Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of plasmid-encoded FLP protein and two recombination sites on the plasmid. The recombination site possesses a specific orientation, which is determined by an asymmetric 8-base pair spacer sequence separating two 13-base pair inverted repeats. The outcome or directionality of site-specific recombination is defined by the alignment of two sites in the same orientation during the reaction. Sites containing point mutations or 1-base pair insertions or deletions within the spacer generally undergo recombination with unaltered sites at reduced levels. In contrast, recombination between the two identical mutant sites (where homology is restored) proceeds efficiently in all cases. Sites containing spacer sequences of 10 base pairs or more are nonfunctional under all conditions. A recombination site in which 5 base pairs are changed to yield an entirely symmetrical spacer sequence again recombines efficiently, but only with an identical site. This reaction, in addition, produces a variety of new products which can only result from random alignment of the two sites undergoing recombination, i.e. the reaction no longer exhibits directionality. These and other results demonstrate that both the efficiency and directionality of site-specific recombination is dependent upon homology between spacer sequences of the two recombining sites. This further implies that critical DNA-DNA interactions between the spacer region of the two sites involved in the reaction occur at some stage during site-specific recombination in this system. The specific spacer sequence itself appears to be unimportant as long as homology is maintained; thus, these sequences are probably not involved in recognition by FLP protein.     

The impact of abasic sites on DNA flexibility

We use internal coordinate molecular mechanics calculations to study the impact of abasic sites on the conformation and the mechanics of the DNA double helix. Abasic sites, which are common mutagenic lesions, are shown to locally modify both the groove geometry and the curvature of DNA in a sequence dependent manner. By controlled twisting and bending, it is also shown that these lesions modify the deformability of the duplex, generally increasing its flexibility, but again to an extent which depends on the nature of the abasic site and on the surrounding base sequence. Both the conformational and mechanical influence of this type of DNA damage may be significant for recognition and repair mechanisms.     

Development of gonadotrophin-binding sites in the immature rat ovary.

There was a temporal relationship between ovarian development and the sites to which 125I-labelled gonadotrophins became bound. Labelled human LH and FSH bound uniformly to 5- and 15-day-old ovaries. At 21 days, heavy FSH and light LH binding was observed over the granulosa cells increased at Day 33 and again at Day 38. Quantitative determination by gamma-ray spectrophotometry of binding to ovarian sections showed that LH binding gradually increased with advancing age, but FSH binding remained relatively constant between Days 5 and 33 with a significant increase between Days 33 and 38.     

Thermodynamic Framework of the Interaction between Protein and Solvent Drives Protein Folding

Abstract

We use internal coordinate molecular mechanics calculations to study the impact of abasic sites on the conformation and the mechanics of the DNA double helix. Abasic sites, which are common mutagenic lesions, are shown to locally modify both the groove geometry and the curvature of DNA in a sequence dependent manner. By controlled twisting and bending, it is also shown that these lesions modify the deformability of the duplex, generally increasing its flexibility, but again to an extent which depends on the nature of the abasic site and on the surrounding base sequence. Both the conformational and mechanical influence of this type of DNA damage may be significant for recognition and repair mechanisms.     

Lipid associated calcium ionophores in islet cell plasma membrane following glucose stimulation

The effect of glucose exposure on lipid associated calcium ionophoretic activity was measured in cultured neonatal rat pancreatic islet cells using two model systems. The first measured the ability of a lipid extract of islet cells to facilitate calcium transfer from an aqueous to organic phase and thus detected lipids which transfer calcium in the manner of authentic ionophores or which chelate the ion. In this system glucose stimulation was followed by an increase in total cell ionophoretic activity and a decrease in the activity associated with the plasma membrane. The second system measured the transfer of calcium across an artificial phospholipid membrane and detected authentic ionophoretic activity. In this model an increase in total ionophoretic activity was again seen following glucose but there was no change in the ionophoretic activity of a plasma membrane extract. The results indicate that the lipid modifications which accompany glucose-induced insulin release may alter cellular calcium stores by decreasing lipid bound calcium at the plasma membrane and increasing the capacity for calcium ionophoresis at intracellular sites.     

Increase in initiation sites for chromatin directed RNA synthesis by acetylation of chromosomal proteins.

Treatment of rat liver chromatin with 0.7 mM acetic anhydride (1) leads to an approximately twofold increase in initiation sites for DNA-dependent RNA polymerase from $\text{E.}\text{coli}$. With reconstituted chromatin, in which only the histone moiety was acetylated, again a twofold increase in initiation sites could be observed, compared to control chromatin which had undergone the dissociation and reassociation procedure, but which had not been exposed to acetic anhydride.     

