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PubMedDate not supplied

The discovery of gene amplification in mammalian cells: to be in the right place at the right time.

The constancy of the genome structure of an organism has been accepted dogma for a number of decades. The genetic variegation of maize as described by McClintock in the 1940s and subsequently shown to be mediated by transposable elements indicated a degree of genomic fluidity not appreciated previously. The discovery of gene amplification in somatic mammalian cells in 1977 has added a new component to the phenomenon of genomic fluidity, which has implications for various subdisciplines of biology.

Animals↗
PubMed1990

Processing of recombination intermediates in vitro.

Genetic recombination involves the exchange of genetic material between chromosomes to produce new assortments of alleles. As such, it affects one of the most fundamental and important components of heredity--the genome itself. To understand the molecular basis of recombination, efforts have been directed to try to determine how simple organisms recombine their DNA. One approach involves the development of in vitro systems in which recombination reactions can be studied using purified enzymes. Detailed studies of these systems, using enzymes isolated from bacteria and bacterial viruses, indicate the formation of unique protein-DNA complexes. The structure of the DNA within these complexes has important consequences for the subsequent formation of recombinant products.

Base Sequence↗
PubMed1985

Major polyadenylated transcripts of cassava latent virus and location of the gene encoding coat protein.

The nucleotide sequences of infectious cloned DNAs 1 and 2 of a Kenyan isolate of cassava latent virus (CLV) have been determined. Five virus-specific polyadenylated transcripts have been identified and mapped either to the viral or complementary sense DNAs of both components of the CLV genome, confirming that transcription is bidirectional on both DNAs. A major mRNA has been translated in vitro to yield a 30 000 mol. wt. product, which is precipitated by antibodies raised against whole virus, and has been mapped by both the S1 nuclease procedure and hybrid-arrested translation to the long open reading frame (ORF) in the viral sense of DNA 1 which encodes the coat protein. Other transcripts were of sufficient size and appropriate origin to encode at last five potential products.

Blotting, Northern↗
PubMed1988

Mitochondrial RNA polymerase of Saccharomyces cerevisiae: composition and mechanism of promoter recognition.

Mitochondrial RNA polymerase of Saccharomyces cerevisiae consists of two different proteins: a core RNA polymerase of 145 kd and a specificity factor of 43 kd, which contributes the capacity to recognize promoters of the various genes encoded in the mitochondrial genome. We purified both components by SDS-PAGE, followed by renaturation to the active state. The two components were used either singly or in combination to study their interactions with promoter-containing DNA fragments. The core component showed random and weak interaction with DNA, the specificity factor none at all, whereas both components together specifically bound to a promoter. In DNase I footprinting experiments, promoter-bound RNA polymerase protected a short region of DNA flanked by hypersensitivity sites and centred around the position at which RNA synthesis starts. The initial phase of transcription gave rise to specific changes in this footprint: the upstream border remained at the same position up to synthesis of a 4-nt RNA chain, whereas at the downstream border progressive disappearance of hypersensitivity sites took place.

Base Sequence↗
PubMed1994

Nuclear matrix and the regulation of gene expression: tissue specificity.

Tissue specific regulation of gene expression by a single transcription factor or group of transcription factors cannot be explained simply by DNA sequence alone. For example, in the same animal a particular transcription factor is capable of interacting with DNA in the nucleus of many different cell types, resulting in unique gene expressions despite the presence of a similar genome in all cells. Historically, these differences in response to a single type of factor within target tissues in the same animal have been suggested to occur through different alterations in chromatin structure. Recent, data has demonstrated that combinations of hormones and transcription factors working together may cooperatively play a role in the regulation of gene expression [Pearce and Yamamoto (1993): Science 259:1161-1165]. However, the molecular mechanisms of this tissue specific regulation of gene expression still remains largely unexplained. Current evidence suggests that in different cell types the interplay between the specific three-dimensional organization of the genome and the structural components of the nucleus, the nuclear matrix, may accomplish the regulation of specific gene expression.

Animals↗
PubMedDate not supplied

Controlled transgene expression by E1-E4-defective adenovirus vectors harbouring a "tet-on" switch system.

