Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.      

A method for quantification of the level of somatic embryogenesis among Norway spruce callus lines

A method for quantitative determination of the level of somatic embryogenesis in Norway spruce embryogenic callus is described. Embryogenic callus was dispersed in liquid by agitation and plated in a thin layer of medium containing 0.6% low melting point agarose. The number of embedded somatic embryos per mg of callus ranged from 0.2 to 1.5 among 11 embryogenic callus lines surveyed. Each callus line was derived from an individual immature embryo explant. Further development occurred as somatic embryos grew out of the agarose layer. This method was useful for identifying highly embryogenic callus lines among phenotypically similar lines, and should be useful for quantitatively determining the effect of medium and growth regulator modifications on somatic embryo density and developmental capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid - ABA abscisic acid     

Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32Pi into cellular adenosine triphosphate (ATP) (Babcock et al., 1975). We report conditions under which 32Pi is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [gamma-32P]ATP (2.3 X 10(4) cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pool. In the absence of glucose or other added substrates, the specific activity of [gamma-32P]ATP was 10-25% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32Pi for 2 h at 37 degrees C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4O, and 5% beta-mercaptoethanol contained in excess of 40 32Pi-labeled proteins as assessed by two-dimensional polyacrylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5 mM 2[N-cyclohexylamino] ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100 degrees C. In contrast to the urea/Nonidet P-40 (NP40)/beta-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphorylated regulatory subunit (RII) of cyclic adenosine 3',5'-monophosphate-dependent protein kinase from bovine heart. Antibodies to RII immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, [32P] RII after two-dimensional electrophoresis. RII, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.     

Protein phosphorylation of plasma membranes from bovine epididymal spermatozoa

Plasma membranes from bovine epididymal spermatozoa possess both cAMP-independent and cAMP-dependent protein kinase activity. With the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly as substrate, the basal activity of the membrane-associated protein kinase(s) was 0.1 nmol phosphate incorporated X min X mg protein. In the presence of 5 microM cAMP, the apparent activity was increased about twofold. The addition of Nonidet P-40 (0.05%) to the assay mixture increased protein kinase activity to 0.4 and 4.0 nmol phosphate incorporated X min X mg protein in the absence or presence of 5 microM cAMP, respectively. Both isozymes of the cAMP-dependent protein kinase were detected in detergent-solubilized membranes but 95% of the activity appeared as a Type II form based on DEAE-Sephacel chromatography. Several polypeptide components of the plasma membrane served as substrates for membrane-associated cAMP-dependent protein kinases, in vitro. In the absence of detergent, two cAMP-dependent phosphoproteins of 41,000 Mr and 60,000 Mr were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When 0.05% Nonidet P-40 was included in the assay mixture, a cAMP-dependent phosphoprotein of 43,000 Mr appeared. Two-dimensional polyacrylamide gel electrophoresis of membranes phosphorylated in the presence of 5 microM and 0.05% Nonidet P-40 revealed phosphoproteins of the following molecular weights/isoelectric points: 56,000/6.7, 56,000/6.9, 51,000/6.2, 42,000/5.9, 42,000/6.0, 38,000/6.1, 38,000/6.4, 14,000/7.2, 12,000/7.4 and a train of five polypeptides appearing at 14,000/5.4-6.0.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites

As an extension of our previous reports that cardiac and skeletal muscle troponin I (Tn-I) and troponin T (Tn-T) are excellent substrates for protein kinase C (PKC) (Katoh, N., Wise, B. C., and Kuo, J. F. (1983) Biochem. J. 209, 189-195; Mazzei, G. J., and Kuo, J. F. (1984) Biochem. J. 218, 361-369), we have now determined that PKC phosphorylated serine 43 (and/or serine 45), serine 78, and threonine 144 in the free Tn-I subunit and threonine 190, threonine 199, and threonine 280 in the free Tn-T subunit of bovine cardiac troponin. PKC appeared to phosphorylate the same sites of the subunits present in the form of the troponin complex, as indicated by the similarity in the two-dimensional phosphopeptide maps. Although some of the phosphorylation sites were shared by other classes of protein kinases, PKC exhibited a distinct substrate specificity. It was also noted that phosphorylated serine and threonine residues in Tn-I and Tn-T had neighboring basic amino acid residues separated by 1 or 2 other residues both at the amino and carboxyl termini, in agreement with the conclusion of House et al. (House, C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol. Chem. 262, 772-777) based upon their studies on other substrate proteins. Several peptides having sequences around the phosphorylating sites have been synthesized. The phosphorylation experiments indicated that these peptides were substrates for PKC, and their relative substrate activity (determined by the ratios of Vmax/Km) compared with other proteins, in descending order, was Tn-I = Tn-I(134-154) greater than Tn-T much greater than histone H1 greater than Tn-I(33-35) approximately Tn-T(268-284) greater than Tn-T(179-198) approximately Tn-T(191-209). It is suggested that PKC phosphorylation of Tn-I and Tn-T could be biologically significant in terms of possible modifications in interactions among the individual contractile protein components as well as the Ca2+ sensitivity and activity of actomyosin ATPase.     

