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An automatic DNA sequencing analysis system has been developed by Scanalytics/CSPI. The strategies used to solve some of the common problems in the automation of DNA sequencing systems will be discussed. Algorithms developed include fast image background correction, automatic base detection, resolution enhancement by deconvolution, desmiling and artificial intelligence algorithms for sequence interpretation.
The whiE gene cluster of Streptomyces coelicolor, which is related to gene sets encoding the biosynthesis of polycyclic aromatic polyketide antibiotics, determines a spore pigment. Southern blotting using probes from three different parts of the whiE cluster revealed related gene sets in about half of a collection of diverse Streptomyces strains. A 5.2-kb segment of one such cluster, sch, previously shown to determine spore pigmentation in Streptomyces halstedii, was sequenced. Seven open reading frames (ORFs), two of them incomplete, were found. Six of the ORFs resemble the known part of the whiE cluster closely. The derived gene products include a ketosynthase (= condensing enzyme) pair, acyl carrier protein and cyclase, as well as two of unidentified function. The seventh ORF diverges from the main cluster and encodes a protein that resembles a dichlorophenol hydroxylase. Comparison with sequences of related gene sets for the biosynthesis of antibiotics suggests that gene clusters destined to specify pigment production diverged from those destined to specify antibiotics early in the evolution of the Streptomyces genus.
To examine the molecular basis for efficient induction of superovulation in the rabbit, we determined the cDNA sequences of the luteinizing hormone beta-subunit (LHB) from Japanese White (JW), New Zealand White (NZW), and Dutch-Belted (Dutch) rabbits, and we compared these LHB sequences with those of other mammals. Using 5'- and 3'-rapid amplification of cDNA ends (RACE) with pituitary cDNA libraries, we found that the LHB cDNAs of all three breeds are the same length (523 bp from the 5'-end to the polyA site) and have putative AATAAA polyadenylation signal sequences at nucleotides 504 to 509. Northern blot analysis indicated that the approximately 600-nt mRNA encoding JW LHB is slightly longer than the LHB mRNAs of the other two breeds. The NZW and Dutch rabbit LHB coding sequences are 426 bp long, and their G+C contents are higher (>73%) than those of other mammalian LHBs (<70%). The predicted 141-amino-acid sequences of the JW and NZW LHB proteins are identical, and the Dutch LHB and JW/NZW sequences differ at only two residues. The exon-intron configuration of the NZW LHB gene (three exons and two introns) is similar to that of other mammalian LHB genes, and the sequences of NZW rabbit and other mammalian LHB promoter regions are highly conserved. Phylogenetic analysis of the deduced amino acid sequences of the three rabbit LHB proteins indicated that the rabbit occupies a phylogenetic position between rodents and domestic animals, and is far from humans. The results suggest that LH prepared from rodents or domestic animals, if available, would be a better inducer for superovulation in rabbits than human LH/CG.
The complete sequence of the yellow fever mosquito, Aedes aegypti, mitochondrial cytochrome c oxidase subunit 1 gene has been identified. The nucleotide sequence codes for a 512 amino acid peptide. The AeCOI sequence is A + T rich (68.6%) and the codon usage is highly biased toward a preference for A- or T-ending triplets. The A. aegypti COI peptide shows high homology, up to 93% identity, with several other insect sequences and a phylogenetic analysis indicates that the A. aegypti sequence is closely related to two other mosquito species, Anopheles gambiae and A. quadrimaculatus. Comparisons of the nucleotide sequence for four A. aegypti laboratory strains revealed single nucleotide polymorphisms, with 25 nucleotide sites showing SNPs between strains. All SNPs occurred as synonymous transitions such that the peptide sequence is conserved among A. aegypti strains. RT-PCR analysis showed that COI is expressed at similar levels in all developmental stages and tissues.
The foraminifera are one of the last major groups of eukaryotes for which no published DNA sequences exist. DNA sequence of ubiquitin repeat units from 5 foraminifera (representing 3 suborders), a diatom, and a dinoflagellate were characterized using the polymerase chain reaction, cloning, and sequencing. The phylogenetic utility of ubiquitin DNA sequence is discussed in reference to a full eukaryote data base. The foraminifera are possibly a polyphyletic group, whereas the phylogenetic placement of the diatom and the dinoflagellate are congruent with existing hypotheses. The lack of a noneukaryote root and limited phylogenetically informative sequence information indicate that polyubiquitin may not be useful for phylogenetic reconstruction of all eukaryotes.
A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primer-directed DNA sequencing, and compare and discuss the relative merits and limitations of these methods.