BACKGROUND: The "tet switch system" was originally described under the tet-off configuration with its components encoded by two separate plasmids. Since then, many virus vectors harbouring tet-off components have been designed and their regulation by tetracycline is widely reported. On the contrary, tet-on regulation by viral vectors is poorly documented. METHODS: E1-E4-defective adenoviruses harbouring either rtTA or the luciferase gene under a minimal inducible promoter (TK* or CMV*) or both components in a single genome were produced. Using either a double or a single virus strategy, induction of luciferase expression was investigated in various cell lines, in mice muscle and in rat brain. RESULTS: Over 400-fold induction can be reached with PC12 and NHA cells using a double virus strategy. Comparison of the background activity of different minimal inducible promoters revealed a significant difference between TK* and CMV* promoters both with the cell culture and the in vivo experiments. Interestingly, a single virus strategy permitted an induction exceeding 600-fold with human astrocyte primary cells. Moreover, the E1-E4-defective adenovirus-mediated tet-on system can be quickly switched off and turned back on again. Depending on the cell line, the level of rtTA derived by the single virus strategy differed, resulting in different efficiencies. Experiments performed in rat striatum and mouse muscle confirmed the importance of rtTA expression and minimal promoter used on both doxycycline-independent expression and induction efficiency. Under appropriated rtTA expression, a 32-fold induction is observed in mouse muscle. CONCLUSIONS: In the recombinant adenovirus context, the CMV* but not the TK* promoter is sensitive to transcriptional interference resulting in high doxycycline-independent expression. By paying attention to the rtTA expression, moderate and high induction can be obtained in vivo and in vitro accordingly.

Adenoviridae↗
PubMed1998

Polyoma middle T antigen activates the Ser/Thr kinase Akt in a PI3-kinase-dependent manner.

Polyoma middle T antigen (PMT) was originally identified as the tumorigenic component of the polyomavirus genome. To investigate whether the serine/ threonine kinase Akt/PKB, which is the proto-oncogene transduced by the transforming AKT8 retrovirus, is activated by PMT, 3T3-L1 fibroblasts were stably transfected with wild type PMT. PMT expression accelerated glucose transport and increased phosphorylation of p70 S6-kinase and MAPK. PMT expression also stimulated Akt kinase activity 7 fold as compared to untreated, mock infected cells. This stimulation rivaled that obtained following insulin treatment of both mock and PMT infected cells. Akt activation and phosphorylation were eliminated in a PMT mutant incapable of interacting with PI3-kinase, but not one which does not interact with Shc, and correlated closely to the amount of PI3-kinase activity in anti-phosphotyrosine immunoprecipitates. These results indicate that the PI3-kinase pathway is requisite, but the Shc pathway is dispensable, for Akt activation. The studies further suggest that Akt may participate in PMT and PI3-kinase's regulation of cellular transformation and tumorigenesis.

3T3 Cells↗
PubMed1993

Notes on the definition and nomenclature of tandemly repetitive DNA sequences.

Tandemly repetitive DNA is a major component of all eukaryotic genomes. This fact has been known for almost 30 years and research on this class of DNA is still being done. Its biology and evolution are therefore now becoming fairly well understood. DNA-fingerprint techniques rely very much on this knowledge. However, the large amount of research on these sequences has inevitably led to a large number of different concepts and theories about their nature. This has also resulted in some confusion as to the nomenclature. The following notes are intended to resolve this confusion somewhat and to give some definitions for the major classes of tandemly repetitive DNA.

Animals↗
PubMed1992

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts.

The effects of methylation on plant viral DNA replication have been studied in Nicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residues in vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequences in vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.

Blotting, Southern↗
PubMed1994

Partial sequencing and mapping of clones from two maize cDNA libraries.

As one component of a maize genome project, we report the analysis of a number of randomly selected cDNAs, by a combination of measuring mRNA expression, 'single-pass' sequencing (SPS), and genome mapping. Etiolated seedling (490) and membrane-free polysomal endosperm cDNA clones (576) were evaluated for their transcription levels by hybridizing with a probe prepared from total mRNA and categorized as corresponding to abundantly or rarely expressed mRNAs and as either constitutive or tissue-specific. A total 313 clones from the two libraries were submitted to 'single-pass' sequencing from the presumed 5' end of the mRNA and the nucleotide sequence compared with the GenBank database. About 61% of the clones showed no significant similarities within GenBank, 14% of the clones exhibited a high degree of similarity, while the remaining 25% exhibited a lesser degree of similarity. The chromosomal location of more than 300 clones was determined by RFLP mapping using standard populations. The results demonstrate that a combination of analyses provides synergistic information in eventually deducing the actual function of these types of clones.

Chromosomes↗
PubMed1995

Inheritance of chromosome-length polymorphisms in Ophiostoma ulmi (sensu lato).

We have investigated the mitotic and meiotic transmission of chromosome-length polymorphisms in Ophiostoma ulmi s.l., the causal agent of Dutch elm disease. The North-American aggressive (NAN) strain CESS16K has an atypical electrophoretic karyotype, carrying two chromosome-sized DNAs (chDNAs) that have not been observed in other members of the NAN biotype. Independent CESS16K chDNA preparations, even after repeated inoculation and recovery from the elm host, and analysis of 16 progeny strains after a cross between the NAN strains FG245Br-O and CESS16K, demonstrated that these unique chDNAs are integral components of the CESS16K genome. Analysis of the progeny, by electrophoretic karyotyping and hybridizations with probes specific to individual chDNAs, presented evidence that genome rearrangements can occur as a consequence of meiosis. Even though novel electrophoretic karyotypes and a novel-sized chromosome were observed in the karyotypes of the progeny strains, the low level of reassortment between the chromosomes carrying length polymorphisms presented evidence that there are constraints to genome plasticity for this fungus.

