Microcomputer programs for DNA sequence analysis.

Computer programs are described which allow (a) analysis of DNA sequences to be performed on a laboratory microcomputer or (b) transfer of DNA sequences between a laboratory microcomputer and another computer system, such as a DNA library. The sequence analysis programs are interactive, do not require prior experience with computers and in many other respects resemble programs which have been written for larger computer systems (1-7). The user enters sequence data into a text file, accesses this file with the programs, and is then able to (a) search for restriction enzyme sites or other specified sequences, (b) translate in one or more reading frames in one or both directions in order to find open reading frames, or (c) determine codon usage in the sequence in one or more given reading frames. The results are given in table format and a restriction map is generated. The modem program permits collection of large amounts of data from a sequence library into a permanent file on the microcomputer disc system, or transfer of laboratory data in the reverse direction to a remote computer system.     

A protein secondary structure prediction scheme for the IBM PC and compatibles

A prediction scheme has been developed for the IBM PC and compatiblescontaining computer programs which make use of the protein secondarystructure prediction algorithms of Nagano (1977a,b), Gamieret al. (1978), Burgess et al. (1974), Chou and Fasman (1974a,b),him (1974) and Dufton and Hider (1977). The results of the individualprediction methods are combined as described by Hamodrakas etal. (1982) by the program PLOTPROG to produce joint predictionhistograms for a protein, for three types of secondary structure:-helix, ß-sheet and ß-turns. The schemerequires uniform input for the prediction programs, producedby any word processor, spreadsheet, editor or database programand produces uniform output on a printer, a graphics screenor a file. The scheme is independent of any additional softwareand runs under DOS 2.0 or later releases. Received on January 26, 1988; accepted on May 24, 1988     

Computer programs to identify and classify amphipathic alpha helical domains.

The amphipathic alpha helix is an often-encountered secondary structural motif in biologically active peptides and proteins. An amphipathic helix is defined as an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix. In a recent review article we grouped amphipathic helixes into seven distinct classes (A, H, L, G, K, C, and M) based upon a detailed analysis of their physical-chemical and structural properties (Segrest, J. P., et al. Amphipathic helix motif: classes and properties. Proteins. 1990. 8: 103-117). We have developed five computer programs that automate analysis and classification of potential amphipathic helical domains from primary amino acid sequence data. Here we describe these five programs and illustrate their usefulness by comparing two data sets of sequences representing different amphipathic alpha helical motifs from the exchangeable apolipoproteins. In a companion review article (Segrest, J. P., et al. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function. J. Lipid Res. 1992. 33: 000-000) these five programs are used to localize and characterize the putative amphipathic helixes in the exchangeable apolipoproteins.     

Two computer programs for rapid entry of DNA sequence data

Two computer programs for the IBM personal computer are describedfor rapid and accurate entry of DNA sequence data. The DNA sequencefiles produced can be used directly by the DNA sequence manipulationprograms by R. Staden (the DataBase system), the Universityof Wisconsin Genetics Computer Group, DNASTAR, or D. Mount.The first program, DIGISEQ, utilizes a sonic digitizer for semi-automationof sequence entry. To enter the DNA sequence each band of agel reading is touched by the stylus of the sonic digitizer.DIGISEQ corrects for both changes in lane width and lane curvature.The algorithm is extremely efficient and rarely requires re-entenngthe centers of the lanes. The second program, TYPESEQ, usesonly the keyboard for input. The keyboard is reconfigured toplace nucleotides and ambiguity codes under the fingers of onehand, corresponding to the order of the nucleotides on the geldefined by the user Both programs produce individual tones foreach nucleotide, and certain ambiguity codes. This verifiesinput of the correct nucleotide or ambiguity code, and thuseliminates the need to visually check the screen display duringsequence entry. Received on November 16, 1986; accepted on June 16, 1987     

The computer program package "SAMSON" for analysis of primary structure of biopolymers

A program package "SAMSON" for the computer analysis of biopolymer primary structures is described. All possible modes of sequence investigation are considered. The programs for sequence comparison are described in some details. The general principles of a program package organisation and of its user interface are also mentioned. For more complete information see Vernoslov S.E. et al. "Program package "SAMSON" for the analysis of the polymer primary structures", parts 1 and 2, Poustchino, ONTI NCBI, 1989.     