Seasonal patterns of population structure in a colonial marine invertebrate (Bugula stolonifera, Bryozoa)

For sessile invertebrates, the degree to which dispersal mechanisms transport individuals away from their natal grounds can have significant ecological implications. Even though the larvae of the marine bryozoan Bugula stolonifera have limited dispersal potential, high levels of genetic mixing have been found within their conspecific aggregations. In this study, we investigated whether this high mixing within aggregations of B. stolonifera also resulted in high mixing between aggregations. Adult colonies were collected from five sites within and one site outside of Eel Pond, Woods Hole, Massachusetts, in August 2009 and genotyped at 10 microsatellite loci. Significant genotypic differentiation was found between most sites, suggesting limited connectivity across sites, even those separated by only 100 m. This investigation was extended to determine if low levels of genetic mixing throughout the reproductive season could result in increased homogeneity between sites. Four of the five sites in Eel Pond were sampled early, mid-, and late in the reproductive season in 2010, and again in early 2011. Inter- and intra-annual genotypic differentiation was then assessed within and between sites. Results from these analyses document that low levels of mixing could result in increased homogeneity between some aggregations, but that barriers to genetic exchange prevented mixing between most sites. Further, results from inter-annual comparisons within sites suggest that any potential homogeneity achieved throughout the reproductive season will likely be lost by the beginning of the next reproductive season due to the annual cycle of colony die-back and regrowth experienced by B. stolonifera colonies in Eel Pond.     

Fusion between myogenic cells in Vivo: An ultrastructural study in regenerating murine skeletal muscle

Fusion of myogenic cells in adult murine skeletal muscle regenerating in vivo was examined at the ultrastructural level. Fusion of myoblast to myoblast, myoblast to myotube, and myotube to myotube was observed by 4 to 5 days after injury. Fusion between myogenic cells (myoblasts or myotubes) lacking a definitive glycocalyx or external lamina (basal lamina) occurred at multiple sites. It was defined by zones of cytoplasmic confluence between apposed cells at sites where contiguous segments of the cell membranes were interrupted while their edges had united resulting in linear continuity; vesicles of varying dimensions were frequent in these areas of fusion. Myoblasts were seen invaginating the surface of myofibres and again vesicles were seen in abundance in such regions. Cilia were often observed at this junctional zone suggesting that they might play a role in fusion. In the one example of probable fusion between a myotube and a myofibre, only a single area of cytoplasmic continuity was apparent.     

Non-defendable resources affect peafowl lek organization: a male removal experiment

A lekking mating system is typically thought to be non-resource based with male providing nothing to females but genes. However, males are thought to clump their display sites on areas where they are more likely to encounter females, which may depend on non-defendable resource location. We tested this hypothesis on a feral population of peacocks. In agreement, we found that, within the lek, display site proximity to food resources had an effect on female visitation rate and male mating success. The attractiveness of display sites to male intruders was explained by the distance to the feeding place and by the female visitation rate. We randomly removed 29 territorial males from their display sites. Display sites that were more attractive to male intruders before removal remained highly attractive after removal and display sites closer to the feeding area attracted the attention of intruders significantly more after removal. Similarly, display sites that were more visited by females before removal remained more visited after removal, suggesting again that the likelihood of encountering females is determined by the display site location. Overall, these results are in agreement with non-defendable resources affecting lek spatial organization in the peafowl.     

Contemporary Land Change Alters Fish Communities in a San Francisco Bay Watershed,California, U.S.A.

Urbanization is one of the leading threats to freshwater biodiversity, and urban regions continue to expand globally. Here we examined the relationship between recent urbanization and shifts in stream fish communities. We sampled fishes at 32 sites in the Alameda Creek Watershed, near San Francisco, California, in 1993–1994 and again in 2009, and we quantified univariate and multivariate changes in fish communities between the sampling periods. Sampling sites were classified into those downstream of a rapidly urbanizing area (“urbanized sites”), and those found in less impacted areas (“low-impacted sites”). We calculated the change from non-urban to urban land cover between 1993 and 2009 at two scales for each site (the total watershed and a 3km buffer zone immediately upstream of each site). Neither the mean relative abundance of native fish nor nonnative species richness changed significantly between the survey periods. However, we observed significant changes in fish community composition (as measured by Bray-Curtis dissimilarity) and a decrease in native species richness between the sampling periods at urbanized sites, but not at low-impacted sites. Moreover, the relative abundance of one native cyprinid (Lavinia symmetricus) decreased at the urbanized sites but not at low-impacted sites. Increased urbanization was associated with changes in the fish community, and this relationship was strongest at the smaller (3km buffer) scale. Our results suggest that ongoing land change alters fish communities and that contemporary resurveys are an important tool for examining how freshwater taxa are responding to recent environmental change.     

Transient modulation and internalization of T4 antigen induced by phorbol esters

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.     

An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1.

A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis. The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacterium bovis BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coli; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.     

Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components

Retinol (vitamin A alcohol) is involved in the proper differentiation of epithelia. The mechanism of this involvement is unknown. We have previously reported that purified cellular retinol-binding (CRBP) will mediate specific binding of retinol to nuclei isolated from rat liver. We now report that pure CRBP delivers retinol to the specific nuclear binding sites without itself remaining bound. Triton X-100-treated nuclei retain the majority of these binding sites. CRBP is also capable of delivering retinol specifically to isolated chromatin with no apparent loss of binding sites, as compared to whole nuclei. CRBP again does not remain bound after transferring retinol to the chromatin binding sites. When isolated nuclei are incubated with [3H]retinol- CRBP, sectioned, and autoradiographed, specifically bound retinol is found distributed throughout the nuclei. Thus, CRBP delivers retinol to the interior of the nucleus, to specific binding sites which are primarily, if not solely, on the chromatin. The binding of retinol to these sites may affect gene expression.     

