PERFILS: a program for the quantitative treatment of footprinting data

PERFILS, a computer program written in Borland TurboPascal,performs quantitative analysis of footprinting experiments usingany IBM PC or compatible microcomputer. The program uses theheight of the bands obtained from densitometric scanning offootprinting autoradiographs to calculate a differential cleavageplot. Such a plot displays, on a logarithmic scale, the differenceof susceptibility of a DNA fragment to DNase I, or any othercleaving agent, in the presence of any ligand versus the sequence.PERFILS calculates the fractional cleavage values for controland ligand, giving a table of values for each internucleotidicbond and rendering the differential cleavage plot in only afew seconds.     

The densitometric study of G bands on human metaphase chromosomes

Summary The method of densitometric measurement of G bands on human metaphase chromosomes is described, and some factors influencing the densitometric patterns are discussed. The densitometric patterns are reproducible and typical for a given chromosomal pair, although they display some degree of variability. Typical patterns of all human metaphase chromosomes are presented, with the patterns obtained from the measurement of some structurally abnormal chromosomes (t13q14q, t14q21q, Xp-).

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Zusammenfassung Wir beschreiben die Methode der photometrischen Darstellung von G-Banden an menschlichen Chromosomen und diskutieren über einigen Faktoren, die das Resultat beeinflussen. Die photometrischen Kurven sind reproduzierbar und für jedes Chromosomenpaar typisch. Selbstverständlich muß man mit geringen Abstufungen ihrer Variabilität rechnen. Wir zeigen unsere typischen Kurven aller menschlichen Chromosomen, die mit Hilfe der ASG-Technik gefärbt wurden. Gleichzeitig geben wir einige Kurven abnormaler Chromosomen wieder, die auch in unserem Labordargestellt wurden (t(13q14q), t(14q21q), Xp-).

     

Slot blot method for the quantification of DNA sequences and mapping of chromosome rearrangements: Application to chromosome 21

As an alternative to the methods of gene dosage based on either RFLP studies or Southern blots using specific and reference probes, we designed a "slot blot" method for the evaluation of the copy number of unique chromosome 21 sequences. Varying amounts of denatured DNA from a normal control, a trisomy 21 patient, and the subject to be analyzed were loaded on the same membrane. Successive hybridizations with reference probes and chromosome 21 probes were then carried out. Intensities of the signals on autoradiograms were quantified by densitometric scanning. Graphic and statistical analysis of the linear regressions between reference and chromosome 21 probe signals were performed, and the conclusion that the DNA from the studied subject had two or three copies for a given chromosome 21 sequence was assessed by statistical comparison of the slopes. As a test for the validation of this method, 10 coded blood DNAs from five normal controls and from five trisomy 21 patients were analyzed, by using two reference (COL1A1 and COL1A2) and two chromosome 21 (D21S11 and D21S17) probes. Among the 10 DNAs analyzed, it was possible to diagnose, with 100% accuracy, normal controls and trisomic 21 individuals. Application of this methodology to the mapping of partial chromosome 21 rearrangements is presented.     

Cleavage in and around the DR1 element of the A sequence of herpes simplex virus type 1 relevant to the excision of DNA fragments with length corresponding to one and two units of the A sequence

The A sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage) to yield unit-length linear HSV-1 DNA prior to or during packaging of HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and 0.5 kb (longer band), the lengths of which correspond to one and two units of the A sequence, was identified using acrylamide gel electrophoresis of HSV-1 DNA preparations extracted by the method of Hirt. Twelve DNA fragments from each band were molecularly cloned, and nucleotide sequences were determined. Both termini of eight (67%) DNA clones from the shorter band corresponded to the specific cleavage site on DR1. Five (41%) DNA clones from the longer band had a terminus corresponding to the specific cleavage site on DR1 on one side, but not on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA clones from the longer band were in and around DR1. Thus, cleavage events of DR1 can be classified into three categories: (i) authentic cleavage; (ii) site-specific cleavage on the third DR1 distal from the S component (secondary site-specific cleavage), which is related to the generation of the shorter DNA band in combination with authentic cleavage; and (iii) less-specific cleavage events in and around other DR1 elements which relate to the generation of the longer DNA band.     

Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.     

