Determination of kinetic parameters related to the piasmid instability: for the recombinant fermentation under repressed condition

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12DeltaH1Deltatrp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (theta) describing the rate at which the plasrnid-harboring cells lose plas-mids and the plasmid-free cells are generated per unit time and the difference in growth rates (Delta) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (Omega) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters theta and Delta were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.     

An alternate method for estimation of cell growth kinetics from batch cultures

An alternative to estimation of cell growth kinetics via continuous culture experiments is proposed in this article. The method employed is based on batch culture experiments with very small inocula (initial cell concentrations being typically less than 5000 cells/mL). Such low initial cell concentrations result in extended exponential cell growth phase during which culture conditions remain unchanged, thereby permitting precise estimation of specific cell growth rates from batch experiments especially for fast-growing microorganisms such as Bacillus species. The effectiveness and utility of this approach are demonstrated via several experiments conducted with a wild-type strain (Bacillus subtilis TN106) and a recombinant strain (B. subtilis TN106[pAT5]). True establishment of exponential growth phase requires insignificant variance of most of the culture conditions during the initial growth phase. Satisfaction of this requirement is demonstrated for microbial systems investigated here. This approach is especially well suited for recombinant microorganisms containing segregationally unstable plasmids, since estimation of growth kinetics of these from continuous cultures is very difficult and highly unreliable due to continual reversion of recombinant ceils to plasmid-free host cells unless some selection pressure is applied at levels sufficient to keep the presence of plasmid-free cells minimal.     

A new method for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates

A new method is described for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates. The reducing ends of various mono-, di-, tri-, and tetra-saccharides were conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (a fluorescent and negatively charged compound) by reductive amination using sodium cyanoborohydride. The sugar conjugates were purified by preparative gradient polyacrylamide gel electrophoresis followed by a newly developed technique involving their semi-dry transfer to positively charged nylon membranes and elution with sodium chloride. The structures of a monosaccharide- and trisaccharide-conjugate were established by f.a.b.-m.s. and 2D n.m.r. Seven linear oligosaccharide-fluorescent conjugates were treated sequentially with exoglycosidases and with endoglycosidases. Analysis of the products by gel electrophoresis provided sequence information. These methods may be useful for sequencing oligosaccharides that are chemically or enzymically (endoglycosidase) released from glycoproteins, glycolipids, and proteoglycans.     

Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle.

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.     

Structure and expression of the attacin genes in Hyalophora cecropia

To study the regulation of the immune genes in insects, we have cloned and sequenced the attacin gene locus of the giant silk moth Hyalophora cecropia. The locus contains one acidic and one basic attacin gene as well as two pseudogenes, which are remnants of basic attacin genes. A small insertion element was found within the locus. The two functional attacin genes are transcribed in opposite directions and have two introns inserted at homologous positions. A common sequence, GGGGATTCCT, is found at nucleotide position -48 in the acidic gene and at nucleotide position -58 in the basic gene. Interestingly, this decanucleotide is similar to the consensus of the NF-k B-binding site. Expression studies revealed that both attacins are strongly induced by phorbol 12-myristate 13-acetate, lipopolysaccharide and bacteria. However, only the acidic attacin gene showed a clear response to injury.     

Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase alpha- and beta-subunits, and identification of beta'-, a putative beta-subunit isozyme produced by alternative splicing of the beta-subunit gene.

We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) alpha- and beta-subunits of mouse retinal rod photoreceptors. The open reading frames predict an alpha-subunit of 100 kDa (856 residues), and a beta-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve beta-subunit clones predicts the presence in the retina of an additional PDE, termed beta', which is generated by alternative splicing of the beta-subunit gene. beta' differs from beta only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the alpha- and beta-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of alpha, beta and beta'.     

Immunohistochemical distribution of MAM-3 and MAM-6 antigens in developing salivary glands of the human fetus

Summary The immunohistochemical expression of MAM-3 and MAM-6 antigens was studied in developing human fetal salivary gland removed at autopsy of 22 normal fetuses of varying maturity (10–40 weeks of gestation). The onset of functional maturation in the fetal gland was seen at 21 weeks of gestational maturity. The acini and ducts then underwent distinct alterations in antigen expression with growth and maturation until the late developmental stage (33–40 weeks of gestation) when they resemble the adult salivary gland. The role of maturing duct cells in histogenesis of salivary gland tumours is discussed.     

Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations.

In previous work, Rhizobium meliloti SU47 produced its alternative exopolysaccharide (EPSb [also called EPS II]) only in strains that were genetically altered to activate EPSb synthesis. Here we report that EPSb synthesis is not entirely cryptic but occurred under conditions of limiting phosphate. This was shown in several different exo mutants that are blocked in the synthesis of the normal exopolysaccharide, succinoglycan. In addition, EPSb biosynthetic gene expression was markedly increased by limiting phosphate. An apparent regulatory mutant that does not express alkaline phosphatase activity was unable to produce EPSb under these conditions. A mucR mutant that was previously shown to produce EPSb instead of the normal exopolysaccharide, succinoglycan, was not sensitive to phosphate inhibition of EPSb synthesis. No evidence was found to indicate that exoX, which affects succinoglycan synthesis, had any influence on EPSb synthesis. In contrast to limiting phosphate, limiting nitrogen or sulfur did not stimulate EPSb synthesis as it does succinoglycan.     

Anomalous carbonylation of [Pd(dppm)(O2CCF3)]2 to give an asymmetric μ-CO complex

The reactive palladium dimer, [Pd(dppm)(O₂CCF₃)]₂, is carbonylated to [Pd(dppm)(O₂CCF₃)]₂(μ-CO) in a reversible reaction with K = c. 7.2(2)x10⁴ atm⁻¹ (P_1/2 = c. 2.4 Torr). This is significantly larger than is expected based on the λ_max = 280 nm in the electronic spectrum. The product can be isolated in analytically pure form by crystallization under a CO atmosphere. It forms crystals in the monoclinic space group Cc with a = 18.584(5), b = 28.65(1), c = 11.164(3) Å and β = 95.16(2)°. The structure is significantly distorted. The bonding about the two palladium atoms is quite asymmetric. While one is close to a square planar geometry with a Pd---C(O) distance of 1.90(2) Å, the other is significantly pyramidalized and has a longer (2.00(2) Å) bond to the bridging CO. The Pd---Pd distance is only 2.896(2) Å, much shorter than that usually observed for formally non-bonded Pd atoms.     

Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.