Inhibition of Real-Time PCR in DNA Extracts from Aquifer Sediment

Variation in inhibition of real-time PCR was investigated with DNA extracts from 50 aquifer sediment core samples of 5 cm length collected through a 2.5 meter vertical profile across a landfill leachate plume. The inhibition was quantified using an internal control of the green fluorescent protein ( gfp ) gene, which was spiked into the real-time PCR reactions. The inhibition was investigated at two gfp gene concentrations: at 1.7 · 10 ⁷ gfp gene copies/g sediment (5.1 · 10 ⁴ gfp gene copies/PCR reaction) and at 1.7 · 10 ⁵ gfp gene copies/g sediment (5.1 · 10 ² gfp gene copies/PCR reaction). Despite the low TOC content of the sediment (average 0.4 mg C/g dw) the average real-time PCR response was partially inhibited, compared to a reference (pure water), at both high and low gfp concentrations. The relative amplification (reference = 1) was 0.85 ± 0.20 (high) and 0.66 ± 0.23 (low), showing significantly (P < 0.05) stronger inhibition at the lower target gene concentration. The inhibition of the real-time PCR did not show a systematic variation in the vertical profile related to plume position but variations were significant on a small scale of 5–15 cm depth intervals. One of the 50 samples failed to produce a signal with either concentration of the gfp internal control and three other samples inhibited real-time PCR at both high and low gfp concentration. These 4 samples, which were the samples with the highest inhibition, were the only DNA extracts with a visible brown colouration, indicating contents of humic-like substances. Elevated absorbance at 400 nm of these samples also indicated that humic-like substances were responsible for inhibition. However, other factors not associated with either absorbance or TOC may have contributed to the inhibition in less inhibited samples since the variation in real-time PCR response could not be sufficiently explained by absorbance or TOC. The results of this study suggest that an internal control is needed in real-time PCR reactions with DNA from environmental samples due to variation in inhibition to correctly quantify the number of target genes, especially at low target gene concentrations, when dilution of DNA extracts is not practical.     

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.     

The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

Zero effect of Bt rice on expression of genes coding for digestion,detoxification and immune responses and developmental performances of Brown Planthopper Nilaparvata lugens (Stål)

Transgenic Cry1Ac, Cry2Aa and Cry1Ca (Bt toxins) rice lines are well developed to manage lepidopteron pests in China. The impact of transgenic Bt rice on the non-target Brown Planthopper (BPH) has become an essential part of environmental risk assessment, however, scanty evidence is found addressing on developmental and molecular responses of BPH to the ingestion of Bt protein from transgenic rice. The focus of the current study is to examine the developmental characteristics and the expression profiles of gene in relation to digestion, detoxification and immune responses were examined. Our study strongly revealed that the tested Bt rice strains have no unfavorable effect on fecundity, survival and growth of BPH. Furthermore, each of the tested genes did not exhibit distinct expression pattern responding to non Bt parental cultivar, thus, it could be concluded that Bt rice have no detrimental effects on the physiological processes of digestion, detoxification and immune responses of BPH.     

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).     

A Shared Endoplasmic Reticulum-associated Degradation Pathway Involving the EDEM1 Protein for Glycosylated and Nonglycosylated Proteins

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF^Fbs2 E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.