Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Absence of 7-acetyl taxol binding to unassembled brain tubulin

The effect of taxol on microtubule proteins at 0 degrees C is controversial. In order to determine if taxol is unable to bind to unassembled tubulin, as has been hypothesized, the binding of [3H]acetyl taxol has been studied using equilibrium microdialysis. Ac-taxol bound to microtubules at 37 degrees C and the binding remained stable when the temperature was lowered to 0 degrees C. Ac-taxol bound also at 0 degrees C to microtubules stabilized with rhazinilam. In contrast, there was no binding of Ac-taxol to unassembled tubulin, either free tubulin at 0 degrees C or tubulin, complexed with several microtubule poisons, at 0 and 37 degrees C.     

A model for short-term control of the bacterioplankton by substrate and grazing

Substrate supply and grazing are the factors with the greatest potential for short-term control of planktonic bacterial density and productivity. A model was developed based on Monod kinetics, where growth rates are limited by food supply in a saturation type equation. In the model, substrate, bacteria, heterotrophic flagellates and zooplankton are state variables linked by trophic transfer and expressed as carbon. The steady state assumption allows calculation of equations indicating the following: (l) bacterial density is determined primarily by the ratio of substrate input to grazing rate; (2) bacterial production is balanced by a combination of losses due to maintenance, death and grazing, and occurs at a rate determined by the rate of substrate input and the growth yield; (3) ambient substrate concentration is directly related to grazing rate. Sensitivity analysis of the model on a computer demonstrates some differences between grazer-controlled and substrate-controlled bacterial systems, and predictions of the model are listed for possible validation in natural systems. The model is potentially useful in evaluating the ‘link vs. sink’ question, as it provides a framework for investigating energy flow through the microbial food web as a function of controlling factors.     

1H NMR studies of human C3a anaphylatoxin in solution: sequential resonance assignments, secondary structure, and global fold

The spin systems that comprise the 1H nuclear magnetic resonance (NMR) spectrum of the complement fragment C3a (Mr 8900) have been completely identified by an approach which integrates data from a wide range of two-dimensional NMR experiments. Both relayed and multiple quantum experiments play an essential role in the analysis. After the first stage of analysis the spin systems of 60 of the 77 residues were assigned to the appropriate residue type, providing an ample basis for subsequent sequence-specific assignments. Elements of secondary structure were identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser effects (NOEs), values of 3JHN alpha, and locations of slowly exchanging backbone amide protons. Three well-defined helical segments are found. Gradients of increasing mobility in distinct segments of the C3a polypeptide are observed, with very high mobilities for several residues near the C- and N-termini, including the complete C-terminal receptor binding site pentapeptide LGLAR. The NMR data, combined with known disulfide linkages and a small number of critical long-range NOEs, provide the global folding pattern of C3a in solution. Identical solution structures were found for both the intact active protein and the largely inactive physiologic product des-Arg77-C3a.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhomogeneous translational diffusion of monoclonal antibodies on phospholipid Langmuir-Blodgett films.

The translational mobility of fluorescent-labeled monoclonal antibodies specifically bound to supported phospholipid bilayers containing hapten-conjugated phospholipids has been measured as a function of the surface concentration of bound antibodies using fluorescence recovery after photobleaching. Fluorescence recovery curves are fit well by a model that assumes the presence of two populations of antibodies with different lateral diffusion coefficients. The larger diffusion coefficient equals 3.5 x 10(-9) cm2/s, the smaller diffusion coefficient ranges from 1.5 x 10(-9) cm2/s to 2.5 x 10(-10) cm2/s, and the fractional fluorescence recovery associated with the smaller coefficient increases from approximately 0 to approximately 0.7 with increasing concentration of bound antibody. These results suggest that complexes of haptenated phospholipids and antibodies in phospholipid Langmuir-Blodgett films form clusters or domains in a concentration-dependent fashion.     

Membrane ordering effects of the anticancer agent VM-26

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.     

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.     

Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase

gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex.     

Some aspects of the biology of the red bass, Lutjanus bohar (Forsskal), from the Tigak Islands, Papua New Guinea

The growth rate, food, reproduction and mortality rates of the red bass, Lutjanus bohar , are described for fish associated with coral reefs in the Tigak Islands of Papua New Guinea. Spawning intensity by month was investigated by examination of gonad maturation stages. The length–at–maturity of female L. bohar was estimated from the relationship between changes in gonad index with increasing size. Female fish matured at about 45–0 cm fork length. The Petersen method was used to obtain age–at–length data from length frequency data of L. bohar captured by hook–and–line fishing. The growth of L. bohar was described by a von Bertalanffy growth curve with the parameters AT=0.27 and L_∞ = 81.7 cm. There was a positive correlation between water depth (D) and the length of L. bohar (L) that was described by L = 318–8+ 3.050 D . The mortality rates ( Z ) of L. bohar from shallow (≤30m) and deep (≤80m) water were estimated from length converted catch curves and were 1–181 and 0–64 respectively. The high Z value for the shallow water population of L. bohar was ascribed to the migration of larger specimens into deeper water. The deep water population of L. bohar remains unfished and the estimate of Z was believed to be equivalent to the natural mortality rate ( M ). An empirical method of calculating M gave an estimate of 0.59. Lutjanus bohar is a carnivorous piscivore that feeds throughout the water column.