Showing papers in "Methods in Enzymology in 1987"

Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes

Abstract: Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.

Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.

Abstract: Publisher Summary This chapter discusses the specific synthesis of deoxyribonucleic acid (DNA) in vitro through the medium of a polymerase-catalyzed chain reaction. A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter. The same method can be used to alter the amplified sequence or to append new sequence information to it. It is necessary that the ends of the sequence be known in sufficient detail that oligonucleotides can be synthesized, which will hybridize to them and that a small amount of the sequence be available to initiate the reaction. The oligonucleotides are complementary to different strands of the desired sequence and at relative positions along the sequence such that the DNA polymerase extension product of the one, when denatured, can serve as a template for the other and vice versa. Oligonucleotides were synthesized using an automated DNA synthesis machine (Biosearch, Inc., San Rafael, California) using phosphoramidite chemistry. “Mispriming"” can be usefully employed to make intentional in vitro mutations or to add sequence information to one or both ends of a given sequence. The chapter explores the possibility of utilizing a heat-stable DNA polymerase so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation

Production of Single-Stranded Plasmid DNA

Abstract: Publisher Summary This chapter discusses the production of single-stranded plasmid DNA. The chapter focuses on M13KO7 and its uses. The chapter reviews M13 biology; M13 mutants play a vital role in the functioning of M13KO7. M13 is a phage that contains a circular single-stranded DNA (ssDNA) molecule of 6407 bases packaged in a filamentous virion, which is extruded from the cell without lysis. It can infect only cells having F-pili to which it binds for entering the cell. The phage genome consists of nine genes encoding 10 proteins and contains an intergenic region of 508 bases. Phage replication consists of three phases: (1) ss-double strand (ds), (2) ds-ds, and (3) ds-ss. The intergenic region (IG) structure contains regions important for four phage processes: (1) the sequences necessary for the recognition of an ssDNA by phage proteins for its efficient packaging into viral particles; (2) the site of synthesis of an RNA primer that is used to initiate strand synthesis; (3) the initiation; and (4) the termination of (+) strand synthesis. In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated. The origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.

5-Fluoroorotic acid as a selective agent in yeast molecular genetics.

Abstract: 5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode vital functions.

Detection and localization of single base changes by denaturing gradient gel electrophoresis.

Abstract: Publisher Summary This chapter discusses the detection and localization of single base changes by denaturing gradient gel electrophoresis For most purposes, single-stranded deoxyribonucleic acid (DNA) probes 140–1000 bases in length are used, which allows about half of this number of base pairs to be screened for base changes in a single gel lane The chapter discusses the use of single-stranded RNA probes It describes the development of two new methods for detecting and localizing single base changes in cloned and genomic DNA In both procedures, a single-stranded, radioactively labeled probe of wild-type sequence is annealed to cloned or genomic DNA If the DNA under test carries a single base change, a hybrid double-stranded species containing a mismatch is formed In the first procedure, either a ribonucleic acid (RNA) or a DNA probe is used, and the mismatched duplex is separated from the perfectly paired, wild type duplex by electrophoresis in a denaturing gradient gel In the second procedure, the probe is composed of single-stranded RNA and the mismatch is cleaved by ribonuclease; the resulting cleaved products are examined by polyacrylamide gel electrophoresis and autoradiography It is estimated that each method will detect approximately 50% of all possible substitutions, insertions, or deletions in a probed region Although there is some overlap in the base changes that are detected with the two procedures, it is likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions and they should be particularly useful in mapping and diagnosing small mutations that result in genetic disease This method has led to the detection of neutral polymorphisms for genetic linkage studies The chapter lists all the equipment needed to prepare and run the gels and also describes the hybridization procedure for RNA probes

Unidirectional digestion with exonuclease III in DNA sequence analysis.

