A powerful approach for generating and sequencing DNA deletions: sequencing from the outside in.
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Sequence Analysis, DNA↗
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Cloning of deleted sequences (CODE): A genomic subtraction method for enriching and cloning deleted sequences.
The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.
Base Sequence↗
Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome.
Characteristic long terminal repeats (LTR) of approximately 700 and 750 bp were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp gag-pol sequence with ACTGCCC repeat, a 113-bp mid-pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse species showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus, all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.
Animals↗
Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array.
The purity of a drug substance can influence its toxicity and potency, so impurities must be specifically determined. In the case of synthetic oligodeoxyribonucleotide drugs, however, product complexity makes complete impurity speciation difficult. The goal of the present work was to develop a new analytical method for speciation of individual internal (n-1)mer impurities arising from formal nucleotide deletion in synthetic oligodeoxyribonucleotides. A complete series of oligodeoxyribonucleotide probes were designed, each complementary to an (n-1)mer deletion sequence of the drug in question. Glass plates were used as a solid support for individually immobilizing the entire probe array. The total mixture of internal (n-1) length impurities was isolated from a synthetic oligodeoxyribonucleotide by PAGE and labeled with 35S. Under stringently optimized conditions, only the perfectly sequence-matched oligodeoxyribonucleotide hybridized to each probe, while all other deletion sequences were removed by washing with buffer. The 35S signal intensity of the bound oligodeoxyribonucleotide was proportional to the concentration of each (n-1)mer deletion sequence in the analyte solution. This method has been applied to a number of synthetic phosphorothioate oligodeoxy-ribonucleotide lots and shown to be reliable for speciation and relative quantitation of the internal (n -1)mer deletion sequences present.
Base Pair Mismatch↗
Enrichment of DNA fragments for deleted sequences in human genome with nuclease S1 digestion and PCR amplification.
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Base Sequence↗
Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development.
Nucleotide sequence and deletion analysis have been used to identify the regulatory and coding sequences comprising the cholera toxin operon (ctx). Incorporation of defined in vitro-generated ctx deletion mutations into Vibrio cholerae by in vivo genetic recombination produced strains which have practical value in cholera vaccine development.
Base Sequence↗
Isolation of DNA sequences deleted in lung cancer by genomic difference cloning.
To identify DNA sequences that are deleted in human lung cancer, genomic subtraction hybridization was used to construct plasmid libraries that are enriched for DNA sequences deleted in the small cell lung carcinoma cell line SK-LC-17. The clones of the libraries contained predominantly single copy sequences, allowing direct screening of normal and tumor DNA by genomic Southern blotting. Of 150 clones tested, three independent clones (del-27, del-118, and del-109) were identified that specifically hybridized with normal human DNA but not with tumor DNA from the cell line SK-LC-17. The corresponding DNA sequences are localized on human chromosomes 5, 8, and X/Y. The DNA regions identified by del-109 and del-118 were also found to be deleted in several other lung carcinoma cell lines. Moreover, del-118 was deleted in a freshly isolated lymph node metastasis of a human lung adenocarcinoma. It is therefore reasonable to speculate that the identified clones are derived from independent genetic loci encoding potential tumor suppressor genes.
Base Sequence↗
Defective forms of cotton leaf curl virus DNA-A that have different combinations of sequence deletion, duplication, inversion and rearrangement.
Tobacco and tomato plants inoculated at least 9 months previously with a Pakistani isolate of cotton leaf curl virus (CLCuV-PK), a whitefly-transmitted geminivirus, contained substantial amounts of circular dsDNA molecules that were mostly about half the size of CLCuV-PK dsDNA-A. They appeared to be derived from CLCuV-PK DNA-A by various combinations of sequence deletion, duplication, inversion and rearrangement and, in a few instances, insertion of sequences of unknown origin. Each of ten tobacco plants contained a different predominant form of such a defective molecule; however, all the forms contained the intergenic region and part of the AC1 (Rep) gene. Some of the forms contained novel open reading frames and might have a role in the evolution of variant geminiviruses. The defective components were not detected at 3 months after the original culture of CLCuV-PK was transmitted by whiteflies (Bemisia tabaci) from cotton to tomato but were present after a further 6 months. They were transmitted, along with full-length DNA-A, between tobacco and tomato plants by grafting and by B. tabaci.
Animals↗
The DNA of a plant retroviroid-like element is fused to different sites in the genome of a plant pararetrovirus and shows multiple forms with sequence deletions.
