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Genes and non-coding DNA sequences.

Many new categories of DNA sequence organization in higher organisms have been discovered in the last 20 years. Most of these are not found in bacterial and each in turn is hailed as a likely candidate for those regulatory elements which are assumed to distinguish higher from lower organisms. This article stresses the similarities between their sequence organizations and the continuing lack of convincing function for any of them. It suggests that these sequences often appear after chance recombinational events and their presence then facilitates further change. Within the constraints of proper function the genome is clearly able to find many organizational solutions of apparently equal advantage to the organism. Finally, an example of rapid genome reorganization resulting from selection for resistance to the drug methotrexate is considered.

Animals↗

PubMed1993

Large arrays of tandemly repeated DNA sequences in the green alga Chlamydomonas reinhardtii.

We describe the characterization of tandemly repeated DNA sequences, which resemble the satellite DNA sequences of multicellular eucaryotes, in the unicellular green alga Chlamydomonas reinhardtii. Restriction enzymes that cleave C. reinhardtii DNA relatively frequently produce a number of high molecular weight DNA fragments in addition to the bulk of low molecular weight DNA fragments. pTANC 1.5 contains a 1.5 kb Sau3A fragment cloned from one of these large bands. pTANC 1.5 hybridized to at least three large arrays (200 to 700 kb) of tandemly repeated DNA sequences in the cell-wall-deficient strain cw1.5. These arrays are composed of repeat units that are each cleaved once by BamHl into bands of 1.5, 1.9, 2.0 and 2.5 kb in size. The copy numbers of the 1.5, 1.9, 2.0 and 2.5 kb BamHl bands vary between different C. reinhardtii strains. Chlamydomonas smithii and a number of C. reinhardtii strains are deficient in all four BamHl bands. Genetic analysis of wild-type strain 137c, which is deficient in the 2.0 kb BamHl band, indicates that the 1.5, 1.9 and 2.5 kb BamHl bands derive from at least five loci. The 1.5, 1.9 and 2.5 kb repeat units are not extensively interspersed with each other in strain 137c. Pulsed-field gel electrophoresis of intact C. reinhardtii chromosomes indicates that TANC arrays are present on more than one chromosome.

Animals↗

PubMed2000

Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima.

The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters, which brings independent evidence for the lateral gene transfer in the genome of T.maritima. The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms of different origin. The typical genomic periodicity for Bacteria is 11 bp whilst it is 10 bp for Archaea. Eukaryotes have more complex spectra but the dominant period in the yeast Saccharomyces cerevisiae is 10.2 bp. These periodicities are most likely reflective of differences in chromatin structure.

Chromatin↗

PubMed1984

DNA sequence of the white locus of Drosophila melanogaster.

The DNA sequence of the white locus of Drosophila melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. We also report the sequence of a complementary DNA clone which established the position of the 3' end of the white RNA on this genomic sequence. The probable exon-intron structure of the gene has been predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with seven insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wzm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11)), one deletion mutant (white-spotted 4 (wsp4)) and one internal duplication mutant (white-ivory (wi)) have been determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.

Animals↗

PubMed1984

Molecular hybridization under conditions of high stringency permits cloned DNA segments containing reiterated DNA sequences to be assigned to specific chromosomal locations.

Identifying the specific DNA sequences involved in the chromosomal abnormalities in developmental and neoplastic diseases may be essential to understanding the molecular biology of these disorders. The use of recombinant DNA techniques in conjunction with rodent-human hybrid cells makes it possible to assign chromosomal locations to specific DNA sequences. However, the ubiquitous presence of reiterated DNA species often complicates the application of straightforward molecular hybridization. To accelerate the mapping of cloned sequences to specific chromosomal locations, we investigated the possibility that cloned sequences containing reiterated DNA might be used without isolating unique sequences. By varying conditions of hybridization (specifically temperature) and using restricted DNA samples from human genomic DNA, Chinese hamster ovary-human chromosome 11 hybrids, and non-chromosome 11 hybrids, we have been able to assign cloned DNA sequences containing reiterated sequences to their chromosome of origin. By hybridization under the high-stringency condition of 55 degrees C, specific banding was produced with both human genomic DNA and the human-chromosome-containing hybrid from which the probe was prepared. Furthermore, using a panel of chromosome 11 deletion mutants, we have been able to assign a cloned sequence to a specific chromosomal location. We believe that this approach will accelerate gene mapping procedures and facilitate identification of DNA sequences involved in chromosomal abnormalities.

