Control and manipulation of gene expression during tomato fruit ripening

Ripening is a complex developmental process involving changes in the biochemistry, physiology and gene expression of the fruit. It is an active process characterised by changes in all cellular compartments. cDNA cloning has been used as an approach to analyse changes in gene expression during fruit ripening. This has revealed that several genes are switched on specifically during fruit ripening, including one encoding polygalacturonase (PG), a major cell wall protein. These cDNA clones have been used to study the expression of the genes in normal and ripening mutant fruits, and under environmental stress conditions.The PG gene has been isolated and it has been demonstrated that 1450 bases 5 of the coding region are sufficient for the tissue- and development-specific expression of a bacterial marker gene in transgenic tomatoes. Antisense RNA techniques have been developed to generate novel mutant tomatoes in which the biochemical function of this enzyme and its involvement in fruit softening has been tested.     

Flow cytometry chamber with 4 pi light collection suitable for epifluorescence microscopes

A compact, solid, spherico-ellipsoidal chamber (SEC), which has approaching 4 pi ("all around") light collection, has been developed for flow cytometry. This was mounted onto the stage of a standard fluorescence photomicroscope, and the camera was replaced by a photomultiplier. Both components can be added or removed in minutes. The increased light collection efficiency of the SEC (about 85%) compared with about 4% from standard chambers enabled a fluorescence microscope with a 50 W mercury vapour lamp to "double" as a flow cytometer. The system was tested with microbeads and cells stained for DNA with ethidium bromide, and results were comparable to those obtained with our laser-based instrument.     

A 6000 kb segment of chromosome 1 is conserved in human and mouse.

A murine linkage map generated from analyses of 428 meiotic events in an interspecific cross and pulsed field gel electrophoresis allowed examination of the genomic organization of a 6000 kb segment of mouse and human chromosome 1. Analysis of five genes within this syntenic segment of both species revealed striking conservation of gene order, intergenic distance and, to a lesser extent, CpG dinucleotides. In the mouse, meiotic crossover events were not evenly distributed; a hot spot for meiotic recombination was coincident with a CpG-island. These studies provide a practical approach to aid physical mapping of the human genome and a model for determining the molecular principles that govern meiotic recombination. In addition, these findings demonstrate profound conservation of genomic organization over mammalian evolution.     

Purification and characterization of a digestive cysteine proteinase from the American lobster (Homarus americanus).

A new cysteine proteinase was isolated from the digestive juice of the American lobster (Homarus americanus). The enzyme was purified by a combination of affinity and ion-exchange chromatography and gel filtration. The cysteine proteinase accounted for 80% of the proteolytic activity in the lumen of the hepatopancreas. The most potent heavy-metal inhibitors were Hg, Cu, and Ag ions. Inhibition by organic proteinase inhibitors, including E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane] and activation of the enzyme by 2-mercaptoethanol and dithiothreitol are characteristic of cysteine proteinases. Several similarities to papain are noted and include the N-terminal sequence, of which 22 of the first 28 amino acids are identical. Some notable differences are the higher Mr of 28,000 compared with 23,350 for papain, and the low isoelectric point (pI 4.5) of the lobster enzyme. The effects of pH and temperature on catalytic activity of the lobster proteinase were studied with benzyloxycarbonylalanine p-nitrophenyl ester as the substrate. The kcat./Km value was effectively temperature-independent between 10 and 60 degrees C. The pH-activity profile for the lobster enzyme revealed four apparent protonation states, of which only two are active.     

