Intron Conservation in a UV-Specific DNA Repair Gene Encoded by Chlorella Viruses

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5′-AG/GTATGT and 3′-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences. Received: 13 May 1999 / Accepted: 20 August 1999     

Interploid St. Augustinegrass [<Emphasis Type="Italic">Stenotaphrum secundatum</Emphasis> (Walt.) Kuntze] hybrids recovered by embryo rescue

St. Augustinegrass is one of the most important warm season turfgrasses in the southern United States because of its shade tolerance. Most cultivars are diploids (2n = 2x = 18) and are susceptible to various diseases and insects. Polyploid cultivars in the species have some resistance to pests, but most lack cold tolerance. In this study, eight polyploid genotypes were crossed with six diploid cultivars to transfer pest resistance to the diploids. Because interploid crosses often result in aborted seed, it was necessary to use in vitro techniques. Using embryo rescue, 268 plants were recovered from 2,463 emasculated and pollinated florets (10.88% crossability). Because of the heterogeneous nature of the species, these purported hybrids could not be verified by phenotype. DNA markers were used for hybrid identification. A subset of 25 plants from crosses between the aneuploid cultivar Floratam (2n = 4x = 32) and five diploid cultivars were analyzed using 144 expressed sequence tags–simple sequence repeats (EST-SSRs) developed from buffelgrass cDNA sequence data. Chi-square tests for paternal-specific markers revealed that all analyzed progeny were true F₁ hybrids and none originated from self-fertilization or unintended outcrossing. In addition to identifying DNA polymorphism, the EST-SSRs revealed that genetic variation exists among all analyzed cultivars and is not partitioned between ploidy levels. The findings demonstrate that these embryo rescue techniques will enable the entire spectrum of St. Augustinegrass genetic variation to be better used through the recovery of interploid hybrids.     

Delayed adenosine A1 receptor preconditioning in rat myocardium is MAPK dependent but iNOS independent

Adenosine A1 receptor delayed preconditioning (PC) against myocardial infarction has been well described; however, there have been limited investigations of the signaling mechanisms that mediate this phenomenon. In addition, there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating A1 late-phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) in in vivo delayed A1 receptor PC and whether this protection at the myocyte level is due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 h after treatment with vehicle or the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 100 microg/kg ip). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB-203580 (1 mg/kg ip) or the MAPK/ERK kinase (MEK) inhibitor PD-098059 (0.5 mg/kg ip). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400 W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 +/- 2 to 28 +/- 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400 W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during H2O2 exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400 W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 h after CCPA treatment. These results indicate that adenosine A1 receptor delayed PC in rats is mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS.     

Temperature controls on aquatic bacterial production and community dynamics in arctic lakes and streams

The impact of temperature on bacterial activity and community composition was investigated in arctic lakes and streams in northern Alaska. Aquatic bacterial communities incubated at different temperatures had different rates of production, as measured by ¹⁴C‐leucine uptake, indicating that populations within the communities had different temperature optima. Samples from Toolik Lake inlet and outlet were collected at water temperatures of 14.2°C and 15.9°C, respectively, and subsamples incubated at temperatures ranging from 6°C to 20°C. After 5 days, productivity rates varied from 0.5 to ～13.7 µg C l^?1 day^?1 and two distinct activity optima appeared at 12°C and 20°C. At these optima, activity was 2‐ to 11‐fold higher than at other incubation temperatures. The presence of two temperature optima indicates psychrophilic and psychrotolerant bacteria dominate under different conditions. Community fingerprinting via denaturant gradient gel electrophoresis (DGGE) of 16S rRNA genes showed strong shifts in the composition of communities driven more by temperature than by differences in dissolved organic matter source; e.g. four and seven unique operational taxonomic units (OTUs) were found only at 2°C and 25°C, respectively, and not found at other incubation temperatures after 5 days. The impact of temperature on bacteria is complex, influencing both bacterial productivity and community composition. Path analysis of measurements of 24 streams and lakes sampled across a catchment 12 times in 4 years indicates variable timing and strength of correlation between temperature and bacterial production, possibly due to bacterial community differences between sites. As indicated by both field and laboratory experiments, shifts in dominant community members can occur on ecologically relevant time scales (days), and have important implications for understanding the relationship of bacterial diversity and function.     

Inferences about the conformation of somatostatin at a biologic receptor based on NMR studies

Examination of two diastereomeric analogs of somatostatin differing in stereochemistry at the tryptophan residue has revealed a high field resonance in the -Trp isomer which is assigned to the γ-methylene of Lys⁹. The extent of correlation of this shift with biologic activity for a series of analogs of somatostatin is discussed. From comparison of close analogs, it is suggested that the biologically active conformation of somatostatin at the receptor controlling insulin release is not the major conformation of this hormone in solution. It is suggested that the conformation of somatostatin at this receptor resembles more closely the solution conformation of analogs having tryptophan in the -configuration. This latter conformation places the Trp⁸-Lys⁹ side chains in close proximity, thus shifting the γ-methylene protons of Lys⁹ upfield.     

Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.     

Structural properties of prion protein protofibrils and fibrils: an experimental assessment of atomic models

Decades after the prion protein was implicated in transmissible spongiform encephalopathies, the structure of its toxic isoform and its mechanism of toxicity remain unknown. By gathering available experimental data, albeit low resolution, a few pieces of the prion puzzle can be put in place. Currently, there are two fundamentally different models of a prion protofibril. One has its building blocks derived from a molecular dynamics simulation of the prion protein under amyloidogenic conditions, termed the spiral model. The other model was constructed by threading a portion of the prion sequence through a beta-helical structure from the Protein Data Bank. Here we compare and contrast these models with respect to all of the available experimental information, including electron micrographs, symmetries, secondary structure, oligomerization interfaces, enzymatic digestion, epitope exposure, and disaggregation profiles. Much of this information was not available when the two models were introduced. Overall, we find that the spiral model is consistent with all of the experimental results. In contrast, it is difficult to reconcile several of the experimental observables with the beta-helix model. While the experimental constraints are of low resolution, in bringing together the previously disconnected experiments, we have developed a clearer picture of prion aggregates. Both the improved characterization of prion aggregates and the existing atomic models can be used to devise further experiments to better elucidate the misfolding pathway and the structure of prion protofibrils.     

Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

Fast Bayesian inference for large occupancy datasets

In recent years, the study of species' occurrence has benefited from the increased availability of large-scale citizen-science data. While abundance data from standardized monitoring schemes are biased toward well-studied taxa and locations, opportunistic data are available for many taxonomic groups, from a large number of locations and across long timescales. Hence, these data provide opportunities to measure species' changes in occurrence, particularly through the use of occupancy models, which account for imperfect detection. These opportunistic datasets can be substantially large, numbering hundreds of thousands of sites, and hence present a challenge from a computational perspective, especially within a Bayesian framework. In this paper, we develop a unifying framework for Bayesian inference in occupancy models that account for both spatial and temporal autocorrelation. We make use of the Pólya-Gamma scheme, which allows for fast inference, and incorporate spatio-temporal random effects using Gaussian processes (GPs), for which we consider two efficient approximations: subset of regressors and nearest neighbor GPs. We apply our model to data on two UK butterfly species, one common and widespread and one rare, using records from the Butterflies for the New Millennium database, producing occupancy indices spanning 45 years. Our framework can be applied to a wide range of taxa, providing measures of variation in species' occurrence, which are used to assess biodiversity change.