Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general pol II transcription factors have been elusive. The protozoan parasite Trypanosoma brucei branched early in eukaryotic evolution and is probably the most divergent well-studied eukaryote. Many discoveries of general interest have been made in T. brucei, emphasizing its value for understanding the evolution of core molecular processes.Transcription of protein-coding genes by RNA polymerase II (pol II) in T. brucei differs in two important aspects from most other eukaryotes. First, transcription is polycistronic: Arrays of sometimes >100 genes are transcribed in polycistronic transcription units (PTUs). This organization is reminiscent of operons in prokaryotes except that there is no evidence to suggest clustering of functionally related genes in T. brucei. Second, mRNAs are separated post-transcriptionally by coupled splicing and polyadenylation reactions that add a 39-nucleotide (nt) ''spliced-leader'' to every mRNA (for review, see Liang et al. 2003). Within a PTU, genes are transcribed from the same strand, but transcription of two neighboring PTUs can either be convergent or divergent. The regions between PTUs are referred to as strand switch regions (SSRs). Strand-specific nuclear run-on assays performed in Leishmania (Martinez-Calvillo et al. 2003), a genus related to T. brucei, have shown that pol II transcription starts at SSRs between two transcriptionally divergent PTUs (divergent SSRs) and ends at SSRs between two transcriptionally convergent PTUs (convergent SSRs). Because 75% of all Leishmania major genes can be found in the same genomic context in T. brucei (El-Sayed et al. 2005), indicating a high degree of synteny, it is reasonable to hypothesize that divergent SSRs in T. brucei are transcription start s...

Evolvability—the capacity to generate beneficial heritable variation—is a central property of biological systems. However, its origins and modulation by environmental factors have not been examined systematically. Here, we analyze the fitness effects of all single mutations in TEM-1 β-lactamase (4,997 variants) under selection for the wild-type function (ampicillin resistance) and for a new function (cefotaxime resistance). Tolerance to mutation in this enzyme is bimodal and dependent on the strength of purifying selection in vivo, a result that derives from a steep non-linear ampicillin-dependent relationship between biochemical activity and fitness. Interestingly, cefotaxime resistance emerges from mutations that are neutral at low levels of ampicillin but deleterious at high levels; thus the capacity to evolve new function also depends on the strength of selection. The key property controlling evolvability is an excess of enzymatic activity relative to the strength of selection, suggesting that fluctuating environments might select for high-activity enzymes.

Transcription of protein-coding genes in trypanosomes is polycistronic and gene expression is primarily regulated by post-transcriptional mechanisms. Sequence motifs in the untranslated regions regulate mRNA trans-splicing and RNA stability, yet where UTRs begin and end is known for very few genes. We used high-throughput RNA-sequencing to determine the genome-wide steady-state mRNA levels (‘transcriptomes’) for ∼90% of the genome in two stages of the Trypanosoma brucei life cycle cultured in vitro. Almost 6% of genes were differentially expressed between the two life-cycle stages. We identified 5′ splice-acceptor sites (SAS) and polyadenylation sites (PAS) for 6959 and 5948 genes, respectively. Most genes have between one and three alternative SAS, but PAS are more dispersed. For 488 genes, SAS were identified downstream of the originally assigned initiator ATG, so a subsequent in-frame ATG presumably designates the start of the true coding sequence. In some cases, alternative SAS would give rise to mRNAs encoding proteins with different N-terminal sequences. We could identify the introns in two genes known to contain them, but found no additional genes with introns. Our study demonstrates the usefulness of the RNA-seq technology to study the transcriptional landscape of an organism whose genome has not been fully annotated.

Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence-encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine.

The internal mechanics of proteins—the coordinated motions of amino acids and the pattern of forces constraining these motions—connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

Oligonucleotide microarrays are based on the hybridization of labeled mRNA molecules to short length oligonucleotide probes on a glass surface. Two effects have been shown to affect the raw data: the sequence dependence of the probe hybridization properties and the chemical saturation resulting from surface adsorption processes. We address both issues simultaneously using a physically motivated hybridization model. Based on publicly available calibration data sets, we show that Langmuir adsorption accurately describes GeneChip hybridization, with model parameters that we predict from the sequence composition of the probes. Because these parameters have physical units, we are able to estimate absolute mRNA concentrations in picomolar. Additionally, by accounting for chemical saturation, we substantially reduce the compressive bias of differential expression estimates that normally occurs toward high concentrations.

Contingency, the persistent influence of past random events, pervades biology. To what extent, then, is each course of ecological or evolutionary dynamics unique, and to what extent are these dynamics subject to a common statistical structure? Addressing this question requires replicate measurements to search for emergent statistical laws. We establish a readily replicated microbial closed ecosystem (CES), sustaining its three species for years. We precisely measure the local population density of each species in many CES replicates, started from the same initial conditions and kept under constant light and temperature. The covariation among replicates of the three species densities acquires a stable structure, which could be decomposed into discrete eigenvectors, or "ecomodes." The largest ecomode dominates population density fluctuations around the replicate-average dynamics. These fluctuations follow simple power laws consistent with a geometric random walk. Thus, variability in ecological dynamics can be studied with CES replicates and described by simple statistical laws.

Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear and kinetoplast DNA. Others have since assumed the consistency of morphological indicators of cellcycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase. KeywordsCell cycle; Deconvolution microscopy; DNA quantification; Fluorescence-activated cell sorting; Histone modification; Trypanosoma brucei Members of the order Kinetoplastidae are characterized by the presence of the kinetoplast, the uniquely structured mitochondrial DNA that consists of an interlocked network of several thousand minicircles and more than twenty maxicircles. Understanding the mechanism of kinetoplast DNA (kDNA) replication has been of great interest [1,2]. Initiation of kDNA and nuclear DNA (nDNA) replication probably coincide, but kDNA segregation is completed before the onset of mitosis [3]. Any cell can hence be assigned to a specific cell-cycle stage by comparing nuclear and kinetoplast morphology. For example, cells with an elongated kinetoplast would be in the G2. However, our recent studies suggested that elongated kinetoplasts exist in nuclear S phase as well as [4].The goal of this study was to determine the cell-cycle stage of individual cells solely by their nDNA content. Using modern fluorescence microscopy and deconvolution algorithms, we * Corresponding author. Tel.: +1 212 327 7571; fax: +1 212 327 7845. E-mail address: george.cross@rockefeller.edu (G.A.M. Cross). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Quantification of images requires images of high quality. Spherical aberration, nonuniform illumination, and differential response of sensor pixels, need to be avoided or corrected for. Spherical aberration occurs when light waves passing through the periphery of...