Abstract: Publisher Summary This chapter discusses the unidirectional digestion with exonuclease III in deoxyribonucleic acid (DNA) sequence analysis. The chapter describes a rapid strategy and a simplified version related to the uniform rate at which Escherichia coli exonuclease III can digest DNA from one end. This strategy can be used with single-stranded phage, plasmid, and synthetic RNA-plasmid systems. The reagents that are used are inexpensive and readily available. The chapter illustrates a figure that outlines the steps involved in generating the ordered sets of deletion breakpoints for DNA sequencing. The method is based on a series of enzymatic treatments of a segment of DNA cloned into a suitable vector. Cloning efficiencies are sufficiently high that standard Ca2+ treated cells are adequate for transfection or transformation. Exonuclease III is an incompletely processive nuclease. The rate of exonuclease III is very sensitive to the temperature of incubation. The procedure described in the chapter has several advantages. Most importantly, it yields cloned breakpoints at high efficiency that are tightly clustered around a particular point in the sequence, greatly reducing the tedious screening steps required to fill in gaps in the sequence.

Use of eukaryotic expression technology in the functional analysis of cloned genes

Abstract: The purpose of this chapter is to describe ways in which eukaryotic expression technology can be used to identify and to analyze the function of cloned eukaryotic genes. The assumption is made that the clone of interest has been sequenced and an open reading frame has been identified. Although expression of genomic sequences will be briefly discussed, in general it is assumed that the sequence of interest is a cDNA. This chapter is divided into three sections. The first section describes several possible strategies for maximizing heterologous gene expression in the cells of higher eukaryotes. The second section deals with potential assays for gene expression based on function, and the third section describes some immunological approaches. Overall, the focus is on the use of techniques which yield information not obtainable from heterologous gene expression in bacteria or yeast.

In vitro RNA synthesis with SP6 RNA polymerase.

Abstract: Publisher Summary This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 RNA polymerase. The chapter describes the use of in vitro transcription systems for the synthesis of RNAs for use as substrates and hybridization probes. The chapter discusses the advantages of the technique and several associated problems. The system of SP6 RNA polymerase is used as an example throughout the chapter, but the observations are stated to be general and can be applied to any of the phage polymerase and promoter systems, including T3 and T7. The chapter discusses the in vitro transcription with bacteriophage RNA polymerase, vectors containing bacteriophage promoters, RNA synthesis reaction, and several related concepts. The optimum conditions for in vitro transcription of deoxyribonucleic acid (DNA) cloned into plasmids containing an SP6 promoter are described in the chapter. The normal product of a transcription reaction contains a triphosphate group at the 5' end of the molecule. RNA is very stable when stored as an ethanol suspension and unlike DNA it does not seem to aggregate. As a result, very reproducible samples of RNA can be taken from an ethanol suspension providing that it is vortexed prior to setting up the reaction. The chapter reviews some of the problems most commonly encountered when using in vitro transcription systems and some potential solutions.

Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis.

Abstract: Publisher Summary This chapter discusses the application of computational simulation of deoxyribonucleic acid (DNA) melting for denaturing gradient gel electrophoresis Calculation of the theoretical pattern of thermal stability of DNA molecules of known sequence, together with calculation of the expected changes in electrophoretic mobility in gels under denaturing conditions, is the first useful step toward searching for sequence changes by means of denaturing gradient gels and in designing a gradient gel for the preparative isolation of mutants after intensive mutagenesis The calculations permit a reasonably reliable test of the experimental prospects for new hypotheses and experimental designs They show, for example, that about 50–70% of all possible single base changes that might occur within the human β-globin gene cluster would be detectable using the denaturing gradient system after the restriction fragments of the genome are hybridized with labeled, single strands corresponding to the same fragments of the normal sequence The calculations are easy to execute for any sequence on a digital computer They provide an indication of the regions within the sequence where base changes are likely to be detected, the magnitude of the effects that can be expected, and guidance as to the choice of restriction sites for fragmentation The theoretical calculation is sufficiently reliable to serve as a means for the evaluation of some thermodynamic parameters relevant to melting from gel data and to signal anomalous properties The chapter discusses theoretical treatment of the helix-random chain transition for complex sequences, the melting map program MELT, the mobility program MU, and so on The melting calculation is useful in several different applications The most elementary result is a description of the probability that each base is helical (or nonhelical) at specified temperatures

High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives.