Carnation small viroid-like RNA (CarSV RNA) and its homologous DNA are the two forms of a unique plant retroviroid-like system. CarSV RNA is a 275-nucleotide noninfectious viroid-like RNA, present in certain carnation plants, which can adopt hammerhead structures in both polarity strands and self-cleave accordingly. CarSV DNA is organized as a series of head-to-tail multimers forming part of extrachromosomal elements in which CarSV DNA sequences are fused to sequences of carnation etched ring virus (CERV), a plant pararetrovirus. Analysis of more than 30 CarSV-CERV DNA junctions showed that distinct regions of the viral genome seem able to interact with CarSV DNA. All these junctions were short nucleotide stretches common to both CarSV and CERV DNAs. This suggests a polymerase-driven mechanism for their origin involving an enzyme with low processivity, most likely the viral reverse transcriptase. This view was further supported by the observation that most of CarSV sequences forming part of the junctions correspond either to strong secondary structure motifs in the conformation proposed for CarSV RNA or to its self-cleavage sites, which may have facilitated polymerase jumping. Accompanying the most-abundant CarSV RNA, a series of CarSV RNAs with sequence deletions were previously characterized. Here we have identified some of their corresponding DNA forms, together with other CarSV DNA forms with deletions not found in any CarSV RNA species identified so far. Some of these CarSV DNA forms have also been detected fused to CERV sequences. The existence of these shortened CarSV DNA versions may provide a continuous input of their corresponding transcripts and explain the persistence of CarSV RNAs with defective hammerhead structures for which an RNA-RNA model of amplification seems unlikely.
Base Sequence↗
Diverse function of aromatase and the N-terminal sequence deleted form.
The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).
Aromatase↗
Homology modeling three-dimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis.
AnxB1, a novel annexin previously isolated from Cysticercus cellulose, shows high thrombi affinity and anticoagulant activity in vivo. In order to investigate the relationship between structure and biological function, a predicted three-dimensional (3D) model of AnxB1 was generated by homology modeling. This model contains four homologous internal-domains and the Ca trace of domain I, II and IV shows high similarity. Based on the structure characterization, four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E. coli. Two of the mutants, GST-M3 and GST-M4 reserved high anticoagulant activity (p<0.01 vs. GST). Furthermore, compared with the wild type GST-AnxB1, the immunogenicity of GST-M3 and GST-M4 was reduced significantly (p<0.01) and the molecular weight was lowered to 27 kD and 34 kD, respectively. These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.
Annexins↗
Deletion sequences of salmon calcitonin that retain the essential biological and conformational features of the intact molecule.
Salmon calcitonin has an amino acid sequences that would allow it to form an amphipathic helix from approximately residue 9 to residue 22. We have synthesized a number of analogues of this peptide hormone with deletions in the carboxyl terminus of this putative amphipathic helix. These analogues include deletions of single amino acid residues at positions 19, 20, 21, or 22 as well as deletions of progressively larger segments starting with residue 19 and including deletions of residues 19 and 20; 19, 20, and 21; or 19, 20, 21, and 22. There is a small decrease in the helical content of these analogues compared with the native hormone, both in the presence and absence of amphiphiles. However, the extent of formation of secondary structure, as measured by circular dichroism, is similar for these deletion sequences as it is for the native hormone. In all cases, there is a large increase in the helical content of the peptide in the presence of dimyristoylphosphatidylglycerol, lysolecithin, or sodium dodecyl sulfate. All of the analogues have hypocalcemic activity in vivo in rats, comparable to the native hormone, except for des-Leu19-salmon calcitonin, which is about twice as active as the unmodified hormone. With use of an in vitro assay of adenylate cyclase activation in purified rat kidney membranes, des-Tyr22-salmon calcitonin, des-Leu19,Gln20,Thr21-salmon calcitonin, and des-Leu19Gln20,Thr21,Thr22-salmon calcitonin exhibited about one-tenth the stimulatory activity of the native hormone. Des-Tyr22-sCT and des-Leu19,Gln20,Thr21,Tyr22-sCT were also tested for their activity in inhibiting prolactin release from isolated rat pituitary cells. Both of these analogues exhibited inhibitory activity. Thus, the region of residues 19-22 does not greatly affect either the conformational or the biological properties of salmon calcitonin.
Adenylyl Cyclases↗
Post-integration stabilization of a transposon vector by terminal sequence deletion in Drosophila melanogaster.
Germline transformation systems for nearly 20 insect species have been derived from transposable elements, allowing the development of transgenic insects for basic and applied studies. These systems use a defective nonautonomous vector that results in stable vector integrations after the disappearance of transiently provided transposase helper plasmid, which is essential to maintain true breeding lines and consistent transgene expression that would otherwise be lost after vector remobilization. The risk of remobilization by an unintended transposase source has so far not been a concern for laboratory studies, but the prospective use of millions of transgenic insects in biocontrol programs will likely increase the risk, therefore making this a critical issue for the ecological safety of field release programs. Here we describe an efficient method that deletes a terminal repeat sequence of a transposon vector after genomic integration. This procedure prevents transposase-mediated remobilization of the other terminal sequence and associated genes, ensuring their genomic stability.
Animals↗
Complete reversions of a gypsy retrotransposon-induced cut locus mutation in Drosophila melanogaster involving jockey transposon insertions and flanking gypsy sequence deletions.
