Deletion Of DNA Sequences Research Articles

SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION

We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.

Oncogenes and tumor-suppressor genes.

The functional role of oncogenes in human lung carcinogenesis has been investigated by transfer of activated oncogenes into normal cells or an immortalized bronchial epithelial cell line, BEAS-2B. Transfection of v-Ha-ras, Ki-ras, or the combination of myc and raf into BEAS-2B cells produced tumorigenic cell lines, while transfection of raf or myc alone produced nontumorigenic cell lines. In addition to studying the pathogenic role of oncogenes, we are attempting to define negative growth-regulating genes that have tumor-suppressive effects for human lung carcinomas. Our strategy to identify tumor-suppressor genes involves loss of heterozygosity studies, monochromosome-cell fusion, and cell-cell fusion studies. Loss of heterozygosity studies have revealed consistent allelic DNA sequence deletions on chromosome 17p in squamous cell carcinomas, while large cell carcinomas and adenocarcinomas retained this locus. Mutations in p53, a tumor-suppressor gene located on chromosome 17p, have been observed. Cell-cell hybrid clones produced from fusion of nontumorigenic BEAS-2B cells with tumorigenic HuT292DM cells generally are nontumorigenic. The mechanistic role of the known tumor-suppressor genes Rb-1 and p53 in the development of human lung carcinomas is being investigated in this epithelial cell model of human bronchogenic carcinogenesis.

A chromosomal hotspot for multiple rearrangements associated with genetic instability of Streptomyces ambofaciens DSM 40697

Genetic instability in Strepfomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.

Multi‐residue gaps, a class of molecular characters with exceptional reliability for phylogenetic analyses

AbstractQualitative methods of obtaining phylogenies seek distinct classes of characters that collectively are common, yet individually are insufficiently improbable that they provide reliable indications of monophyly. Multi‐residue gaps (insertions or deletions) in DNA and protein sequences are easily recognised and are available in diverse organisms and loci. They are likely to experience few homoplasies, because these require two separate events to be matched in both starting position and length. Two tests of the ability of multi‐residue gaps to recognise clades are conducted. (1) Among the 10.76 kb of noncoding DNA sequences from the ψη‐globin region of 5 primates, there are 7 analysable multi‐nucleotide gaps. These provide a corroborated and noncontradictory, fully resolved gene tree of hominoids. (2) Among the 35 amino acid sequences of globin chains available in 1978, there are 9 nonoverlapping multi‐amino acid gaps. These indicate 8 noncontradictory divisions that agree with recognised taxa and gene categories. In the 412 realigned globin sequences available in 1989, 8 of the gaps are still analysable. These show a maximum of two homoplasies among the 8 × 412 = 3296 species‐characters. Multi‐residue gaps have a high potential to indicate clades. They could be used to reduce the computing labour required in a quantitative study or pooled from different data sets to examine particularly important or difficult phylogenetic questions.

Variable regions on the genome of Malawi isolates of African swine fever virus

Restriction enzyme site mapping showed that most BamHI and all ClaI sites were conserved on the genomes of 17 African swine fever virus isolates from separate disease outbreaks that occurred between 1982 and 1989 in Malawi. However, frequent variation between virus genomes did occur due to addition or deletion of DNA sequences at various positions along the genome and 11 virus genotypes could thus be distinguished among the 17 isolates analysed. Length variations occurred at 10 different loci on the virus genome. These variable regions were located between the left DNA terminus and a position up to 48 kb from that terminus, in the centre of the genome 90 to 93 kb from the left DNA terminus and between the right DNA terminus and a position 22 kb from that terminus. Length variations in most of these regions were small (less than 1 kb) but variations of about 4 kb occurred in a region up to 20 kb from the left DNA terminus.

Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers

A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required. The efficiency of heteroduplex formation between mutagenic primers directing large deletions and singlestranded template is discussed.

Dynamic changes of DNA sequences in Schistosoma mansoni in the course of development

Deletion and/or amplification of DNA sequences in Schistosoma mansoni were demonstrated by Southern blot analysis. Total cellular DNAs and genomic clones derived from S. mansoni miracidia, adult males and females were used as probes. Endonuclease BamHI-restricted DNAs from miracidia, adult males and females of both S. mansoni and S. japonicum were reacted to each probe. Hybridization with a total cellular DNA from S. mansoni miracidia as a probe showed elimination of signals in S. mansoni adults. On the other hand, blot analysis using a total cellular DNA from S. mansoni adult males as a probe revealed elimination of hybridization signals in S. mansoni miracidia. Hybridization with a clone SmE15 DNA from S. mansoni miracidia as a probe showed no signal in the DNAs from S. mansoni adults, indicating these sequences deleted in adults. Hybridization experiments using the probes SmF25 and SmM51 which are 1.3 and 2.2 kb fragments cloned from S. mansoni adult females and males respectively, demonstrated no signal to DNA from S. mansoni miracidia. Our data suggested the existence of stage-specific DNA sequences in S. mansoni. We propose a model for multiple-step rearrangement of DNA sequences in S. mansoni during the course of development.

