Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example

A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.     

RSITE: a computer program to predict the recognition sequence of a restriction enzyme.

A computer program (RSITE) was developed which predicts the recognition sequence of a restriction endonuclease. The sizes of fragments experimentally determined on cleavage of a DNA of known sequence were input. Possible recognition sequences producing fragments of sizes matching those determined empirically were printed out. The program faithfully predicted the specificity of restriction enzymes of known recognition sequence and also determined the recognition sequence of a new restriction enzyme from Haemophilus influenzae GU (HinGU II).     

Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.     

Construction and application of a modified “gene machine”: A circular concentrating preparative gel electrophoresis device employing discontinuous elution

A modified version of a preparative circular gel electrophoresis apparatus, first described by Edwin Southern (Medical Research Council, University of Edinburgh, Edinburgh, Scotland), has been constructed. The apparatus fractionates a large volume of sample into concentric bands which migrate toward a small circular collection chamber. Samples exiting the gel into the collection chamber are concentrated against a dialysis membrane which encloses the inner electrode and are pumped from this center chamber into a fraction collector at fixed time intervals. The apparatus has been employed to fractionate samples of DNA (10 mg) by electrophoresis through either agarose or acrylamide gels. Two examples of nucleic acids which have been successfully fractionated are given: restriction endonuclease cleavage fragments of total soybean DNA, and a heterogeneous mixture of covalently closed circular plasmid DNA from Bacillus megaterium. Franctionated DNA is suitable for molecular cloning directly from acrylamide and, after one additional treatment, from agarose. The run time for DNA treated with restriction endonuclease is from 24 to 48 h. Purification of 60- to 200-fold is common for a DNA restriction fragment from a total genome.     

Physical mapping of bacteriophage lambda DNA with restriction endonuclease HpaI

The restriction endonuclease from Haemophilus parainfluenzae, endoR·HpaI cleaves λcI857s7 DNA into 14 fragments. The sizes of these fragments were determined and a physical map was constructed. The ordering of the fragments was carried out using different deletion and substitution mutants of λ phage, double cleavages with another restriction enzyme, endoR·BamHI, and partial protection of individual HpaI recognition sites by the antibiotics distamycin A and actinomycin D. HpaI produces fragments from the left arm of the λ DNA genome, which may help in investigating the structure and function of this part of the phage.     

Analysis of polypeptide molecular weights by electrophoresis in urea

Ten proteins of differing disulfide contents and isoionic points were subjected to disc gel electrophoresis in the presence of 8 urea-0.9 acetic acid to evaluate the use of this technique in determining polypeptide molecular weights. Comparison of the electrophoretic mobilities before and after reduction of the proteins' disulfide bonds demonstrated that only after all disulfide bonds were broken, could their molecular weights be estimated with any degree of accuracy. The expression of the electrophoretic mobilities as a function of the proteins' effective hydrodynamic sizes, thereby taking into account the extent of constraint by disulfide bonds, allowed a comparison of disulfide cross-linked and linear forms of the protein polypeptides. The extent to which intrinsic charge affects a protein's electrophoretic mobility was estimated by comparing alpha-lactalbumin and lysozyme, two proteins of identical size but vastly different isoionic points. They exhibited a 20% difference in mobilities. An apparent slow reduction of disulfide bonds was observed to occur when proteins were exposed to reducing agent at low pH in 8 urea.     

Hemoglobin synthesis in isolated erythroid colonies from the chick embryo.

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived.Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [³H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P′, and P″) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed.     

Identification and restriction endonuclease mapping of the threonine operon regulatory region.

A DNA fragment carrying the thrP region of Escherichia coli has been identified and characterized. Starting from a secondary site lysogen of bacteriophage λ, where the position of the prophage is either within thrP or between thrP and thrP structural genes (Gardner et al., 1974; Gardner &; Smith, 1975) it has been possible to isolate transducing phages which carry bacterial DNA from either side of the prophage. By Int-Xis mediated site-specific recombination we have generated recombinant transducing phages which carry an intact thrP region. A phage carrying a regulatory mutation, which maps in the thrP region, has also been constructed.Mapping with several restriction endonucleases has allowed us to construct a map of the cleavage sites of the thrP region. A 1700 base-pair HaeIII fragment carrying the secondary attachment site (attΔOΔ′) was isolated and its position was localized within a 180 base-pair TaqI-HhaI restriction fragment. The location of attΔOΔ′ in the HaeIII fragment suggests that the entire thrP region is carried by this fragment.     

Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (>10⁶), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.     

Antigenic and electrophoretic variants of vitellogenin in Oncopeltus blood and their control by juvenile hormone

Antigenic analysis of adult female-specific blood and yolk proteins in Oncopeltus demonstrated an incomplete vitellogenin (A), which appears in the blood prior to yolk deposition and is later modified or joined by an antigenically complete molecule (AB). Vitellogenin AB is antigenically indistinguishable from the major yolk protein of mature eggs, though the electrophoretic mobilities of the two differ in 6% acrylamide gels. Vitellogenin A alone appears in the blood of adult females in which the corpora allata are known to be inactive, i.e., during starvation or photoperiodically induced diapause. Stimulation of these females with a juvenile hormone analog restores yolk deposition, and also induces the appearance of AB in the blood. While juvenile hormone is needed for the termination of diapause and the maturation of vitellogenin in this species, diapause begins with the vitellogenin-producing mechanism already partially assembled.     