Determining SNP allele frequencies in DNA pools

Single nucleotide polymorphisms (SNPs) are among the most common types of polymorphism used for genetic association studies. A method to allow the accurate quantitation of their allele frequencies from DNA pools would both increase throughput and decrease costs for large-scale genotyping. However, to date, most DNA pooling studies have concentrated on the use of microsatellite polymorphisms. In the case of SNPs that are restriction fragment length polymorphisms (RFLPs), studies have tended to use methods for the quantitation of allele frequency from pools that rely on densitometric evaluation of bands on an autoradiograph. Radiation-based methods have well-known drawbacks, and we present two alternative methods for the determination of SNP allele frequencies. For RFLPs, we used agarose gel electrophoresis of digested PCR products with ethidium bromide staining combined with densitometric analysis of gel images on a PC. For all types of SNP, we used allele-specific fluorescent probes in the Taqman assay to determine the relative frequencies of two different alleles. Both methods gave accurate and reproducible results, suggesting they are suitable for use in DNA pooling experiments.     

Analysis of DNA sequences using a single chemical cleavage procedure

A novel approach to sequence analysis of end-labeled, defined DNA fragments, using a single chemical cleavage procedure and electrophoretic separation in a single lane, has been developed. Prolonged treatment with hot aqueous piperidine results in partial cleavage of the DNA at all positions; the relative propensity for this cleavage is different for the various bases in the DNA. The hydrolysate is resolved on a DNA sequencing gel, and the distribution of radioactivity in the electrophoretic lane is analyzed (a) in terms of differential peak heights of the radioactive bands and (b) in terms of the spacings between successive bands. Simultaneous application of these two base-characteristic criteria allows the deduction of the nucleotide sequence with an accuracy approaching that of the established four-lane methods of DNA sequencing.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria

A direct method for calculating the average number of replicationforks per chromosome in an exponentially growing bacterial cultureand the fraction of reinitiation after an inductive treatmentof the initiation step is presented. This method has allowedthe development of REPLJCON, a computer program designed forthe resolution of the algorithm and simulation of the bacterialchromosome replication. Using REPLJCON the following parameterscan be obtained: average number of replication forks per chromosome,time required for the complete replication cycle, average amountof DNA per nucleotide, gene frequency of any chromosomal locusand reinitiation fraction. The use of this analysis also permitsthe determination of the uni- or bidirectionality of replication. Received on September 30, 1987; accepted on May 10, 1988     

The effect of replication errors on the mismatch analysis of PCR-amplified DNA.

The mismatch analysis of PCR-amplified DNA has generally assumed the absence of artificially introduced base substitutions in a significant proportion of the amplification product. This technique, however, differs from the direct sequencing of amplified DNA in that non-specific substitutions will render a molecule useless in analysis. The expected signal-to-noise ratio is heavily influenced by several parameters viz. initial template copy number, number of replication cycles, eventual product yield and the type of experimental system adopted. Mathematical modelling can be used to optimize fragment length with respect to the method applied and suggests as yet undescribed improvements such as partial modification or cleavage to optimize signal detection.     

Automatic quantitative evaluation of autoradiographic band films by computerized image analysis

The present paper describes a new image processing method for automatic quantitative analysis of autoradiographic band films. It was developed in a specific image analysis environment (IBAS 2.0), but the algorithms and methods can be utilized elsewhere. The program is easy to use and presents some particularly useful features for evaluation of autoradiographic band films, such as the choice of whole film or single lane background determination; the possibility of evaluating bands with film scratch artifacts and the quantification in absolute terms or relative to reference values. The method was tested by comparison with laser-scanner densitometric quantifications of the same autoradiograms. The results show the full compatibility of the two methods and demonstrate the reliability and sensitivity of image analysis. The method can be used not only to evaluate autoradiographic band films, but to analyze any type of signal bands on other materials (e.g. electrophoresis gel, chromatographic paper, etc.).     

Modification of ribosomal RNA by ribosome-inactivating proteins from plants.

We have surveyed 14 different toxic and nontoxic ribosome-inactivating proteins from plants for the ability to act on the RNA of the eucaryotic 60 S ribosomal subunit. All of these proteins act to introduce a specific modification into 26-28 S RNA which renders the RNA sensitive to cleavage by aniline. Sequence analysis of the 5'-termini of the fragments produced by ricin and saporin following aniline cleavage indicate that both proteins possess identical specificity. Our observations support the conclusion of Endo and Tsurugi (J. Biol. Chem. 262, 8128-8130, 1987) that ricin is a specific N-glycosidase and we have located the site of this cleavage by direct sequence analysis. Our results further suggest that all plant ribosome-inactivating proteins function as specific N-glycosidases with the same specificity.     

