William G. Spollen

Root Growth Maintenance at Low Water Potentials: Increased Activity of Xyloglucan Endotransglycosylase and Its Possible Regulation by Abscisic Acid

Previous work suggested that an increase in cell wall-loosening contributes to the maintenance of maize (Zea mays L.) primary root elongation at low water potentials ([psi]w). It was also shown that root elongation at low [psi]w requires increased levels of abscisic acid (ABA). In this study we investigated the effects of low [psi]w and ABA status on xyloglucan endotransglycosylase (XET) activity in the root elongation zone. XET is believed to contribute to wall-loosening by reversibly cleaving xyloglucan molecules that tether cellulose microfibrils. The activity of XET per unit fresh weight in the apical 10 mm (encompassing the elongation zone) was constant at high [psi]w but increased by more than 2-fold at a [psi]w of -1.6 MPa. Treatment with fluridone to decrease ABA accumulation greatly delayed the increase in activity at low [psi]w. This effect was largely overcome when internal ABA levels were restored by exogenous application. Spatial distribution studies showed that XET activity was increased in the apical 6 mm at low [psi]w whether expressed per unit fresh weight, total soluble protein, or cell wall dry weight, corresponding to the region of continued elongation. Treatment with fluridone progressively inhibited the increase in activity with distance from the apex, correlating with the pattern of inhibition of elongation. Added ABA partly restored activity at all positions. The increase in XET activity at low [psi]w was due to maintenance of the rate of deposition of activity despite decreased deposition of wall material. The loss of activity associated with decreased ABA was due to inhibition of the deposition of activity. The results demonstrate that increased XET activity is associated with maintenance of root elongation at low [psi]w and that this response requires increased ABA.

Effects of Vendor and Genetic Background on the Composition of the Fecal Microbiota of Inbred Mice

The commensal gut microbiota has been implicated as a determinant in several human diseases and conditions. There is mounting evidence that the gut microbiota of laboratory mice (Mus musculus) similarly modulates the phenotype of mouse models used to study human disease and development. While differing model phenotypes have been reported using mice purchased from different vendors, the composition and uniformity of the fecal microbiota in mice of various genetic backgrounds from different vendors is unclear. Using culture-independent methods and robust statistical analysis, we demonstrate significant differences in the richness and diversity of fecal microbial populations in mice purchased from two large commercial vendors. Moreover, the abundance of many operational taxonomic units, often identified to the species level, as well as several higher taxa, differed in vendor- and strain-dependent manners. Such differences were evident in the fecal microbiota of weanling mice and persisted throughout the study, to twenty-four weeks of age. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota and thus possibly reduce the number of animals required to perform studies with sufficient statistical power.

Spatial distribution of transcript changes in the maize primary root elongation zone at low water potential

BackgroundPrevious work showed that the maize primary root adapts to low Ψw (-1.6 MPa) by maintaining longitudinal expansion in the apical 3 mm (region 1), whereas in the adjacent 4 mm (region 2) longitudinal expansion reaches a maximum in well-watered roots but is progressively inhibited at low Ψw. To identify mechanisms that determine these responses to low Ψw, transcript expression was profiled in these regions of water-stressed and well-watered roots. In addition, comparison between region 2 of water-stressed roots and the zone of growth deceleration in well-watered roots (region 3) distinguished stress-responsive genes in region 2 from those involved in cell maturation.ResultsResponses of gene expression to water stress in regions 1 and 2 were largely distinct. The largest functional categories of differentially expressed transcripts were reactive oxygen species and carbon metabolism in region 1, and membrane transport in region 2. Transcripts controlling sucrose hydrolysis distinguished well-watered and water-stressed states (invertase vs. sucrose synthase), and changes in expression of transcripts for starch synthesis indicated further alteration in carbon metabolism under water deficit. A role for inositols in the stress response was suggested, as was control of proline metabolism. Increased expression of transcripts for wall-loosening proteins in region 1, and for elements of ABA and ethylene signaling were also indicated in the response to water deficit.ConclusionThe analysis indicates that fundamentally different signaling and metabolic response mechanisms are involved in the response to water stress in different regions of the maize primary root elongation zone.

The Maize Root Transcriptome by Serial Analysis of Gene Expression

Serial Analysis of Gene Expression was used to define number and relative abundance of transcripts in the root tip of well-watered maize seedlings (Zea mays cv FR697). In total, 161,320 tags represented a minimum of 14,850 genes, based on at least two tags detected per transcript. The root transcriptome has been sampled to an estimated copy number of approximately five transcripts per cell. An extrapolation from the data and testing of single-tag identifiers by reverse transcription-PCR indicated that the maize root transcriptome should amount to at least 22,000 expressed genes. Frequency ranged from low copy number (2–5, 68.8%) to highly abundant transcripts (100→1,200; 1%). Quantitative reverse transcription-PCR for selected transcripts indicated high correlation with tag frequency. Computational analysis compared this set with known maize transcripts and other root transcriptome models. Among the 14,850 tags, 7,010 (47%) were found for which no maize cDNA or gene model existed. Comparing the maize root transcriptome with that in other plants indicated that highly expressed transcripts differed substantially; less than 5% of the most abundant transcripts were shared between maize and Arabidopsis (Arabidopsis thaliana). Transcript categories highlight functions of the maize root tip. Significant variation in abundance characterizes transcripts derived from isoforms of individual enzymes in biochemical pathways.

