Selected by Tony Cass

Imperial College of Science, Technology and Medicine, London, UK

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Brenner S, Johnson M, Bridgman J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M et al.: Nat Biotechnol 2000, 18:630-634.

•• Significance: The completion of the human genome sequence is focussing attention on identifying variation in gene sequences and in expression levels. In these applications, the ability to analyse accurately and quickly large numbers of genes is the key requirement.

Findings: The authors describe an approach that combines signature sequencing with a microbead-array-based cloning method. Although only short signature sequences are read (16–20 bases), the inherent parallelism of the method, involving simultaneous analysis of millions of beads (i.e. millions of templates) gives a very high-throughput with good statistics. The method was validated by sequencing over 269,000 signatures from a yeast cDNA library and by measuring gene expression in a human cell line

Atomic force microscopy reveals the mechanical design of a modular protein. Li H, Oberhauser AF, Fowler SB, Clarke J, Fernandez JM: Proc Natl Acad Sci USA 2000, 97:6527-6531.

• Significance: The mechanical properties of proteins are important in processes such as cell adhesion and muscle action. Proteins that are involved in such processes are often composed of tandem repeat structures and understanding their behaviour requires measurement of their force-extension profiles. In this paper, a method for measuring force-extension profiles using atomic force microscopy is described.

Findings: Atomic force microscopy was used to directly measure the force-extension curves of recombinant polyproteins composed of either homo- or hetero-tandem repeats of Ig domains. Typical unfolding forces were 200–300 pN with individual profiles being observed for each domain. It was found through comparison with chemical unfolding experiments that kinetic rather than thermodynamic factors were important in the mechanical stabilisation of the different domains.

Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Deniz AA, Laurence TA, Beligere GS, Dahan M, Martin AB, Chemla DS, Dawson PE, Schultz PG, Weiss S: Proc Natl Acad Sci USA 2000, 97:5179-5184.

•• Significance: The ensemble character of proteins in solution has lead to an increasing interest in measurement of the structure and function of single molecules. This is particularly so for protein folding studies where the ensemble nature is exhibited by folded, denatured and intermediate states. Current models of protein folding are derived from ensemble properties and it would be valuable to test them against single-molecule measurements. This paper describes a method for making such single-molecule measurements.

Findings: A small single domain protein, CI 2, was site-specifically labelled with a FRET donor–acceptor pair and single-molecule energy transfer efficiencies were determined with freely diffusing species. Frequency distributions of fluorescence events clearly revealed two populations of events whose relative proportions varied with denaturant concentration. The effect of a known destabilising mutation was also clearly shown in the frequency distribution. Calculated ensemble properties agreed closely with the experimental values.

A new microvolume technique for bioaffinity assays using two-photon excitation. Hänninen P, Soini A, Meltola N, Soini J, Soukka J, Soini E: Nat Biotechnol 2000, 18:548-550.

• Significance: Binding assays are arguably the most commonly used and versatile of bioanalytical methods. They find application in areas such as clinical diagnostics and drug discovery, and in both fields the demands are for faster procedures that consume smaller amounts of reagents. Here, a novel miniaturized bioaffinity assay is described that exploits two-photon laser induced fluorescence.

Findings: An assay format using 3 mm latex beads was developed in which an antibody is bound to the bead and a fluorescent antigen is present in solution. Two-photon laser fluorescence measurements could be used to determine the amount of antigen bound to the bead without the need to separate antigen free in solution. A dynamic range for determination of the antigen of four orders of magnitude was achieved and the performance of the assay was unaffected by reducing the volume from 25 ml to 2 ml, suggesting that assay volumes could be made even smaller.

Structural basis for the interaction of the fluorescence probe 8-anilino-1-naphthalene sulfonic acid (ANS) with the antibiotic target MurA. Schönbrunn E, Eschenburg S, Luger K, Kabsch W, Amrhein N: Proc Natl Acad Sci USA 2000, 97:6345-6349.