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This work is part of the effort for sequencing chromosome XIV of Saccharomyces cerevisiae. Cosmid 14-13b contains a 37.8 kb insert derived from a partial Sau3A digestion of the genome, cloned into the BamHI site of the vector Pou6. The strategy used for sequencing is based on the fragmentation of the whole cosmid by sonication, followed by shotgun sequencing on an Applied Biosystem DNA sequencer. The clones with inserts corresponding to the vector were identified by dot-blot hybridization, without the need of sequencing. The analysis of the DNA sequence reveals 29 open reading frames (ORFs) longer than 300 bases. Nine ORFs are internal to some other ORFs. Similarity searches against DNA and protein data banks show that six ORFs correspond to already known yeast genes (OMP1, PSU1, MLS1, RPC19, DBP2, CYB5) and one ORF matches the sequence of a putative yeast gene (ESBP6).
An international effort is now underway to obtain the DNA sequence for the entire human genome (Watson and Jordan, 1989, Genomics 5, 654-656; Barnhart, 1989, Genomics 5, 657-660). This Human Genome Initiative will generate sequence data from several species other than humans, and will result in several copies per species of at least some regions of the genome. Although the project has generated much interest, it is but one aspect of the widespread effort to generate DNA sequence data. Published sequences are collected in common databases, and release 63 of GenBank in March 1990 contained 40,127,752 bases from 33,337 reported sequences (News from GenBank 3; Mountain View, California: Intelligenetics, Inc., 1990). Large though this database is, it is only about 1% of the number of bases in the human genome. Interpretations of data of such magnitude are going to require the collaborative efforts of biometricians and molecular biologists, and an aim of this paper is to show that there is also a role for readers of this journal in the design of surveys of DNA sequences. Discussion here will center on the use of sequence data in evolutionary studies, where some region of DNA is sequenced in several different species. The object is to infer the evolutionary history of that particular region, or of the species themselves. Statistical issues in the very important studies on sequences to locate and characterize regions responsible for human diseases will not be addressed here. We will discuss appropriate ways of measuring distances between DNA sequences and of predicting the sampling properties of the distances. There are procedures for inferring evolutionary histories for a set of elements that depend on a matrix of distances between each pair of elements, and the precision of resulting trees must be influenced by the precision of the distances. We will show that account needs to be taken of two sampling processes--the sampling of sequences by the investigator ("statistical sampling"), and the sampling of genetic material involved in the formation of offspring from a parental population ("genetic sampling").
Mitochondrial genes for cytochrome oxidase I (COI) from hydrothermal-vent-endemic gastropods of the genus Alviniconcha were sequenced to determine the phylogenetic relationships among specimens from three areas in the western Pacific. Individuals of Alviniconcha hessleri were collected at two vent fields (depths 1470 m and 3600 m) in the Mariana Trough. Specimens collected in the North Fiji Basin could be divided into two genetically distinct groups, both of which also differed from A. hessleri from the Mariana Trough. None of the specimens of the genus Alviniconcha collected in the Manus Basin differed genetically from the dominant group from the North Fiji Basin. We suggest that the specimens of the genus Alviniconcha analyzed in the present study can be tentatively classified into A. hessleri and two undescribed species.
We report here a simple method of directly visualizing in automated DNA sequencing chromatograms DNA methylations of different types including cytosine methylations in Hpa II and dcm sites as well as adenine methylations in dam sites. This is made possible by the observation that the extent of incorporation of fluorescently labeled dideoxynucleotides is influenced by the methylated bases in template DNA. This simple approach involves routine automated DNA sequencing without any prior treatment of DNA specific for detecting DNA methylation.
The complete DNA sequence of equine herpesvirus-4 (EHV-4) strain NS80567 was determined. The genome is 145597 bp in size and consists of a long unique region (UL, 112398 bp) flanked by a short inverted repeat (TRL/IRL, 27 bp) linked to a short unique region (Us, 12789 bp) flanked by a substantial inverted repeat (TRs/IRs, 10178 bp). EHV-4 is predicted to contain 76 different genes; three of these are present twice in TRs/IRs, giving a total of 79 genes. The closely related virus equine herpesvirus-1 (EHV-1) also possesses 76 different genes corresponding to those of EHV-4, but has a total of 80 genes because four are present twice in TRs/IRs. Interpretations of the coding capacity of the EHV-4 and EHV-1 genomes were refined by comparing the complete DNA sequences.
The evolution of highly polymorphic gene loci is following routes that cannot be extrapolated from the existing knowledge of single copy genes. In addition, interpreting the evolution of the most polymorphic loci in vertebrates requires a plethora of data from different taxa. We evaluate here the rules for the evolution of Major Histocompatibility Complex (MHC-)DRB genes recently established in humans and other primates on the basis of sequences from several artiodactyl species. MHC genes encode essential molecules for self/altered-self/non-self discrimination in the interaction of the organism with its environment. The necessity to effectively present various different antigens to immunocompetent cells causes positive selection pressure on the variability of these genes in the population. Artiodactyls represent the third mammalian order in which this phenomenon was evidence independently. A further incentive to investigate also the surroundings of MHC-DRB loci was the presence of a particular repetitive sequence stretch in the vicinity of the polymorphic exon--in addition to the evolutionarily old alleles, ancient polymorphisms and the mechanisms for their generation and/or maintenance. Besides their utility for indirect gene diagnosis (MHC-DRB typing), the closely linked stretches of simple repetitive DNA in the neighborhood of the highly polymorphic MHC-DRB genes are also interesting remains of the evolutionary history. Evolutionary development is different in genetically inert intronic DNA compared to the exonic counterparts, despite their close vicinity. The persistence of these simple repeats over nearly 100 million years in one location preserving the same basic motif structure is startling. Indirect evidence is weighed that biological meaning should be considered for these elements. The combined analysis of the polymorphic DRB genes and the (highly variable but persistent) simple repeat stretches deepen our understanding of the complexities within a unique genomic compartment encoding essential molecules for self/non-self differentiation in the interaction of the organism with its environment.