Developmental Expression of 2489 Gene Clusters During Pig Embryogenesis: An Expressed Sequence Tag Project

Abstract Identification of mRNAs that are present at early stages of embryogenesis is critical for a better understanding of development. To this end, cDNA libraries were constructed from germinal vesicle-stage oocytes, in vivo-produced four-cell- and blastocyst-stage embryos, and from in vitro-produced four-cell- and blastocyst-stage embryos. Randomly picked clones (10 848) were sequenced from the 3′ end and those of sufficient quality (8066, 74%) were clustered into groups of sequence similarity (>95% identity), resulting in 2489 clusters. The sequence of the longest representative expressed sequence tag (EST) of each cluster was compared with GenBank and TIGR. Scores below 200 were considered unique, and 1114 (44.8%) did not have a match in either database. Sequencing from the 5′ end yielded 12 of 37 useful annotations, suggesting that one third of the 1114 might be identifiable, still leaving over 700 unique ESTs. Virtual Northerns compared between the stages identified numerous genes where expression appears to change from the germinal vesicle oocyte to the four-cell stage, from the four-cell to blastocyst stage, and between in vitro- and in vivo-derived four-cell- and blastocyst-stage embryos. This is the first large-scale sequencing project on early pig embryogenesis and has resulted in the discovery of a large number of genes as well as possible stage-specific expression. Because many of these ESTs appear to not be in the public databases, their addition will be useful for transcriptional profiling experiments conducted on early pig embryos.

Transcriptional Profiling by Deep Sequencing Identifies Differences in mRNA Transcript Abundance in In Vivo-Derived Versus In Vitro-Cultured Porcine Blastocyst Stage Embryos

In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.

High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase

Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

Effects of exposure to bisphenol A and ethinyl estradiol on the gut microbiota of parents and their offspring in a rodent model

ABSTRACT Gut dysbiosis may result in various diseases, such as metabolic and neurobehavioral disorders. Exposure to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), especially during development, may also increase the risk for such disorders. An unexplored possibility is that EDC-exposure might alter the gut microbial composition. Gut flora and their products may thus be mediating factors for the disease-causing effects of these chemicals. To examine the effects of EDCs on the gut microbiome, female and male monogamous and biparental California mice (Peromyscus californicus) were exposed to BPA (50 mg/kg feed weight) or EE (0.1 ppb) or control diet from periconception through weaning. 16s rRNA sequencing was performed on bacterial DNA isolated from fecal samples, and analyses performed for P0 and F1 males and females. Both BPA and EE induced generational and sex-dependent gut microbiome changes. Many of the bacteria, e.g. Bacteroides, Mollicutes, Prevotellaceae, Erysipelotrichaceae, Akkermansia, Methanobrevibacter, Sutterella, whose proportions increase with exposure to BPA or EE in the P0 or F1 generation are associated with different disorders, such as inflammatory bowel disease (IBD), metabolic disorders, and colorectal cancer. However, the proportion of the beneficial bacterium, Bifidobacterium, was also elevated in fecal samples of BPA- and EE-exposed F1 females. Intestinal flora alterations were also linked to changes in various metabolic and other pathways. Thus, BPA and EE exposure may disrupt the normal gut flora, which may in turn result in systemic effects. Probiotic supplementation might be an effective means to mitigate disease-promoting effects of these chemicals.

Identification of small RNAs associated with meiotic silencing by unpaired DNA.

In Neurospora crassa, unpaired genes are silenced by a mechanism called meiotic silencing by unpaired DNA (MSUD). Although some RNA interference proteins are necessary for this process, its requirement of small RNAs has yet to be formally established. Here we report the characterization of small RNAs targeting an unpaired region, using Illumina sequencing.

Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status

Bacteria harbored in the male reproductive system may influence reproductive function and health of the male and result in developmental origins of adult health and disease (DOHaD) effects in his offspring. Such effects could be due to the seminal fluid, which is slightly basic and enriched with carbohydrates; thereby, creating an ideal habitat for microbes or a potential seminal fluid microbiome (SFM). Using wild-type (WT) and estrogen receptor-alpha (ESR1) knockout (KO) male mice, we describe a unique SFM whose inhabitants differ from gut microbes. The bacterial composition of the SFM is influenced according to whether mice have functional Esr1 genes. Propionibacterium acnes, causative agent of chronic prostatitis possibly culminating in prostate cancer, is reduced in SFM of ESR1 KO compared to WT mice (P ≤ 0.0007). In certain genetic backgrounds, WT mice show a greater incidence of prostate cancer than ESR1 KO, which may be due to increased abundance of P. acnes. Additionally, select gut microbiome residents in ESR1 KO males, such as Lachnospiraceae and Christensenellaceae, might contribute to previously identified phenotypes, especially obesity, in these mutant mice. Understanding how genetics and environmental factors influence the SFM may provide the next frontier in male reproductive disorders and possibly paternal-based DOHaD diseases.