• Significance: 8-anilino-1-naphthalene sulfonic acid (ANS) has been used for many decades as a fluorescence probe of proteins, particularly for hydrophobic binding sites. Typically, there is a significant increase in the intensity of the dye when bound to protein. Despite the analytical usefulness of the dye, molecular details of its interactions with proteins are sketchy.

Findings: The structure of the antibiotic target protein MurA complexed with ANS was determined at a resolution of 1.7 Å. The ANS-binding site was found to be at a solvent-exposed loop. As a consequence of the interaction of the dye with the protein, a large reorganisation of the loop takes place to create a specific binding site for ANS that is solvent shielded. In this protein at least, the mechanism of ANS binding involves both polar and hydrophobic interactions, thus reconciling the two differing views of ANS–protein interactions.

Selected by Jim Dunwell

University of Reading, Berkshire, UK

Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides. Zhu T, Mettenburg K, Peterson DJ, Tagliani L, Baszczynski CL: Nat Biotechnol 2000, 18:555-558.

•• Significance: This report confirms that oligonucleotide-mediated gene manipulation can be applied to crop improvement. Such a method does not involve genomic integration of transgenes and therefore avoids problems related to position effects or transgene silencing.

Findings: Maize plants resistant to imidazolinone herbicides were produced by targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. Specifically, a precise single nucleotide mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a location within the gene that was known to confer resistance against this class of herbicide. Herbicide painting assays confirmed the resistance phenotype in the plants regenerated from resistant calli.

Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells. James EA, Wang C, Wang Z, Reeves R, Shin JH, Magnuson NS, Lee JM: Protein Expr Purif 2000, 19:131-138.

• Significance: These results extend the range of important human growth factors that can be produced in plant cells, and confirm the potential of plant production systems.

Findings: A gene encoding the human granulocyte-macrophage colony-stimulating factor (GM-CSF), a haemopoietic growth factor, was introduced into tobacco cells. The transgenic cell suspensions produced and secreted the recombinant protein. Initial batch cultivation studies indicated a maximum of 250 μg/L extracellular and 150 μg/L intracellular GM-CSF. This plant-produced GM-CSF was biologically active and could be separated using a nickel affinity matrix.

Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis. Belbahri L, Chevalier L, Bensaddek L, Gillet F, Fliniaux MA, Boerjan W, Inze D, Thomas D, Thomasset B: Biotechnol Bioeng 2000, 69:11-20.

• Significance: The results presented here demonstrate that the flux through the biosynthetic pathway of a plant secondary product can be modified by means of genetic engineering. This is of importance in any attempt to change the profile of secondary products produced by transgenic plants.

Findings: The authors generated transformed callus cultures of Nicotiana tabacum in which the SAM-1 gene from Arabidopsis thaliana encoding S-adenosylmethionine synthetase (SAM-S), under the control of the 35S promoter, had been integrated. The presence of the SAM-1 gene was detected in all tested transformants and the SAM-S activity correlated with the accumulation of SAM in the callus cultures. The modifications in SAM-S activity were also reflected in the pattern of secondary products present in the different cell lines produced.

Selected by Andrew J Daugulis and the Biochemical Engineering Laboratory

Queens University, Kingston, Ontario, Canada

Estimation of gas hold-up in aerated vessels. Paglianti A, Takenaka K, Bujalski W, Takahshi K: Can J Chem Eng 2000, 78:386-392.

• Significance: The authors have been able to formulate a semi-empirical equation, based on a mass balance for the gas phase, of the predicted gas hold-up in stirred tank vessels, which will be very useful to those with an interest in designing fermentation vessels.

Findings: By dividing a well-mixed tank into three mixing zones, and for radial flow turbine impellers, the authors undertook to derive mass balance equations, leading to an expression for gas hold-up. Using data from others as well as from their own work, it was shown that the correlation provided good predictions for various tank diameters, aspect ratios, multiple impellers, and fluid properties, including viscosity.

