Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the Simian virus 40 T-antigen.

Selective nuclear protein transport was analyzed in single living cells. Hybrid proteins consisting of short stretches of the Simian virus 40 T-antigen and of the almost complete beta-galactosidase moiety were generated by molecular genetic methods and injected into the cytoplasm of rodent hepatoma cells. A histochemical assay showed that all proteins containing the karyophilic signal of the T-antigen (residues 126/127-132) were equally well accumulated by the nucleus within 15 h after injection. Microfluorimetric measurements of nuclear transport kinetics, however, revealed large differences. Proteins containing the karyophilic signal without flanking sequences were taken up by the nucleus on a time scale of hours. The same held for a protein containing T-antigen residues 127-147. However, a protein containing T-antigen residues 111-135 was accumulated by the nucleus with a half-time of 8-10 min reaching an equilibrium nucleocytoplasmic concentration ratio of greater than or equal to 15. Photobleaching measurements showed that, independently of subcellular localization, the mobility of all proteins was quite large. Thus, our measurements revealed a striking effect of T-antigen residues 111-125 on the kinetics of nuclear transport. Residues 111-125 do not seem to contain a second karyophilic signal. Conspicuously, however, they comprise a cluster of phosphorylation sites.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

Sublethal effects of aldicarb on the behaviour of Aphis fabae and two clones of Myzus persicae and on the transmission of beet mosaic virus by these aphids

In laboratory experiments treatment of sugar-beet plants with aldicarb stimulated the mobility of Aphis fabae and two clones of Myzus persicae which were susceptible (S) and resistant (R) to carbamate-based insecticides, respectively. On the other hand, the number of aphids probing and the total number of probes made was reduced, and hence the transmission of beet mosaic virus (BMV) was restricted. In outdoor experiments the spread of BMV from aldicarb-treated plants by naturally infesting aphids was also restricted. The number of infected plants decreased with increasing distance from the sources of infection.

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Résumé Des plantes de betterave traitées au laboratoire avec de l'aldicarbe ont stimulé la mobilité d'Aphis fabae et de deux clones de Myzus persicae, l'un sensible et l'autre résistant à des insecticides contenant des carbamates. Par ailleurs, le nombre de pucerons en train de sonder les feuilles ainsi que le nombre total de sondages ont été réduits et ainsi la transmission du virus de la mosaïque de la betterave (BMV) a été limitée. Dans des expériences à l'extérieur, la vitesse de propagation de BMV par des pucerons sur des plantes traitées à l'aldicarbe a été aussi plus limitée. Le nombre de plantes contaminées diminuait avec la distance de la source de contamination.

     

Disruption of the Rad52 Gene Alters the Spectrum of Spontaneous Sup4-O Mutations in Saccharomyces Cerevisiae

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Field Study Comparing Growth and Viability of a Population of Phototrophic Bacteria

The growth and viability of an anoxygenic, phototrophic bacterial community in the hypolimnion of Zaca Lake, Calif., were compared throughout the summer. The community is dominated by a single species, “Thiopedia rosea,” that inhabits the entire hypolimnion (6 to 8 m) for approximately 11 months. Suboptimal conditions in the hypolimnion (extremely low light intensity, high or low H₂S levels) result in zero or extremely low growth rates (doubling times > 1 month) for most of the population, most of the time, yet cells remain viable and capable of high specific growth rates (doubling times of 1 to 10 days) when placed under favorable conditions (higher light intensities and temperatures). We first conclude that phototrophic bacterial populations in situ may frequently exist in a viable yet nongrowing state. Second, the viability of cells is likely to be reduced with depth owing to higher concentrations of potentially toxic chemicals and to changes in the physiological state associated with the prolonged periods of darkness commonly found at the bottom of bacterial plates.     

Effects of stream order and season on mineralization of [14C]-phenol in streams

Mineralization of trace levels of [¹⁴C]-phenol by heterotrophic microorganisms was quantified at 4 sites along a river continuum in southwestern Virginia. Significant phenol mineralization rates were detected in surface sediment and seston samples at all sites from August 1985 through May 1986. Phenol degradation was strongly affected by season (ANOVA; P < 0.0001). From a baseline rate in August (range: 1.19 × 10^-5 to 897 × 10^-4 mg phenol mineralized mg AFDW^-1 h^-1) phenol mineralization rose to a yearly maximum in October (range: 1.21 × 10^-4 to 1.16 × 10^-3 mg phenol mineralized mg AFDW^-1 h^-1) despite decreasing stream temperatures. This autumnal peak in phenol degradation was attributed to the pulsed input of allochthonous detritus, especially leaf litter, which contains substantial quantities of phenols and related compounds. Although phenol mineralization was significant in these streams, phenols were metabolized at much slower rates than more labile compounds present in the dissolved organic matter (DOM) pool. Estimates of turnover rates for three major components of DOM revealed that glucose and glutamate turnover rates (0.064–0.140 h^-1 mg sediment AFDW^-1 and 0.140–0.610 h^-1 mg sediment AFDW^-1, respectively) were, respectively, 2.2–4.7 × and 9.6–16.9 × greater than phenol turnover rates (0.015–0.064 h^-1 mg sediment AFDW^-1). Although the relatively low rates of utilization of refractory phenolic materials suggest that these compounds may accumulate and become more prevalent components of the DOM pool, phenol concentrations at the 4 study sites remained below detectable levels (i.e., < 1 g 1^-1) throughout the study. Consequently, it seems that although phenolic materials are metabolized more slowly than labile DOM, phenols are degraded at rates which preclude accumulation in the water column.