Multiple sites of replication initiation in the human beta-globin gene locus

The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5′ to the β-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined β-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site ~20 kb upstream is ~4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.     

Seed-specific,developmentally regulated genes of peanut

Four cDNAs of seed-specific and developmentally regulated peanut (Arachis hypogaea L.) genes were identified by differential screening of a peanut-seed cDNA library using cDNA probes constructed from mRNAs isolated from immature and mature stages of the seed. Northern analysis, probed with the four cloned cDNAs, indicated that the genes represented by two cDNAs were expressed abundantly early in seed development, while another two were abundantly expressed later at the cell-expansion stages of seed development. These four genes did not show expression in roots, pegs or leaves. However, one of the early expressed genes was seed coat-specific. One of the clones, Psc11, had significant sequence similarity to subtilisin-like genes in Arabidopsis and soybean. Clones Psc32 and Psc33 had significant similarity to the peanut allergen genes Ara h II and Ara h 6, respectively. The sequence of clone Psc12 was unique and did not show significant similarity to any sequence in the databases. One of the four seed-specific clones showed restriction fragment length polymorphism (RFLP) among peanut lines representing the four peanut botanical varieties. These findings indicate that polymorphism exists in peanut seed-storage genes. This contrasts with other genes previously used for genetic mapping of cultivated peanut. Received: 1 September 2000 / Accepted: 4 May 2001     

Endocytosis of cholera toxin by human enterocytes is developmentally regulated

Many secretory diarrheas including cholera are more prevalent and fulminant in young infants than in older children and adults. Cholera toxin (CT) elicits a cAMP-dependent chloride secretory response in intestinal epithelia, which accounts for the fundamental pathogenesis of this toxigenic diarrhea. We have previously reported that the action of this bacterial enterotoxin is excessive in immature enterocytes and under developmental regulation. In this study, we tested the hypothesis that enhanced endocytosis by immature human enterocytes may, in part, account for the excessive secretory response to CT noted in the immature intestine and that enterocyte endocytosis of CT is developmentally regulated. To test this hypothesis, we used specific inhibitors to define endocytic pathways in mature and immature cell lines. We showed that internalization of CT in adult enterocytes is less and occurs via the caveolae/raft-mediated pathway in contrast to an enhanced immature human enterocyte CT uptake that occurs via a clathrin pathway. We also present evidence that this clathrin pathway is developmentally regulated as demonstrated by its response to corticosteroids, a known maturation factor that causes a decreased CT endocytosis by this pathway.