IBM microcomputer programs that analyze DNA sequences for tRNA genes

A set of four computer programs that search DNA sequence datafiles for transfer RNA genes have been written in IBM (Microsoft)BASIC for the IBM personal computer. These programs locate andplot predicted secondary structures of tRNA genes in the cloverleafconformation. The set of programs are applicable to eukaryotictRNA genes, including those containing intervening sequences,and to prokaryotic and mitochondrial tRNA genes. In addition,two of the programs search up to 150 residues downstream oftRNA gene sequences for possible eukaryotic RNA polymerase IIItermination sites comprised of at least four consecutive T residues.Molecular biologists studying a variety of gene sequences andflanking regions can use these programs to search for the additionalpresence of tRNA genes. Furthermore, investigators studyingtRNA gene structure-to-function relationships would not needto do extensive restriction mapping to locate tRNA gene sequenceswithin their cloned DNA fragments. Received on October 29, 1985; accepted on January 28, 1986     

A description of micro- and mainframe-computer programs to summarize frequency,duration and sequences of behavior

Computer programs were developed to hasten the summarization of behavioral data. Behavioral data may be collected by hand (pencil, paper and watch), strip-chart mechanical event recorder, electronic event recorder or by a computer. These behavioral data, in raw form, enter (electronically or manually) a microcomputer (IBM-PC, 128K) or mainframe computer. The microcomputer version summarizes the number of occurrences (frequency), duration and sequence of each behavior. As a microcomputer memory is limited, a program to summarize larger data sets containing more behavior patterns was developed on our mainframe computer (CDC Cyber 730/760). This sequential analysis program can accumulate up to 10 behavioral sequences (9 orders of transition) of up to 50 behaviors in a data set containing up to 10 000 elements. The computer-summary of each treatment may be combined to determine if treatment differences exist. An example data set is provided.     

The cell cycle time in intestinal crypts by simulation of FLM experiments

A computer program is developed that permits simulation of the dynamic behaviour of cells in intestinal crypts (Meinzer & Sandblad, 1985). Here we present the simulation of FLM data which is compared with the experimental findings of Al-Dewachi et al. (1974). The phase durations and total cycle times of cells in the jejunal crypts of rats were calculated. Additionally, the influence of various control parameters on the simulation output is discussed, e.g. the standard errors of phase times and the grain dilution at mitosis.     

A package of computer programs for handling taxonomic databases

A package of programs is described which processes taxonomicdata, suitable to use when preparing monographs, handbooks,Floras or Faunas, in which species or other taxa are describedand identified. There is also an interactive program for specimenidentification, and conversion routines which prepare data fornumerical taxonomy (clustering and cladistics). The programsare equally suitable for botany or for zoology, or even fornon-biological data. Received on September 20, 1985; accepted on November 11, 1985     

Genetic localization of diuron- and mucidin-resistant mutants relative to a group of loci of the mitochondrial DNA controlling coenzyme QH2-cytochrome c reductase in Saccharomyces cerevisiae.

Summary Diuron-resistance, DIU (Colson et al., 1977), antimycin-resistance, ANA (Michaelis, 1976; Burger et al., 1976), funiculosin-resistance, FUN (Pratje and Michaelis, 1977; Burger et al., 1977) and mucidin-resistance, MUC (Subik et al., 1977) are each coded by a pair of genetic loci on the mit DNA of S. cerevisiae. In the present paper, these respiratory-competent, drug-resistant loci are localized relative to respiratory-deficient BOX mutants deficient in coenzyme QH₂-cytochrome c reductase (Kotylak and Slonimski, 1976, 1977) using deletion and recombination mapping. Three drug-resistant loci possessing distinct mutated allelic forms are distinguished. DIU1 is allelic or closely linked to ANA2, FUN1 and BOX1; DIU2 is allelic or closely linked to ANA1, MUC1 and BOX4/5; MUC2 is allelic to BOX6. The high recombinant frequencies observed between the three loci (13% on the average for 33 various combinations analyzed) suggest the existence of either three genes coding for three distinct polypeptides or of a single gene coding for a single polypeptide but subdivided into three easily separable segments. The resistance of the respiratory-chain observed in vitro in the drug-resistant mutants and the allelism relationships between respiratory-competent, drug-resistant loci and coQH₂-cyt c reductase deficient, BOX, loci strongly suggest that each of the three drug-resistant loci codes for a structural gene-product which is essential for the normal coQH₂-cyt c reductase activity and is obviously a good candidate for a gene product of the drug-resistant loci mapped in this paper. Polypeptide length modifications of cytochrome b were observed in mutants deficient in the coQH₂-cyt c red and localized at the BOX1, BOX4 and BOX6 genetic loci (Claisse et al., 1977, 1978) which are precisely the loci allelic to drug resistant mutants as shown in the present work. Taken together these two sets of data provide a strong evidence in favor of the idea that there exist three non contiguous segments of the mitochondrial DNA sequence which code for a single polypeptide sequence of cytochrome b. In each segment mutations which modify the polypeptide sequence can occur leading to the loss (BOX mutants) or to a modification (drug resistant mutants) of the enzyme activity.Chercheur qualifié du Fonds National de la Recherche Scientifique     