Comparisons of Mitochondrial DNA from the Sibling Species Heterodera glycines and H. schachtii

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.     

The c-terminal DNA-binding domain ofChironomus BR gene products shows preferential affinity for (dA,dT)-rich sequences

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midgeChironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived fromBR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the largeBRs (BR1 andBR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of theBR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of theBR genes.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis

Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 10³ to 10⁶. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (M_r), and the logarithm of the relative mobility, log (R_l), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, K_R, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (M_r) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (R_l). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (M_r) and log (R_l) or the one between log (M_r) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis.     

Improved single-strand DNA sizing accuracy in capillary electrophoresis.

Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.     

Action of Escherichia coli P1 restriction endonuclease on simian virus 40 DNA

The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA. In the presence of cofactors S-adenosylmethionine and ATP the enzyme cleaves 70% of the closed circular SV40 DNA molecules once to produce unit-length linear molecules and renders the remaining 30% resistant to further cleavage. No molecules were found by electron microscopy or by gel electrophoresis that were cleaved more than once. It would appear that the double-strand break is made by two nearly simultaneous single-strand breaks, since no circular DNA molecules containing one single-strand break were found as intermediates during the cleavage reaction. The EcoP1 endonuclease-cleaved linear SV40 DNA molecules are not cleaved at a unique site, as shown by the generation of about 65% circular molecules after denaturation and renaturation. These EcoP1 endonuclease-cleaved, renatured circular molecules are resistant to further cleavage by EcoP1 endonuclease.The EcoP1 endonuclease cleavage sites on SV40 DNA were mapped relative to the partial denaturation map and to the EcoRI and HpaII restriction endonuclease cleavage sites. These maps suggest there are a minimum of four unique but widely spaced cleavage sites at 0.09, 0.19, 0.52, and 0.66 SV40 units relative to the EcoRI site. The frequency of cleavage at any particular site differs from that at another site. If S-adenosylmethionine is omitted from the enzyme reaction mix, SV40 DNA is cleaved into several fragments.An average of 4.6 ± 1 methyl groups are transferred to SV40 DNA from S-adenosylmethionine during the course of a normal reaction containing the cofactors. Under conditions which optimize this methylation, 7 ± 1 methyl groups can be transferred to DNA. This methylation protects most of the molecules from further cleavage. The methyl groups were mapped relative to the Hemophilus influenzae restriction endonuclease fragments. The A fragment receives three to four methyl groups and the B and G fragments each receive one to two methyl groups. These fragments correspond to those in which cleavage sites are located.     

Oligo[d(C).(G)] runs exhibit a helical repeat of 11.1 bp in solution and cause slight DNA curvature when properly phased.

We have inserted d(C)10 in a set of DNA fragments with bent segments on both ends, which are rotated with respect to each other by base pair wise increasing insertions. The electrophoretic mobilities on polyacrylamide gels of these DNA fragments were used to identify insertion sizes with cis conformations of the bent ends. These experiments revealed a helical repeat in solution of d(C).d(G) tracts of 11.1 +/- 0.08 bp. The electrophoretic mobilities of ligation ladders with properly phased d(C)5 and d(C)16 runs demonstrate a small but clearly detectable curvature of these fragments.     

Pulse-field electrophoresis indicates full-length Mycoplasma chromosomes range widely in size.

Full-size linear chromosomes were prepared from mycoplasmas by using gamma-irradiation to introduce one (on average) double-strand break in their circular chromosomes. Chromosome sizes were estimated by pulsed-field gel electrophoresis (PFGE) from the mobilities of these full-length molecules relative to DNA size references. Sizes estimated for Ureaplasma urealyticum T960 and 16 Mycoplasma species ranged from 684 kbp (M. hominis) to 1315 kbp (M. iowae). Using this sample, we found no correlation between the mobility of the full-size linear chromosomes and their G + C content. Sizes for A. laidlawii and A. hippikon were within the range expected from renaturation kinetics. PFGE size estimates are in good agreement with sizes determined by other methods, including electron microscopy, an ordered clone library, and summation of restriction fragments. Our estimates also agree with those from renaturation kinetics for both the largest and some of the smallest chromosomes, but in the intermediate size range, renaturation kinetics consistently provides lower values than PFGE or electron microscopy. Our PFGE estimates show that mycoplasma chromosomes span a continual range of sizes, with several intermediate values falling between the previously recognized large and small chromosome size clusters.     

A detailed restriction endonuclease site map of theZea mays plastid genome

Fragments produced by partial digestion of plastid DNA fromZea mays withEco RI were cloned in Charon 4A. A circular, fine structure physical map of the plastid DNA was then constructed from restriction endonucleaseSal I,Pst I,Eco RI, andBam HI recognition site maps of cloned overlapping segments of the plastid genome. These fragments were assigned molecular weights by reference to size markers from both pBR322 and lambda phage DNA. Because of the detail and extent of the derived map, it has been possible to construct a coordinate system which has a unique zero point and within which all the restriction fragments and previously described structural features can be mapped. A computer program was constructed which will display in a circular fashion any of the above features using an X-Y plotter.