Sequence-function relationships in E. coli transcriptional regulation

Understanding how transcriptional regulatory sequence maps to regulatory function remains a difficult problem in regulatory biology. Given a particular DNA sequence for a bacterial promoter region, we would like to be able to say which transcription factors bind there, how strongly they bind, and whether they interact with each other and/or RNA polymerase, with the ultimate objective of integrating knowledge of these parameters into a prediction of gene expression levels. The theoretical framework of statistical thermodynamics provides a useful framework for doing so, enabling us to predict how gene expression levels depend on transcription factor binding energies and concentrations. We used thermodynamic models, coupled with models of the sequence-dependent binding energies of transcription factors and RNAP, to construct a genotype to phenotype map for the level of repression exhibited by the lac promoter, and tested it experimentally using a set of promoter variants from E. coli strains isolated from different natural environments. For this work, we sought to ``reverse engineer'' naturally occurring promoter sequences to understand how variations in promoter sequence affects gene expression. The natural inverse of this approach is to ``forward engineer'' promoter sequences to obtain targeted levels of gene expression. We used a high precision model of RNAP-DNA sequence dependent binding energy, coupled with a thermodynamic model relating binding energy to gene expression, to predictively design and verify a suite of synthetic E. coli promoters whose expression varied over nearly three orders of magnitude.

However, although thermodynamic models enable predictions of mean levels of gene expression, it has become evident that cell-to-cell variability or ``noise'' in gene expression can also play a biologically important role. In order to address this aspect of gene regulation, we developed models based on the chemical master equation framework and used them to explore the noise properties of a number of common E. coli regulatory motifs; these properties included the dependence of the noise on parameters such as transcription factor binding strength and copy number. We then performed experiments in which these parameters were systematically varied and measured the level of variability using mRNA FISH. The results showed a clear dependence of the noise on these parameters, in accord with model predictions.

Finally, one shortcoming of the preceding modeling frameworks is that their applicability is largely limited to systems that are already well-characterized, such as the lac promoter. Motivated by this fact, we used a high throughput promoter mutagenesis assay called Sort-Seq to explore the completely uncharacterized transcriptional regulatory DNA of the E. coli mechanosensitive channel of large conductance (MscL). We identified several candidate transcription factor binding sites, and work is continuing to identify the associated proteins.

Mitochondrial control region sequence variation within the argali wild sheep (Ovis ammon): evolution and conservation relevance

The phylogenetic relationship of several subspecies of Ovis ammon were analyzed by comparing DNA sequences within the entire mitochondrial D-loop region. Five putative subspecies of ammon (dalai-lamae, darwini, hodgsoni, sairensis and adamerzi) were sampled from four provinces in China [Xinjiang, Qinghai, Gansu and Xizang (Tibet)] and two (servertzovi and nigrimontana) from Uzbekistan. The argalis sampled represent most of the currently recognized putative Subspecies of argali. Analysis of mtDNA sequences revealed high variability within ammon (7.7%), ranging from 2.4 to 11.5%. MaxiMUM-Parsimony tree indicated that nigrimontana from Uzbekistan diverged First, followed by severtzovi from Uzbekistan. The dispersal of argalis into China gave rise to three clades, suggesting that the argali originated in Western Asia and then dispersed throughout the central Asian highlands on a southeastward course. Among the Chinese argalis, mtDNA analysis places dalailamae genetically closer to hodgsoni than to darwini. Severtzovi and.. nigrimontana are two separate subspecies and genetically distinct from the Chinese argali.

High variability of the gvpA-gvpC region in Microcystis

Gas vesicles provide buoyancy to Microcystis and other common cyanobacterial bloom-forming species. gvpA and gvpC are structural genes encoding gas vesicle proteins. Phylogenetic analyses of 10 Microcystis strains/uncultured samples showed that gvpC and each intergenic segment of the gvpA-gvpC region can be divided into two types. The combination of different types of gvpC and intergenic segments is an important factor that diversifies this genomic region. Some Microcystis strains isolated in China possess a 172 to 176 bp sequence tag in the intergenic segment between gvpA and gvpC. The gvpA-gvpC region in Microcystis can be divided into at least 4 classes and more numbers of subclasses. Compared to rbcLX and other regions, the high variability of the gvpA-gvpC region should be more useful in identifying geographical isolates or ecotypes of Microcystis.