On the control of an unstable periodic state in the repeated fed-batch operation of a biochemical system. Jyany A, Subramaniam P: Can J Chem Eng 2000, 78:423-427.

• Significance: In systems subject to substrate inhibition, there exists an unstable operating region that can not normally be sustained. This paper, however, describes a feedback control strategy that permits operation in this area. This approach may be of value in fed-batch fermentations.

Findings: Simple Haldane kinetics and mass-balance equations were used along with a shooting method and the Floquet Theory to formulate a proportional feedback control strategy for operating in the positive slope region depicted in a bifurcation diagram. Through simulation the authors were able to show the values of the gain in a feedback controller that was able to successfully achieve stable operation. Not all gain values provided stability using this proportional feedback control algorithm.

Selected by Thomas A Kost and Patrick Condreay

GlaxoWellcome, North Carolina, USA

Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors. Nakai N, Storm TA, Kay MA: Nat Biotechnol 2000, 18:527-532.

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A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. Duan D, Yue Y, Yan Z, Engelhardt JF: Nat Med 2000, 6:595-598.

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Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Sun LI, Li J, Xioa X: Nat Med 2000, 6:599-602.

•• Significance: The methodologies reported in these papers provide a means for increasing the packaging capacity of recombinant adeno-associated virus (rAAV) vectors and extending the potential application of rAAV gene therapy.

Findings: The described strategies to increase the packaging capacity of AAV rely on the heterodimerization of two separate vectors in a head-to-tail orientation during concatamer formation. The experimental results indicate that it is possible to increase the packaging capacity of AAV from < 5 kb to 10 kb. This system was used successfully for directing gene expression in mouse muscle and liver tissue. These results provide a methodology for generating rAAVs with increased packaging capacity for the inclusion of larger regulatory elements and cDNAs.

Somatic integration and long-term transgene expression in normal and hemophilic mice using a DNA transposon system. Yant SR, Meuse L, Chiu W, Ivics Z, Izsvak Z, Kay MA: Nat Genet 2000, 25:35-41.

• Significance: This report describes the successful application of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA.

Findings: Two plasmids were constructed: one containing a neomycin–phosphotransferase expression cassette flanked by the Sleeping Beauty (SB) transposon inverted repeats, and a second that encodes the SB transposase. Co-transfection of the plasmids into cultured cells resulted in an 80-fold increase in G418^R transformants as compared to cells transfected with a control plasmid expressing a mutant transposase. To quantitate the efficiency of transposon integration in vivo the plasmids were administered to immune deficient mice. The SB transposase was able to efficiently insert transposon DNA into the mouse genome in approximately 5–6% transfected mouse liver cells. Chromosomal transposition was shown to result in the long-term expression of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B.

Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens. Oess S, Hildt E: Gene Ther 2000, 7:750-758.

• Significance: This report describes the identification of a novel hepatitis B (HepB) virus peptide derived from the PreS2-domain that confers cell permeability to PreS proteins and unrelated proteins fused to the domain.

Findings: A 12 amino acid peptide (PLSSIFSRIGDP) located in the PreS2-domain of the hepatitis B virus surface protein efficiently translocates in an energy-independent manner into cells and is distributed evenly in the cytosol. Fusion of the peptide sequence to the amino-terminus of enhanced green fluorescent protein resulted in the translocation of the E. coli-expressed, purified protein into the cytoplasm. Infusion of the fusion protein into the portal vein of anesthetized rats demonstrated fusion protein translocation into liver cells. Replacement of three amino acid residues within the amphipathic α-helix by serine (PSSSSSRIGDP) resulted in a significant reduction in cell permeability.

Assembly of human papilloma virus type 16 pseudovirions in Saccharomyces cerevisiae. Hum Gene Ther 2000, 11:1165-1176.

• Significance: S. cerevisiae provides a system for easily producing large quantities of human papillomavirus virus-like particles (VLPs) containing a target expression plasmid for gene delivery.