Maternal messenger RNA distribution in silkmoth eggs. I. Clone Ec4B is associated with the cortical cytoskeleton

We have constructed a cDNA library from mature egg RNA of the silkmoth, Hyalophora cecropia. Differential screening of the library using cDNA made against mRNAs from the yolky cytoplasm (soluble fraction) and the cortical cytoplasm (cytoskeletal-associated or cortical fraction) resulted in several clones that hybridized to a higher degree to the cDNA from the cytoskeletal-associated fraction. We selected and analyzed the clone giving the strongest signal (designated Ec4b) for its distribution in situ and found that it bound to mRNAs in the nurse cell cytoplasm, in the cortex and in the follicle cells of oocytes. Hybridization of the insert from Ec4b to both detergent-soluble and -insoluble (cortical) RNA on dot blots further supported the observation that the mRNA corresponding to Ec4b was enriched in this cytoskeletal fraction. The mRNA for Ec4b was approximately 500 bases long and the gene seems to be a member of a large multigene family in the H. cecropia genome. Analyses of the nucleotide and amino acid sequences reveal similarity to lepidopteran chorion genes and a lesser but convincing similarity to vertebrate cytokeratins. The filter and in situ hybridization data point to the association of specific messenger RNAs with the cortical cytoskeleton of silkmoth oocytes. Aspects of the structure of the protein encoded by this mRNA suggest that it is a structural component necessary for formation of the cellular blastoderm of the embryo. The association of this maternal mRNA with the cortical cytoskeleton presents the interesting possibility that mRNA bound to the cytoskeleton may be capable of participating in the synthesis of new cytoskeleton or related structures during blastoderm formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).     

Site-directed mutagenesis of yeast phosphoglycerate kinase. The 'basic-patch' residue arginine 168

There is evidence, some of it of questionable authenticity, which suggests that phosphoglycerate kinase takes up a more compact form following the binding of substrates. Using this evidence it has been assumed that a conformational rearrangement is required for phosphoryl transfer to occur and that this is brought about by moving the enzyme's two domains towards each other. In order to test this hypothesis we have modified, by site-directed mutagenesis, an arginine residue thought to be involved in stabilising the transition-state intermediate. Although some 1.3 nm away from the site of phosphoryl transfer, as seen in the crystallographically determined structure, the substitution of arginine 168 by lysine (R168K) more than halves the specific activity of the enzyme. Substituting the arginine with a methionine (R168M) reduces activity further, but not completely, thus proving that the charge associated with this residue is not essential for catalytic activity. Both mutations raise the Michaelis constants (Km) for ATP and glycerate 3-phosphate. The largest change is observed with the triose substrate and the methionine mutant, suggesting that the primary function of arginine 168 is to influence the environment of this substrate. The effect on activity of adding sulphate to R168K and R168M mutant enzyme has also been investigated. The sulphate activation effect at low substrate concentrations is reduced for the methionine substitution but almost abolished for the lysine substitution. The most reasonable explanation of all these findings is that, in the wild-type enzyme, the guanidinium group of arginine 168 forms a hydrogen bond with one of the triose substrate's C1 oxygens. This steric arrangement would not be possible in the 'open form' of this enzyme as observed in the crystal structure.     

Activation of sexual behavior by implantation of testosterone propionate and estradiol benzoate into the preoptic area of the male Japanese quail (Coturnix japonica)

Intracranial implantation of minute pellets of gonadal steroids was performed to determine neuroanatomical loci at which steroids activate sexual behavior in the Japanese quail (Coturnix japonica). In this species, systemic treatment of castrated males with either testosterone propionate (TP) or estradiol benzoate (EB) restores male-typical copulatory behavior (head grabbing, mounting, and cloacal contact movements). In addition, EB activates female-typical receptive behavior (crouching). Adult male castrated quail were implanted intracranially with 300-micrograms pellets containing TP, EB, or cholesterol (CHOL) and behavior was tested with intact males and females. Either TP or EB pellets in the preoptic area (POA) activated male-typical copulatory behavior. Mounting was specifically activated without concomitant activation of other steroid-sensitive sexual and courtship behaviors. TP and EB implants in adjacent nuclei containing receptors for these steroids and CHOL implants in POA had no effect on male-typical copulatory behavior. Eighteen percent of all males tested for female-typical receptivity crouched, but no specific effect of EB was seen at any site. The similarity of the POA sites for activation of mounting by TP and EB is consistent with the hypothesis that cells within the POA aromatize testosterone to estrogens, which directly stimulate the cellular processes leading to behavioral activation.