Showing papers in "Molecular Biotechnology in 1998"
TL;DR: The essence of the method is to amplify messenger RNA 3′ termini using a pair of anchored oligo-dT primer and a short primer with an arbitrary sequence to identify up- and downregulation genes of interest.
Abstract: Characterization of regulated gene expression in eukaryotic cells is essential for studying cell growth and differentiation as well as for understanding the molecular mechanisms of diseases. Differential display was developed for such comparative studies by allowing a systematic and nonbiased screening for molecular differences at the level of mRNA expression between or among different cells or tissues. The essence of the method is to amplify messenger RNA 3' termini using a pair of anchored oligo-dT primer and a short primer with an arbitrary sequence. The amplified cDNAs labeled with radioisotope are then distributed on a denaturing polyacrylamide gel and visualized by autoradiography. Side-by-side comparison of mRNA species from two or more related samples allows identification of both up- and downregulation genes of interest. Some of the most recent improvements have been incorporated into this general protocol for differential display.
136 citations
TL;DR: In this review, procedures for handling and storage of retinoids and biological samples containing them have been described, and simplified procedures for derivatizations and purification, and methods for quantitation ofretinoids have been presented.
Abstract: Retinoids are unstable compounds being readily oxidized and/or isomerized to altered compounds, especially in the presence of oxidants including air, light, and excessive heat. They are labile toward strong acids and solvents that have dissolved oxygen or peroxides. In this review, procedures for handling and storage of retinoids and biological samples containing them have been described. The physical and chemical properties of retinoids have been reported. Simplified procedures for derivatizations and purification, and methods for quantitation of retinoids have been presented.
72 citations
TL;DR: It is demonstrated that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and a simple procedure is described that allows the level of transformation-competent cells to be maximized.
Abstract: The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).
68 citations
TL;DR: DD is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR) to identify genes that are differentially expressed in different cell populations.
Abstract: Differential display of mRNA (DD) is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). PCR primers and conditions are chosen so that any given reaction yields a limited number of amplified cDNA fragments, permitting their visualization as discrete bands following gel electrophoresis. This robust and relatively simple procedure allows identification of genes that are differentially expressed in different cell populations. Here we review DD including some recent modifications, and compare it with other techniques for analyzing differential mRNA expression.
64 citations
TL;DR: The evolution of PFGE is traced, the understanding of its theoretical basis is summarized, and a comprehensive description of the methodology is provided.
Abstract: Pulsed-field gel electrophoresis (PFGE) was originally developed as a technique for providing electrophoretic karyotypes of micro-organisms. Since then the technique has evolved and diversified in many new directions. This review traces the evolution of PFGE, summarizes our understanding of its theoretical basis, and provides a comprehensive description of the methodology. Established and novel applications are explored and the reader is provided with an extensive list of references.
53 citations
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TL;DR: The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA, and is presently unique among RNA catalysts.
Abstract: The hairpin ribozyme is a member of a family of small RNA endonucleases, which includes hammerhead, human hepatitis delta virus, Neurospora VS, and the lead-dependent catalytic RNAs. All these catalytic RNAs reversibly cleave the phosphodiester bond of substrate RNA to generate 5′-hydroxyl and 2′,3′-cyclic phosphate termini. Whereas the reaction products from family members are similar, large structural and mechanistic differences exist. Structurally the hairpin ribozyme has two principal domains that interact to facilitate catalysis. The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA. In this regard it is presently unique among RNA catalysts. Targeting rules for cleavage of substrate have been determined and required bases for catalysis have been identified. The hairpin ribozyme has been developed and used for gene therapy and was the first ribozyme to be approved for human clinical trials.
52 citations
TL;DR: The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially.
Abstract: An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent are Phenol, Isoamyl alcohol, Guanidinium isothiocyanate, and Beta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.
51 citations
TL;DR: At the present stage, many papers on MEKC, which include fundamental characteristics and applications, have been available, and it is necessary to refer some of those literature when the detailed information is required.
Abstract: At the present stage, many papers on MEKC, which include fundamental characteristics and applications, have been available. Because only brief discussion on some aspects of MEKC was described in this article, it is necessary to refer some of those literature when the detailed information is required. Especially, for optimization strategies of MEKC, which was not discussed in this article, theoretical discussions by Foley(85), Vindevogel and Sandra(86), and Khaledi and coworkers(87,88) should be cited, along with the review article(11).
