I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

http://www.biotechlab.ch/UserFiles/File/787339_Ref_CererghinoCregg2000.pdf

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

Survival strategies of bacteria in the natural environment.

In this paper minimal standards for the description of new genera and cultivable species in the family Flavobacteriaceae are proposed in accordance with Recommendation 30b of the Bacteriological Code (1990 Revision). In addition to specified phenotypic characteristics, the description of new species should be based on DNA-DNA hybridization data, and the placement of new taxa should be consistent with phylogenetic data derived from 16S rRNA sequencing. An emended description of the family is also proposed as several new taxa have been described since 1996. These proposals have been endorsed by the members of the Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes.

/pdf/proposed-minimal-standards-for-describing-new-taxa-of-the-3hq66dh1yw.pdf

Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family.

Foreign gene expression in yeast: a review.

Revue des effets cellulaires dus a une nutrition limitee des microorganismes pour le carbone, l'azote, le phosphore, le soufre, le potassium et le magnesium

/pdf/physiological-responses-to-nutrient-limitation-4eaocsu0o3.pdf

Physiological Responses to Nutrient Limitation

The development of microbodies in the yeast Saccharomyces cerevisiae was studied in response to different conditions of growth. Various strains of S. cerevisiae were investigated, using cells from the exponential growth phase on glucose as an inoculum in all transfer experiments. Electron microscopy, including serial sectioning, revealed that these cells generally contained one to four small microbodies which were localized in the vicinity of the cell wall and characterized by the presence of catalase. Transfer of these glucose-grown cells into media supplemented with various compounds known to induce microbody proliferation in other yeasts--i.e. uric acid, alkylated amines, amino acids, C2-compounds such as ethanol or acetate, in the presence or absence of compounds that induce oxygen radical formation--did not result in a significant change in the number of microbody profiles observed. Marked microbody proliferation was, however, observed after a shift of cells into media containing oleic acid and was associated with the induction of activities of beta-oxidation enzymes. In addition, catalase and isocitrate lyase were present in enhanced levels. Kinetic experiments suggested that these microbodies developed from those originally present in the inoculum cells. In thin sections up to 14 microbody profiles were occasionally observed, often present in small clusters. Their ultimate volume fraction amounted to 8-10% of the cytoplasmic volume.

Proliferation of microbodies in Saccharomyces cerevisiae

In natural and man-made environments microorganisms often grow in the presence of a diversity of functionally similar substrates. The pattern of utilization of these mixed substrates is generally dependent upon their concentration. When substrates are present in high (not growth-limiting) concentrations, sequential utilization and diauxic growth is often observed and the substrate that supports the highest growth rate is utilized preferentially from the mixture. When the substrate concentrations are growth-limiting, simultaneous utilization of the various compounds present in the mixture appears to be the general response. Recent studies on mixed substrate utilization in both batch and continuous cultures have thrown light on the strategies of the control mechanisms that, in microbes, govern the utilization of the various substrates. But perhaps more importantly these studies have indicated the possible significance of mixed substrate utilization in microbial competition in nutrient-limited natural ecosystems.

Strategies of mixed substrate utilization in microorganisms

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h-1 showed a maximal capacity to oxidize excess methanol (QO
 2
 max
 ) which was 1.6 times higher than the rate required to sustain the growth rate (Q
O2). When the dilution rate was decreased to 0.03 h-1, QO
 2
 max
 of the cells increased to a value of more than 20 times that of Q
O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K
mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K
mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM. The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.

Growth of hansenula-polymorpha in a methanol-limited chemostat - physiological-responses due to involvement of methanol oxidase as a key enzyme in methanol metabolism

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7 x 10(6)/micrograms plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per microgram DNA) for H. polymorpha remained high when large amounts (up to 10 micrograms) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.

https://pure.rug.nl/ws/files/14644737/1994CurrGenetFaber.pdf

Wim Harder

Papers

Physiological Responses to Nutrient Limitation

Proliferation of microbodies in Saccharomyces cerevisiae

Strategies of mixed substrate utilization in microorganisms

Growth of hansenula-polymorpha in a methanol-limited chemostat - physiological-responses due to involvement of methanol oxidase as a key enzyme in methanol metabolism

Highly-efficient electrotransformation of the yeast Hansenula polymorpha