Findings: S. cerevisiae was transformed with a plasmid to allow the expression of human papilloma VLPs together with a second plasmid containing HPV regulatory elements and the green fluorescent protein (GFP) gene sequence. A population of VLPs was isolated that could be used to effectively transduce a wide variety of cultured cells with resultant expression of GFP. Intramuscular injection of the pseudovirions into mice resulted in GFP expression as detected by confocal microscopy in sections of mouse tissue. Pseudovirions produced by a 200 ml culture of the yeast packaging strain were sufficient for the in vitro transduction of 10⁹ cells.

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R. Kichler A, Pages J-C, Leborgne C, Druillennec S, Lenoir C, Couland D, Delain E, Le Cam E, Roques BP, Danos O: J Virol 2000, 74:5424-5431.

• Significance: This report describes a unique example of a natural peptide that is capable of binding DNA and transporting it into cells.

Findings: A plasmid containing the green fluorescent protein (GFP) gene and the CMV promoter was incubated with the carboxy-terminal half of the human immunodeficiency virus viral protein R (Vpr). This resulted in the formation of a peptide–DNA complex that could mediate transfection of a variety of human and non-human cell lines. Cell survival after transfection was between 70 and 90%. The transfection efficiency of this system was significantly greater than that observed with either polylysine or DOTAP but less than that of polyethylenimine. Further comparisons with other transfection agents and additional cell lines will be required to determine the true utility of this transfection method.

Sendai virosomes revisited: reconstitution with exogenous lipids leads to potent vehicles for gene transfer. Ponimaskin E, Bareesel KKH, Markgraf K, Reska R, Lehmann K, Gelderblom HR, Gawaz M, Schmidt MFG: Virology 2000, 269:391-403.

•• Significance: The use of virus-like particles that contain no viral vector genetic material to package and deliver DNA to cells as is outlined in this report is desirable to avoid the biosafety concerns of using defective viruses as gene delivery tools.

Findings: Sendai virus envelope proteins were extracted with Triton X-100 followed by reconstitution of the proteins with an exogenously added lipid mix. This gave rise to virosome particles of uniform appearance when examined by electron microscopy, and these virosomes demonstrated fusogenic ability. Furthermore, when DNA that had been condensed by incubation with poly-l-lysine was added to the reconstitution mix, the resulting virosomes packaged the DNA. The DNA-containing virosomes were able to efficiently deliver the DNA to cells for subsequent gene expression.

A phage integrase directs efficient site-specific integration in human cells. Groth AC, Olivares EC, Thyagarajan B, Calos MP: Proc Natl Acad Sci USA 2000, 97:5995-6000.

•• Significance: The site-specific integration of recombinant genes into host genomes could help overcome problems such as transcriptional silencing of stably integrated expression cassettes.

Findings: The integrase protein from Streptomyces phage φC31 carries out the site-specific integration reaction between the attP DNA sequence in the phage genome and the attB sequence in the bacterial host genome. A plasmid was constructed with a lacZ gene flanked by an attB and an attP sequence. When co-transformed into E. coli along with a plasmid expressing the φC31 integrase, the lacZ gene is excised because of intramolecular recombination between the att sites. The same reaction is carried out at ∼50% efficiency in human cells indicating that the integrase has no other co-factor requirements. When the att sites are located on different DNA molecules, the integrase is able to recombine the plasmid containing the attP site into the molecule containing the attB site in human cells.

An efficient recombination system for chromosome engineering in Escherichia coli. Yu D, Ellis HM, Lee E-C, Jenkins NA, Copeland NG, Court DL: Proc Natl Acad Sci USA 2000, 97:5978-5983.

• Significance: The method outlined here can be used to introduce specific changes in the E. coli chromosome and can possibly be adapted for construction of bacterial artificial chromosomes for genomic analysis.