We present the nucleotide sequence of a 5207-bp-long region of the mitochondrial genome of the dermatophyte Trichophyton rubrum. This represents about 1/5th of the total genome and extends a previous study. From the 5' end of the present sequence, the order of genes is as follows: the end of the ND4 gene, the gene coding for subunit 6 of ATPase, the gene coding for the small ribosomal RNA (SSU rRNA), the tyrosyl tRNA gene, the ND6 gene, the COXIII gene, the ATPase 8 subunit gene and a cluster of tRNAs genes corresponding respectively to the lysine, glutamine, asparagine, isoleucine and tryptophan isoacceptors. The interesting features of this region are its compact organisation, the presence of subunit 8 of the ATPase gene and the secondary structure of SSU rRNA which is close to that of Aspergillus nidulans. On the basis of the order of the genes, which is essentially similar to that of A. nidulans, we can also assume that the LSU rRNA subunit gene should be just upstream of this sequenced region.
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OBJECTIVE: To investigate the influence of several factors on isothermal automated DNA sequencing. METHODS: The effects of DNA purity and DNA concentration, as well as the quality of water used during sequencing on automated DNA sequencing results were retrospectively analyzed. RESULTS: When the DNA was not pure enough, the nucleotide curve showed lower ratio of signal to noise, or compression would occur, leading to difficulty in correctly judging the nucleotides; the readable length of nucleotides was shorter when low concentration of DNA was used; when the quality of water used in sequencing reaction and polyacrylamide gel solution was not high enough, the peaks of nucleotides became wider and flatter, or double peaks would occur, resulting in unreadable sequences. CONCLUSION: DNA purity and concentration as well as quality of water used in sequencing reaction and polyacrylamide gel solution had apparent influence on isothermal automated DNA sequencing. This suggests that high purity of DNA and high quality of water should be used in isothermal automated DNA sequencing; if longer DNA sequence needs to be obtained, DNA concentration should not be too low.
Two tandemly repeated DNA sequences, the 81-bp family and pOS218, have been isolated from a Sau3AI Olea europaea ssp. sativa partial genomic library. Sequencing of the 81-bp element showed the monomer to be between 78 and 84 bases long and to contain 51-58% adenine and thymidine residues. Comparison between the monomers revealed heterogeneity of the sequence primary structure. The clone pOS218 is 218 bases long, and sequence comparison between the two elements revealed that an internal region of the pOS218 repeated DNA sequence had 79% homology to the 81 bp repeat sequence. A breakage-reunion mechanism, involving the CAAAA sequence, could be responsible for the derivation of pOS218 from the 81 bp family element. By using double target in situ hybridization, co-localization of the two sequences on Olea chromosomes was observed. The sequences were present at DAPI stained heterochromatic regions, as major or minor sites having a subtelomeric or interstitial location. Methylation studies using two sets of isoschizomers, Sau3AI-MboI and MspI-HpaII, demonstrated that most cytosine residues in the GATC sites and the internal cytosine in the CCGG sites of both elements were methylated in O. europaea ssp. sativa. No major difference in methylation was apparent between DNA extracted from young leaves or from callus of O. europaea ssp. sativa. Both elements are also present in Olea chrysophylla, Olea oleaster, and Olea africana, but are absent from other Oleaceae genera, including Phillyrea, Forsythia, Ligustrum, Parasyringa, and Jasminum.
A 3D chaos game is shown to be a useful way for encoding DNA sequences. Since matching subsequences in DNA converge in space in 3D chaos game encoding, a DNA sequence's 3D chaos game representation can be used to compare DNA sequences without prior alignment and without truncating or padding any of the sequences. Two proposed methods inspired by shape-similarity comparison techniques show that this form of encoding can perform as well as alignment-based techniques for building phylogenetic trees. The first method uses the volume overlap of intersecting spheres and the second uses shape signatures by summarizing the coordinates, oriented angles, and oriented distances of the 3D chaos game trajectory. The methods are tested using: (1) the first exon of the beta-globin gene for 11 species, (2) mitochondrial DNA from four groups of primates, and (3) a set of synthetic DNA sequences. Simulations show that the proposed methods produce distances that reflect the number of mutation events; additionally, on average, distances resulting from deletion mutations are comparable to those produced by substitution mutations.