphocytes, using a fluorescent probe, was done before exposure to the SMF, and after 15 min, 1 and 2 h of exposure to the SMF or a corresponding incubation time. For SMF exposure, 0 mT (50 µT magnetic field induction opposite to the geomagnetic field) and 5 mT fields were chosen. The trend of SMF effects for 0 mT was always opposite that of 5 mT. The first one decreased the rate of fluorescence change, while the latter one increased it. Bioelectromagnetics 34:333–336, 2013. © 2012 Wiley Periodicals, Inc.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.