THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

OBSERVATIONS ON A CHAMAESIPHONACEOIS ALGA (CYANOPHYCEAE) WITH SPECIAL REFERENCE TO EXOCYTE FORMATION1,2

A unicellular cyanophycean culture contaminant had features of both Geitleribactron Kom. and Cyanophanon Geitl. The cells were elongated, sheathless, mostly similar in diameter throughout their length, and attached polarly in rosettes or groups and produced only a single elongated exocyte (=exospore). Young cells were moderately elongated and resembled Geitleribactron. As cells aged, they greatly elongated and then resembled Cyanophanon. Some cells formed Y-shaped bifurcations, features of C. mirabile Geitl. and C. minus Geitl., but they lacked the basal sheath (pseudovagina) of C. mirabile. During exocyte formation, a thick and localized L-II wall layer protuberance extended the exocyte away from the parent cell. This terminal wall thickening then appeared to move to one side from subsequent and unequal cell wall growth. Cells sovnetimes bent abruptly, occasionally opposite a thickening in the L-II wall layer. Further studies in culture of putative Geitleribactron and Cyanophanon isolates are necessary to ascertain the breadth of their structural diversity and the identity of the present taxon.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Individualistic responses of bird species to environmental change

We investigated how the population dynamics of the same bird species varied in different environments, and how the population dynamics of different species varied in the same environment, by calculating long-term population trends for 59 insectivorous songbird species in 22 regions or strata of eastern and central North America using data from the North American Breeding Bird Survey. Of the 47 species that occurred in more than one region 77% increased in some regions and declined in others. Of the 22 regions 91% had some species that increased and others that decreased. There were only slightly more significant correlations between strata in species trends and between species for stratum trends than would be expected by chance. Because of nonlinearities in the data, the actual patterns of population fluctuations of the same species in different regions and of different species in the same region were even more heterogeneous than suggested by our analyses of linear trends. We conclude that these bird species respond to spatial and temporal variation in their environment in a very individualistic fashion. These individualistic responses show that the extrapolation of population trends gained from a few local studies to a larger spatial scale, and the use of a few indicator species to monitor the status of a broader community, are suspect.     

Drought stress of apple trees alters leaf emissions of volatile compounds

Actively growing potted apple trees ( Malus domestica [L.] Borkh. ev. Delicious) unacclimated to drought stress were subjected to drought to determine changes in emissions of leaf volatile compounds. Drought stress was imposed over a 2-week period by weighing pots every 2 or 3 days and adding water hack to an arbitrary and decreasing traction of the original pot weight. Stem water potential was -2.7. -2.0 and -0.8 MPa for the severely stressed, moderately stressed and control trees, respectively. 13 days alter watering treatments were begun. Water use the last 4 days of the experiment was about one-half for the moderately and severely stressed trees compared to that of the controls Twenty-nine volatile compounds were identified by using gas chromatography and mass spectroscopy. Emission rates of hexanal, (E)-2-hexenal, (E)-2-hexen-1-ol, 1-hexenol, hexyl acetate and (E)-2-hexenyl acetate were 5 to 310 times higher for severely stressed trees compared to those of the controls with the moderately stressed trees intermediate. The large increases in hexanal. (E)-2-hexenal and l-hexanol may be related. In enhanced lipoxygenase activity. Volatile compounds are products of metabolism and measurement of their changes after biotic or abiotic stresses will increase understanding of the relationship of changes in plant metabolism by those stresses.     

Root water uptake of field-growing plants indicated by measurements of natural-abundance deuterium

Measurements of stable-isotope ratios of water extracted from stems and, in some studies, soils are increasingly being used to study the integrated root function of field-growing plants. This study explored if additional measurements on water extracted from roots could indicate the activity of roots in different areas of the soil profile and their influence on canopy water sources, so providing advantages over more common sampling strategies. Studies were conducted on trees and shrubs located in diverse habitats: a saline, semi-arid floodplain, a subhumid mountain-range front and a cold desert. At each site, roots, soil immediately surrounding the roots, and plant stems were sampled. Roots were taken from different depths in the soil, to approximately 2 m at one site. Overall, 80% of roots sampled had H isotope ratios different from the surrounding soil. The differences up to 37, were significant (p<0.05) at two of the sites. Thus water in most of the roots sampled did not come entirely, if at all, from the surrounding soil, illustrating movement and possible mixing of water within the root system. This condition was not simply related to the availability of water surrounding the soil, which was also measured. There were also differences in root and stem H isotope ratios (up to 17) in 67% of samples, although the difference was only significant in shallow samples from the floodplain. The general similarity in stem and root ²H values indicates that most roots sampled were involved in the main supply of water to the canopy. Patterns of root function varied between the individual sites. Trees were primarily using groundwater at the floodplain and mountain front sites, as the surface soils had mean matric potentials of-1800 kPa. At the mountain front site, the surface roots were transporting groundwater to the canopy in isolation form the surrounding soil. In contrast, surface roots at the floodplain were taking up water from the surrounding soil, although this water was not a significant source in the trees' overall water supply. This activity of surface roots would not have been evident from the ²H data without the root samples. At the cold desert the roots in moist surface soil provided the main source of water for the shrubs. There too the root data indicated different water uptake patterns than otherwise would have been assumed. The root data showed that groundwater could not have been a water source, a conclusion which had been reached in a previous study. Thus measurements of stable isotope ratios in root water may be a valuable tool in assessing water uptake patterns and root function.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

Phosphorus limitation of forest leaf area and net primary production on a highly weathered soil

We tested the hypothesis that P was the nutrient limiting net primary production of a nativeMetrosideros polymorpha forest on a highly weathered montane tropical soil in Hawaii. A factorial experiment used all combinations of three fertilizer treatments: nitrogen (N), phosphorus (P) and a mix of other essential nutrients (OE), consisting primarily of mineral derived cations and excluding N and P. P addition, but not N or OE, increased leaf area index within 12 months, foliar P concentration measured at 18 months, and stem diameter increment within 18 months. Stem growth at 18 months was even greater when trees fertilized with P also received the OE treatment. N and P additions increased leaf litterfall and N and P in combination further increased litterfall. The sequence of responses suggests that increased available P promoted an increase in photosynthetic area which led to increased wood production. P was the essential element most limiting to primary production on old volcanic soil in contrast to the N limitation found on young volcanic soils.