The cell cycle program of polypeptide labeling in Chlamydomonas reinhardtii

The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter- conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide

Studies of the Organization and Expression of Individual Repetitive Sequence Families of the Sea Urchin Genome

Individual repetitive DNA sequence families of the sea urchin Strongylocentrotus purpuratus were investigated with regard to their genomic organization, the internal structure of their members, and the structural and developmental characteristics of their RN A transcripts. Analysis by gel blot hybridization and reassociation kinetics of cloned genomic DNA fragments containing members of three specific repeat families reveals a different pattern of organization in each case. One family is organized into long regions of repeated DNA, usually containing several members of the family in a tandem or clustered arrangement. A second family exists as long repeated elements occurring only once in a local genomic region. The third family consists of short repetitive sequence elements which are generally flanked on either side by single-copy sequences. The internal structure of eight cloned repetitive sequence elements was examined by determination of their nucleotide sequences. The lack of sequence homology among the eight elements indicates that they are representative of distinct repeat families. For the most part they consist of complex sequence internally, with a minor fraction of the length of five of the eight occupied by direct or inverse sequence repetitions. Six of the eight sequences are not translatable. Comparison of the nucleotide sequences of three different members of the same repeat family reveals that they are not simply colinear sequence variants, but that they differ in the presence and/or arrangement of small sequence subelements. Hybridization with cloned repetitive sequence elements was used to demonstrate that the level of representation of specific repeat sequences is quantitatively similar in the egg RNA of two sea urchin species, S. purpuratus and S. franciscanus. Egg and embryo polyadenylated RNAs bearing specific repetitive sequences were analyzed by cDNA cloning, DNA and RNA gel blot hybridization, and DNA sequencing. It was found that the two complements of a given repeat are carried on different sets of polyadenylated transcripts, which are generally quite long (&gt; 3 kilobases, with an estimated number average length of 5-6 kilobases). Within these transcripts, specific short repetitive sequence elements are found interspersed either with single-copy sequences or with other repeat sequences. It is demonstrated by sequencing that one such repeat-containing region is not translatable. The sets of polyadenylated transcripts deriving from several individual repeat families undergo substantial quantitative and probably qualitative modulation during early sea urchin development. Analysis of specific transcripts with single-copy probes from repeat-containing cDNA clones indicates that the embryo genome is transcribed to produce at least some of the same interspersed RNAs as are stored in the oocyte during oogenesis. Finally, the transcripts bearing specific repeat sequences in the polyadenylated egg RNA of two related sea urchin species were found to be qualitatively dissimilar.</p

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

Identification of functional elements and regulatory circuits by Drosophila modENCODE

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns

Repetitive sequence transcripts in development

Interspersed repetitive sequences are represented widely in animal cell nuclear RNAs, in the poly(A) RNA stored in eggs and in some mRNAs. Their expression is developmentally modulated. Although the genomic location of repetitive sequences may change rapidly during evolution, the patterns of their transcription suggest a variety of possible functions

Repetitive sequence transcripts in development

Interspersed repetitive sequences are represented widely in animal cell nuclear RNAs, in the poly(A) RNA stored in eggs and in some mRNAs. Their expression is developmentally modulated. Although the genomic location of repetitive sequences may change rapidly during evolution, the patterns of their transcription suggest a variety of possible functions

Role of Architecture in the Function and Specificity of Two Notch-Regulated Transcriptional Enhancer Modules

<div><p>In <em>Drosophila melanogaster</em>, <em>cis</em>-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities.</p> </div