Calcium-Regulated in Vivo Protein Phosphorylation in Zea mays L. Root Tips

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(β-aminoethyl ether)-N-N′-tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with ³²P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent protein kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.     

The Role of Gravity in Apical Dominance : Effects of Clinostating on Shoot Inversion-Induced Ethylene Production, Shoot Elongation, and Lateral Bud Growth

Shoot inversion-induced release of apical dominance in Pharbitis nil is inhibited by rotating the plant at 0.42 revolutions per minute in a vertical plane perpendicular to the axis of rotation of a horizontal clinostat. Clinostating prevented lateral bud outgrowth, apparently by negating the restriction of the shoot elongation via reduction of ethylene production in the inverted shoot. Radial stem expansion was also decreased. Data from experiments with intact tissue and isolated segments indicated that shoot-inversion stimulates ethylene production by increasing the activity of 1-aminocyclopropane-1-carboxylic acid synthase. The results support the hypothesis that shoot inversion-induced release of apical dominance in Pharbitis nil is due to gravity stress and is mediated by ethylene-induced retardation of the elongation of the inverted shoot.     

Use of the granulosa cell aromatase bioassay for measurement of bioactive follicle-stimulating hormone in urine and serum samples of diverse species

Ovarian steroids and growth factors are intragonadal modulators which augment a key endpoint of follicle-stimulating hormone (FSH) action in granulosa cells: the induction of aromatase activity. Studies of these paracrine hormones that enhance FSH-stimulated estrogen biosynthesis by cultured rat granulosa cells, have led to the development of a sensitive and specific bioassay for FSH. This newly developed granulosa cell aromatase bioassay (GAB) allows for the measurement of bioactive FSH levels in serum and urine of humans and animals with various physiological and pathological conditions. These studies have demonstrated that the GAB assay is useful in detecting possible changes in the molecular forms of FSH. The adaptation of this method for urine samples allows for the measurement of bio-FSH levels in situations where venipuncture is not practical or in species for which specific radioimmunoassays are not available.     

Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

Exposure of cultured rat hepatoma (HTC) cells to a 43 degrees C heat shock transiently accelerates the degradation of the long-lived fraction of cellular proteins. The rapid phase of proteolysis which lasts approximately 2 h after temperature step-up is followed by a slower phase of proteolysis. During the first 2 h after temperature step-up there is a wave of ubiquitin conjugation to cellular proteins which is accompanied by a fall in ubiquitin and ubiquitinated histone 2A (uH2A) levels. Upon continued incubation at 43 degrees C the levels of ubiquitin conjugates fall with a corresponding increase of ubiquitin and uH2A to initial levels. The burst of protein degradation and ubiquitin conjugation after temperature step-up is not affected by the inhibition of heat shock protein synthesis. Cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild-type FM3A mouse cells do. This observation indicates that the ubiquitin system is involved in the degradation of heat-denatured proteins. Sequential temperature jump experiments show that the extent of proteolysis at temperatures up to 43 degrees C is related to the final temperature and not to the number of steps taken to attain it. Temperature step-up to 45 degrees C causes the inhibition of intracellular proteolysis. We propose the following explanation of the above observations. Heat shock causes the conformational change or denaturation of a subset of proteins stable at normal temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.     

Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria.

cDNA encoding porin of Neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. The deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. According to secondary structure predictions, mitochondrial porin consists mainly of membrane-spanning sided beta-sheets. Porin was efficiently synthesized in vitro from the cDNA; this allowed us to study in detail its import into mitochondria. Thereby, three characteristics of import were defined: (i) import depended on the presence of nucleoside triphosphates; (ii) involvement of a proteinaceous receptor-like component on the surface of the mitochondria was demonstrated; (iii) insertion into the outer membrane was resolved into at least two distinct steps: specific binding to high-affinity sites and subsequent assembly to the mature form.     

The nuclear migration signal of Xenopus laevis nucleoplasmin.

Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes. Its ability to target to the nucleus when microinjected into the cytoplasm has been the subject of many studies central to our understanding of how proteins segregate into nuclei. Using a cDNA clone we constructed beta-galactosidase-nucleoplasmin hybrids in modified bacterial expression vectors. The fusion proteins were expressed in Escherichia coli, purified and injected into the cytoplasm of X. laevis oocytes. The distribution of the fusion proteins between the cytoplasmic and nuclear compartments were analysed after incubation of various lengths of time. The results show that the signal sequence for nuclear transport is located close to the carboxy terminus of the protein. The signal sequence has been mapped to a small stretch of amino acids, containing a stretch of four lysines analogous to the SV40 large-T antigen signal.