49 citations
TL;DR: In this article, various techniques for generation of peptide and peptidomimetic libraries are summarized in various formats, including Multipin, tea bag, and split-couple-mix techniques.
Abstract: Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.
48 citations
TL;DR: The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus and the Fc fragment of rabbit IgG has been investigated and it was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity.
Abstract: The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.
43 citations
TL;DR: The data showed that a dicistronic vector containing an internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) was superior to a conventional expression vector in both levels of protein expression and amplification efficiency.
Abstract: Recombinant proteins are useful tools in biological research, drug development, and drug screening. Specially designed expression vectors have been developed to introduce cDNA for recombinant protein expression in mammalian cells. We have combined a dicistronic mRNA design for expression of the recombinant protein, using glutamine synthetase (GS) for selection. A soluble form of human interleukin-4 receptor alpha chain was used as the model protein. The dicistronic vectors were compared to a standard expression vector in CHO-K1 cells in parallel experiments. Our data showed that a dicistronic vector containing an internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) was superior to a conventional expression vector in both levels of protein expression and amplification efficiency. The productivity of these clones was stable without selection pressure for an extended period of time. The GS selection system within a dicistronic vector design can achieve rapid and efficient gene amplification for protein production.
TL;DR: This review describes the current status of the understanding of the regulation of cell division in eukaryotes and especially in plants and features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.
Abstract: Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.
TL;DR: Diagrams of fragmentation for straight-chain, branched- chain, unsaturated and cyclic fatty acids are described together with those containing hydroxy-, epoxy-, keto-, and ether groups.
Abstract: The position of unsaturation, chain branching, and other structural features of fatty acids are not often apparent from the mass spectra of common derivatives such as methyl esters because of factors such as charge location at the carboxy termiunus and migration of double bonds. The spectra of picolinyl esters, on the other hand, contain fragment ions that provide this information. The esters are synthesized by reaction of the acids with thionyl chloride to form the acid chloride that is reacted with 3-pyridylcarbinol to give the ester. Under electron impact conditions in the mass spectrometer, an electron is removed from the nitrogen of the pyridine ring and a hydrogen atom is abstracted from the alkyl chain to this electron-deficient site. This process produces a radical site in the chain that initiates chain cleavage. Hydrogen atoms can be removed from any position of the chain with varying probability, depending on the chain structure. Thus, diagnostic ions are produced from each type of fatty acid whose masses and relative abundances reflect the structure of the alkyl chain and any substituents. Patterns of fragmentation for straight-chain, branched-chain, unsaturated and cyclic fatty acids are described together with those containing hydroxy-, epoxy-, keto-, and ether groups.
TL;DR: In this review, procedures for handling and storage of retinoids and biological samples containing them have been described, and simplified procedures for derivatizations and purification, and methods for quantitation ofretinoids have been presented.
Abstract: Retinoids are unstable compounds being readily oxidized and/or isomerized to altered compounds, especially in the presence of oxidants including air, light, and excessive heat. They are labile toward strong acids and solvents that have dissolved oxygen or peroxides. In this review, procedures for handling and storage of retinoids and biological samples containing them have been described. The physical and chemical properties of retinoids have been reported. Simplified procedures for derivatizations and purification, and methods for quantitation of retinoids have been presented.
TL;DR: The two asparagine-linked glycosylation sites of recombinant coagulation factor VIIa have been characterized by glycosidase digestions, size-exclusion chromatography (SEC), and mass spectrometry (MS).
Abstract: The two asparagine-linked glycosylation sites of recombinant coagulation factor VIIa have been characterized by glycosidase digestions, size-exclusion chromatography (SEC), and mass spectrometry (MS). Nine structures were characterized as core fucosylated bi- and triantennary structures with 0-3 sialic-acid residues, which were α2-3 linked to galactose exclusively. Three of the structures had one or two galactose residues substituted by N-acetylgalactosamine. Significant differences were found between the oligosac-charide profiles for the two glycosylation sites in rFVIIa. At Asn322, the degree of sialylation was lower and higher amounts of structures containing N-acetylgalactosamine were found compared to Asn l45.
