A barcode screen for epigenetic regulators reveals a role for the NuB4/HAT-B histone acetyltransferase complex in histone turnover

Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover, we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics.     

Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.     

Chromosome 14 and late-onset familial Alzheimer disease (FAD)

Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analyses were used, in 49 families with a mean age at onset ≥60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependent penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score ≤ –2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P < .02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52, however, was found for a subgroup of families of intermediate age at onset (mean age at onset ≥60 years and <70 years). These results indicate that the chromosome 14 locus is not responsible for Alzheimer disease in most late-onset FAD kindreds but could play a role in a subset of these kindreds.     

Application of DNA barcodes in Hedyotis L. (Spermacoceae,Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH–psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. assimilis and H. mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH–psbA, but we could amplify and sequence trnH–psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for matK and rbcL combined.     

Associations between White Matter Hyperintensities and β Amyloid on Integrity of Projection,Association, and Limbic Fiber Tracts Measured with Diffusion Tensor MRI

The goal of this study was to assess the relationship between Aβ deposition and white matter pathology (i.e., white matter hyperintensities, WMH) on microstructural integrity of the white matter. Fifty-seven participants (mean age: 78±7 years) from an ongoing multi-site research program who spanned the spectrum of normal to mild cognitive impairment (Clinical dementia rating 0–0.5) and low to high risk factors for arteriosclerosis and WMH pathology (defined as WMH volume >0.5% total intracranial volume) were assessed with positron emission tomography (PET) with Pittsburg compound B (PiB) and magnetic resonance and diffusion tensor imaging (DTI). Multivariate analysis of covariance were used to investigate the relationship between Aβ deposition and WMH pathology on fractional anisotropy (FA) from 9 tracts of interest (i.e., corona radiata, internal capsule, cingulum, parahippocampal white matter, corpus callosum, superior longitudinal, superior and inferior front-occipital fasciculi, and fornix). WMH pathology was associated with reduced FA in projection (i.e., internal capsule and corona radiate) and association (i.e., superior longitudinal, superior and inferior fronto-occipital fasciculi) fiber tracts. Aβ deposition (i.e., PiB positivity) was associated with reduced FA in the fornix and splenium of the corpus callosum. There were interactions between PiB and WMH pathology in the internal capsule and parahippocampal white matter, where Aβ deposition reduced FA more among subjects with WMH pathology than those without. However, accounting for apoE ε4 genotype rendered these interactions insignificant. Although this finding suggests that apoE4 may increase amyloid deposition, both in the parenchyma (resulting in PiB positivity) and in blood vessels (resulting in amyloid angiopathy and WMH pathology), and that these two factors together may be associated with compromised white matter microstructural integrity in multiple brain regions, additional studies with a longitudinal design will be necessary to resolve this issue.     

Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.