Animals↗

PubMed1976

Investigation of the organization of mammalian chromosomes at the DNA sequence level.

New developments in DNA sequencing techniques permit rapid progress in the determination of both repetitious and single-copy mammalian sequences. Three distinct families of highly repetitious satellite DNA's from the kangaroo rat Dipodomys ordii have been sequenced. With the MS satellite it was possible to show that the basic repeat sequence and its variants were arranged in a nonrandom order suggesting a hierarchy of repeats. The HS-alpha satellite from D. ordii was shown to resemble the guinea pig alpha satellite, a long term evolutionary persistence inconsistent with previous models. Sequences from hemoglobin mRNA were determined using hemoglobin complementary DNA as template for transcription in vitro. Seven of the largest fragments have been assigned to untranslated regions of the mRNA whereas 15 others have been tentatively located within the structural genes. From correlations with sequences from corresponding regions in the human hemoglobin mRNA's we have been able to make the first direct measurements of the rate of fixation of mutations that do not change the amino acid sequence. The minimum estimate for this rate is greater than the highest previously estimated rates of fixation of neutral mutations (calculated for fibrinopeptide A. A new technique, deoxysubstitution sequencing, which should speed determination of the complete mRNA sequences, is described.

Animals↗

PubMed1989

Cloning and characterization of a tandemly repeated DNA sequence in the crane family (Gruidae).

A tandemly repeated DNA sequence possessing a unique PstI site has been characterized in several species of the crane family. The "Pst family" comprises at least 8800 monomer units 187 base pairs (bp) in length and constitutes 0.14% of the genome of the sarus crane (Grus antigone). The array is located in the centromeric heterochromatin of chromosome 2 in the two species where in situ hybridizations of a cloned monomer to metaphase chromosome spreads were carried out. DNA sequence comparisons between five monomer units from G. antigone revealed a high degree of homology between four of the individual repeats, while the fifth was somewhat divergent. The G + C content deduced from the DNA sequence makes it likely that the Pst family constitutes part of a density satellite seen in profiles of crane DNA centrifuged to equilibrium in CsCl. The common occurrence of tandem arrays such as the Pst family, with repeat lengths close to 200 bp, leads us to an hypothesis implicating nucleosomes in the evolution of such families.

Animals↗

PubMed1984

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

Base Sequence↗

PubMed1984

Use of short DNA oligonucleotides for determination of DNA sequence modifications induced by benzo[a]pyrene diol epoxide.

Various organic agents that alkylate DNA are known to induce mutations in bacterial and animal cells. The precise nature and location of modified DNA sequences in such mutants are often difficult to ascertain. In this report, a 10-base-pair oligomer (BamHI linker) is treated with (+/-)-trans-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and inserted into replicative form DNA of phage M13 by ligation at a specific restriction site. Escherichia coli are transfected with the recombinant DNA containing the alkylated target, progeny viral plaques are selected, and their DNAs are subjected to DNA sequence analysis at the region of oligomer insertion. For the alkylated inserts used in this study, the DNA sequence analysis of progeny viral DNA showed that nucleotide deletions were present in every clone examined. These deletions occurred primarily, but not exclusively, at G-C cluster regions, varied from 1 to 24 base pairs in length, and included both target and nontarget nucleotides. A second type of repair, which restores most of the original nucleotide bases in the alkylated insert, is also implied by the DNA sequence data obtained.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1995

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Animals↗

PubMed1991

Transformation of DNA sequence data into geometric symbols.