Abstract: Publisher Summary High-performance liquid chromatography (HPLC) provides a rapid, sensitive, and reproducible means of separating and quantifying simultaneously a variety of sulfur-containing amino acids and related derivatives. The HPLC method described in this chapter is modified and is based on the initial formation of S-carboxy-methyl derivatives of free thiols followed by the conversion of free amino groups to 2,4-dinitrophenyl (DNP) derivatives. Following derivatization, nanomole levels of individual sulfur-containing amino acids are measured using UV detection at 365 nm after separation by reverse-phase ion-exchange HPLC. Because of the versatility of this HPLC method, biological specimen preparation as well as derivatization and HPLC analysis procedures are discussed. DNP derivatives of acidic amino acids (including thiol-containing compounds) are separated on a 3-aminopropyl column by reversed-phase ion-exchange HPLC. In the mobile phase, methanol is used to elute rapidly the excess 2,4-dinitrophenol and the DNP derivatives of basic and neutral amino acids. Acetic acid is present in the mobile phase to maintain the bonded-phase amino groups in the protonated form. By increasing the sodium acetate concentration of the mobile phase, selective elution of acidic DNP derivatives is accomplished. The eluted DNP derivatives are measured by detection at 365 nm.

[52] Preparation of high-purity plasma membranes

Abstract: Publisher Summary This chapter describes a method using the preparation of plasma membranes from light-grown oat ( Arena sativa L. ) leaves as an example. Using the batch procedure, two fractions containing purified plasma membrane (U 3 and U 3 , ) and one fraction containing intracellular membranes depleted of plasma membrane (L 1 ) are obtained. Specific staining with phosphotungstic acid or silicotungstic acid seems to be the only universal marker for the plant plasma membrane and the only one that permits a real estimation of the purity of the preparations. Based on this staining the purity is estimated to be higher than 90% and often close to 100%, plasma membrane and similar purities are reported for plasma membrane from maize roots. (>90%) for plasma membrane preparations obtained both by phase partitioning and free flow electrophoresis.

Genetic engineering of the cyanobacterial chromosome

Abstract: Publisher Summary This chapter describes methods for the direct genetic engineering of the cyanobacterial chromosome and techniques for the analysis of DNA and RNA from the resulting transformants. The DNA in lane B described in the chapter is a by-product of the RNA isolation procedure. The genetic engineering of the cyanobacterial chromosome includes the techniques described in the chapter. The chapter focuses on methods that employ wholly homologous transforming DNA or heterologous sequences, which are flanked by cyanobacterial DNA on either side. The analysis of DNA from transformants is necessary to determine the chromosomal structure following recombination. The recombination events illustrated in the chapter are those that are usually seen, but aberrant events have been observed when the cloned region homologous to the chromosome is small. These events include the deletion of a chromosomal segment and integration at a related locus in the case of a gene family. Southern hybridization can determine whether the expected event has in fact, occurred. It is also important to analyze RNA from transformants, which have been engineered by these methods. A homologous recombination event, which replaces a wild-type gene with another functional allele, should result in the production of a full-length mitochondrial RNA (mRNA), which initiates and terminates properly. The chapter also mentions the instructions of the procedure that describe the preparation of glass fines used in the cyanobacterial total DNA miniprep procedure, which was adapted from the method of Vogelstein and Gillespie.

Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method.

Abstract: Publisher Summary This chapter discusses the chemical synthesis of deoxy oligonucleotides by the phosphoramidite method. It discusses the synthesis methodology; detailed protocols for preparing the silica support, the phosphoramidite, and deoxy oligonucleotides; and the purification of synthetic deoxyribo nucleic acid (DNA). The phosphite triester approach to DNA synthesis using deoxynucleoside phosphoramidite as synthons has become the method of choice for the preparation of deoxy oligonucleotides. The general synthetic strategy involves adding mononucleotides sequentially to a deoxynucleoside, attached covalently to a silica-based insoluble polymeric support. Reagents, starting materials, and side products are then removed simply by filtration. At the conclusion of the synthesis, the deoxy oligonucleotide is chemically freed of blocking groups, hydrolyzed from the support, and purified to homogeneity by either polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (HPLC). These preformed synthons are especially attractive for preparing DNA on automatic or semiautomatic DNA synthesizers, or for those who plan to manually synthesize a large number of deoxy oligonucleotides.

Oligonucleotide-directed construction of mutations via gapped duplex DNA.