TL;DR: High identity of DNA sequences suggests that β-galactosidases from P. woesei andPyrococcus furiosus are closely related, and isolated enzyme from E. coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85°C.
Abstract: Pyrococcus woesei (DSM 3773) β-galactosidase gene amplified by polymerase chain reaction was cloned intoKpnI andHindIII binding sites of pET-30LIC expression plasmid. The obtained pGal2 (6785 bp) transcription vector was then transferred toEscherichia coli B121 (DE3) cells. High identity (99.9%) of DNA sequences suggests that β-galactosidases fromP. woesei andPyrococcus furiosus are closely related. This enzyme fromE. coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85°C. The crude β-galactosidase remaining in the solution comprises about 21% of the total amount of proteins extracted fromE. coli cells and has maximal activity at pH 5.4 and temperature of 93°C. Isolated enzyme is active at temperatures up to 110°C and the activity loss after 4h of incubation at 85 and 93°C did not exceed 11 and 15% of the initial value, respectively.
TL;DR: By lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.
Abstract: Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.
TL;DR: A simple method to remove rRNA from total RNA of Staphylococcus aureus by cloning a PCR-amplified S. aureUS rRNA gene fragment into a plasmid, and synthesizing biotin-labeled antisense rRNA to subtract rRNA is developed.
Abstract: Isolation of prokaryotic mRNA by the poly(dT) method has been difficult, primarily due to the great instability of the poly(A) sequence in its mRNA. We developed a simple method to remove rRNA from total RNA of Staphylococcus aureus by cloning a PCR-amplified S. aureus rRNA gene fragment into a plasmid, and then synthesizing biotin-labeled antisense rRNA to subtract rRNA. By using this method, S. aureus rRNA is significantly reduced and mRNA is enriched. This method may be used to prepare prokaryotic mRNA for many molecular biology applications.
TL;DR: The BIAcore instrument as mentioned in this paper uses surface plasmon resonance (SPR) to characterize an antigen or a group of specific monoclonal antibodies (MAbs) or both.
Abstract: An epitope may be defined as a specific site on an antigen module characterized by the binding of one monoclonal antibody (MAb). Epitope mapping by surface plasmon resonance in the BIAcore biosensor may be performed to characterize an antigen or a group of specific MAbs or both. This article describes the BIAcore instrument and methods for such mapping. Examples include molecular interaction studies with simple and complex proteins, such as myoglobin and calprotectin, respectively.
TL;DR: A complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions is presented, making the system amenable to routine clinical use and use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.
Abstract: Patients infected with human irnmunodeficiency virus type 1 (H1V-1) are being treated with a number of different combinations of antiretroviral compounds that target the essential viral enzymes reverse transcriptase and protease. Different sets of HIV-1 mutations that confer drug resistance have been well defined; they allow reasonabe prediction of the drug sensitivity pattern from analysis of the HIV-1 genotype in vivo. Since periodical monitoring of genotypic resistance is expected to improve clinical management in a large number of infected patients, practical and cost-effective methods are highly desirable to set at least medium-scale sequencing in clinical diagnostic settings. We present a complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions. Features making the system amenable to routine clinical use include:
1.
Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR);
2.
Direct use of the crude unpurified PCR product as the sequencing template; and
3.
Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.
TL;DR: Novel gene-expression systems in lactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.
Abstract: Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described in L. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems in Lactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.
TL;DR: A simple and rapid method for the preparation of genomic DNA from multiple samples is reported that produces clean DNA for use in most molecular biology methods in <90 min.
Abstract: Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on. Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in < 90 min.
TL;DR: The suitability of DNA of high molecular mass from blackcurrant and other soft-fruit species for Southern analysis and genomic-library construction is demonstrated.
Abstract: A method is described for isolating DNA of high molecular mass (M(r)) from blackcurrant and other softfruit species. Following a hexacethylytimethyl ammonium bromide (CTAB)-based extraction procedure, samples are treated with a glycosidic hydrolase mixture and RNase, and then purified. The suitability of this DNA for Southern analysis and genomic-library construction is demonstrated.