Comparative analysis of related DNA sequences has been simplified by the transformation of data in the standard A, G, C, T format into a set of geometric symbols that promote pattern recognition. Previously, comparing more than 2 or 3 sequences simultaneously has been difficult because of the monotonous patterns established by letters. Here 33 sequences are simultaneously compared to demonstrate the ease with which nucleotide substitutions are accurately identified. This has been accomplished by writing a Word-Perfect macro program to facilitate this transformation. Since this word processing program is widely used, performing this kind of analysis is readily achievable in most laboratories involved in DNA sequence analysis.

Animals↗

PubMed1994

Cloning and characterisation of a repetitive DNA sequence from Theileria mutans: application as a species-specific probe.

Repetitive DNA sequences were isolated from a Theileria mutans genomic library by screening with T. mutans total DNA. DNA sequence analysis demonstrated that a section of one of the clones contained a complex series of overlapping perfect repeats ranging between 99 and 20 bp in size. The T. mutans repetitive sequence did not contain large open reading frames (ORFs), unlike T. parva Tpr repetitive DNA sequences. When used as a hybridisation probe the repetitive sequence revealed restriction-fragment-length polymorphisms (RFLPs) between the EcoRI-digested DNAs of two T. mutans stocks. The T. mutans repetitive probe gave a signal with 1 ng of purified T. mutans piroplasm DNA and detected T. mutans sequences in whole-blood DNA isolated from an experimentally infected animal when the piroplasm parasitaemia was equal to or above 0.4%. Oligonucleotide primers derived from the repetitive sequence allowed more sensitive detection of T. mutans DNA by polymerase chain reaction (PCR) amplification. Using the PCR, T. mutans DNA was amplified from an experimentally infected animal with a parasitaemia of < 0.1%.

Animals↗

PubMed1991

Identification of a silent point mutation in the LDL-receptor gene by direct DNA sequencing.

To study the allelic variation at the human LDL-receptor gene locus in a number of individuals with familial hypercholesterolemia, a protocol was applied for direct sequence analysis of genomic DNA. The asymmetric polymerase chain reaction (PCR) was used to synthesize single-stranded DNA. Sequencing was carried out with modified T7 DNA polymerase (Sequenase version 2.0, United States Biochemical) after purification of the amplification product with a glass powder adhesion method. The method is sensitive enough to identify the heterozygous state of allelic variation and bypasses cumbersome cloning and subcloning procedures. Here we report the occurrence of a guaninine-to-adenine change in the codon for amino acid 655. However, the glycine residue is not replaced by the base change at this position, and the mutation observed here does not represent the underlying genetic defect of the LDL-receptor defect in this individual. Preliminary data suggest that this mutation represents a rare genetic variation.

Amino Acid Sequence↗

PubMed1998

Detection of SV40-like DNA sequences in pleural mesothelioma, bronchopulmonary carcinoma and other pulmonary diseases.

Mesotheliomas are pleural-based tumours mainly associated with asbestos exposure (70% of cases) and the incidence is still raising. Recently, a possible viral connection was reported and 60% of mesotheliomas were demonstrated to contain and express SV40-like DNA sequences. In this study, the presence of SV40-like DNA sequences were investigated in mesotheliomas (15 tissue samples and six cell lines) and in 63 additional bronchopulmonary carcinomas, one parietal osteosarcoma and non-malignant lung samples as well as in organizing pleuritis (8). Finally, 163 samples were analysed by the polymerase chain reaction (PCR) with a set of primers PYV.for and PYV.rev to amplify a 173 bp region of the SV40 Tag. and a 179 bp region JC virus (JCV) as well as a 182 bp region BK virus (BKV). PCR amplification and hybridization with a probe specific for SV40 Tag revealed that 47.6% of mesotheliomas, 28.6% of primary bronchopulmonary carcinomas and 16% of non-neoplasic lung diseases contained SV40-like DNA sequences. No statistically significant difference in the occurrence of these DNA sequences was found between malignant mesothelioma and bronchopulmonary carcinoma. However, a significantly higher number of mesothelioma cases exhibited SV40- like DNA sequences in comparison with non-malignant pleural and pulmonary tissues. The DNA sequences were not related to BK and JC virus sequences. These results indicate that SV40-like DNA sequences are present in mesotheliomas as well as in bronchopulmonary carcinomas and non-malignant pleuropulmonary diseases.