Two 46,XX,t(X;Y) females with linear skin defects and congenital microphthalmia: a new syndrome at Xp22.3.

We describe two females with de novo X;Y translocations, who presented at birth with irregular linear areas of erythematous skin hypoplasia involving the head and neck, along with eye findings...

Differential DNA sequence deletions from chromosomes 3, 11, 13, and 17 in squamous-cell carcinoma, large-cell carcinoma, and adenocarcinoma of the human lung.

Activation of protooncogenes and inactivation of putative tumor suppressor genes are genetic lesions considered to be important in lung carcinogenesis. Fifty-four cases of non-small-cell lung cancer (23 adenocarcinomas, 23 squamous-cell carcinomas, and 8 large-cell carcinomas) were examined for loss of DNA sequences at 13 polymorphic genetic loci. Loss of heterozygosity was seen more frequently in squamous-cell carcinoma than in adenocarcinoma. The loss of DNA sequences from the short arm of chromosome 17 (D17S1 locus) was detected in 8 of 9 heterozygous cases of squamous-cell carcinoma and in only 2 of 11 heterozygous cases of adenocarcinomas. Furthermore, in 7 of these 8 squamous-cell carcinomas, loss of heterozygosity from chromosome 17 was accompanied by loss of DNA sequences from chromosome 11. The spectrum of allelic sequences lost from chromosome 11 was, however, similar in every type of carcinoma studied, and the data show two regions commonly deleted from chromosome 11 (11pter-p15.5 and 11p13-q13) that may have a role in the pathogenesis of all these types of non-small-cell bronchogenic carcinoma. Loss of DNA sequences from chromosome 3 was seen in 16 of 31 cases where the constitutive DNA was heterozygous-i.e., informative. These data included only 6 of 16 cases where loss of heterozygosity involved a chromosomal locus previously shown to be lost consistently in small-cell lung cancer (DNF15S2). Loss of heterozygosity at the chromosome 13q locus, D13S3, was seen in 9 of 21 informative cases, and in 2 cases, both adenocarcinomas, duplication of the intact DNA sequences suggested the possibility that mitotic recombination had occurred. Frequent DNA sequence deletions, including those from chromosome 17, in squamous-cell carcinomas may reflect the extensive mutagenic and clastogenic effects of tobacco smoke that may lead to inactivation of putative tumor-suppressor genes.

Summary of the workshop on DNA methylation

Summary of the workshop on DNA methylation

The transcriptional control regions of thecopiaretrotransposon

We have analysed the sequence elements that control expression of the copia retrotransposon. Expression of copia fusion constructs containing DNA sequence deletions and rearrangements was assayed by transient expression analysis. Progressive deletion and linker substitution identifies two regions on either side of the major transcriptional start sites in the copia long terminal repeat These regions are both required for high level expression in a cultured Drosophila melanogaster cell line but only the upstream region is required for copia expression in a Drosophila hydei cell line. A third control region lies downstream of the long terminal repeat in a region previously believed to contain no cis-acting regulatory sequences. We show by displacement and inversion of this region that it contains a transcriptional enhancer.

An important step in radiation carcinogenesis may be inactivation of cellular genes

The loss of genetic material may result in a predisposition to malignant disease. The best studied example is retinoblastoma where deletion or transcriptional inactivation of a specific gene is associated with the development of the tumor. When hereditary retinoblastoma patients are treated with radiation, the incidence of osteosarcoma within the treatment field is extremely high compared to other cancer patients treated with radiotherapy. These data, together with cytogenetic and molecular data on the development of acute non-lymphocytic leukemia secondary to radiotherapy and chemotherapy treatment suggest that radiation-induced deletions of critical DNA sequences may be an important event in radiation carcinogenesis. Therefore, we propose that radiation-induced tumors may result from deletion of tissue specific regulatory genes. Base alterations caused by radiation in dominantly transforming oncogenes may also contribute to radiation carcinogenesis.

Plasmodium falciparum: Comparison of the genomic organization of the knob protein gene in knobby and knobless variants

The genomic organization of the knob protein (KP) gene of knobby (K +) and knobless (K −) variants of the Thai isolate NT 108 and the Gambian isolate FCR-3 are compared. The restriction enzyme maps and the chromosomal location of the KP gene of K + variants of both isolates are apparently identical. A comparison of the susceptibility of the KP gene to deletions and the extent of deletion of chromosomal DNA in K − variants of each geographical isolate suggests isolate-specificity of the stability of the deleted DNA sequences. With the exception of a mutant K − population of FCR-3, which could not be distinguished from K + FCR-3, in all other K − variants of both geographical isolates the deletion of the KP gene was accompanied by the loss of several hundred base pairs of DNA from chromosome 2. The deletion resulted in the localization of the mutant KP gene in proximity to telomeric DNA sequences.

Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C.F. Austerberry, C.D. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.

DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis.

The replication terminus, terC, of Bacillus subtilis is the chromosomal site at which movement of the clockwise replication fork is blocked. The effect of deletion or modification of DNA sequences on either side of terC (defined by the sequence location of the arrested clockwise fork junction) has been investigated. Deletion of sequences ahead of terC to within 250 base pairs (bp) had no effect on fork arrest, whereas removal of a further 130 bp abolished it. The 250-bp segment immediately ahead of terC encompassed the previously identified inverted repeat region as well as potential promoters for the transcription of an adjoining open reading frame (ORF). Deletion of DNA from the other side of terC up to 80 bp from it also abolished fork arrest. This deletion removed the bulk of the ORF. Disruption of this ORF by the insertion of 4 bp also abolished fork arrest. A model for clockwise fork arrest at terC, implicating both the inverted repeat region and the protein product of the ORF, is proposed.

Sequence Structures of Two Developmentally Regulated, Alternative DNA Deletion Junctions in Tetrahymena thermophila

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C.F. Austerberry, C.D. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.

Molecular and cytogenetic evidence for the location of Tdy and Hya on the mouse Y chromosome short arm.

Using a combination of in situ mapping and DNA analysis with recombinant DNA probes specific for the Sxr region of the mouse Y chromosome, we show that both the gene(s) controlling sex determination and the expression of the male-specific antigen H-Y (Tdy and Hya, respectively) are located on the minute short arm of the mouse Y chromosome. We demonstrate that the H-Y- variant of Sxr (Sxr') arose by a partial deletion within the Sxr region. Also, we show that intrachromosomal recombination between the Y short arm and Sxr' can sometimes occur during male meiosis, restoring the deleted DNA sequences and resulting in an H-Y+ mouse (male 719 in this paper). Based on these results, we propose a model for the generation of the original Sxr region and the Sxr' and Sxr719 variants.

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants.

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.

Deletion of DNA sequences flanking an Mr 19,000 zein gene reduces its transcriptional activity in heterologous plant tissues.

Analysis of a series of clones containing deletions in the 5' noncoding sequence of a gene encoding an Mr 19,000 zein allowed identification of a region required for maximal transcription. Transcriptional activity was assayed in two heterologous plant systems. In one system, the Ti plasmid was used to introduce the modified zein genes into the sunflower genome. In the other system, electroporation was used to transform carrot protoplasts with plasmids containing the zein genes. For the electroporation experiments, the 5' noncoding sequences from the zein clones were linked to the protein coding sequence of chloramphenicol acetyl transferase. The results showed that an upstream sequence, delimited by nucleotides -337 and -125 with respect to the mRNA cap site, is required for maximal transcription of the gene. In contrast, very low levels of transcription were directed by constructs that contained 125 bp of 5' noncoding sequence that included the CAAT and TATA boxes, suggesting that the additional sequences (-337 to -125) further 5' exert a quantitative effect on transcription. Examination of the additional 5' sequences showed five regions that share homology with the SV40 enhancer core sequence.

Comparisons of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra baculovirus mutants

Few polyhedra (FP) mutants of Autographa californica nuclear polyhedrosis virus (AcNPV) and the closely related strain Galleria mellonella (Gm) NPV have been reported which contain Trichoplusia ni host cell DNA sequences inserted into the viral genome between map units 35.0 and 37.7. New FP mutants are described with alterations of the HindIII-I restriction enzyme fragment (33.8 to 37.7 map units) of AcNPV, either deletions of viral DNA sequences or insertions of Spodoptera frugiperda host cell DNA sequences. S. frugiperda DNA insertions from FP mutants were compared to T. ni host DNA insertions from FP mutants previously isolated ( M. J. Fraser, G. E. Smith, and M. D. Summers (1983) J. Virol. 47, 287–300). Two host cell DNA sequences isolated from FP mutants, one of T. ni origin and one from S. frugiperda DNA, were transcribed in infected cells. Deletions of viral DNA sequences and insertions of host DNA sequences produce altered transcripts at the site of mutation as determined by both Northern and S1 nuclease analysis. Cell-free translation of cRNAs transcribed from wild-type viral DNA revealed an open reading frame coding for a 25-kDa protein at the site where host cell DNA insertions have been mapped. This was the same size as an infected-cell protein missing from most FP mutants examined.