Findings: A λ lysogen of E. coli was constructed in which the PL operon is controlled solely by a temperature-sensitive λ repressor. When induced by incubation at 42°C and electroporated with linear DNA containing short stretches of homology with the E. coli chromosome, the linear DNAs are integrated into the chromosome by homologous recombination. This method was used to introduce point mutations into genes and to replace entire genes. The reaction can occur with as little as 30 bp of sequence identity flanking the region to be introduced, and is dependent on expression of the exo, bet, and gam genes from the λ prophage.

Noninvasive measurement of gene expression in skeletal muscle. Walter G, Barton ER, Sweeney HL: Proc Natl Acad Sci USA 2000, 97:5151-5155.

• Significance: The ability to determine gene expression in vivo mediated by therapeutic vectors is essential to the development of gene therapy.

Findings: The invertebrate arginine kinase (AK) gene catalyzes the formation of phosphoarginine and is the correlate of the creatine kinase gene in vertebrates. Mice were injected in the hindlimb with a recombinant adenovirus containing the AK gene. Expression of AK in treated mice was followed by detection of phosphoarginine using ³¹P-magnetic resonance spectroscopy. Expression of AK could be detected repeatedly in the treated animals up to eight months after injection.

Activation of gene expression by small molecule transcription factors. Mapp AK, Ansari AZ, Ptashne M, Dervan PB: Proc Natl Acad Sci USA 2000, 97:3930-3935.

• Significance: The ability to design artificial molecules that can regulate transcription is an important advance for gene therapeutics.

Findings: A transcriptional activator was constructed by synthesis of a DNA-binding domain, a dimerization domain, and an amphipathic helical activation domain, which were then linked together. The DNA-binding module was made up of a hairpin polyamide that specifically binds to the sequence TGTTAT in the minor groove. The artificial transcription factor binds to DNA with a dissociation constant of 11 nM and activates transcription from a minimal promoter containing three binding sites. Furthermore, the dimerization peptide could be replaced with a nonpeptide linker with only a 50% reduction in transcriptional activation.

A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis. Smith GA, Enquist LW: Proc Natl Acad Sci USA 2000, 97:4873-4878.

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Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Almazán F, González JM, Pénzes Z, Izeta A, Calvo E, Plana-Durán J, Enjuanes L: Proc Natl Acad Sci USA 2000, 97:5516-5521.

•• Significance: The ability to genetically manipulate viral genomes and subsequently derive infectious virus makes it possible to study the pathogenesis of viruses with large genomes.

Findings: In the first report, a bacterial artificial chromosome (BAC) was constructed by placing the miniF replicon flanked by loxP sites into an intergenic space of the 142 kb pseudorabies genome. Upon co-transfection with a Cre-expressing plasmid into PK15 porcine kidney cells the viral genome is excised from the BAC resulting in the production of infectious pseudorabies virus. To improve the efficiency of Cre-mediated excision, an intron-containing cre gene was recombined into the miniF region of the BAC. In the second report, a cDNA of a 30 kb coronavirus genome was constructed in a BAC with the transcription of the cDNA controlled by the CMV promoter. When cells are transfected with the BAC the cDNA is transcribed in the nucleus and migrates to the cytoplasm where it launches an infection that gives rise to infectious virus.

Selected by Jeroen Hugenholtz

Wageningen Centre for Food Sciences, The Netherlands

Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae. Hancock RD, Galpin JR, Viola R: FEMS Microbiol Lett 2000, 186:245-250.

•• Significance: Clear evidence is presented that baker’s yeast can produce vitamin C, but only under unphysiological fermentation conditions. This opens interesting possibilities for the use of this yeast as a cell factory for the production of this essential vitamin.

Findings: Many fungi, including baker’s yeast, have the ability to synthesize d-erythroascorbic acid (EAA), a redox compound with similar properties as l-ascorbic acid. In this paper, the authors describe the synthesis of l-ascorbic acid (vitamin C) by Saccharomyces cerevisiae, using this EAA-pathway, when the cells are presented with unusual fermentation substrates, such asl-galactose and several lactones.