Antigens, Polyomavirus Transforming↗

PubMed1987

Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearrangements of target plant DNA sequences.

The transfer DNA (T-DNA) mobilized into plant cells by Agrobacterium tumefaciens seems to integrate rather randomly into the plant genome. We analyzed a target site in the genome of Nicotiana tabacum before and after integration of a T-DNA. Clones presenting right and left T-DNA/plant DNA junctions were used as probes to identify and isolate a unique 1.8-kilobase EcoRI fragment corresponding to the plant DNA target site for a T-DNA insertion event. Comparison of the nucleotide sequences of the plant DNA portions of the T-DNA junction clones with the original plant DNA target revealed that several types of rearrangements resulted from insertion of the T-DNA. The most dramatic alteration was a 158-base-pair direct repeat of target plant sequences at the left and right T-DNA junctions. In addition, there were deletion and insertion events at the ends of the right and left copies of the 158-base-pair repeat. The variety of target-site rearrangements suggests that T-DNA insertion is a multistep process of recombination accompanied by local replicative and repair activities mediated by host-cell enzymes.

Journal Article↗

PubMed2004

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II.

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.

Bacterial Proteins↗

PubMed1991

Frequency and organization of papA homologous DNA sequences among uropathogenic digalactoside-binding Escherichia coli strains.

The frequency of selected papA DNA sequences among 89 digalactoside-binding, uropathogenic Escherichia coli strains was evaluated with 12 different synthetic 15-base probes corresponding to papA genes from four digalactoside-binding piliated recombinant strains (HU849, 201B, and 200A). The papA probes encode amino acids which are common at the carboxy terminus of all strains, adjacent to the proximal portion of the intramolecular disulfide loop of strain 210B, or predicted to constitute the type-specific epitope for each of the four recombinant strains or other epitopes of strain HU849. The presence among the strains of DNA sequence homology to the papA probes was determined by in situ colony hybridization. Hybridization data suggest that there is a high frequency of homologous papA DNA sequences corresponding to selected regions of the papA gene from strain HU849 among the clinical strains. The following nucleotide locations which encode portions of the mature HU849 PapA are detected in a high percentage (42 to 70%) of clinical isolates: 208 to 222, 310 to 324, 478 to 492, 517 to 531, 553 to 567, and 679 to 693. These sequences encode portions of the predicted protective, immunogenic, and/or antigenic epitopes of this PapA. The data also indicate considerable heterogeneity of papA sequences among the strains, especially in the region of nucleotide bases corresponding to positions 391 to 418. These oligonucleotides encode the predicted PapA type-specific immunogenic dominant epitope. Determination of the extent of genetic variability in the papA gene among digalactoside-binding strains will require more extensive DNA sequencing of prototypic papA genes, additional hybridization studies employing other papA gene oligonucleotide probes, and assessment of the different pap operons and their copy number in each strain.

Amino Acid Sequence↗

PubMed1990

Probe mapping to facilitate transposon-based DNA sequencing.

A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. We demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon gamma delta (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal gamma delta segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine gamma delta insertion sites. The insertions were quite randomly distributed, even though the target DNA fragment had both A + T-rich and G + C-rich regions; in G + C-rich DNA, the insertions were found in A + T-rich "valleys." These data demonstrate that gamma delta is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

Animals↗