We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

/pdf/identification-and-analysis-of-functional-elements-in-1-of-2o640l8ikl.pdf

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project

A unified view of polymer, dumbbell, and oligonucleotide nearest-neighbor (NN) thermodynamics is presented DNA NN DG° 37 parameters from seven laboratories are presented in the same format so that careful comparisons can be made The seven studies used data from natural polymers, synthetic polymers, oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN parameters; used dif- ferent methods of data analysis; used different salt concen- trations; and presented the NN thermodynamics in different formats As a result of these differences, there has been much confusion regarding the NN thermodynamics of DNA poly- mers and oligomers Herein I show that six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies re- main Further, a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodynamics Empirical salt dependencies are also derived for oligonucleotides and polymers

/pdf/a-unified-view-of-polymer-dumbbell-and-oligonucleotide-dna-1r0s8psdu0.pdf

A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics

Transcription of the c-fos proto-oncogene is greatly increased within minutes of administering purified growth factors to quiescent 3T3 cells. This stimulation is the most rapid transcriptional response to peptide growth factors yet described, and implies a role for c-fos in cell-cycle control. Transformation by c-fos may result from a temporal deregulation of this control.

Stimulation of 3T3 cells induces transcription of the c- fos proto-oncogene

Although the importance of the polyelectrolyte character of DNA has been recognized for some time (Felsenfeld & Miles 1967), few of the implications have been explored, primarily because of a lag in translating the breakthroughs in polyelectrolyte theory of the last decade into a form that is well adapted to the analysis of the specialized problems of biophysical chemistry. Perhaps an analogous situation existed in the field of protein chemistry during the period after the formulation and confirmation of the Debye—Huckel theory of ionic solutions but before Scatchard's incorporation of the theory into his analysis of the binding properties of proteins. An achievement for polynucleotide solutions parallel to Scatchard's was recently presented by Record, Lohman, & de Haseth (1976) and further developed and reviewed by Record, Anderson & Lohman (1978).

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0033583500002031

The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides.

Epstein-Barr virus.

https://delangelab.rockefeller.edu/pubs/111_Sfeir_Cell2009.pdf

Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication

Data on the decrease of the DNA melting temperature Tm with the salt concentration M are reported and discussed. The electrostatic free energy change in the helix–coil transition, ΔFe is related to the potential, ψ, which represents the electrostatic repulsion between the phosphate charges; ψ is calculated as a function of M and of the distances between the charges of the two strands. The Debye-Huckel approximation is shown to overestimate ψ. It is suggested that the high local concentration of the counterions in the immediate vicinity of the fixed charges screen these charges from interacting with other fixed charges, to the extent that the system behaves as if the fixed ions carry a reduced charge. The notion of a reduced charge represents in a single parameter the deviation of the Debye-Huckel approximation from the true potential. A plot of Tm versus ΔFe gives a straight line as predicted. ΔH0 is calculated from the slope and found to be consistent with experimentally determined values. Our calculations support the hypothesis that the change of Tm with salt concentration is due to changes in the screened interactions between the fixed phosphate charges. In analyzing the results of these calculations, we are able on the one hand to indicate some of the limitations of the theoretical model and, on the other hand, draw some conclusions about the order of magnitude of the nonelectrostatic interaction energy of formation of the double helix.

Dependence of the melting temperature of DNA on salt concentration

https://www.cell.com/cell/pdf/0092-8674(89)90433-9.pdf

The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication

In a comprehensive study, the temporal replication of tissue-specific genes and flanking sequences was compared in nine cell lines exhibiting different tissue-specific functions. Some of the rules we have determined for the replication of these tissue specific genes include the following. (i) Actively transcribed genes usually replicate during the first quarter of the S phase. (ii) Some immunoglobulin genes replicate during the first half of S phase even when no transcriptional activity is detected but appear to replicate even earlier in cell lines where they are transcribed. (iii) Nontranscribed genes can replicate during any interval of S phase. (iv) Multigene families arranged in clusters of 250 kilobases or less define a temporal compartment comprising approximately one-quarter of S phase. While these rules, and others that are discussed, apply to the tissue-specific genes studied here, all tissue-specific genes may not follow this pattern. In addition, housekeeping genes did not follow some of these rules. These results provide the first molecular evidence that the coordinate timing of replication of contiguous sequences within a multigene family is a general property of the mammalian genome. The relationship between replication very early during S phase and the transcriptional activity within a chromosomal domain is discussed.

Replication program of active and inactive multigene families in mammalian cells.

We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating molecules was achieved by isolating S-phase enriched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedure that enriches for molecules containing single-stranded regions, a characteristic of replication forks. Two-dimensional agarose gel electrophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscribed spacer (NTS) of human ribosomal DNA but not within the 13-kb transcription unit or adjacent regulatory elements. Although initiation events were detected throughout the majority of the NTS, some regions may initiate more frequently than others. Termination of replication, the convergence of opposing replication forks, was found throughout the ribosomal DNA repeat units, and, in some repeats, specifically at the junction of the 3' end of the transcription unit and the NTS. This site-specific termination of replication is the result of pausing of replication forks near the sites of transcription termination. The naturally occurring multicopy rRNA gene family offers a unique system to study mammalian DNA replication without the use of chemical synchronization agents.

Carl L. Schildkraut

Papers

Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication

Dependence of the melting temperature of DNA on salt concentration

The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication

Replication program of active and inactive multigene families in mammalian cells.

Initiation and termination of DNA replication in human rRNA genes.