Biologic properties and nucleotide sequence analysis of human papillomavirus type 51.

Human papillomaviruses (HPVs) may be grouped according to the site from which they are isolated and the disease with which they are associated. We recently identified and cloned HPV type 51 (HPV-51) from a low-grade precancerous lesion (G. Nuovo, E. DeVilliers, R. Levine, S. Silverstein, and C. Crum. J. Virol. 62:1452-1455, 1988). Molecular epidemiologic analysis of cervical lesions, including condylomata and low- and high-grade precancers, revealed that HPV-51 was present in about 5% of the samples we examined. We have now determined the complete nucleotide sequence of this virus and compared it with other sequenced HPVs. Our analysis reveals that the 7,808-bp genome is composed of eight open reading frames which are encoded on the same strand and that this virus is most closely related to HPV-31. Sequence comparisons place this virus in the group of high-risk viruses (those with an increased risk of progressing to malignancy) along with HPV-16, -18, -31, and -33. Morphologic transformation experiments demonstrated that HPV-51 had transformation potential and that transformed cells contained RNAs homologous to E6 and E7.     

Two Sample Logo: a graphical representation of the differences between two sets of sequence alignments

SUMMARY: Two Sample Logo is a web-based tool that detects and displays statistically significant differences in position-specific symbol compositions between two sets of multiple sequence alignments. In a typical scenario, two groups of aligned sequences will share a common motif but will differ in their functional annotation. The inclusion of the background alignment provides an appropriate underlying amino acid or nucleotide distribution and addresses intersite symbol correlations. In addition, the difference detection process is sensitive to the sizes of the aligned groups. Two Sample Logo extends WebLogo, a widely-used sequence logo generator. The source code is distributed under the MIT Open Source license agreement and is available for download free of charge.     

A test for nucleotide sequence homology

Two macromolecular sequences which have evolved from a common ancestor sequence will tend to include a large number of elements unaffected by replacement mutations in both sequences, as long as the evolutionary rate is not too high or the divergence time is not too great. The positions of corresponding elements may have changed in either daughter sequence due to deletion/insertion mutations involving other sequence elements, but their order can be expected to be the same in both sequences. These sets of correspondences, called matches, may be computed by a recursive algorithm which incorporates constraints on the number of deletion/insertion mutations hypothesized to have occurred. A test is developed which computes the significance of each deletion/insertion hypothesized, based on Monte-Carlo sampling of random sequences with the same base composition as the experimental sequences being tested. Applying the test to 5 S RNAs confirms the relation of Escherichia coli and KB carcinoma 5 S RNAs and establishes the previously undetected homology between Pseudomonas fluorescens and KB 5 S RNAs.     

Correlation between fibroin amino acid sequence and physical silk properties

The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet.     

Thermodynamic parameters for an expanded nearest-neighbor model for the formation of RNA duplexes with single nucleotide bulges

Thirty-four RNA duplexes containing single nucleotide bulges were optically melted, and the thermodynamic parameters deltaH degrees, deltaS degrees, deltaG degrees (37), and T(M) for each sequence were determined. Data from this study were combined with data from previous thermodynamic data [Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990) Biochemistry 29, 278-85] to develop a model that will more accurately predict the free energy of an RNA duplex containing a single nucleotide bulge. Differences between purine and pyrimidine bulges as well as differences between Group I duplexes, those in which the bulge is not identical to either neighboring nucleotide, and Group II duplexes, those in which the bulge is identical to at least one neighboring nucleotide, were considered. The length of the duplex, non-nearest-neighbor effects, and bulge location were also examined. A model was developed which divides sequences into two groups: those with pyrimidine bulges and those with purine bulges. The proposed model for pyrimidine bulges predicts deltaG degrees (37,bulge) = 3.9 kcal/mol + 0.10deltaG degrees (37,nn) + beta, while the model for purine bulges predicts deltaG degrees (37,bulge) = 3.3 kcal/mol - 0.30deltaG degrees (37,nn) + beta, where beta has a value of 0.0 and -0.8 kcal/mol for Group I and Group II sequences, respectively, and deltaG degrees (37,nn) is the nearest-neighbor free energy of the base pairs surrounding the bulge. The conformation of bulge loops present in rRNA was examined. Three distinct families of structures were identified. The bulge loop was either extrahelical, intercalated, or in a "side-step" conformation.     

Complete nucleotide sequence of the Streptomyces lividans plasmid pIJ101 and correlation of the sequence with genetic properties.

The complete nucleotide sequence of the multicopy Streptomyces plasmid pIJ101 has been determined and correlated with previously published genetic data. The circular DNA molecule is 8,830 nucleotides in length and has a G+C composition of 72.98%. The use of a computer program, FRAME, enabled identification in the sequence of seven open reading frames, four of which, tra (621 amino acids [aa]), spdA (146 aa), spdB (274 aa), and kilB (177 aa), appear to be genes involved in plasmid transfer. At least two of the above genes are predicted to be transcribed by known promoters that are regulated in trans by the products of the korA (241 aa) and korB (80 aa) loci on the plasmid. The segment of the plasmid capable of autonomous replication contains one large open reading frame (rep; 450 aa) and a noncoding region presumed to be the origin of replication. Four other small (less than 90 aa) open reading frames are also present on the plasmid, although no function can be attributed to them. The sequence of the pIJ101 replication segment present in several widely used cloning vectors (e.g., pIJ350 and pIJ702) has also been determined, so that the complete nucleotide sequences of these vectors are now known.     

