ORGANOGENESIS IN THE CARPELLATE FLOWER OF DRIMYS LANCEOLATA

This study of floral development in Drimys lanceolata in Section Tasmannia provides a basis for comparison with D. winteri, a member of the section Wintera, which has been described previously. The carpellate flowers of D. lanceolata have 2 sepals, 4–6 petals, and a solitary carpel, which form in acropetal succession. In symmetry the flower and its apical meristem are bilateral rather than radial, as in the flower of Drimys winteri. The floral apex of D. lanceolata is zonate while that of D. winteri is organized as a mantle and core. Preceding carpel initiation the floral apex of D. lanceolata is narrowly wedge-shaped, while that of D. winteri is low-convex. The entire apex is utilized in carpel initiation in D. lanceolata, involving many subsurface cell divisions over the entire summit. No apical residuum remains, and the carpel is terminal. In this feature the contrast with D. winteri is particularly marked, since in the latter, carpels are initiated laterally around the floral apex, which c an be recognized as an apical residuum after all appendages have formed.     

Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.     

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction site mapping for three or more enzymes

Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined. Received on July 30, 1989; accepted on April 4, 1990     

Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase.

A genomic clone encoding flavanone 3-hydroxylase (F3H) was isolated from Arabidopsis thaliana. The deduced amino acid sequence is 72 to 94% identical to all previously reported F3H proteins. Low-stringency DNA blot analysis indicated that F3H is encoded by a single gene in Arabidopsis. The F3H locus was mapped to the bottom of chromosome 3 and therefore does not correspond to any of the 13 flavonoid-deficient transparent testa mutants for which a map position is known. Analysis of gene expression in etiolated seedlings exposed to white light and in two putative regulatory mutants, ttg and tt8, demonstrated that the Arabidopsis F3H gene is coordinately expressed with chalcone synthase and chalcone isomerases is seedlings, whereas dihydroflavonol reductase expression is controlled by distinct regulatory mechanisms. The F3H gene may represent a pivotal point in the regulation of flavonoid biosynthesis because its expression is coordinated with different subsets of genes in different plant species.     

Bcl-2 blocks p53-dependent apoptosis.

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.     

Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.     

Spectral heterogeneity and time-resolved spectroscopy of excitation energy transfer in membranes of Heliobacillus mobilis at low temperatures.

Transient absorption difference spectra in the Qy absorption band from membranes of Heliobacillus mobilis were recorded at 140 and 20 K upon 200 fs laser pulse excitation at 590 nm. Excitation transfer from short wavelength absorbing forms of bacteriochlorophyll g to long wavelength bacteriochlorophyll g occurred within 1-2 ps at both long wavelength bacteriochlorophyll g occurred within 1-2 ps at both temperatures. In addition, a slower energy transfer process with a time constant of 15 ps was observed at 20 K within the pool of long wavelength-absorbing bacteriochlorophyll g. Energy transfer from long wavelength antenna pigments to the primary electron donor P798 was observed, yielding the primary charge-separated state P798+A0-. The time constant for this process was 30 ps at 140 K and about 70 ps at 20 K. A decay component with smaller amplitude and a lifetime of up to hundreds of picoseconds was observed that was centered around 814 nm at 20 K. Kinetic simulations using simple lattice models reproduce the observed decay kinetics at 295 and 140 K, but not at 20 K. The kinetics of energy redistribution within the spectrally heterogeneous antenna system at low temperature argue against a simple "funnel" model for the organization of the antenna of Heliobacillus mobilis and favor a more random spatial distribution of spectral forms. However, the relatively high rate of energy transfer from long wavelength antenna bacteriochlorophyll g to the primary electron donor P798 at low temperature is difficult to explain with either of these models.