Predicting Effects of Water Regime Changes on Waterbirds: Insights from Staging Swans

Predicting the environmental impact of a proposed development is notoriously difficult, especially when future conditions fall outside the current range of conditions. Individual-based approaches have been developed and applied to predict the impact of environmental changes on wintering and staging coastal bird populations. How many birds make use of staging sites is mostly determined by food availability and accessibility, which in the case of many waterbirds in turn is affected by water level. Many water systems are regulated and water levels are maintained at target levels, set by management authorities. We used an individual-based modelling framework (MORPH) to analyse how different target water levels affect the number of migratory Bewick’s swans Cygnus columbianus bewickii staging at a shallow freshwater lake (Lauwersmeer, the Netherlands) in autumn. As an emerging property of the model, we found strong non-linear responses of swan usage to changes in water level, with a sudden drop in peak numbers as well as bird-days with a 0.20 m rise above the current target water level. Such strong non-linear responses are probably common and should be taken into account in environmental impact assessments.     

A luminescence probe for metallothionein in liver tissue: emission intensity measured directly from copper metallothionein induced in rat liver

We report the first use of an emission probe based on the Cu(I)-thiolate chromophore, for the direct observation of copper metallothionein located in samples of rat liver. Elevated synthesis of Cu-MT in the rat liver was induced by subcutaneous injections of a series of aqueous CuCl2 solutions containing increasing amounts of Cu(II). Luminescence intensity in the 600 nm region, detected from frozen solutions of Cu-MT and from slices of the liver frozen at 77 K, following excitation in the 300 nm region, was dependent on the concentration of the Cu(II) used in the inducing solution. No such luminescence intensity was found for control samples obtained from the livers of rats not exposed to copper salts. It is suggested that this new method will allow direct visualization of Cu-MT in tissue where genetic disorders impare copper metabolism.     

Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro

The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described. CAF-I is a multisubunit protein that, when added to a crude cytosol replication extract, promotes chromatin assembly on replicating SV40 DNA. Chromatin assembly by CAF-I requires and is coupled with DNA replication. The minichromosomes assembled de novo by CAF-I consist of correctly spaced nucleosomes containing the four core histones H2A, H2B, H3, and H4, which are supplied in a soluble form by the cytosol replication extract. Thus, by several criteria, the CAF-I-dependent chromatin assembly reaction described herein reflects the process of chromatin formation during DNA replication in vivo.     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.     

Identification of cellular components required for SV40 DNA replication in vitro

To investigate the cellular proteins involved in simian virus 40 (SV40) replication, extracts derived from human 293 cells have been fractionated into multiple components. When such fractions are combined with the virus-encoded T antigen (TAg) and SV40 origin containing plasmid DNA, efficient and complete replication is achieved, while each fraction alone is inactive. At present, a minimum of eight such cellular components have been identified. Previous experiments have demonstrated one of these to be the cell-cycle-regulated proliferating-cell nuclear antigen (PCNA). As PCNA has been identified as a processivity factor for DNA polymerase delta, we suggest that both polymerases alpha and delta are involved in this system. Three further fractions have been identified. One is a partially purified fraction which, under certain conditions, is required with TAg for the formation of a pre-synthesis complex of proteins at the replication origin. The second of these factors, RF-A, is a complex of three polypeptides which may function as a eucaryotic SSB. The third, RF-C, is a factor which is required, with PCNA, for coordinated leading- and lagging-strand synthesis at the replication fork. Complete synthesis and segregation of the daughter molecules also requires the presence of topoisomerases I and II. These results suggest a model for DNA synthesis which involves multiple stages prior to and during replicative DNA synthesis.     

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.     

In vitro culture of tissue from the tunicateStyela clava

Summary Pharyngeal explants and circulatory hemocytes from the tunicateStyela clava were cultured in a medium containing tunicate plasma, artificial seawater, RPMI 1640, and antibiotics. Pharnngeal tissue remained viable and proliferated for up to 72 d in vitro. Proliferative activity maintained the pool of hemocytes within explants and facilitated the migration of pharyngeal hemocytes from explants into culture supernatants. The diversity of morphologically distinct cell types within the hemocyte pool of pharyngeal cultures indicated that cell division was followed by regulated differentiation. In contrast to pharyngeal cultures, suspensions of circulatory hemocytes did not survive for prolonged periods in vitro. Proliferative activity could not be detected in circulatory hemocyte cultures. These results are discussed in terms of the differentiation state of hemocytes and the efficacy of culture conditions. This study was supported by the National Science Foundation, Washington, DC (grant DCB 85 19848) and by BRSG funds from UCLA Schools of Medicine and Dentistry. Flow cytometric facilities were sponsored in part by a Johnson Cancer Center Core Grant (CA 16042). David A. Raftos is a Fulbright Postdoctoral Fellow and recipient of a Frederik B. Bang Scholarship in Marine Invertebrate Immunology administered by the American Association of Immunologist. Dan L. Stillman was supported by an REU supplement from the National Science Foundation.