The Cell Cycle Time In Intestinal Crypts By Simulation of Flm Experiments

Abstract. A computer program is developed that permits simulation of the dynamic behaviour of cells in intestinal crypts (Meinzer & Sandblad, 1985). Here we present the simulation of FLM data which is compared with the experimental findings of Al-Dewachi et al. (1974). the phase durations and total cycle times of cells in the jejunal crypts of rats were calculated. Additionally, the influence of various control parameters on the simulation output is discussed, e.g. the standard errors of phase times and the grain dilution at mitosis.     

Isolation of a D-genome specific repeated DNA sequence fromAegilops squarrosa

Three repeated sequence clones, pAS1(1.0 Kb), pAS2(1.8 Kb) and pAS12(2.5 Kb), were isolated fromAegilops squarrosa (Triticum tauschii). The inserts of the three clones did not hybridize to each other. Two of the clones, pAS2 and pAS12, contain repeated sequences which were distributed throughout the genome. The clone pAS1 sequence was more restricted and was located in specific areas on telomeres and certain interstitial sites along the chromosome length. This cloned sequence was also found to be restricted to the D genome at the level ofin situ hybridization. The pAS1 sequence will be useful in chromosomal identification and phylogenetic analysis. All three clones will allow assessment of genome plasticity inAegilops squarrosa. Nuclear DNA content varies over a range of 10,000 fold among all organisms (Nagl et al., 1983). Among angiosperms, at least a 65-fold range in genome size occurs in diploid species (Sparrow, Price and Underbrink, 1972; Bennett, Smith and Heslop-Harrison, 1982). This DNA variation has been reported within families, genera, and species (Rothfels et al., 1966; Rees and Jones, 1967; Miksche, 1968; Price, Chambers and Bachmann, 1981). Much of the interspecific variation in genome size among angiosperms appears to be due to amplification and/or deletion of DNA within chromosomes. The variation in genome size does not appear to result in changes in the number of coding genes (Nagl et al., 1983). While the number of coding genes, with the exception of rDNA in specific examples, appears to remain constant, the remaining non-coding regions are quite flexible. This non-coding DNA encompasses over 99% of the plant genome and consists of sequences that exist as multiple copies throughout the genome and are identified as repeated DNA sequences (Flavell et al., 1974). Flavell et al. (1974) have reported that increasing genome size in higher plants is associated with increasing repetitive DNA amounts. Subsequent reports have substantiated this correlation (Bachmann and Price, 1977; Narayan, 1982). In various cereals, heterochromatin, which has been demonstrated to be correlated with the location of specific repeated DNA sequences, has been positively correlated with genome size (Bennett, Gustafson and Smith, 1977; Rayburn et al., 1985). Furuta, Nishikawa and Makino (1975) found significant DNA content variation among different accessions ofAegilops squarrosa L. This species contains the D genome, a pivotal genome in several polyploid species and also found in hexaploid wheat (AABBDD). The importance of this genome to the study of bread wheat genomes makes the mechanism(s) of this genomic plasticity of particular interest. In order to determine which sequences are varying, one must first have a way to identify specific types of chromatin and/or DNA. Specific types of chromosome banding such as C- and N-banding have been used to identity types of chromatin in previous studies. C-banding of the D genome results in very lightly staining bands whose pattern is somewhat indistinct. N-banding alternatively has been shown to be useful in identifying certain chromosomes of hexaploid wheat but is limited by the lack of major bands in the D genome (Endo and Gill, 1984). Specific DNA sequences have been isolated fromTriticum aestivum cultivar “Chinese Spring” (hexaploid wheat). However, these sequences are representatives of the A and/or B genomes of hexaploid wheat and are not found in significant quantities in the D genome (Hutchinson and Lonsdale, 1982). Various other repeated DNA sequences have been successfully isolated from rye (Bedbrook et al., 1980) and identified on rye chromosomes (Appels et al., 1981; Jones and Flavell, 1982). Certain of these sequences are found in wheat genomes, but the sequences are representative of only a minor fraction of the D genome (Bedbrook et al., 1980; Rayburn and Gill, 1985). The purpose of this report is to describe three distinct repeated DNA sequences isolated fromA. squarrosa (D genome). Two clones appear to be distributed throughout the total genome, and the third clone is restricted to specific sites along the chromosomes. This latter clone will prove useful in cytologically defining the D genome chromosomes. These sequences appear representative of two types of repeated DNA genome organization: 1) sequences distributed throughout the genome and 2) specific arrays of repeated sequences. The availability of such repeated DNA sequence clones along with the known intraspecific DNA content variation inA. squarrosa will allow the study of genomic plasticity of this species.     

