A Technique for Predicting the Solvent-Producing Ability of Clostridium acetobutylicum

Changes in colony morphology were associated with the degeneration of solvent-producing strains of Clostridium acetobutylicum. The most efficient solvent-producing strains gave rise exclusively to colonies with dense centers containing large numbers of spores. Many outgrowths of various morphologies developed from the perimeter of such colonies after several days of incubation. The most degenerate cultures did not produce solvents and gave rise to large diffuse colonies that did not contain spores. These diffuse colonies did not produce outgrowths. Intermediate colony types were also observed. These could be derived from liquid cultures that were relatively poor solvent producers or from the outgrowths of colonies of efficient solvent-producing strains. Some of these intermediate types produced spores but did so less frequently than the high-solvent-producing strains. The spores of the intermediate types could not be distinguished from those of the most efficient solvent producers on the basis of heat sensitivity. The relationship observed between colony morphology and solvent production provides a method for predicting the solvent-producing potential of C. acetobutylicum cultures.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost,Gillichthys mirabilis

Summary A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/ UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.     

Mode of Action Studies on Nitrodiphenyl Ether Herbicides : II. The Role of Photosynthetic Electron Transport in Scenedesmus obliquus

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI) induces light- and O₂-dependent lipid peroxidation and chlorophyll (Chl) bleaching in the green alga Scenedesmus obliquus. Under conditions of O₂-limitation, these effects are diminished by prometyne and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), both inhibitors of photosynthetic electron transport. Mutants in which photosynthetic electron transport is blocked are also resistant to DPEI under conditions of O₂-limitation. Light- and O₂-dependent lipid peroxidation and Chl bleaching are also induced by 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methoxyphthalide (DPEII), a diphenyl ether whose redox properties preclude reduction by photosystem I. However, these effects of DPEII are also inhibited by DCMU. Under conditions of high aeration, DCMU does not protect Scenedesmus cells from Chl bleaching induced by DPEI, but does protect against paraquat. DPEI, but not paraquat, induces tetrapyrrole formation in treated cells in the dark. This is also observed in a mutant lacking photosystem I but is suppressed under conditions likely to lead to O₂ limitation. Our results indicate that, in contrast to paraquat, the role of photosynthetic electron transport in diphenyl ether toxicity in Scenedesmus is not to reduce the herbicide to a radical species which initiates lipid peroxidation. Its role is probably to maintain a sufficiently high O₂ concentration, through water-splitting, in the algal suspension.     

Increased protoplast yield from oat leaves and bean internodes by non-injurious mechanical perturbation

Summary A simple new technique has been developed to greatly increase the yield of protoplasts from plant organs without injury to the plant. Mechanical perturbation (MP) by non-stressful rubbing of oat leaf segments and bean internodes yielded ten to twenty times more viable protoplasts than did controls. The increase in protoplast yield due to MP is best manifested, if the organs are excised and transferred to the cellulytic enzymes immediately after MP is given to the intact organ. The enzymes begin digesting from the lower end of the bean internodes and proceed acropetally. Vacuum infiltration of control oat leaf segments for 15 min with enzyme solution resulted in increased yield but less than due to MP. Increased levels of calcium (10 mM) in the medium decreased the yield of protoplasts from both control and MP-treated plant organs. EGTA significantly increased the yield of protoplasts from control oat leaf segments and marginally over that found in the control bean internodes. Cycloheximide increased the yield of protoplasts from oat leaf segments but not from bean internodes. It is suggested that MP may increase the susceptibility of cell wall polymers to cellulytic enzymes by reducing calcium cross linking. MP is thus a tool for increasing the yield of protoplasts from plant organs without causing injury.Abbreviations CHI cycloheximide - EGTA ethyleneglycol-bis-(ß-aminoethyl ether)-N,N-tetraacetic acid - FDA fluorescein diacetate - MP mechanical perturbation     

Glycosylation reduces the bioactivity of calcitonin

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

Binding of a pancreatic nuclear protein is correlated with amylase enhancer activity.

The mouse amylase gene Amy-2.2 is expressed at high levels specifically in the acinar cells of the pancreas. The region between -172 and -110 of this gene includes sequence elements common to pancreas-specific genes. Nuclear proteins with specific affinity for this region were partially purified from rat pancreas. The consensus element of another pancreas-specific gene, elastase 1, competes for protein binding to the amylase sequences. Binding was localized by DNase I protection to the sequence -156 to -122. Site-directed mutagenesis of this sequence resulted in concomitant loss of protein binding and enhancer activity. Photo-affinity labelling of pancreatic nuclear extracts identified one predominant binding protein with a molecular weight of approximately 75 kDa. The data indicate that binding of this nuclear protein is essential for the enhancer activity of this pancreas-specific element.     

Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate.

To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.