Temperature effects on seed imbibition and leakage mediated by viscosity and membranes

The possible involvement of membranes and water viscosity in the temperature effects on imbibition and solute leakage of radish (Raphanus sativa var. Early Scarlet Globe) seeds and excised sugar pine (Pinus lambertiana Dougl.) embryos was evaluated. In these two seed materials, the temperature effect on initial rates of imbibition and solute leakage could be accounted for primarily by changes in water viscosity, the relationship being approximately linear. It appears that membranes are involved both in water uptake and solute leakage. Heat-killed radish seeds and sugar pine embryos exhibited significantly higher rates of imbibition and solute leakage than did viable ones. In addition, sugar pine embryos exhibited an abrupt change in rates of imbibition and solute leakage between 15 and 20°C, resulting in abnormally high water uptake and solute leakage above this temperature.     

Purification and partial characterization of plasma membranes from bovine spermatozoa

Bovine epididymal spermatozoa were subjected to nitrogen cavitation (600 psi for 10 min) to remove plasma membrane. Examination of the cavitated cells by electron microscopy revealed that the plasma membrane was preferentially removed from the periacrosomal and flagellar regions. Nuclear, mitochondrial and acrosomal membranes remained intact and attached to the spermatozoa, but the cytoplasmic droplets were frequently disrupted and their internal membrane-bound vesicles were released. Lower pressures (less than 200 psi) were relatively ineffective in removing the periacrosomal plasma membrane, while an intermediate pressure (400 psi) removed this membrane from about 70% of the spermatozoa. No apparent selectivity for removal of the periacrosomal and flagellar plasma membrane was observed as a function of cavitation pressure. The cavitated cells were separated from the plasma membranes by differential followed by linear sucrose density gradient centrifugation. Two distinct membrane populations were resolved on sucrose gradients and were designated Band I and Band II. Band I contained only spherical vesicles which arose from the plasma membrane. Surface labeling of intact cells confirmed the plasma membrane as the origin of Band I. The membranes of higher density comprising Band II were heterogeneous consisting of both spherical and flattened vesicles. When purified cytoplasmic droplets were cavitated and centrifuged on the sucrose gradient only Band II was obtained. These studies indicate that nitrogen cavitation of bovine epididymal spermatozoa can result in significant contamination of plasma membrane fractions by cytoplasmic droplet membranes unless appropriate differential centrifugation is used to separate the membrane fractions.     

Demonstration of distinct forms of testicular adenylate cyclases associated with germinal and Leydig cell fractions

Decapsulated testes from adult rats were digested with collagenase, and the fraction enriched in germinal and Leydig cells was applied to a 0-4% continuous metrizamide gradient and centrifuged. This leads to separation of a germinal cell fraction and two putative Leydig cell populations that bind human choriogonadotropin, but only one of which responds to the gonadotropin with marked increase in testosterone production. Adenylate cyclase activity was present in these three fractions, and Mn2+ was more effective than Mg2+ as a divalent cation. The adenylate cyclase activity associated with the germinal cell fraction was just marginally stimulated by fluoride and by the non-hydrolyzable GTP analog 5'-guanylimidodiphosphate, while that associated with the Leydig cell populations was stimulated to a greater degree depending upon the type of divalent cation. Only the Leydig cell populations exhibited marked human choriogonadotropin-sensitive stimulation of adenylate cyclase activity in the presence of 5'-guanylimidodiphosphate above that observed with the GTP analog alone. These results suggest the presence of distinct adenylate cyclases in adult rat testis and indicate that both populations of Leydig cells are capable of producing cyclic AMP in response to gonadotropins such as human choriogonadotropin.