DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]₁₁ microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol κ) inserts dGMP and dCMP within the [T]₁₁ mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol κ and polymerase α-primase within a [T]₁₁ allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.     

Persistent alterations in heart rate variability, baroreflex sensitivity, and anxiety-like behaviors during development of heart failure in the rat

Depressed heart rate variability and mood are associated with increased mortality in patients with congestive heart failure (CHF). Here autonomic indexes were assessed 3 and 7 wk after left coronary artery ligation in telemetered rats, after which anxiety-like behaviors were assessed in an elevated plus maze. Low frequency (LF) and high frequency (HF) heart rate variability were reduced in CHF rats 3 wk after infarction (LF, 1.60 +/- 0.52 vs. 6.97 +/- 0.79 ms(2); and HF, 1.53 +/- 0.39 vs. 6.20 +/- 1.01 ms(2); P < 0.01). The number of sequences of interbeat intervals that correlated with arterial pressure was decreased in CHF rats at 3 and 7 wk (week 3, 26.60 +/- 10.85 vs. 59.75 +/- 11.4 sequences, P < 0.05; and week 7, 20.80 +/- 8.97 vs. 65.38 +/- 5.89 sequences, P < 0.01). Sequence gain was attenuated in CHF rats by 7 wk (1.34 +/- 0.06 vs. 2.70 +/- 0.29 ms/mmHg, P < 0.01). Coherence between interbeat interval and mean arterial blood pressure variability in the LF domain was reduced in CHF rats at 3 (0.12 +/- 0.03 vs. 0.26 +/- 0.05 k(2), P < 0.05) and 7 (0.16 +/- 0.02 vs. 0.31 +/- 0.05 k(2), P < 0.05) wk. CHF rats invariably entered the open arm of the elevated plus maze first and spent more time in the open arms (36.0 +/- 15% vs. 4.6 +/- 1.9%, P < 0.05). CHF rats also showed a tendency to jump head first off the apparatus, whereas controls did not. Together the data indicate that severe autonomic dysfunction is accompanied by escape-seeking behaviors in rats with verified CHF.     

Tight junction biogenesis during early development

The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na⁺/K⁺-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.     

Chaperone-aided in vitro renaturation of an engineered E1 envelope protein for detection of anti-Rubella virus IgG antibodies

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.     

Cryopreservation of Echinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones)

Four isolates of larval Echinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2-4 degrees C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (-196 degrees C). After storage for one week the samples were rapidly thawed at +37 degrees C for approximately 3 min, washed in fresh EMEM/A (37 degrees C) and transferred into the peritoneal cavity of Meriones for viability testing. As judged by histological examinations and metacestode weights of each 24 Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at -28 degrees C, 30 min at -80 degrees C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7 degrees C min-1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65 degrees C min-1 for step 3. These results demonstrate that metacestodes of E. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.     

An active role of inferior frontal cortex in conscious experience

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