Responses of Dryas octopetala to ITEX environmental manipulations: a synthesis with circumpolar comparisons

We have examined organismic responses of Dryas octopetala to simulated changes in the summer climate at four tundra sites as part of the International Tundra Experiment (ITEX). Our study sites are located in the High Arctic, on Svalbard, Norway, in the Low Arctic at Abisko, Sweden, and at Toolik Lake, Alaska, USA and our temperate alpine site is at Niwot Ridge, Colorado, USA. These sites represent a range of tundra temperature and precipitation regimes, being generally cold and dry in the High Arctic and warmer and wetter at Toolik Lake and Niwot Ridge. Results from our studies indicate organismic attributes such as flowering shoot length varies by 30% between low and high arctic populations and that experimental warming results in significant increases in shoot height at three of four sites. We find that phenological development of Dryas is accelerated under experimentally warmed conditions which corresponds with a lengthening of the growing season in autumn, greater degrees of seed set and a higher likelihood of colonization of bare ground. We also observe that Dryas dominated ecosystems which are exposed to experimental manipulations are capable of exhibiting net carbon sequestration in late autumn, and that Dryas photosynthesis and green leaf biomass is significantly greater under warmer as opposed to ambient temperature conditions. Dryas leaf nitrogen is also significantly lowered under warmer conditions resulting in senescent leaves having a higher C:N ratio than those under ambient conditions. Together these findings indicate that Dryas phenology and carbon flux may be altered to the greatest degree in spring and again in autumn by higher summer temperatures and that simultaneously both positive and negative feedback effects may result from changes in plant and ecosystem performance.     

Scanning independent ribosomal initiation of the Sendai virus Y proteins in vitro and in vivo.

The Sendai virus P/C mRNA contains five ribosomal initiation sites between positions 81 and 201 from the 5' end. One of these sites initiates in the P open reading frame (ORF) (ATG/104), whereas four initiate in the C ORF (ACG/81 and ATGs/114, 183, 201), to give a nested set of C proteins (C', C, Y1, Y2). Introduction of new ATGs or physically breaking the mRNA upstream of these natural sites was used in vitro to prevent ribosomal scanning downstream. The results suggest that a minority of the ribosomes which initiate C (ATG/114) and all of those which initiate Y1 and Y2 (ATGs/183 and 201) do so by a scanning independent mechanism. When the leaky ACG/81 site is changed to a non-leaky ATG site in in vivo experiments, ribosomal initiation at Y is again not diminished, whereas that at C as well as at P becomes undetectable. Ribosomal initiation at Y appears to be scanning independent in vitro and in vivo. That at C is partly independent in vitro, but completely dependent in vivo. These results are discussed in terms of a model of internal initiation at Y.     

The importance of incorporating age and sex when backcalculating length in bullhead Cottus gobio

The present study backcalculated body length for a data set of a bullhead Cottus gobio population located at different sampling sites in a river network. Model comparison between various growth models, which included successively new parameters, showed the effect and importance of taking sex, age and the location in the river network into account. The data sets obtained by backcalculation were fitted by the von Bertalanffy growth function, which revealed the effect of the backcalculation formula on the estimation of the von Bertalanffy growth parameters. Fitting results and parameter estimates showed again the importance of incorporating age and sex when backcalculating body length in the C. gobio population studied.     

The binding of metal ions to bovine factor IX

The binding isotherms of Ca2+ and Mn2+ to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25 degrees C. At pH 7.4, at least 2 strong Ca2+ sites, with an average KDISS of 0.1 +/- 0.02 mM, are found. An additional 10 to 11 weaker Ca2+ binding sites, with an average KDISS of 1.3 +/- 0.2 mM are noted, at high levels of Ca2+. At pH 6.5, again at least 2 strong Ca2+ sites on factor IX are evident, with an average KDISS of 0.11 +/- 0.02 mM; but slightly fewer (7 to 8) weaker sites, with an average KDISS of 1.9 +/- 0.3 mM, are obtained. Qualitatively, the binding of Mn2+ to bovine factor IX appears similar to that of Ca2+. At pH 6.5, approximately 2 strong Mn2+ binding sites, with an average KDISS of 13 +/- 1.5 micrometer, and an additional 5 to 6 weak sites, with an average KDISS of 160 +/- 15 micrometer, are present. Manganese does not completely displace Ca2+; and Ca2+ does not completely displace Mn2+ from their respective binding sites. On the other hand, Tb3+ and Sm3+ readily displace Ca2+, at pH 6.5, from its sites on factor IX. The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system. Substitution of Sr2+ for Ca2+ leads to approximately 50% of the maximum rate of factor IXa formation, and final yield of factor IXa, in this activation system. Manganese does not substitute for Ca2+ in this activation, but does inhibit the stimulatory effect of Ca2+. Both Tb3+ and Sm3+ are effective inhibitors of Ca2+ in factor IX activation by factor XIa.