Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5

Transgenic tomato plants expressing antisense RNA to a ripening-related cDNA clone (pTOM5) had yellow ripening fruit and pale coloured flowers. Carotenoid levels in fruit of these plants were reduced by up to 97%. In order to determine the step of carotenoid biosynthesis which was blocked, a cell-free system active in the synthesis of carotenoid intermediates was prepared. Incubations with radiolabelled carotenoid precursors led to the identification of the block between GGDP and phytoene. Analysis of carotenoids in different tissues of transgenic and control plants indicated that although ripe fruit and flower carotenoid levels were reduced in the modified plants, leaf carotenoid levels were not decreased. This implies that the pTOM5 gene product is not involved in carotenoid synthesis in the leaf.     

Effect of polydimethylsiloxane oxygen carriers on the biological bleaching of hardwood kraft pulp by Trametes versicolor

Summary Improving the availability of oxygen by adding polydimethylsiloxanes (PDMS) oxygen carriers to Trametes versicolor cultures increased pulp brightening. The presence of the oxygen carriers in cultures of T. versicolor with hardwood kraft pulp increased the growth rate of the fungus, but not the ultimate biomass yield. The PDMS also stimulated brightening of hardwood kraft pulp by it T. versicolor immobilized in polyurethane foam. A threefold increase in the oxygen uptake rate in T. versicolor cultures with PDMS was observed. This increase can be explained by elevated oxygen transfer rate and attributed to the surfactant properties of PDMS. Offprint requests to: E. ZiomekIssued as NRCC 32760     

Assumption validity in human optimal foraging: The Barí hunters of Venezuela as a test case

Optimal foraging theory is being used increasingly as a means of understanding human foraging behavior. One of the central assumptions of optimal foraging theory is that prey items or patches are encountered sequentially and as a Poisson process. Using empirical data gathered on the Barí hunters of Venezuela, we assess the validity of this central assumption. We compare our observed distribution of encounter frequencies with an expected Poisson distribution, utilizing chisquare tests and graphic representations. The results are strikingly consonant with the expected Poisson distribution and lend support to the applicability of optimal foraging models to human hunting behavior.     

Analysis of X-chromosome inactivation and presumptive expression of the Wiskott-Aldrich syndrome (WAS) gene in hematopoietic cell lineages of a thrombocytopenic carrier female of WAS

Summary We report on a thrombocytopenic female belonging to a pedigree with the Wiskott-Aldrich syndrome (WAS). Restriction fragment length polymorphism (RFLP) analysis with probe M27, closely linked to the WAS gene, demonstrated that she is a carrier of WAS. Both small-sized and normal-sized platelets were present, suggesting that, unlike the vast majority of WAS carriers, she does not manifest nonrandom X-chromosome inactivation in the thrombopoietic cell lineage. Study of X-chromosome inactivation by means of RFLP and methylation analysis demonstrated that the pattern of X-chromosome inactivation was nonrandom in T lymphocytes, but random in granulocytes. While this is the first complete report on the occurrence of thrombocytopenia in a carrier female of WAS as the result of atypical lyonization, it also suggests that expression of the WAS gene occurs at (or extends up to) a later stage than the multipotent stem cell along the hematopoietic differentiation pathway.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

Karyotype and meiosis studies in three South African Pyrgomorpha species (Orthoptera: Pyrgomorphidae)

Two South African Pyrgomorpha species have reduced chromosome numbers, due to centric fusions between the largest autosomes and the medium and small autosomes. P. rugosa has 2n=11(XO) (4 pairs of submetacentric and 1 pair of acrocentric autosomes) and P. granulata has 2n=13(XO) (3 pairs of submetacentric and 3 pairs of acrocentric autosomes). A third South African species has a typical Pyrgomorphidae number of 2n=19(XO) (acrocentrics). The mean chiasma frequency of the 2n=19 species is higher than that of the other two, although the frequencies of distal chiasmata in all three are similar. The recombination potential of the two species with lower chromosome numbers has been reduced, due to fewer crossovers in comparison to the 2n=19 species, as well as to independent assortment.     

Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli

Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.     

Mixtures of a series of homologous hydrophobic peptides with lipid bilayers: a simple model system for examining the protein-lipid interface

The interactions of several members of a homologous series of peptides with the phospholipid bilayer have been examined by using fluorescence and deuterium NMR spectroscopy, differential scanning calorimetry, and measurements of water-to-bilayer partition coefficients. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers and tripeptides of the form Ala-X-Ala-O-tert-butyl are used as a model system to probe the influence of amino acid side-chain substitution on the insertion of peptides into membranes and the behavior of peptide/bilayer mixtures. Tripeptides with X = Gly, Ala, Phe, and Trp have been examined. All of the tripeptides are water soluble, and all partition into DMPC bilayer vesicles to some extent. The Gly-containing peptide is the least soluble and the Trp-containing peptide the most soluble in the bilayer. The extent of perturbation of the bilayer structure induced by the peptides parallels their bilayer solubility: the Gly and Ala peptides act as simple impurities while peptides containing bulky aromatic rings cause a phase separation. Changes in the fluorescence properties of the Trp analogue upon incorporation into the bilayer indicate that the Trp side chain is probably immersed in the hydrocarbon region of the bilayer. Peptides of this form should serve as easily modifiable model systems with which to examine details of how the bilayer environment affects peptide conformation, as well as how hydrophobic peptides affect the bilayer structure.