Sequence analysis of the ITS region and 5.8S rDNA of Porphyra haitanensis

The sequences of the ITS (internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli (NB, PT and ST) were amplified, sequenced and analyzed. In addition, the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated. The results are as follows: the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains. The sequences of ITS l were 331 by to 334 bp, while those of the 5.8S rDNA were 158 by and the sequences of ITS2 ranged from 673 by to 681 bp. The sequences of the ITS had a high level of homology (up to 99.5%) with that of P. haitanensis (DQ662228) retrieved from GenBank, but were only approximately 50% homologous with those of other species of Porphyra. The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis. These results suggest that the three cultivated strains of P. haitanensis evolved conservatively and that the ITS showed evolutionary consistency. However, the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations. Therefore, the relationship of Porphyra interspecies phyletic evolution could be judged, which provides the proof for Porphyra identification study. However, proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.

A Phylogenetic Mixture Model for Detecting Pattern-Heterogeneity in Gene Sequence or Character-State Data

Mark Pagel, Andrew Meade (2004). A phylogenetic mixture model for detecting pattern-heterogeneity in gene sequence or character-state data. Systematic Biology, 53(4), 571-581. RAE2008

Intergenic and genic sequence lengths have opposite relationships with respect to gene expression.

Eukaryotic genomes are mostly composed of noncoding DNA whose role is still poorly understood. Studies in several organisms have shown correlations between the length of the intergenic and genic sequences of a gene and the expression of its corresponding mRNA transcript. Some studies have found a positive relationship between intergenic sequence length and expression diversity between tissues, and concluded that genes under greater regulatory control require more regulatory information in their intergenic sequences. Other reports found a negative relationship between expression level and gene length and the interpretation was that there is selection pressure for highly expressed genes to remain small. However, a correlation between gene sequence length and expression diversity, opposite to that observed for intergenic sequences, has also been reported, and to date there is no testable explanation for this observation. To shed light on these varied and sometimes conflicting results, we performed a thorough study of the relationships between sequence length and gene expression using cell-type (tissue) specific microarray data in Arabidopsis thaliana. We measured median gene expression across tissues (expression level), expression variability between tissues (expression pattern uniformity), and expression variability between replicates (expression noise). We found that intergenic (upstream and downstream) and genic (coding and noncoding) sequences have generally opposite relationships with respect to expression, whether it is tissue variability, median, or expression noise. To explain these results we propose a model, in which the lengths of the intergenic and genic sequences have opposite effects on the ability of the transcribed region of the gene to be epigenetically regulated for differential expression. These findings could shed light on the role and influence of noncoding sequences on gene expression.

Response variability in Attention Deficit Hyperactivity Disorder: Evidence for neuropsychological heterogeneity

Response time (RT) variability is a common finding in ADHD research. RT variability may reflect frontal cortex function and may be related to deficits in sustained attention. The existence of a sustained attention deficit in ADHD has been debated, largely because of inconsistent evidence of time-on-task effects. A fixed-sequence Sustained Attention to Response Task (SART) was given to 29 control, 39 unimpaired and 24 impaired-ADHD children (impairment defined by the number of commission errors). The response time data were analysed using the Fast Fourier Transform, to define the fast-frequency and slow-frequency contributions to overall response variability. The impaired-ADHD group progressively slowed in RT over the course of the 5.5 min task, as reflected in this group's greater slow-frequency variability. The fast-frequency trial-to-trial variability was also significantly greater, but did not differentially worsen over the course of the task. The higher error rates of the impaired-ADHD group did not become differentially greater over the length of the task. The progressive slowing in mean RT over the course of the task may relate to a deficit in arousal in the impaired-ADHD group. The consistently poor performance in fast-frequency variability and error rates may be due to difficulties in sustained attention that fluctuate on a trial-to-trial basis. (c) 2006 Elsevier Ltd. All rights reserved.