Abstract: Publisher Summary This chapter discusses the oligonucleotide-directed construction of mutations via gapped duplex deoxyribo nucleic acid (DNA). Oligonucleotide-directed construction of mutations has become the method of choice to introduce predetermined structural changes into DNA. This chapter discusses the gapped duplex DNA approach to oligonucleotide-directed mutation construction. The key intermediate in this process is a partial DNA duplex of a recombinant M13 genome, gapped duplex DNA (gdDNA), which has only the target region of mutation construction exposed in single-stranded form, and which furthermore, carries distinguishable genetic markers in the two DNA strands in such a way that, a rigorous selection can be applied in favor of phage progeny arising from the shorter strand— that is, (the minus strand of the MI3 genome). Two alternative variants of the gdDNA method are applicable: the “fill-in,” which incorporates DNA polymerase/ DNA ligase reactions in vitro , and the simplified “mix-heat-transfect” protocol. It bypasses these enzymatic manipulations.

Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography.

Abstract: Methods are described for the preparation and high-performance liquid chromatography (HPLC) analysis of monobromobimane derivatives of low molecular weight thiols in extracts of biological samples. Typical problems encountered in the development and application of these methods are discussed. Analysis of mung bean extract is used as an example.

Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli.

Abstract: Publisher Summary The chapter describes a general method for producing eukaryotic proteins in Escherichia coli using the fusion protein expression vector pLcII(nic – ) and subsequent purification and cleavage methods using blood coagulation factor X a The foreign coding sequence can be joined in phase to a short coding sequence of a highly expressed E coli gene so that a hybrid protein is produced The short segment of E coli gene directs the folding of the mRNA in the vicinity of the ribosome-binding site, and thereby ensures high translational efficiency If the fusion protein, thus, produced can be cleaved specifically at the junction of the two sequences by an appropriate enzymatic or chemical method, the authentic protein will then be liberated A fusion protein expression vector pLclI(nic-) is constructed This plasmid has multiple cloning sites downstream of a short coding sequence from the λ cII gene, which is under the control of the strong leftward (PL) promoter of λ phage The following proteins have been produced in this way at the level of 5–10% of total E coli cellular protein: human β-globin and human α-globin, human myoglobin In a second stage, a sequence encoding the tetrapeptide Ile-Glu-Gly-Arg at the junction in the fusion proteins is inserted The chapter discusses cloning into the pLclI(nic – ) fusion protein expression vector, protein purification, the isolation of factor X from bovine blood, and several other related concepts The pLcII(nic – ) fusion protein expression vector is a straightforward and general method for production of eukaryotic proteins in E coli

Binary ti vectors for plant transformation and promoter analysis

Abstract: Publisher Summary This chapter discusses binary Ti vectors for plant transformation and promoter analysis. In recent years, several studies have demonstrated the use of the naturally occurring Ti plasmids as transformation vectors for higher plants. In the technique, exogeneous DNA is either inserted into the tDNA region of the Ti plasmid by homologous recombination using an intermediate vector system or directly into one of the recently designed binary vectors. The binary vector system is a convenient way to transform higher plants, because foreign DNA can first be inserted into the vector and manipulated using E. coli as a host. The chapter illustrates an example of binary Ti vector and the important features of the molecule, pGA482, which is 13.2 kbp long for simple and efficient plant transformation. The chapter describes the two different methods that are used to move the binary vector to Agrobacterium triparental mating and direct DNA transfer. The chapter mentions that clonal transformation is enhanced by the protoplast cocultivation method. Cultured tobacco calli can be transformed by the Ti vector system.

Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template.

Abstract: The important features of the protocol described here are as follows: First, the procedure consists of a few simple steps and results in a reasonably high frequency of mutagenesis. Second, using two primers, there is no need to isolate covalently closed double-stranded molecules as in our previous method. Third, the use of vectors derived from single-stranded phage facilitates template preparation, mutagenesis efficiency, screening, and DNA sequencing. Fourth, the same basic steps can be directly applied when using the single-stranded pUC derivatives.

Purification, specific fragmentation, and separation of large DNA molecules.