Regulation of urease gene expression by Streptococcus salivarius growing in biofilms. Li Y-H, Chen Y-YM, Burne RA: Environ Microbiol 2000, 2:169-177.

• Significance: Clear upregulation of urease activity at low pH was detected in biofilms of the oral bacterium Streptococcus salivarius. The urease gene promotor is presented as a biological probe for sensing pH in situ. The reported regulation of urease can also be expected in yoghurt production, where the related microorganism Streptococcus thermophilus plays a central role in the acidification process.

Findings: Direct measurement of urease activity in biofilms and gene expression studies using fusions of the urease promoter and the cat gene show clear low-pH-induced activity of urease in Streptococcus salivarius. Apparently, urease activity plays a direct role in pH-homeostasis. Interestingly, urease activity was much higher in biofilms than in fluid cultures. As much as 130-fold increased activities of urease were observed in S. salivarius biofilms at low pH.

Investigation of the relationship between lysogeny and lysis of Lactococcus lactis in cheese using prophage-targeted PCR. O’Sullivan D, Ross RP, Fitzgerald GF, Coffey A: Appl Environ Microbiol 2000, 66:2192-2198.

•• Significance: Lysis of starter bacteria, leading to release of intracellular enzymes essential for flavour formation, is an essential part of cheese production. This paper demonstrates the direct relationship between the presence of prophages in the cells and their ability to lyse.

Findings: A large collection of Lactococcus lactis strains were screened for their ability to lyse and for the presence of prophages. All rapid lysing strains, measured by release of the intracellular enzyme lactate dehydrogenase, appeared to contain prophages, identified by a specific PCR-method. This knowledge is very useful in selecting suitable strains for cheese manufacture.

Selected by Andrew J Daugulis and the Biochemical Engineering Laboratory

Queens University, Kingston, Ontario, Canada

Biofiltration of air contaminated by styrene: effect of nitrogen supply, gas flow rate, and inlet concentration. Jorio H, Bibeau L, Heitz M: Environ Sci Technol 2000, 34:1764-1771.

• Significance: Biofiltration is a relatively ‘low tech’ but promising means of removing volatile organic compounds (VOCs) from gas streams. The characterization of biofilter performance, particularly as applied to a challenging VOC such as styrene, is a useful contribution to extending this technology to other applications.

Findings: Nitrogen supplementation, either as ammonia or nitrate, along with gas flows and inlet styrene concentrations were varied, and the effect on the performance of the biofilters was determined. Ammonia proved to be the superior nutrient supplementation, although overgrowth of biomass and bed clogging were observed. Switching to nitrate was not entirely successful in alleviating this problem. The complete biodegradation of styrene in the biofilters was demonstrated.

Thermostability of proteins by diglycerol phosphate a new compatible solute from the hyperthermophile Archaeoglobus fulgidus. Lamosa P, Burke A, Peist R, Huber R, Lu M, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C, Santos H: Appl Environ Microbiol 2000, 66:1974-1979.

• Significance: Diglycerol phosphate (DGP), a solute produced under conditions of salt stress in the archaeon Archaeoglobus fulgidis, or synthetically produced, can play a role in thermostabilization of proteins, and hence maintenance of protein performance at high temperatures. This ability makes DGP a potentially useful protein stabilizer for biotechnological applications.

Findings: Addition of incremental concentrations of DGP results in increased protection of proteins to high temperature exposure (with a near linear dependence). In many cases, the beneficial DGP effect is particularly impressive when compared with drastic losses in activity, and very short half lives, observed in the absence of solute. Experimentation shows that DGP does in fact confer protection from denaturation rather than just promoting correct protein refolding. The extent of protection depends on the particular solute–protein pair and specific structural features of the target protein. Use of DGP as a stabilizer is advantageous relative to common solutes (e.g. glycerol, trahalose) in that these are effective primarily in the molar range, whereas DGP can confer a high degree of protection at even millimolar concentrations.