We have analysed the structures of three phenotypic revertant alleles of a gypsy retrotransposon-induced mutation at the cut locus of Drosophila melanogaster. All three revertants are associated with the insertion of jockey transposons into a common region of gypsy. Two of these alleles are complete reversions to wild type. One complete revertant (ct+D) is derived from a third allele, a partial revertant (ctMRpD) by a deletion of part of the gypsy sequence flanking the jockey transposon. Sequence differences between the jockey elements in ctMRpD and ct+D suggest that this deletion may have been created by the insertion of a second jockey near to the first, followed by recombinational excision of a composite jockey and the region between the two genetic elements. The other complete revertant also carries a deletion of gypsy DNA flanking the jockey insertion. The deleted regions of both complete revertants and the target region for all the jockey insertions contain a repeated sequence that resembles a transcriptional enhancer. The strength of the cut phenotype in these mutants correlates with the proportion of this region remaining near the gypsy transcriptional start site, suggesting that the jockey insertions relieve the gypsy-induced mutation at cut by interfering with a region which is required for the transcriptional competence of gypsy.
Alleles↗
Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements.
Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines.
Animals↗
Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted.
A mutant of herpes simplex virus type 1 (HSV-1) in which glycoprotein H (gH) coding sequences were deleted and replaced by the Escherichia coli lacZ gene under the control of the human cytomegalovirus IE-1 gene promoter was constructed. The mutant was propagated in Vero cells which contained multiple copies of the HSV-1 gH gene under the control of the HSV-1 gD promoter and which therefore provide gH in trans following HSV-1 infection. Phenotypically gH-negative virions were obtained by a single growth cycle in Vero cells. These virions were noninfectious, as judged by plaque assay and by expression of beta-galactosidase following high-multiplicity infection, but partial recovery of infectivity was achieved by using the fusogenic agent polyethylene glycol. Adsorption of gH-negative virions to cells blocked the adsorption of superinfecting wild-type virus, a result in contrast to that obtained with gD-negative virions (D. C. Johnson and M. W. Ligas, J. Virol. 62:4605-4612, 1988). The simplest conclusion is that gH is required for membrane fusion but not for receptor binding, a conclusion consistent with the conservation of gH in all herpesviruses.
Animals↗
Several new bacteriophage T4 genes, mapped by sequencing deletion endpoints between genes 56 (dCTPase) and dda (a DNA-dependent ATPase-helicase) modulate transcription.
We have analyzed DNA of wild-type T4 and of 13 independent large viable deletions isolated by Homyk and Weil (Virology 61 (1974) 505-523) and by Little (Virology 53 (1973) 47-59), by sequencing, cloning, and expression studies. The deletions can be explained by illegitimate recombination between short (4- to 15-bp) ectopic repeats. In four deletions, adjacent regions are partially homologous, and in at least one of them, the base adjacent to the overlap was deleted during recombination. The sequence 5'-GGGC, which has not been associated with T4 deletions in other map regions, occurs within three repeats, and near the repeats in four more of the 13 deletions. Five previously named genes, 69, soc, mrh, modA, and dda were mapped relative to the deletion endpoints. Nine additional ORFs were found interspersed between them. One of these shares some similarities with mrh (modulates rpoH; Frazier and Mosig, Gene 88 (1990) 7-14), and another one resembles modA (coding for an ADP-ribosyl-transferase that modifies RNA polymerase alpha subunits, Skórko et al., Eur. J. Biochem. 79 (1977) 55-66) respectively. We found that the host's heat shock sigma factor, sigma32, is phosphorylated, and that Mrh protein modulates this phosphorylation. The ORF dda.9 downstream of mrh has a patchy similarity with conserved C-terminal segments (motifs) of sigma32; therefore, we call it srh. Another ORF, dda.2 located between modA and dda, shares sequence similarity with sigma70, and we call it srd. We consider the possibility that Srh and Srd act as decoys for sigma32, or sigma70, respectively. Expression of several of the ORFs from cloned DNA appears to be toxic to the host bacteria. Mutant clones only could be constructed from gene 69 and from modA. Moreover, dda.2 (srd)-containing bacteria grow extremely slowly, and they form filaments in liquid cultures. Clones carrying mrh and srh show less severe filamentation. Our results highlight the importance of 'non-essential' genes for phage development and evolution.
Amino Acid Sequence↗
Isolation of a conserved sequence deleted in Duchenne muscular dystrophy patients.
We have isolated a DNA sequence (HIP25) by subtraction- hybridisation which is deleted in a number of Duchenne muscular dystrophy (DMD) patients. HIP25 is conserved in evolution and hybridises to human fetal and adult muscle mRNA. HIP25 is absent in human fetal fibroblast mRNA. Physical mapping data localise this sequence within Xp21 between the breakpoints of X;autosome translocations found in two females suffering from the disease. HIP25 is a candidate exon sequence for the basic defect in DMD boys deleted at this locus.
Adult↗