Abstract: Publisher Summary This chapter discusses the processes related to the purification, specific fragmentation, and separation of large deoxyribonucleic acid (DNA) molecules. The chapter mentions the development of three new techniques that allow the fractionation and analysis of DNA molecules on a size scale much larger than previously possible. The current methods permit the routine handling of DNAs up to 1.5 million base pairs (bp). The first technique involves the preparation of unbroken genomic DNAs inside agarose gels. The second involves the digestion of such DNAs in agarose with restriction nucleases that produce discrete, large fragments. The third technique, pulsed-field gel (PFG) electrophoresis, allows the size separation of DNAs ranging from 10,000 bp (10 kb) to more than 1.5 million bp (1.5 Mb). The chapter describes the new techniques and demonstrates examples of their applicability for the analysis of bacterial genomes and unicellular eukaryotic genomes. The chapter describes the procedure for preparing intact DNA. Ordinary DNA preparative procedures are carried out in solution. In a typical DNA preparation, the walls of the cells are first removed by appropriate enzymatic treatment. The resulting spheroblasts or protoplasts are then broken open by the destruction of their cell membranes with detergents and a metal chelator. This produces a complex mixture of DNA, ribonucleic acid (RNA), and proteins. Treatment of the mixture with proteases and RNases may remove some of the unwanted components. Additional proteins are removed by chemical extraction of the DNA solution with phenol, and the DNA is concentrated by alcohol precipitation and centrifugation. To obtain clean DNA preparations that do not have contaminants, which interfere with subsequent analytical or preparatory procedures, it is frequently necessary to repeat some of these steps. It is also essential that, during preparation, DNAs are not exposed unnecessarily to DNases. The chapter also discusses the pulsed-field gel electrophoretic separation of intact chromosomal DNA molecules, the specific fragmentation of high-molecular-weight DNA, and the applications of large DNA technology for the study of organisms with simple genomes.

Collagen cross-linking amino acids.

Abstract: Publisher Summary This chapter emphasizes on the methods for detecting and quantifying the mature hydroxypyridinium cross-links, but also describes the procedures for quantifying them together with the borohydride-reducible cross-linking residues in the same sample of collagen. To measure cross-linking amino acids in a collagen hydrolyzate using a conventional amino acid analyzer with ninhydrin detection, the bulk amino acids must be largely removed to avoid plugging the reaction coil. Tissue is thoroughly reacted with sodium borohydride to convert all intermediate cross-linking residues to their reduced, acid-stable forms. More vigorous reduction conditions may be used than when preparing profiles of tritium-labeled cross-links using NaB3H4 and direct amino acid analysis. This chapter presents the direct quantitation of hydroxypyridinium cross-links in tissue hydrolyzates by HPLC and fluorescence detection. The following method based on reverse-phase high-performance liquid chromatography can resolve and quantify the HP and LP forms of hydroxypyridinium residue down to 1 pmol directly in hydrolyzates of whole connective tissue, without any preliminary clean-up or enrichment step.

Improved oligonucleotide-directed mutagenesis using M13 vectors.

Abstract: Publisher Summary This chapter discusses the improved oligonucleotide-directed mutagenesis using M 13 vectors. An oligonucleotide is synthesized, which is complementary to part of the DNA template but contains an internal mismatch to direct the required change (point mutation, multiple mutation, insertion, or deletion). The simplest approach to mutagenesis in MI3 is “single priming,” where the mutagenic primer is annealed with the single-stranded M13 template and extended with DNA polymerase I. Single-stranded DNA is then prepared from individual plaques and sequenced by the dideoxy method to verify the mutation. In constructing point mutations, often only the required mutation is obtained. However in constructing deletions, usually only a fraction of the hybridization positive clones carry the required mutation. The specificity of priming of the mutagenic oligonucleotide may be checked by using the oligonucleotide in a dideoxy sequencing reaction or in a primer-directed extension reaction followed by restriction endonuclease digestion. The E.coli (EcoK/ EcoB) selection system provides a convenient way of constructing a series of mutations at high yield.

High-efficiency cloning of full-length cDNA; construction and screening of cDNA expression libraries for mammalian cells.

