Undergraduate research in a biotechnology teaching laboratory: expression and purification of a tyrosine kinase

The class consisted of senior molecular biology majors who had previously taken cell biology, microbiology, biochemistry and molecular biology lecture courses but who had little or no previous lab experience. These students were asked to design and create an expression vector and purify the expressed protein. This project provides the students with the opportunity to appreciate interconnections between experiments while learning the necessary techniques. Journal of Industrial Microbiology & Biotechnology (2000) 24, 359–363. Received 02 April 1999/ Accepted in revised form 10 November 1999     

Sweet home actinomycetes: The 1999 MDS Panlabs Lecture

For the past 25 years, I have devoted most of my research efforts to the application of molecular genetics to yield improvement and production of novel secondary metabolites in actinomycetes. My group at Lilly Research Laboratories worked with a variety of Streptomyces species and with strains of Amycolatopsis and Saccharopolyspora. We developed molecular genetic tools to manipulate actinomycete genes, and applied them to important secondary metabolites, including tylosin, daptomycin, vancomycin, chloroeremomycin, and spinosyns. In the early years, I helped establish recombinant DNA technology to manufacture mammalian proteins, and more recently, helped implement microbial genomics as a research tool for antibiotic discovery. In this paper, I review some highlights, primarily from the actinomycete work. Journal of Industrial Microbiology & Biotechnology (2000) 24, 79–88. Received 25 October 1999/ Accepted in revised form 12 November 1999     

Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype

Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170–1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica. Received: 19 January 1999 / Revised version: 3 November 1999 / Accepted: 22 November 1999     

Integration of laboratory exercises in developmental biology and neurobiology courses using the Xenopus oocyte expression system

This collaborative laboratory exercise integrates two upper division laboratory courses (Developmental Biology and Neurobiology) offered to biology majors at Wake Forest University. The laboratory exercise involves the use of the Xenopus oocyte expression system to study the function of specific membrane receptors and ligand-activated channels. cDNA or mRNA for receptor proteins is injected into Xenopus oocytes. The oocytes are assayed for expression of receptor proteins and two-electrode voltage clamping is done to determine whether the expressed proteins are functional in the oocyte system. This series of laboratory exercises is innovative in its interdisciplinary and collaborative approach to undergraduate teaching, and in its use of sophisticated molecular biological and physiological techniques in the undergraduate teaching laboratory. Students learn first-hand how these techniques have been used to achieve a new level of understanding of both development and neurobiology. Journal of Industrial Microbiology & Biotechnology (2000) 24, 353–358. Received 02 April 1999/ Accepted in revised form 10 November 1999     

The virtual workplace: integration of research approaches and fundamental skills into an upper level biology course

Undergraduate science education is based on a model developed in the decade following World War II. It has undergone no fundamental changes since then with courses that combine lectures and laboratory experiments. Traditional courses are typically based on individual performance and much of that performance is evaluated by tests and examinations. At the same time, the modern workplace has undergone revolutionary changes that are characterized by: interdisciplinary approaches; work in teams; the exponential growth of scientific information; the rapid turnover in projects; the need for continued retraining; multiple career tracks; the globalization of science and industry; and the pervasive use of electronic communications and information systems. Journal of Industrial Microbiology & Biotechnology (2000) 24, 310–313. Received 02 April 1999/ Accepted in revised form 01 January 2000     

The genetic heterogeneity of deer mouse populations (Peromyscus maniculatus) in an insular landscape

A survey of the genetic variability in deer mouse populations was performed using specimens collected from six different islands on a lake covering approximately 50 km². Random amplified polymorphic DNA (RAPD) was used to measure the extent of the genetic differences in this insular system. An analysis of molecular variance (AMOVA) revealed that populations are clearly separated at this microgeographic scale (F _st = 0.13863; P < 0.001). The homogeneity of molecular variance test (HOMOVA) indicated that within-population levels vary greatly (B _p = 0.76831; P < 0.001). The within-population molecular variance was found to be mainly correlated with the accessibility of the islands, computed as the inverse of the geographic distance separating an island from the lakeshore (r = 0.916; P < 0.003). Received: March 5, 1999 / Accepted: July 16, 1999     

