Increasing cell biomass in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

Background  

Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

Differential response of fetal and neonatal myoblasts to topographical guidance cues in vitro

 Fusion of mononucleated myoblasts into parallel arrays of mutinucleated myotubes is an essential step in skeletal myogenesis. The formation of such a highly ordered structure requires myoblasts to come together, orient and align in the correct location prior to fusion. We report here that fetal and neonatal myoblasts can use topographical features as strong guidance cues in vitro. Myoblasts were cultured on multiple grooved substrata of varying dimensions, and the axial orientations of individual cells were recorded. Both fetal and neonatal myoblasts aligned parallel with the direction of deep grooves (2.3–6.0 μm), which is correlated well with the location of myoblasts in similar sized grooves during secondary myogenesis. Fetal myoblasts also responded to shallower grooves (0.04–0.14 μm) by aligning parallel or perpendicular to the direction of the grooves, indicating the ability of these cells to respond to fine elements normally encountered within the developing muscle architecture. In contrast, neonatal myoblasts failed to respond to shallow grooves, adding to the suggestion that fetal and neonatal myoblasts may represent separate populations of myoblasts. Overall, the results demonstrate that myoblasts respond to large and small features of the physical topography in vitro and indicate that structural elements in the microenvironment of the muscle may play a critical role in myoblast spatial organization during myogenesis. Received: 29 May 1998 / Accepted: 17 February 1999     

Pseudomonas putida Mutants Defective in the Metabolism of the Products of meta Fission of Catechol and Its Methyl Analogues

A selection procedure is described which was used to isolate mutants of Pseudomonas putida strain U in the following enzymes of the meta-fission pathway of phenol and cresols: hydrolase, tautomerase, and decarboxylase. Strains deficient in the hydrolase are unable to use either o- or m-cresol as a sole carbon source and were shown to accumulate 2-hydroxy-6-keto-2,4-heptadienoate when incubated in the presence of o- or m-cresol. When 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form) was metabolized by phenol-induced extracts of tautomerase-deficient strains, the enol tautomer of 4-oxalocrotonate accumulated and was then converted slowly to the keto tautomer by a nonenzymatic reaction. Phenol-induced extracts of decarboxylase-deficient strains accumulated the keto tautomer of 4-oxalocrotonate from 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form). Strains with an inactive decarboxylase are unable to completely metabolize either phenol or p-cresol. Tautomerase-defective strains are unable to grow with p-cresol as the sole carbon source and grow only very slowly on phenol.     

BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.