TL;DR: Langitudinally sliced embryonic axes from pea and lentil mature seeds cocultivated with A. tumefaciens carrying agus reporter gene in its T-DNA provided a convenient means to evaluate the efficiency of gene transfer to tissues in different cultivars and cocultivation conditions.
Abstract: Longitudinally sliced embryonic axes from pea and lentil mature seeds cocultivated with A. tumefaciens carrying a gus reporter gene in its T-DNA provided a convenient means to evaluate the efficiency of gene transfer to tissues in different cultivars and cocultivation conditions. Use of this technique demonstrated wide variation in susceptibility to Agrobacterium among several pea and lentil commercial genotypes.
TL;DR: This article presents the state-of-the-art, with an emphasis on similarity search methods and the resources available through Internet, for interpreting vertebrate genomic sequences.
Abstract: Computer methods for the complete and accurate detection of genes in vertebrate genomic sequences are still a long way to perfection. The intermediate task of identifying the coding moiety of genes (coding exons) is now reasonably well achieved using a combination of methods. After reviewing the intrinsic difficulties in interpreting vertebrate genomic sequences, this article presents the state-of-the-art, with an emphasis on similarity search methods and the resources available through Internet.
TL;DR: Two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol®BD are described, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.
Abstract: This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol BD. In the first procedure, DNA is isolated from 1-20 microL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 microL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.
TL;DR: It was shown that in their OFF stage the authors' two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stagetetR/PtetOtet-lacZ vectors.
Abstract: We have developed a tightly controlled, two-stage expression system. It is based on a single plasmid that carries the TetR repressor/Ptet promoter/Otet operator for the first-stage control, and the Flp recombinase/ FRT sites for the second-stage control. The gene to be expressed (GENE) is cloned in an inverted orientation (with respect to the stationary promoter) into a multiple-cloning site (MCS) located between two convergent FRT1 and FRT2 sites. In the OFF stage, no inadvertent transcription can enter the 5' end of cloned GENE because of four rrnBT1 terminators, located just outside the FRT1-MCS-FRT2 cassette and because the FRT2 construct was deprived of any promoter function. When using the lacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stage tetR/PtetOtet-lacZ vectors. To enter the ON stage, the tetR/PtetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts the FRT1-MCS-FRT2 module together with the cloned GENE. This results in the massive GENE expression from one (pInvMS) or two (pImpMS) stationary promoters.
TL;DR: The multiple cloning site of pUC19 was replaced by a multiple cloning site possessing a double Xcm1 restriction site as mentioned in this paper, which provides the easiest way of creating a vector in which PCR fragments produced by Taq polymerase can be directly cloned without further modifications.
Abstract: The multiple cloning site of pUC19 was replaced by a multiple cloning site possessing a double Xcm1 restriction site. Digestion with XcmI gives a linear vector with a single 3'-overhanging T-residue at both ends. This provides the easiest way of creating a vector in which PCR fragments produced by Taq polymerase can be directly cloned without further modifications.
TL;DR: Using this assay, Scatchard analysis was performed on the data for the specific binding of iron-loaded transferrin to its receptors on mouse fibroblasts and yieldedKd values similar to those obtained with other published methods.
Abstract: A simple, nonradioactive method is presented that can be used for performing large numbers of binding assays of cell membrane receptors with their ligands. The method adopts the simple membrane preparation and biotin-based quantitation methods of the semi-intact cell endocytosis assays. After binding of the biotinylated ligand to its receptors on the semi-intact cell membranes, a rapid centrifugation step separates the membranes from unbound ligand. Bound ligand is subsequently released by detergent, captured by a specific antibody coated on teh surface of microwells, and quantitated with peroxidase-conjugated streptavidin in a colorimetric assay. Using this assay, Scatchard analysis was performed on the data for the specific binding of iron-loaded transferrin to its receptors on mouse fibroblasts and yielded Kd values similar to those obtained with other published methods. The assay is sensitive, rapid, and also convenient, because aliquots of semi-intact cells can be stored frozen. The perforated plasma membrane of the cells offers the additional possibility of screening factors that interact with the cytoplasmic domain of the receptors for their possible effects on the parameters of the extracellular ligand-receptor interaction.