CSI 2264: Simultaneous Optical and Infrared Light Curves of Young Disk-bearing Stars in NGC 2264 with CoRoT and Spitzer—Evidence for Multiple Origins of Variability

We present the Coordinated Synoptic Investigation of NGC 2264, a continuous 30 day multi-wavelength photometric monitoring campaign on more than 1000 young cluster members using 16 telescopes. The unprecedented combination of multi-wavelength, high-precision, high-cadence, and long-duration data opens a new window into the time domain behavior of young stellar objects. Here we provide an overview of the observations, focusing on results from Spitzer and CoRoT. The highlight of this work is detailed analysis of 162 classical T Tauri stars for which we can probe optical and mid-infrared flux variations to 1% amplitudes and sub-hour timescales. We present a morphological variability census and then use metrics of periodicity, stochasticity, and symmetry to statistically separate the light curves into seven distinct classes, which we suggest represent different physical processes and geometric effects. We provide distributions of the characteristic timescales and amplitudes and assess the fractional representation within each class. The largest category (>20%) are optical "dippers" with discrete fading events lasting ~1-5 days. The degree of correlation between the optical and infrared light curves is positive but weak; notably, the independently assigned optical and infrared morphology classes tend to be different for the same object. Assessment of flux variation behavior with respect to (circum)stellar properties reveals correlations of variability parameters with Hα emission and with effective temperature. Overall, our results point to multiple origins of young star variability, including circumstellar obscuration events, hot spots on the star and/or disk, accretion bursts, and rapid structural changes in the inner disk. Based on data from the Spitzer and CoRoT missions. The CoRoT space mission was developed and is operated by the French space agency CNES, with participation of ESA's RSSD and Science Programmes, Austria, Belgium, Brazil, Germany, and Spain.

Young Stellar Object VARiability (YSOVAR): Long Timescale Variations in the Mid-infrared

The YSOVAR (Young Stellar Object VARiability) Spitzer Space Telescope observing program obtained the first extensive mid-infrared (3.6 and 4.5 μm) time series photometry of the Orion Nebula Cluster plus smaller footprints in 11 other star-forming cores (AFGL 490, NGC 1333, Mon R2, GGD 12-15, NGC 2264, L1688, Serpens Main, Serpens South, IRAS 20050+2720, IC 1396A, and Ceph C). There are ~29,000 unique objects with light curves in either or both IRAC channels in the YSOVAR data set. We present the data collection and reduction for the Spitzer and ancillary data, and define the "standard sample" on which we calculate statistics, consisting of fast cadence data, with epochs roughly twice per day for ~40 days. We also define a "standard sample of members" consisting of all the IR-selected members and X-ray-selected members. We characterize the standard sample in terms of other properties, such as spectral energy distribution shape. We use three mechanisms to identify variables in the fast cadence data—the Stetson index, a χ2 fit to a flat light curve, and significant periodicity. We also identified variables on the longest timescales possible of six to seven years by comparing measurements taken early in the Spitzer mission with the mean from our YSOVAR campaign. The fraction of members in each cluster that are variable on these longest timescales is a function of the ratio of Class I/total members in each cluster, such that clusters with a higher fraction of Class I objects also have a higher fraction of long-term variables. For objects with a YSOVAR-determined period and a [3.6]-[8] color, we find that a star with a longer period is more likely than those with shorter periods to have an IR excess. We do not find any evidence for variability that causes [3.6]-[4.5] excesses to appear or vanish within our data set; out of members and field objects combined, at most 0.02% may have transient IR excesses.