EZ-ASSIGN, a program for exhaustive NMR chemical shift assignments of large proteins from complete or incomplete triple-resonance data

For several of the proteins in the BioMagResBank larger than 200 residues, 60 % or fewer of the backbone resonances were assigned. But how reliable are those assignments? In contrast to complete assignments, where it is possible to check whether every triple-resonance Generalized Spin System (GSS) is assigned once and only once, with incomplete data one should compare all possible assignments and pick the best one. But that is not feasible: For example, for 200 residues and an incomplete set of 100 GSS, there are 1.6 × 10²⁶⁰ possible assignments. In “EZ-ASSIGN”, the protein sequence is divided in smaller unique fragments. Combined with intelligent search approaches, an exhaustive comparison of all possible assignments is now feasible using a laptop computer. The program was tested with experimental data of a 388-residue domain of the Hsp70 chaperone protein DnaK and for a 351-residue domain of a type III secretion ATPase. EZ-ASSIGN reproduced the hand assignments. It did slightly better than the computer program PINE (Bahrami et al. in PLoS Comput Biol 5(3):e1000307, 2009) and significantly outperformed SAGA (Crippen et al. in J Biomol NMR 46:281–298, 2010), AUTOASSIGN (Zimmerman et al. in J Mol Biol 269:592–610, 1997), and IBIS (Hyberts and Wagner in J Biomol NMR 26:335–344, 2003). Next, EZ-ASSIGN was used to investigate how well NMR data of decreasing completeness can be assigned. We found that the program could confidently assign fragments in very incomplete data. Here, EZ-ASSIGN dramatically outperformed all the other assignment programs tested.     

Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods.

Nine currently available methods for HRP neurohistochemistry have been compared with each other on matching tissue sections from four rats and four rhesus monkeys. The nine methods investigated in this report are the diaminobenzidine (DAB) procedures of LaVail JH and LaVail MM (J Comp Neurol 157:303, 1974), of Adams JC (Neuroscience 2:141, 1977) and of Streit P and Reubi JC (Brain Res 126:530, 1977); the benzidine dihydrochloride (BDHC) procedures of Mesulam M-M (J Histochem Cytochem 24:1273, 1976) and of De Olmos J and Heimer L (Neurosci Lett 6:107, 1977); the o-dianisidine (O-D) procedure of De Olmos J (Exp Brain Res 29:541, 1977); the p-phenylenediamine dihydrochloride and pyrocatechol (PPD-PC) procedure of Hanker JS et al., (Histochem J 9:789, 1977) and the tetramethyl benzidine (TMB) procedures of Mesulam M-M (J Histochem Cytochem 26:106, 1978) and of De Olmos J et al. (J Comp Neurol 181:213, 1978). Quantitative comparisons were based on counts of retrogradely labeled perikarya. The extent of anterograde transport and the size of the injection site were also compared at a more qualitative level. The results indicate that one TMB procedure (Mesulam M-M, J Histochem Cytochem 26:106, 1978) is distinctly superior to each of the other eight procedures in the number of labeled perikarya that it can demonstrate. Furthermore, these differences are statistically significant at better than the 0.05 level of confidence. Differences in sensitivity are most evident when the perikarya contain small quantities of transported HRP. The same TMB method also demonstrates more anterograde transport and a larger injection site than all the other procedures. If less sensitive procedures are employed, afferent or efferent connections that are clearly demonstrated by this TMB procedure are either underestimated or completely overlooked. It is suggested that sensitivity in HRP neurohistochemistry is determined by multiple factors which include the method of fixation, post-fixation storage, the choice of chromogen, the incubation parameters, the type of HRP enzyme that is administered, and the postreaction treatment.     

