Improved methods of estimating root length using a photocopier,a light box and a bar code reader

Photocopying was found to be a rapid method of making a permanent record of a root sample. The method used produced a copy with white roots against a black background. Manual estimates of root length were made from photocopies using a light box. The number of intersections visible when laid over a copy of a white on black regular square grid was counted. Automated estimates of root length were made by scanning a photocopy with a bar code reader in place of a pen in a computer-driven graph plotter. Roots >0.2 mm diameter were resolved with precision and speed.     

Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L

Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO₂ exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine.     

The differential protection by WR2721 of skin versus growing cartilage following irradiation in weanling rats

The potential for radioprotection of growing cartilage by the thiophosphate WR2721 was evaluated in weanling rats using single fractions of irradiation. Protection of acute skin toxicity was monitored simultaneously. Single doses of 600, 1200, 1800, or 2400 cGy were administered to the left tibia of CrL:CD(SD)BR female rats in groups of 12. Identically treated groups were injected with 310 mg/kg WR2721 (2/3 the determined LD50/30) in a concentration of 26 mg/ml intraperitoneally 15 min prior to irradiation. Rats untreated or given WR2721 without radiation served as control groups. Radiographs of the irradiated and unirradiated tibiae for each animal were obtained weekly to the date of sacrifice at 80 days following the initial treatment. Skin toxicity was assessed weekly starting on the second week using Moulder's scale (J.E. Moulder, J.J. Fischer, and A. Casey, Radiology 115, 465-470 (1975]. No significant difference in bone growth as measured by tibial lengths for the WR2721-treated or untreated animals was observed. Skin toxicity including moist desquamation occurred in irradiated limbs and was substantially less in rats treated with WR2721. As opposed to previous work with cysteamine, WR2721 as administered had no significant radioprotective effect on tibial growth in weanling rats but substantially reduced the accompanying skin toxicity.     

Effect of incubation temperature on the hatching of rotifer resting eggs collected from sediments

Sediment samples from Loch Leven, Scotland, were incubated at 5°C, 10°C and 15°C to induce hatching of rotifer resting eggs. The emergent animals were identified and counted. The temperature which induced hatching varied among the nine species studied. These results are discussed in relation to seasonality and temperature preferences previously recorded for the most abundant species. Resting egg densities of 2.2–13.9 eggs cm^–3 were recorded in the upper 5 cm of sediment.     

Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment

The gene sequence encoding porcine preprogastrin is known; in order to clarify pathways of post-translational processing of the predicted precursor peptide we have characterized material reacting with antibodies to a synthetic peptide corresponding to the expected extreme COOH-terminal portion of the precursor. Radioimmunoassay was used to identify and monitor the purification of peptides in porcine antral mucosa. Two peptides (I and II) were isolated to homogeneity by steps involving gel filtration, ion exchange, and reversed-phase high performance liquid chromatography. The two co-eluted on gel filtration but were separated on anion-exchange chromatography. The more acidic peptide (II) was less hydrophobic on high performance liquid chromatography. Automated gas-phase microsequencing revealed the less acidic peptide (I) to have the sequence of porcine preprogastrin 96-104 (SAEEGDQRP); it would be produced by tryptic-like cleavage of Arg95-Ser96. The second peptide did not yield a phenylthiohydantoin-derivative on the first cycle but thereafter it sequenced as the first peptide (i.e. -AEEGDQRP). Incubation in alkali liberated almost equimolar amounts of phosphate from peptide II but not from I. In addition, alkaline phosphatase liberated phosphate and converted the acidic peptide to the less acidic one. The results suggest that serine in the first position is phosphorylated in peptide II but not I. The tripeptide -Ser(P)-Ala-Glu- also occurs in adrenocorticotropic hormone; this tripeptide is a substrate for physiological casein kinase. Potential phosphorylation sites occur at comparable positions in the precursors of a number of regulatory peptides.     

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.     

Linkage Relationships Reflecting Ancestral Tetraploidy in Salmonid Fish

Fifteen classical linkage groups were identified in two salmonid species (Salmo trutta and Salmo gairdneri) and three fertile, interspecific hybrids (S. gairdneri X Salmo clarki, Salvelinus fontinalis X Salvelinus namaycush and S. fontinalis X Salvelinus alpinus) by backcrossing multiply heterozygous individuals. These linkage relationships of electrophoretically detected, protein coding loci were highly conserved among species. The loci encoding the enzymes appeared to be randomly distributed among the salmonid chromosomes. Recombination frequencies were generally greater in females than in males. In males, certain linkage groups were pseudolinked with other linkage groups, presumably because of facultative multivalent pairing and directed disjunction of chromosomes. Five such pseudolinkage groups were identified and they also appeared to be common among species and hybrids. Duplicate loci were never classically linked with each other, although some exhibited pseudolinkage and some showed evidence of exchanging alleles. Gene-centromere recombination frequencies estimated from genotypic distributions of gynogenetic offspring were consistent with map locations inferred from female intergenic recombination frequencies. These linkage relationships support the contention that all extant salmonids arose from a common tetraploid progenitor and that this progenitor may have been a segmental allotetraploid.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Genetic diversity in Cucumis sativus L. assessed by variation at 18 allozyme coding loci

Summary The genetic diversity of the U.S. Cucumis sativus L. germplasm collection [757 plant introductions (PI) representing 45 countries] was assessed using 40 enzymes which represented 74 biochemical loci. Polymorphisms were observed at 18 loci (G2dh-1, Gpi-1, Gpi-2, Gr-1, Gr-2, Idh, Mdh-1, Mdh-2, Mdh-3, Mpi-2, Pepla-2, Peppap-2, Per-4, Pgd-1, Pgd-2, Pgm-1, Pgm-3, and Skdh). Two PIs (285606 and 215589) contained alleles [G2dh-1(1) and Per-4(2), respectively] which did not occur in any other PI. Other alleles which occurred in low frequencies (in < 1% of the PIs) included Gpi-1(3), Gpi-2(3), Gr-1(3), Gr-2(1), Idh(1), Mdh-1(2), Mdh-2(1), Peppap-2(1), and Pgd-1(1). Individual loci containing more than one allele in greater than 20% of the PIs included Mpi-2, Pepla-2, Pgd-2, and Pgm-1. Multivariate analyses aided in the reduction of data (principle components), depicted relationships among PIs (cluster), and identified the most discriminating enzyme loci (Pgm-1, Pepla-2, Gr-1, Pgd-2, Mpi-2, and Skdh) (classification and regression tree).Research partially supported by Asgrow, DeRuiter, Nickerson-Zwaan, Nunhems, and Sun Seed Companies; and the Graduate School, University of Wisconsin, Madison