Apoptosis-like DNA fragmentation in leaves and floral organs precedes their developmental senescence

ABSTRACT

We investigated the senescence of flag leaves of durum wheat (Triticum durum) during grain-filling, and of petal-like ray flowers of Jerusalem artichoke (Helianthus tuberosus) at anthesis. In both systems, we observe cleavage of DNA to high molecular weight fragments, followed by further degradation to nucleosomal fragments (laddering), a classical hallmark of apoptosis. We show that DNA fragmentation in such specialised leaves is triggered early in organ development, before the appearance of visual symptoms of senescence. Our observations support the hypothesis that senescence and cell death are part of the plant developmental program, activated by developmental cues.     

The auto-orientation in high magnetic fields of oxidized cytochrome b562 as source of constraints for solution structure determination

¹⁵N-¹H ¹J couplings were measured at 500 MHz and 800 MHz for ¹⁵N enriched oxidized cytochrome b ₅₆₂ from E. coli. The magnetic field dependence of 70 ¹J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc). The rdc were used as further constraints to improve the existing structure [Arnesano et al. (1999) Biochemistry, 38, 8657–8670] with a protocol which uses the rhombic anisotropy [Banci et al. (1998) J. Am. Chem. Soc., 120, 12903–12909]. The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions.     

Bioinformatics in the community college

Biotechnology is becoming an information-based field. In this article we describe some resources available to instructors, show how these resources are used in the biotechnology training program, and provide examples of activities used by non-science majors to increase their understanding of biology. We discuss some of the challenges we have encountered using these tools in the classroom. Journal of Industrial Microbiology & Biotechnology (2000) 24, 314–318. Received 08 April 1999/ Accepted in revised form 10 November 1999     

Integrating biotechnology into a non-majors biology curriculum

A general education biology course entitled ‘Biotechnology Transforms Our World’ has been developed to illustrate biological concepts with advances from biotechnology. The contributions of molecular biology to understanding human genetics, evolution, plant and animal (including human) biology and ecology are illustrated with specific case studies. Journal of Industrial Microbiology & Biotechnology (2000) 24, 308–309. Received 02 April 1999/ Accepted in revised form 11 November 1999     

Study of some physico-chemical characteristics of a Saccharomyces cerevisiae endopolygalacturonase: a possible use in beverage industry

Zymograms of a crude protein extract from S. cerevisiae strain SCPP containing endopolygalacturonase were studied and compared to the purified enzyme by determining their physico-chemical properties. The results obtained with crude extract were similar to those of the purified enzyme. The endopolygalacturonase from both sources displayed a pH optimum between 3.0 and 4.0, and was active at temperatures between 4 and 50°C on a large panel of substrates. These characteristics make this S. cerevisiae endopolygalacturonase an attractive tool for the beverage industry. Journal of Industrial Microbiology & Biotechnology (2000) 24, 296–300. Received 24 September 1999/ Accepted in revised form 29 January 2000     

Use of the green fluorescent protein as an educational tool

The green fluorescent protein (GFP) is a bioluminescent protein that can be expressed and easily detected as a fully fluorescent protein in both bacterial and eukaryotic cells. These properties, along with its ability to withstand exposure to denaturants, organic solvents, high temperature and a wide pH range, make GFP an ideal educational tool. To that end, two GFP-based laboratory modules are described that can be used to teach recombinant DNA and protein purification techniques to high school and undergraduate college students. Journal of Industrial Microbiology & Biotechnology (2000) 24, 323–326. Received 02 April 1999/ Accepted in revised form 20 November 1999     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Interaction of an anticancer benzopyrane derivative with DNA: Biophysical,biochemical, and molecular modeling studies