Lethal short limb dwarfism with dysmorphic face, omphalocele and severe ossification defect: Piepkorn syndrome or severe "boomerang dysplasia"?

The authors report a case of lethal neonatal dwarfism characterized by striking micromelia, fused rudimentary and supernumerary digits, large, soft head, pronounced hypertelorism, protruding eyes set laterally, enormous omphalocele and severe deficiency of tubular bone and spine ossification. Histologic examination showed lack of ossification of the cartilaginous anlage of many tubular bones. The cartilage had irregularly distributed chondrocytes. The matrix contained hypocellular and degenerated areas with scattered large chondrocytes. In a few bones a very disorganized growth cartilage was present. The case is similar to that described by Piepkorn et al. (1977) and may represent a severe form of "boomerang dysplasia" (Kozlowski et al., 1981; Tenconi et al., 1983; Kozlowski et al., 1985; Winship et al., 1990).     

All are not equal: a benchmark of different homology modeling programs

Modeling a protein structure based on a homologous structure is a standard method in structural biology today. In this process an alignment of a target protein sequence onto the structure of a template(s) is used as input to a program that constructs a 3D model. It has been shown that the most important factor in this process is the correctness of the alignment and the choice of the best template structure(s), while it is generally believed that there are no major differences between the best modeling programs. Therefore, a large number of studies to benchmark the alignment qualities and the selection process have been performed. However, to our knowledge no large-scale benchmark has been performed to evaluate the programs used to transform the alignment to a 3D model. In this study, a benchmark of six different homology modeling programs- Modeller, SegMod/ENCAD, SWISS-MODEL, 3D-JIGSAW, nest, and Builder-is presented. The performance of these programs is evaluated using physiochemical correctness and structural similarity to the correct structure. From our analysis it can be concluded that no single modeling program outperform the others in all tests. However, it is quite clear that three modeling programs, Modeller, nest, and SegMod/ ENCAD, perform better than the others. Interestingly, the fastest and oldest modeling program, SegMod/ ENCAD, performs very well, although it was written more than 10 years ago and has not undergone any development since. It can also be observed that none of the homology modeling programs builds side chains as well as a specialized program (SCWRL), and therefore there should be room for improvement.     

On antisense peptides: the parathyroid hormone as an experimental example and a critical theoretical view

We followed an approach which predicts that translation of two complementary RNA strands into protein generates pairs of "antisense" peptides which bind each other with specific and high affinity (Bost et al. Proc. Natl. Acad. Sci. (1985) 82, 1372). We used human parathormone as an experimental example, and we analysed by computer homologies between antisense peptide sequences and their published receptor sequences. We conclude that there is no experimental indication that parathormone binds to a synthetic peptide, the sequence of which was derived from the antisense RNA sequence. Based on homology scores and antigenicity indexes (Hopp) the analysis shows that the peptide ligand itself, or a random artificial peptide, are as good candidates as the antisense peptide in producing antibodies, presumably recognizing the receptor. We therefore question the general applicability of this approach.     

Signal sequence of alkaline phosphatase of Escherichia coli.

The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.     

Focus on the patentability of computer programs

The Nuts and Bolts section of our Journal (mirrored on the ICCNS society web site), is meant to provide a very practical way to share useful information, that goes beyond the scope of cell signaling and basic CCN protein biology. Considering the number of requests we have had for information related to protection of Intellectual Property (IP), I am pleased to initiate what will be a series of articles that will focus on various IP topics. The inaugural topic is the protection of computer programs. Some colleagues may wonder how and why the patentability of computer programs is a topic of interest for scientists working on CCN proteins . . . As a matter of fact, to assist us in analyzing the potential involvement of CCN3 in human genetic diseases, we considered developing a computer program designed to analyze large amounts of data. Sharing the concepts and the computer program raised concerns regarding IP and protection of the software that we would handle. We believe that many colleagues have encountered similar problems. This article provides a short focus on computer program patentability. It is aimed to provide basic legal information, and to help our readers in understanding the process. It is not intended to replace IP counselors or technology transfer departments. Future articles will address other practical aspects of IP protection.