The Location and Protection Status of Earth’s Diminishing Marine Wilderness

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Bidirectional termination of chromosome replication in Escherichia coli

Summary Autoradiography was used to study the termination of replication of the circular chromosome of Escherichia coli. The experiments were conducted with cells in which termination occurred with a moderate amount of synchrony. Grain tracks were observed that demonstrated the approach at the replication terminus of the two replication forks involved in bidirectional replication. Other grain tracks were formed by replication forks that had met at the replication terminus. The frequency at which these patterns were observed indicates that most, if not all, terminations occur with both replication forks reaching the terminus at approximately the same time.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Location of sites that inhibit progression of replication forks in the terminus region of Escherichia coli.

We used a Southern hybridization assay to locate precisely the sites at which DNA replication is arrested in the terminus region of the Escherichia coli chromosome. The assay was based on the properties of restriction fragments that contain stalled replication forks. Replication forks that entered the terminus from the clockwise direction with respect to the genetic map were inhibited near manA at a site called T2, which we located at kilobase 442 on the physical map of Bouché (J. P. Bouché, J. Mol. Biol. 154:1-20, 1982). Those that entered the terminus region traveling in the counterclockwise direction were inhibited near pyrF at a site called T1, which we located at kilobase 90. In each case we found only a single, precise site of arrest. Inhibition at T1 was not detectable in our assay in strains lacking the trans-acting locus tus, which is located near T2 and is required for T1 to function. We demonstrated that the sites of inhibition are also used during termination of replication in exponentially growing, wild-type cells. In all previous studies on the terminus of E. coli, inhibition has only been detected in strains that were modified so that the origin used was placed near the terminus to force the use of the sites of inhibition.     

Intramembrane client recognition potentiates the chaperone functions of calnexin

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.     

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.     

P1 transduction map spanning the replication terminus of Escherichia coli K12

Summary The region of the E. coli chromosome that contains the replication terminus has not previously been spanned by P1 cotransduction. We have used Tn5, Tn9 and Tn10 transposons inserted in this region as genetic markers, and have constructed a genetic map that extends from fnr (min 29.3) to manA (min 35.7). The relevant transposons that have been mapped in this region and which are described in this report are trgl::Tn5 (min 31.1), zdc-235::Tn10 (min 32.3), zdd-230::Tn9 (min 33.3), and zde-234::Tn10 (min 34.2). The size of this region as determined by P1 cotransduction is very similar to previous estimates obtained by bacterial conjugation.