BackgroundSIMR1281 is a potent anticancer lead candidate with multi- target activity against several proteins; however, its mechanism of action at the molecular level is not fully understood. Revealing the mechanism and the origin of multitarget activity is important for the rational identification and optimization of multitarget drugs.MethodsWe have used a variety of biophysical (circular dichroism, isothermal titration calorimetry, viscosity, and UV DNA melting), biochemical (topoisomerase I & II assays) and computational (molecular docking and MD simulations) methods to study the interaction of SIMR1281 with duplex DNA structures.ResultsThe biophysical results revealed that SIMR1281 binds to dsDNA via an intercalation-binding mode with an average binding constant of 3.1 × 10⁶ M⁻¹. This binding mode was confirmed by the topoisomerases' inhibition assays and molecular modeling simulations, which showed the intercalation of the benzopyrane moiety between DNA base pairs, while the remaining moieties (thiazole and phenyl rings) sit in the minor groove and interact with the flanking base pairs adjacent to the intercalation site.ConclusionsThe DNA binding characteristics of SIMR1281, which can disrupt/inhibit DNA function as confirmed by the topoisomerases' inhibition assays, indicate that the observed multi-target activity might originate from ligand intervention at nucleic acids level rather than due to direct interactions with multiple biological targets at the protein level.General significanceThe findings of this study could be helpful to guide future optimization of benzopyrane-based ligands for therapeutic purposes.     

Breeding of a non-urea producing sake yeast with killer character using a kar1-1 mutant as a killer donor

Arginase-deficient (car1/car1) sake yeasts can brew sake without urea, a main precursor of ethyl carbamate, which is a suspected carcinogen in various fermented beverages. For the use of car1/car1 yeasts in sake production, contamination by wild-type (CAR1/CAR1) yeasts is a major problem. To protect sake mash against such contamination, killer character was introduced into the car1/car1 sake yeast HL163 by rare mating and protoplast fusion, using a kar1–1 haploid harboring killer dsRNA plasmids as a killer donor. All killer yeasts obtained showed no arginase activity and the same DNA content per cell as strain HL163, and produced sake with ordinary quality and very low levels of urea. We also demonstrated that one of these killer yeasts could effectively eliminate contaminant cells of a CAR1/CAR1 yeast from sake mash. Journal of Industrial Microbiology & Biotechnology (2000) 24, 203–209. Received 09 August 1999/ Accepted in revised form 07 December 1999     

Selection of international molecular standards for DNA fingerprinting of <Emphasis Type="Italic">Theobroma cacao</Emphasis>

A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.     

Light-dependent transformation of anthranilate to indole by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 transformed anthranilate (2 mM) to an indole (0.7 mM) in a light-dependent process. Photobiotransformation was enhanced by tricarboxylic acid cycle intermediates and the indole formed was identified as 2,3 dihydroxy indole. Journal of Industrial Microbiology & Biotechnology (2000) 24, 219–221. Received 16 September 1999/ Accepted in revised form 20 December 1999     

Chloroform degradation by butane-grown cells of Rhodococcus aetherovorans BCP1

The ability of a Rhodococcus aetherovorans strain, BCP1, to grow on butane and to degrade chloroform in the 0–633 μM range (0–75.5 mg l⁻¹) via aerobic cometabolism was investigated by means of resting-cell assays. BCP1 degraded chloroform with a complete mineralization of the organic Cl. The resulting butane and chloroform maximum specific degradation rates were equal to 118 and 22 μmol , respectively. Butane inhibition on chloroform degradation was satisfactorily interpreted by means of a model of competitive inhibition, with an inhibition constant equal to 38 % of the estimated butane half-saturation constant, whereas chloroform (at 11 μM) did not inhibit butane utilization. Acetylene (1,720 μM) induced an almost complete inactivation of the degradation of both butane and chloroform, indicating that the studied cometabolic process is mediated by a monooxygenase enzyme. BCP1 proved capable of degrading vinyl chloride and 1,1,2-trichloroethane, but not 1,2-trans-dichloroethylene. BCP1 could grow on the intermediates of the most common butane metabolic pathways and on the aliphatic hydrocarbons from ethane to n-heptane. After growth on n-hexane, it was able to deplete chloroform (13 μM) with a degradation rate higher than that obtained, at the same chloroform concentration, after growth on butane.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA

An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 °C instead of 30 °C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10⁸ cfu μg⁻¹) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 × 10⁶ cfu μg⁻¹ xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum. Received: 26 March 1999 / Received revision: 9 June 1999 / Accepted: 11 June 1999