YSOVAR: Mid-infrared Variability in the Star-forming Region Lynds 1688

The emission from young stellar objects (YSOs) in the mid-infrared (mid-IR) is dominated by the inner rim of their circumstellar disks. We present IR data from the Young Stellar Object VARiability (YSOVAR) survey of ~800 objects in the direction of the Lynds 1688 (L1688) star-forming region over four visibility windows spanning 1.6 yr using the Spitzer Space Telescope in its warm mission phase. Among all light curves, 57 sources are cluster members identified based on their spectral energy distribution and X-ray emission. Almost all cluster members show significant variability. The amplitude of the variability is larger in more embedded YSOs. Ten out of 57 cluster members have periodic variations in the light curves with periods typically between three and seven days, but even for those sources, significant variability in addition to the periodic signal can be seen. No period is stable over 1.6 yr. Nonperiodic light curves often still show a preferred timescale of variability that is longer for more embedded sources. About half of all sources exhibit redder colors in a fainter state. This is compatible with time-variable absorption toward the YSO. The other half becomes bluer when fainter. These colors can only be explained with significant changes in the structure of the inner disk. No relation between mid-IR variability and stellar effective temperature or X-ray spectrum is found.

YSOVAR: Mid-infrared Variability of Young Stellar Objects and Their Disks in the Cluster IRAS 20050+2720

We present a time-variability study of young stellar objects (YSOs) in the cluster IRAS 20050+2720, performed at 3.6 and 4.5 μm with the Spitzer Space Telescope; this study is part of the Young Stellar Object VARiability (YSOVAR) project. We have collected light curves for 181 cluster members over 60 days. We find a high variability fraction among embedded cluster members of ca. 70%, whereas young stars without a detectable disk display variability less often (in ca. 50% of the cases) and with lower amplitudes. We detect periodic variability for 33 sources with periods primarily in the range of 2–6 days. Practically all embedded periodic sources display additional variability on top of their periodicity. Furthermore, we analyze the slopes of the tracks that our sources span in the color–magnitude diagram (CMD). We find that sources with long variability time scales tend to display CMD slopes that are at least partially influenced by accretion processes, while sources with short variability timescales tend to display extinction-dominated slopes. We find a tentative trend of X-ray detected cluster members to vary on longer timescales than the X-ray undetected members.

YSOVAR: Mid-infrared Variability Among YSOs in the Star Formation Region GGD12-15

We present an IR-monitoring survey with the Spitzer Space Telescope of the star-forming region GGD 12-15. More than 1000 objects were monitored, including about 350 objects within the central 5′, which is found to be especially dense in cluster members. The monitoring took place over 38 days and is part of the Young Stellar Object VARiability project. The region was also the subject of a contemporaneous 67 ks Chandra observation. The field includes 119 previously identified pre-main sequence star candidates. X-rays are detected from 164 objects, 90 of which are identified with cluster members. Overall, we find that about half the objects in the central 5′ are young stellar objects (YSOs) based on a combination of their spectral energy distribution, IR variability, and X-ray emission. Most of the stars with IR excess relative to a photosphere show large amplitude (>0.1 mag) mid-infrared (mid-IR) variability. There are 39 periodic sources, and all but one is found to be a cluster member. Almost half of the periodic sources do not show IR excesses. Overall, more than 85% of the Class I, flat spectrum, and Class II sources are found to vary. The amplitude of the variability is larger in more embedded YSOs. Most of the Class I/ II objects exhibit redder colors in a fainter state, which is compatible with time-variable extinction. A few become bluer when fainter, which can be explained with significant changes in the structure of the inner disk. A search for changes in the IR due to X-ray events is carried out, but the low number of flares prevented an analysis of the direct impact of X-ray flares on the IR light curves. However, we find that X-ray detected Class II sources have longer timescales for change in the MIR than a similar set of non-X-ray detected Class IIs.

