Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli.

Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom.     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

The relationship between polyploidy and organogenetic potential in embryo- and root-derived tissue cultures of Vicia faba L.

The cell cycle was examined in embryo and root explants of Vicia faba in culture to test whether or not polyploidy and aneuploidy affected organogenetic potential. Nuclear DNA contents and the mitotic index were measured in the 0–1 mm apical segment of primary roots of 5-day old seedlings and at various times following transfer to modified MS in darkness or Chu's N6 medium in an 8 h light/16h dark cycle (N6-MS programme) at 20°C. Mature embryos were dissected and cut longitudinally. Each half was cultured on the N6-MS programme. Root explants grown on MS in darkness developed into callus but there was no subsequent organogenesis. Only on the N6-MS programme were new roots initiated from root-derived callus. Using the N6-MS programme, embryo-derived callus became green and after 3 to 4 months, produced roots and shoots. Approximately 40% of these cultures regenerated plantlets. Polyploidy occurred within 24 h of culture irrespective of both tissue source and culture protocol. Variations in chromosome number from 2n=2x=12 were also routinely observed. Thus, calluses had the ability to initiate roots and shoots regardless of persistent polyploidy and aneuploidy. Compared with the baseline of cell cycle data for roots in vivo, the proportions of cells in the different cell cycle phases remained constant. Thus, in V. faba induction of organogenesis seems more related to culture protocols than to specific changes to the cell cycle. The mitotic index was significantly lower in vitro compared with meristems of intact roots.     

Changes in ribulosebisphosphate carboxylase/oxygenase and ribulose 5-phosphate kinase abundances and photosynthetic capacity during leaf senescence

The abundances of ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco) and ribulose-5-phosphate (Ru5P) kinase in field-grown soybean (Glycine max L. Merr.) leaves were quantified by a Western blot technique and related to changes in chlorophyll and photosynthetic capacity during senescence. Even though the leaf content of Rubisco was approximately 80-fold greater than that of Ru5P kinase, the decline in the levels of these two Calvin cycle enzymes occurred in parallel during the senescence of the leaves. Moreover, the decrease in the content of Rubisco was accompanied by parallel decreases of both the large and small subunits of this enzyme but not by an accumulation of altered large or small subunit isoforms. With increasing senescence, decreases in abundances of Rubisco, Ru5P kinase and chlorophyll were closely correlated with the decline in photosynthetic capacity; thus, the specific photosynthetic capacity when expressed per abundance of any of these parameters was rather constant despite an 8-fold decrease in photosynthetic capacity. These results suggest that during senescence of soybean leaves the chloroplast is subject to autolysis by mechanisms causing an approximately 80-fold greater rate of loss of Rubisco than Ru5P kinase.Jointly supported by the United States Department of Agricultural Research Service and the Kentucky Agricultural Experiment Station, Lexington (paper No. 88 3 286).Mention of a commercial product does not constitute endorsement by the United States Department of Agriculture.     

Giving between generations in American families

This paper documents the types and amounts of aid exchanged between adults and their non-coresidential parents. Data for the study are drawn from a representative national sample survey of Americans age 19 and older conducted in 1987–1988. Exchanges of monetary and material resources, childcare, household assistance, and companionship and advice are considered. Patterns of intergenerational exchange are found to differ by gender, family structure, age, ethnicity, and socioeconomic situation. Differences in exchange between males and females and between whites and Mexican-Americans are related to other life-course characteristics, and to the availability and proximity of kin. Blacks and persons living in poverty are shown to be less involved than other groups in intergenerational exchanges. Finally, patterns of prior assistance and the available needs and resources of the respondents and their parents are found to influence current patterns of exchange. Support for this research was provided by NICHD Grant No. 1 R01 HD26070-01, “Intergenerational Exchanges in Families with Children,” Dennis P. Hogan, Principal Investigator. Funds for the computer analysis were provided by the Pennsylvania State University Intercollege Research Programs. David Eggebeen is an Assistant Professor of Human Development in the Department of Human Development and Family Studies and a research associate at the Population Issues Research Center at Pennsylvania State University. He trained in sociology and demography at the University of North Carolina. His current research interests, besides those related to intergenerational relations, are the recent changes in the demographic structure of childhood in America and their implications for children’s social and economic well-being. Dennis P. Hogan is a professor of sociology and the director of the Population Issues Research Center at Pennsylvania State University. His current research interests, besides those related to intergenerational relations, are in the interrelation of social structures and the demographic life course. He is coauthor with David I. Kertzer ofFamily, Political Economy, and Demographic Change: The Transformation of Life in Casalecchio, Italy, 1861–1921, University of Wisconsin Press, 1989.     

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].     

Identification of fungi for sweet whey permeate utilization and eicosapentaenoic acid production

Summary Eighteen selected organisms of theEumycota division of the fungi kingdom were examined for eicosapentaenoic acid production and utilization of sweet whey permeate. The organisms belong to the subdivisionsMastigomycotina, Zygomycotina, Ascomycotina andDeuteromycotina. Seven organisms were initially identified as lactose utilizers (the predominant sugar in sweet whey permeate_ and eicosapentaenoic acid (EPA) producers. Utilization of lactose was demonstrated and EPA production was confirmed for four organisms, all of the subdivisionMastigomycotina. Growth studies showed thatP. ultimum had the best potential for future work.     

Reproductive Functions Illustrating Direct and Indirect Effects of Genes on Behavior

Effects of gene products on reproductive behavior which are relatively direct include those of the estrogen receptor and progesterone receptor. For example, work with estrogen receptor-deficient (ERKO) female mice has extended previous evidence contributing to the neurochemical analysis of lordosis behavior. On the other hand, sex differences in behavior present a classic example of indirect effects of genes on behavior. Work with ERKO male mice shows the necessity of ER gene expression for normal masculinization of the brain. In particular, behavioral assay results distinguish apparent motivational performance of ERKO males from male mating reflexes: the former is similar to that of wild-type males in important respects, while the latter are deficient in ERKO males. The present paper first reviews a small number of clear genetic contributions to reproductive behaviors, and then reports one experiment pertinent to the interpretation of the behavioral status of ERKO male mice.