Abstract: Publisher Summary This chapter describes detailed procedures for the construction of full-length complementary deoxyribo nucleic acid (cDNA) expression libraries and the screening of the libraries for particular clones based on their transient expression in mammalian cells. Methods for library transduction and screening based on stable expression are described in this chapter. In the steps that have just been enumerated, double-stranded, full length deoxyribo nucleic acid (DNA) copies of the original messenger ribo nucleic acids (mRNAs) are efficiently synthesized and inserted into the vector to form a functional composite gene with the protein coding sequence derived from the cDNA and the transcriptional and RNA processing signals from the SV40 genome. Successful construction of full-length cDNA libraries depends heavily on the quality of the mRNA preparation. The use of intact, uncontaminated mRNA is essential for generating full-length clones.To screen for or selects a particular clone on the basis of the function it encodes, the library is acutely transfected or stably transduced into cultured cells. The screening method relies on transient expression of cDNA clones in mammalian cells.

Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts.

Abstract: Publisher Summary This chapter discusses the utilization of the high resolution method “hydroxyl radical footprinting” for mapping protein–deoxyribonucleic acid (DNA) contacts. Hydroxyl radical cleaves DNA by abstracting a hydrogen atom from the deoxyribose sugars along the DNA backbone. As hydroxyl radical is exceedingly short lived and reactive and attacks sites on the surface of the DNA molecule, there is almost no sequence dependence or base dependence in the cleavage reaction. Every position along the backbone is cleaved nearly equally. This chemistry is used to determine the helical periodicity of a DNA-restriction fragment bound to an inorganic crystal, because hydroxyl radical could cut efficiently only the DNA backbone sugars that were directed away from the inorganic surface. The chapter illustrates that this same chemistry can be applied to produce the footprints of proteins bound to DNA as cutting of the DNA backbone by hydroxyl radical is blocked by bound protein. The chapter illustrates hydroxyl radical footprinting by its application to a well studied DNA–protein complex that of the bacteriophage λ repressor with the O R 1 operator DNA sequence. It also illustrates the way the reaction conditions for hydroxyl radical footprinting may be altered to accommodate other DNA-protein complexes.

Spectrophotometric assay of thiols.

Abstract: Publisher Summary This chapter summarizes the chief methods in use for thiols in general. These methods exploit either of the two principal properties of the SH group—namely, its capacity for oxidation or substitution. Oxidizing agents may be of analytical value if they do not oxidize thiols further than disulfides (that is, to sulfinic or sulfonic acids) 75 Se-labeled and also give absorbance changes of sufficient magnitude. The chapter describes aromatic disulfides that fulfill these requirements best and, they are the reagents of first choice. Aromatic disulfides dominate the field because they are easily reduced by thiol-disulfide exchange, the equilibrium over-whelmingly favoring the formation of aromatic thiol. Mixed disulfides are stable intermediate products with protein thiols, but with nonprotein thiols they are themselves rapidly reduced. In either case, the overall stoichiometry is one aromatic thiol generated for each biological thiol originally present. Various organic oxidants methods described are lodosobenzoic acid, diphenylpicrylphenylhydrazine, benzofuroxan, quinines, 4, 4’-bisdimethylaminodiphenylcarbinol, and trinitrobenzenesulfonic acid. Various inorganic oxidants methods are also described. The chapter explores substitution products exploited for spectrophotometric assay that includes mercurials, nitrous acids, maleimides, halides, and others.

A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo

Abstract: Publisher Summary This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo . The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic ( E. coli ) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro . Translation of such RNAs in the presence of [ 35 S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro , (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.

Expression and secretion vectors for yeast.

Abstract: Publisher Summary The chapter focuses on methods used to efficiently express and secrete biologically active proteins from Saccharomyces cereoisiae The chapter describes yeast expression vectors utilizing episomal vectors The expression cassettes from these vectors can also be integrated into the yeast chromosome, where they are presented at controlled copy number, exhibiting a high degree of mitotic stability The chapter discusses extrachromosomal replication vectors, centromere plasmids, and the assembly of expression cassettes One of the advantages of a secretion system is the production of heterologous proteins with authentic NH 2 termini The methods utilized to analyze heterologous protein secretion are the same as those used for proteins expressed directly in the cytoplasm (direct expression vectors) The chapter describes several related procedures including the Mung Bean nuclease digestion, the S1 nuclease digestion of cohesive termini, and the analysis of carbohydrate additions to heterologous proteins