Inter- and intramolecular reductive coupling reactions: an approach to the phorbol skeleton.

[reaction: see text] An expeditious convergent route to the ABC-tricyclic core of the phorbol esters is described. The chemistry capitalizes upon both inter- and intramolecular reductive coupling processes promoted electrochemically and via the use of samarium diiodide.

Atom transfer reactions of TMM diyls directed toward the synthesis of rudmollin.

Intramolecular hydrogen atom transfer to a trimethylenemethane (TMM) diradical has been explored as a route to the antileukemial agent, rudmollin (1).

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line.

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.

Replicative intermediates of human papillomavirus type 11 in laryngeal papillomas: site of replication initiation and direction of replication.

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180 degrees opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

Isochores and CpG islands in YAC contigs in human Xq26.1-qter.

GC levels were assessed at 37 loci across 30 Mb of Xq26.1-qter, a region physically mapped in overlapping yeast artificial chromosome clones. In 8 Mb of R band Xq26, GC is relatively high (up to 44%) in the proximal 4 Mb and relatively low (40-41%) in the distal 4 Mb. Consistently low GC values (38-41%) are observed in G band Xq27. In contrast, further toward the telomere in Xq28, the GC level rises progressively to reach 52% at 2 to 4 Mb from the end of the chromosome; this region is delimited by low GC loci. Across these regions of Xq, the content of rare-cutter restriction enzyme sites containing CpG, including "CpG islands" in the most completely mapped Xq26-27.1 region, is correlated with GC level. Isochore mapping can thus provide one index of putative gene content across mapped regions.

Genomic organization of human 5 S rDNA and sequence of one tandem repeat.

An organization of human 5 S rDNA repeats is inferred from Southern analyses of restriction digests of genomic DNA fractionated by pulsed-field and conventional gel electrophoreses. A single unit of 2.2 kb is repeated approximately 90 times within a 200-kb fragment (defined by enzymes that do not cleave within individual units, i.e., EcoR1, BglII, HindIII, and PvuII); a comparable number of 5 S sequences are scattered elsewhere in the genome. A lambda clone containing six complete 5 S repeats was obtained from a human placental DNA library. One repeat contains 2231 bp and includes poly(dG-dT).(dC-dA), tracts of polypyrimidine, and an Alu sequence in the spacer region. Also, 5-S-hybridizing clones, containing DNA inserts with an average size of 250 kb, have been obtained as yeast artificial chromosomes. Thus far, four clones have been partially characterized and shown to be 5 S sequences from loci separate from the tandem repeat units.