A measure of DNA sequence dissimilarity based on free energy of nearest-neighbor interaction

We develop a novel method of asserting the similarity between two biological sequences without the need for alignment. The proposed method uses free energy of nearest-neighbor interactions as a simple measure of dissimilarity. It is used to perform a search for similarities of a query sequence against three complex datasets. The sensitivity and selectivity are computed and evaluated and the performance of the proposed distance measure is compared. Real data analysis shows that is a very efficient, sensitive and high-selective algorithm in comparing large dataset of DNA sequences.     

Portable microcomputer software for nucleotide sequence analysis.

The most common types of nucleotide sequence data analyses and handling can be done more conveniently and inexpensively on microcomputers than on large time-sharing systems. We present a package of computer programs for the analysis of DNA and RNA sequence data which overcomes many of the limitations imposed by microcomputers, while offering most of the features of programs commonly available on large computers, including sequence numbering and translation, restriction site and homology searches with dot-matrix plots, nucleotide distribution analysis, and graphic display of data. Most of the programs were written in Standard Pascal (on an Apple II computer) to facilitate portability to other micro-, mini-, and and mainframe computers.     

Automated constraint-based nucleotide sequence selection for DNA computation

We present techniques for automating the design of computational systems built using DNA, given a set of high-level constraints on the desired behavior and performance of the system. We have developed a program called SCAN that exploits a previously implemented computational melting temperature primitive to search a 'nucleotide space' for sequences satisfying a pre-specified set of constraints, including hybridization discrimination, primer 5' end and 3' end stability, secondary structure reduction, and prevention of oligonucleotide dimer formation. The first version of SCAN utilized 24 h of computer time to search a space of over 7.5 billion unary counter designs and found only nine designs satisfying all of the pre-specified constraints. One of SCAN's designs has been implemented in the laboratory and has shown a marked improvement in performance over the products of previous attempts at manual design. We conclude with some novel ideas for improving the overall speed of the program that offer the promise of an efficient method for selecting optimal nucleotide sequences in an automated fashion.     

Derivation of nearest-neighbor properties from data on nucleic acid oligomers. I. Simple sets of independent sequences and the influence of absent nearest neighbors

The constraints on combinations of nearest neighbors in nucleic acid sequences and the numbers of independent sequences needed to describe nearest-neighbor properties of oligomers and polymers are derived and summarized. It has been pointed out in previous work [D. M. Gray and I. Tinoco, Jr. (1970) Biopolymers, Vol. 9, pp. 223-244; R. F. Goldstein and A. S. Benight (1992) Biopolymers, Vol. 32, pp. 1679-1693] that these constraints restrict the information available from measurements of properties of sequence combinations. The emphasis in this paper is on the properties of oligomer sequences that vary in length, where each nucleotide or base pair at the end of the sequence makes a significant contribution to the measured property by interacting with its boundary of fixed sequence or solvent. In such cases it is not be possible to determine values of properties of individual nearest neighbors, except for the like neighbors [e.g., d(A-A), d(G-G), d(T-T), and d(C-C) nucleotide neighbors in single-stranded DNA or d(A-A)/d(T-T) and d(G-G)/d(C-C) base pair neighbors in double-stranded DNA], solely from measurements of properties of different sequences. Even values for properties of the like neighbors cannot be determined from such oligomeric sequences if the sequences are all of the same length. Nearest-neighbor properties of oligomer sequences that vary in length can be summarized in terms of the values for independent sets of sequences that are nearest neighbors and monomers all with boundaries of the fixed sequence or solvent. Straightforward combinations of the values for the independent sequences will give the values of the property for any dependent sequence, without explicit knowledge of the individual nearest-neighbor values. These considerations have important consequences for the derivation of widely used thermodynamic parameters, as discussed in the following paper.     

Joint modeling of DNA sequence and physical properties to improve eukaryotic promoter recognition

We present an approach to integrate physical properties of DNA, such as DNA bendability or GC content, into our probabilistic promoter recognition system McPROMOTER. In the new model, a promoter is represented as a sequence of consecutive segments represented by joint likelihoods for DNA sequence and profiles of physical properties. Sequence likelihoods are modeled with interpolated Markov chains, physical properties with Gaussian distributions. The background uses two joint sequence/profile models for coding and non-coding sequences, each consisting of a mixture of a sense and an anti-sense submodel. On a large Drosophila test set, we achieved a reduction of about 30% of false positives when compared with a model solely based on sequence likelihoods.     

Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene

A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.     

Complete nucleotide sequence of Tn10

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.     

Mutanase from a Paenibacillus isolate: nucleotide sequence of the gene and properties of recombinant enzymes

A mutanase (alpha-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 degrees C to 50 degrees C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.     

Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens.

The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.