YSOVAR: Mid-infrared Variability in NGC 1333

As part of the Young Stellar Object VARiability (YSOVAR) program, wemonitored NGC 1333 for ∼35 days at 3.6 and 4.5 μm using theSpitzer Space Telescope. We report here on the mid-infrared variabilityof the point sources in the ∼10‧ × ∼20‧ areacentered on 03:29:06, +31:19:30 (J2000). Out of 701 light curves ineither channel, we find 78 variables over the YSOVAR campaign. Abouthalf of the members are variable. The variable fraction for the mostembedded spectral energy distributions (SEDs) (Class I, flat) is higherthan that for less embedded SEDs (Class II), which is in turn higherthan the star-like SEDs (Class III). A few objects have amplitudes(10–90th percentile brightness) in [3.6] or [4.5] > 0.2 mag; amore typical amplitude is 0.1–0.15 mag. The largest color changeis >0.2 mag. There are 24 periodic objects, with 40% of them beingflat SED class. This may mean that the periodic signal is primarily fromthe disk, not the photosphere, in those cases. We find 9 variableslikely to be “dippers,” where texture in the disk occultsthe central star, and 11 likely to be “bursters,” whereaccretion instabilities create brightness bursts. There are 39 objectsthat have significant trends in [3.6]–[4.5] color over thecampaign, about evenly divided between redder-when-fainter (consistentwith extinction variations) and bluer-when-fainter. About a third of the17 Class 0 and/or jet-driving sources from the literature are variableover the YSOVAR campaign, and a larger fraction (∼half) are variablebetween the YSOVAR campaign and the cryogenic-era Spitzer observations(6–7 years), perhaps because it takes time for the envelope torespond to changes in the central source. The NGC 1333 brown dwarfs donot stand out from the stellar light curves in any way except there is amuch larger fraction of periodic objects (∼60% of variable browndwarfs are periodic, compared to ∼30% of the variables overall).

A phylogenetic mixture model for detecting pattern-heterogeneity in gene sequence or character-state data

We describe a general likelihood-based 'mixture model' for inferring phylogenetic trees from gene-sequence or other character-state data. The model accommodates cases in which different sites in the alignment evolve in qualitatively distinct ways, but does not require prior knowledge of these patterns or partitioning of the data. We call this qualitative variability in the pattern of evolution across sites "pattern-heterogeneity" to distinguish it from both a homogenous process of evolution and from one characterized principally by differences in rates of evolution. We present studies to show that the model correctly retrieves the signals of pattern-heterogeneity from simulated gene-sequence data, and we apply the method to protein-coding genes and to a ribosomal 12S data set. The mixture model outperforms conventional partitioning in both these data sets. We implement the mixture model such that it can simultaneously detect rate- and pattern-heterogeneity. The model simplifies to a homogeneous model or a rate- variability model as special cases, and therefore always performs at least as well as these two approaches, and often considerably improves upon them. We make the model available within a Bayesian Markov-chain Monte Carlo framework for phylogenetic inference, as an easy-to-use computer program.

Inter-simple sequence repeat and aggressiveness analyses revealed high genetic diversity, recombination and long-range dispersal in Fusarium culmorum

Inter-simple sequence repeat (ISSR) analysis and aggressiveness assays were used to investigate genetic variability within a global collection of Fusarium culmorum isolates. A set of four ISSR primers were tested, of which three primers amplified a total of 37 bands out of which 30 (81%) were polymorphic. The intraspecific diversity was high, ranging from four to 28 different ISSR genotypes for F. culmorum depending on the primer. The combined analysis of ISSR data revealed 59 different genotypes clustered into seven distinct clades amongst 75 isolates of F. culmorum examined. All the isolates were assayed to test their aggressiveness on a winter wheat cv. 'Armada'. A significant quantitative variation for aggressiveness was found among the isolates. The ISSR and aggressiveness variation existed on a macro- as well as micro-geographical scale. The data suggested a long-range dispersal of F. culmorum and indicated that this fungus may have been introduced into Canada from Europe. In addition to the high level of intraspecific diversity observed in F. culmorum, the index of multilocus association calculated using ISSR data indicated that reproduction in F. culmorum cannot be exclusively clonal and recombination is likely to occur.