A Strategy for Rotation of Different Bacteriophage Defenses in a Lactococcal Single-Strain Starter Culture System

A new strategy for starter culture rotations was developed for a series of phage-resistant clones genetically derived from a single strain of Lactococcus lactis subsp. lactis. Phage-resistant derivatives carrying different defense systems were constructed via conjugation with various plasmids encoding abortive infection (Abi/Hsp) and/or restriction and modification (R/M) systems of different specificity. The plasmids included pTR2030 (Hsp⁺ R⁺/M⁺), pTN20 (Abi⁺ R⁺/M⁺), pTRK11 (R⁺/M⁺), and pTRK68 (R⁺/M⁺). Selected phage-resistant transconjugants or transformants were evaluated in different rotation sequences through cycles of the Heap-Lawrence starter culture activity test in milk contaminated with phage and whey from the previous cycle. When used in consecutive sequence, derivative strains carrying the R/M systems encoded by pTN20, pTRK11, and pTRK68 retarded phage development when the initial levels of phage contamination were below 10² PFU/ml but not when levels were increased to 10³ PFU/ml. Use of a derivative bearing pTR2030 (Hsp⁺ R⁺/M⁺) at the beginning of the rotation prevented phage development, even when the initial levels of phage contamination were high (10⁶ PFU/ml). Alternating the type and specificity of R/M and Abi defenses through the rotation prevented phage proliferation and in some cases eliminated contaminating phages. A model rotation sequence for the phage defense rotation strategy was developed and performed successfully over nine cycles of the Heap-Lawrence starter culture activity test in the presence of high-titer commercial phage composites. This phage defense rotation strategy is designed to protect a highly specialized Lactococcus strain from phage attack during continuous and extended use in the dairy industry.     

Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth. (c) 1993 John Wiley & Sons, Inc.     

Pigmented Soybean (Glycine max) Seed Coats Accumulate Proanthocyanidins during Development

The dominant I gene inhibits accumulation of anthocyanin pigments in the epidermal layer of soybean (Glycine max) seed coats. Seed-coat color is also influenced by the R locus and by the pubescence color alleles (T, tawny; t, gray). Protein and RNA from cultivars with black (i,R,T) and brown (i,r,T) seed coats are difficult to extract. To determine the nature of the interfering plant products, we examined seed-coat extracts from Clark isogenic lines for flavonoids, anthocyanins, and possible proanthocyanidins by thin-layer chromatography. We show that yellow seed-coat varieties (I) do not accumulate anthocyanins (anthocyanidin glycosides) or proanthocyanidins (polymeric anthocyanidins). Mature, black (i,R,T) and imperfect-black (i,R,t) seed coats contained anthocyanins, whereas mature, brown (i,r,T) and buff (i,r,t) seed coats did not contain anthocyanins. In contrast, all colored (i) genotypes tested positive for the presence of proanthocyanidins by butanol/ HCl and 0.5% vanillin assays. Immature, black (i,R,T) and brown (i,r,T) seed coats contained significant amounts of procyanidin, a 3[prime],4[prime]-hydroxylated proanthocyanidin. Immature, black (i,R,T) or brown (i,r,T) seed-coat extracts also tested positive for the ability to precipitate proteins in a radial diffusion assay and to bind RNA in vitro. Imperfect-black (i,R,t) or buff (i,r,t) seed coats contained lesser amounts of propelargonidin, a 4[prime]-hydroxylated proanthocyanidin. Seed-coat extracts from these genotypes did not have the ability to precipitate protein or bind to RNA. In summary, the dominant I gene controls inhibition of not only anthocyanins but also proanthocyanidins in soybean seed coats. In homozygous recessive i genotypes, the T-t gene pair determines the types of proanthocyanidins present, which is consistent with the hypothesis that the T locus encodes a microsomal 3[prime]-flavonoid hydroxylase.     

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.     

Evolutionary rescue under demographic and environmental stochasticity

Populations suffer two types of stochasticity: demographic stochasticity, from sampling error in offspring number, and environmental stochasticity, from temporal variation in the growth rate. By modelling evolution through phenotypic selection following an abrupt environmental change, we investigate how genetic and demographic dynamics, as well as effects on population survival of the genetic variance and of the strength of stabilizing selection, differ under the two types of stochasticity. We show that population survival probability declines sharply with stronger stabilizing selection under demographic stochasticity, but declines more continuously when environmental stochasticity is strengthened. However, the genetic variance that confers the highest population survival probability differs little under demographic and environmental stochasticity. Since the influence of demographic stochasticity is stronger when population size is smaller, a slow initial decline of genetic variance, which allows quicker evolution, is important for population persistence. In contrast, the influence of environmental stochasticity is population-size-independent, so higher initial fitness becomes important for survival under strong environmental stochasticity. The two types of stochasticity interact in a more than multiplicative way in reducing the population survival probability. Our work suggests the importance of explicitly distinguishing and measuring the forms of stochasticity during evolutionary rescue.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PHYTOPLANKTON PIGMENTS USING A POLYMERIC REVERSED-PHASE C18 COLUMN1

A high-performance liquid chromatographic (HPLC) method is described that allows improved resolution of several chemotaxonomically significant phytoplankton pigments. The protocol, which employs two pumps and a modified Mantoura and Llewellyn (1983) solvent system, can be easily adapted for many HPLC systems currently in use. The most unique aspect of the method is the use of a polymeric C₁₈ reversed phase HPLC column (VYDAC 201TP). In comparison to the monomeric C₁₈ columns typically used in the characterization of phytoplankton pigments, polymeric C₁₈ columns offer superior selectivity for structurally similar compounds. The protocol was evaluated for the ability to resolve most of the phytoplankton pigments of diagnostic importance using algal cultures from nine classes. Pigment pairs that were resolved by the method include a) lutein and zeaxanthin, b) neoxanthin and 19′-hexanoyloxyfucoxanthin, and c) α-carotene and β-carotene, and partial resolution of chlorophyll c₁ and chlorophyll c₂.     

VASOMOTOR RESPONSES OF ISOLATED PERIPHERAL BLOOD VESSELS FROM BOWHEAD WHALES: THERMOREGULATORY IMPLICATIONS

Temperature regulation in bowhead whales, Balaena mysticetus , is supported by the characteristic cetacean peripheral circulation, especially notable in the tail flukes. Blood vessels serving this function consist of countercurrent heat exchangers (network of veins surrounding a central artery) favoring heat conservation and an alternate routing via arteriovenous anastomoses (AVAs) providing for heat dissipation. We tested the vasomotor responses of isolated segments of countercurrent arteries and AVAs from the bowhead tail flukes to norepinephrine (NOR), the sympathetic adrenergic neurotransmitter. Isometric tension developed during exposure to a micromolar concentration of NOR was consistently higher in AVAs than in arteries. Accordingly, the AVAs are subject to sympathetic vasoconstriction, and this activation directs blood flow to countercurrent heat exchangers and results in heat conservation. In contrast, AVA relaxation by reduced sympathetic activation favors increased blood flow through AVAs and consequent peripheral heat loss.