Sequence analysis of end-labeled DNA fragments by solvolysis in aqueous solutions of different amines.

Cleavage of 3'-end-labeled DNA in hot aqueous solutions of different amines is comparatively examined for overall rate of DNA scission as well as for potential differences in the preference of the various amines for cleavage at the different bases. Under comparable conditions (0.5 M amine, 0.3 M NaCl, 90 degrees C), piperidine, diethylamine, morpholine, and ethylenediamine produce the same set of labeled fragments, at approximately equal overall cleavage rates. The same set of fragments is also obtained with diisopropylamine, triethylamine, and 1,4-diazabicyclo[2.2.2]octane, but at markedly lower overall cleavage rates. Solvolysis in aqueous piperidine or aqueous diethylamine leads to DNA scission predominantly at A sites, followed by G and C sites, and least frequently at T sites. In contrast, morpholine, ethylenediamine, diisopropylamine, triethylamine, and diazabicyclo[2.2.2]octane cleave the DNA predominantly at G sites. Therefore, use of one of the latter amines allows clear distinction of G bands and C bands, which could not be distinguished by the criterion of band intensity in the original one-lane sequencing method based on cleavage in hot aqueous piperidine (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194-6200). The effect of varying the salt concentration on the cleavage distribution obtained with various amines is also examined, and a rationale is given for the influence of salt concentration and amine basicity on the relative rate of cleavage at G sites.     

Spectrophotometric method of studying the cleavage of DNA-duplexes by restriction endonucleases]

A spectrophotometric method for continuous monitoring the cleavage of DNA duplexes by type II restriction endonucleases was proposed. The time course of cleavage of a 14-membered DNA duplex by MvaI endonuclease was obtained. The spectrophotometric method is characterized by rapidity and high precision in determining the kinetic parameters of the reaction. It can be recommended for testing the preparations for the presence of restriction endonucleases, rapid determination of the activity of any restriction endonucleases, highly precise quantitative analysis of the restriction enzyme catalysed reactions.     

Detection of 81 of 81 Known Mouse β-Globin Promoter Mutations with T4 Endonuclease VII—The EMC Method

The enzyme mismatch cleavage (EMC) method relies on the use of the resolvase T4 Endonuclease VII to cleave and thus detect mismatches in heteroduplex DNA formed by annealing normal DNA with mutant DNA. Detection is based on cleavage 3′ to the mismatch within a few nucleotides. We report the detection of all 81 different homozygous single-basepair changes tested and present in the mouse β-globin promoter by using the EMC method with a single set of conditions. Efficiency of cleavage was rated as strong, medium, or weak based on the intensity of the cleavage product(s) compared with background bands on autoradiography. We expect this method to detect near 100% of mutations.     

PCR‐DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were charac‐terized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communi‐ties, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eu‐karyotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE‐patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the con‐centration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR‐DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optico-structural machine analysis of the nuclear chromatin and cytoplasmic SH-groups of epitheliocytes of the oral mucosa of healthy subjects and in peptic ulcer of the stomach and duodenum

By means of optico-structural machine analysis, epitheliocytes of mucous membrane of the oral walls have been studied in 21 healthy persons and in 30 persons suffering from ulcerous disease of the stomach and duodenum. Changes in densitometric parameters of epitheliocytic nuclei at the ulcerous disease demonstrate increasing amount of heterochromatin and, hence, increasing degree of cell differentiation. Changes in contents and distribution of SH-groups in cell cytoplasm indicate that keratin is accumulated in it and that keratinization processes in the mucous membrane epithelium increase. In this way it is shown that the densitometric parameters are informative on distribution of hetero- and euchromatin in nuclei and SH-groups in epitheliocytic cytoplasm of the mucous membrane in the oral wall for estimation of morphofunctional state of these cells, normal and under pathological conditions.     

Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts.

Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer. The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light. Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine. The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct. By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning. In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75%. Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50%. The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.     

Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.

Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.