A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.     

Observations of hamster sperm-egg fusion in freeze-fracture replicas including the use of filipin as a sterol marker

We have extended the observations of previous transmission electron microscopy studies of sperm-egg fusion to include those of freeze-fracture replicas showing sperm-egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5-30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds beta-OH-sterols to form visible complexes in freeze-fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome-reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm-egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin-rich egg membrane and the filipin-poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin-sterol complexes are not found in sperm-derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.     

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.     

Experimental evaluation of realized niche models for predicting responses of plant species to a change in environmental conditions

Abstract. We estimated, using logistic regression techniques, the realized niches of the four dominant species in an experimental marsh complex located in the Delta Marsh, Manitoba, Canada. These models were then used to predict the probability of occurrence of these species in selected elevation ranges when water levels were raised in 1985 either 0, 30 or 60 cm above the long-term normal water level. These realized-niche models were calculated using elevation and species data collected in 1980. After having been eliminated by two years of deep flooding, the emergent vegetation in this complex had been re-established during a drawdown beginning in either 1983 or 1984. Our hypothesis was that from 1985 to 1989 the frequencies of occurrence of species in selected elevation ranges would converge to their probabilities predicted from the 1980 logistic models. This was not borne out by our results. Actual frequencies and predicted probabilities of occurrence of a species were similar at best less than 40% and then mostly in the control (0 cm) treatment. The realized-niche models were not adequate to predict the distribution of emergents after an increase in water level in the short term because the emergent species did not migrate upslope. Emergent species in the medium and high treatments either (1) died out - Scolochloa festucacea and Scirpus lacustris - after 3 yr because they could not survive permanent flooding, (2) stayed where they were - Phragmites australis - because they were unable to move upslope through clonal growth, or (3) became more widespread - Typha glauca only because of the expansion of small local populations already established in 1985 in areas dominated formerly by other species.     

Mesoderm-specific B104 expression in the Drosophila embryo is mediated by internal cis-acting elements of the transposon

The Drosophila melanogaster genome contains about 100 copies of the B104 transposable element, which is strongly expressed during embryogenesis. Here we show that B104 expression is restricted to the esophageal and amnioproctodeal regions of the embryo and to the developing mesoderm. Mesoderm-specific B104 expression requires the activity of the mesoderm-determining factors twist and snail. Virtually the same expression patterns were observed in Drosophila yakuba, a species that a separated from D. melanogaster by some 15 million years of evolution. We show that B104 expression is directed by internal sequences of the retrotransposon that are capable of acting as a cis-acting regulatory element in front of a heterologous Drosophila promoter. Our findings suggest that retrotransposon insertions can affect the expression patterns of endogenous genes by adding and distributing specific cis-acting control elements throughout the host genome. We therefore propose that transposable elements in addition to reducing the fitness of their hosts may also provide a rich pool of cis-acting sequences that contribute to the long-term evolutionary potential of the population in a beneficial manner.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength     

Entry of immotile spermatozoa into zona-free hamster ova

We studied six men whose spermatozoa were immotile and possessed a variety of sperm tail structural abnormalities by electron microscopy. The semen of all six subjects had a normal percentage of oval forms and sperm undergoing capacitation and acrosome reaction. Despite the absence of motility, when incubated sperm from these subjects was added to a microdrop of medium containing zona pellucida-free hamster ova, sperm penetration or entry into the cytoplasm of from 1–9% of the eggs was evident with phase contrast microscopy. This latter finding suggests that, at least in this system, oocytes actively facilitate sperm incorporation. Penetration was absent when sperm of fertile men were rendered immotile, though still viable, by heat treatment.