Keith W. Hutchison

Diversity and abundance of the bacterial community of the red Macroalga Porphyra umbilicalis: did bacterial farmers produce macroalgae?

Macroalgae harbor microbial communities whose bacterial biodiversity remains largely uncharacterized. The goals of this study were 1) to examine the composition of the bacterial community associated with Porphyra umbilicalis Kützing from Schoodic Point, ME, 2) determine whether there are seasonal trends in species diversity but a core group of bacteria that are always present, and 3) to determine how the microbial community associated with a laboratory strain (P.um.1) established in the presence of antibiotics has changed. P. umbilicalis blades (n = 5, fall 2010; n = 5, winter 2011; n = 2, clonal P.um.1) were analyzed by pyrosequencing over two variable regions of the 16 S rDNA (V5–V6 and V8; 147,880 total reads). The bacterial taxa present were classified at an 80% confidence threshold into eight phyla (Bacteroidetes, Proteobacteria, Planctomycetes, Chloroflexi, Actinobacteria, Deinococcus-Thermus, Firmicutes, and the candidate division TM7). The Bacteroidetes comprised the majority of bacterial sequences on both field and lab blades, but the Proteobacteria (Alphaproteobacteria, Gammaproteobacteria) were also abundant. Sphingobacteria (Bacteroidetes) and Flavobacteria (Bacteroidetes) had inverse abundances on natural versus P.um.1 blades. Bacterial communities were richer and more diverse on blades sampled in fall compared to winter. Significant differences were observed between microbial communities among all three groups of blades examined. Only two OTUs were found on all 12 blades, and only one of these, belonging to the Saprospiraceae (Bacteroidetes), was abundant. Lewinella (as 66 OTUs) was found on all field blades and was the most abundant genus. Bacteria from the Bacteroidetes, Proteobacteria and Planctomycetes that are known to digest the galactan sulfates of red algal cell walls were well-represented. Some of these taxa likely provide essential morphogenetic and beneficial nutritive factors to P. umbilicalis and may have had unexpected effects upon evolution of macroalgal form as well as function.

Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries.

Genome plasticity in the mouse oocyte and early embryo

In dissecting the molecules and molecular mechanisms that control mammalian oocyte-to-embryo transition, we found abundant transcripts representing developmentally regulated ERVs (endogenous retroviruses) in mouse oocyte and two-cell stage embryo cDNA libraries. These retrotransposons can act as alternative promoters and first exons for diverse genes, synchronizing their expression. Heritable genetic change due to replication of these retrotransposons probably occurs specifically in oocytes and early embryos. ERVs are usually epigenetically silenced, through DNA methylation and chromatin-based mechanisms. Their activation and silencing indicates a change in the epigenetic state of the genome. The thousands of endogenous retro-elements in the mouse genome provides potential scope for large-scale co-ordinated epigenetic fluctuations and leads to the hypothesis that differential transposable element expression triggers sequential reprogramming of the embryonic genome during the oocyte-to-embryo transition.

Differential Gene Expression During Maturation-Caused Decline in Adventitious Rooting Ability in Loblolly Pine (Pinus taeda L.)

Loss of adventitious rooting ability occurs very quickly in loblolly pine seedlings, where hypocotyl cuttings from 50-day-old seedlings root readily in response to auxin, while epicotyl cuttings from the same seedling do not, despite their anatomical similarity (Diaz-Sala et al., 1996a). In hypocotyl cuttings, auxin causes root meristems to organize in the cambial region centrifugal to the former primary xylem poles over a 12-day period. In epicotyl cuttings, the cambium dedifferentiates in response to auxin, but roots rarely form, and then only after 50 or more days. A brief exposure (a 5 min pulse) to auxin is sufficient to promote rooting. While both types of cutting transport auxin in a polar manner, a pulse of N-(l-napthl)phthalamic acid (NPA), which inhibits auxin efflux, also delays rooting in hypocotyls only if applied within the first 3 days after the auxin pulse; thereafter it has no effect. These observations show that key events that determine root meristem formation occur in the first 72 hours, and that auxin binding to the efflux carrier that mediates polar auxin transport, may be necessary for rooting (Diaz-Sala et al., 1996a).

The Molecular Genetics of Maturation in Eastern Larch ( Larix Laricina [Du Roi] K. Koch)

Maturation in woody plants has recently received much attention because of the maturation-related decrease in the ability to clone selected individuals using expiants from mature plants (Hackett, 1985; Greenwood, 1987). Furthermore, the phenotypic changes that accompany maturation make it difficult for the tree breeder to select superior genetic families or individuals at the seedling stage (e.g., Lambeth, 1980). The changes associated with maturation include growth rate, branching characteristics and growth habit, reproductive behavior, and the morphology and physiology of foliage. The significance of the latter will be emphasized in this brief report, with emphasis on the role of gene expression in the maturational process. Changes in foliage associated with maturation have been discussed (e.g., Zimmerman et al., 1985; Bauer and Bauer, 1980) and do not follow a completely consistent pattern among species.

Molecular Approaches To Maturation-Caused Decline In Adventitious Rooting Ability in Loblolly Pine (Pinus Taeda L.)

New approaches towards understanding the control of adventitious root formation include studies at the level of gene expression. We discuss attempts to determine the identity of as yet unknown genes which are specific to formation of roots, as well as studies on the expression of genes known to be involved in the wound response. A number of genes appear to be specific to adventitious root formation in several species including loblolly pine, and the expression of these genes changes with maturation. Keywords: adventitious rooting, gene expression, loblolly pine

Gene Expression during Growth and Maturation

The expression of cab and rbcS gene families were measured in RNA extracted from needles from larch trees ranging in age from 1 y to 75 y. Steady state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared with mature plants. Later in the season no consistent difference in cab expression between juvenile and mature plants was detected. Unlike cab gene expression, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions is not due to increased net photosynthesis or cab expression.