Ascomycota↗
PubMed1986

DNA synthesis in purified maize mitochondria.

We have developed an in organelle DNA synthesis system using isolated mitochondria from maize. The organelles used in this assay are shown to be intact by a number of criteria. Both the high molecular weight components and the smaller plasmid-like components of the mitochondrial genome are used as templates; however, the plasmid-like elements are relatively more active as templates. The termini of the linear plasmids--S-1, S-2 and the 2.3 kbp plasmids--are more highly labelled than internal regions, probably as a result of filling in of gaps at the termini mediated by a DNA polymerase or to exonuclease degradation of the 3' OH termini, with subsequent filling in. Although most of the DNA synthesis observed in this system most likely results from this type of synthesis at DNA nicks or termini, a small amount of specific, potentially replication-associated, synthesis is also detected.

Cell-Free System↗
PubMed1983

Evolution of Drosophila repetitive-dispersed DNA.

We have examined the phylogenetic distribution of a spectrum of Drosophila repetitive-dispersed DNAs ranging from structurally complex transposable elements to scrambled middle repetitive sequences. Our data suggest that unlike typical "genes" these DNAs are unstable components of the drosophilid genome. The unusual behavior of these repetitive-dispersed DNAs raises the possibility that this type of sequence may have an important role in the evolution of the family Drosophilidae.

Animals↗
PubMed1989

Mu transposable elements are structurally diverse and distributed throughout the genus Zea.

The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of the Mu family of transposable elements. All characterized Mu elements contain similar approximately 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20-100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to the Mu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously cloned Mu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termed Mu4 and Mu5, have an organization characteristic of transposable elements and possess approximately 200-bp Mu terminal inverted repeats that flank internal DNA, which is unrelated to other cloned Mu elements. Mu4 and Mu5 are both flanked by 9-bp direct repeats as has been observed for other Mu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions of Mu4 and Mu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally from Mu-similar termini. The distribution of these elements in maize lines and related species suggests that Mu elements are an ancient component of the maize genome. Moreover, the structure of the Mu termini and the fact that Mu termini are found flanking different internal sequences leads us to speculate that Mu termini once may have been capable of transposing as independent entities.

Base Sequence↗
PubMed1999

Evolution of tandemly repeated sequences: What happens at the end of an array?

Tandemly repeated sequences are a major component of the eukaryotic genome. Although the general characteristics of tandem repeats have been well documented, the processes involved in their origin and maintenance remain unknown. In this study, a region on the paternal sex ratio (PSR) chromosome was analyzed to investigate the mechanisms of tandem repeat evolution. The region contains a junction between a tandem array of PSR2 repeats and a copy of the retrotransposon NATE, with other dispersed repeats (putative mobile elements) on the other side of the element. Little similarity was detected between the sequence of PSR2 and the region of NATE flanking the array, indicating that the PSR2 repeat did not originate from the underlying NATE sequence. However, a short region of sequence similarity (11/15 bp) and an inverted region of sequence identity (8 bp) are present on either side of the junction. These short sequences may have facilitated nonhomologous recombination between NATE and PSR2, resulting in the formation of the junction. Adjacent to the junction, the three most terminal repeats in the PSR2 array exhibited a higher sequence divergence relative to internal repeats, which is consistent with a theoretical prediction of the unequal exchange model for tandem repeat evolution. Other NATE insertion sites were characterized which show proximity to both tandem repeats and complex DNAs containing additional dispersed repeats. An "accretion model" is proposed to account for this association by the accumulation of mobile elements at the ends of tandem arrays and into "islands" within arrays. Mobile elements inserting into arrays will tend to migrate into islands and to array ends, due to the turnover in the number of intervening repeats.

Base Sequence↗
PubMed2001

Isolation and characterization of repetitive DNA sequences from Panax ginseng.

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization and evolution of the genome of interest. We report the isolation and characterization of the major classes of repetitive sequences from the genome of Panax ginseng. The isolation of repetitive DNA from P. ginseng was achieved by the reannealing of chemically hydrolyzed (200 bp-1 kb fragments) and heat-denatured genomic DNA to low C(o)t value. The low C(o)t fraction was cloned, and fifty-five P. ginseng clones were identified that contained repetitive sequences. Sequence analysis revealed that the fraction includes repetitive telomeric sequences, species-specific satellite sequences, chloroplast DNA fragments and sequences that are homologous to retrotransposons. Two of the retrotransposon-like sequences are homologous to Ty1/ copia-type retroelements of Zea mays, and six cloned sequences are homologous to various regions of the del retrotransposon of Lilium henryi. The del retrotransposon-like sequences and several novel repetitive DNA sequences from P. ginseng were used to differentiate P. ginseng from P. quinquefolius, and should be useful for evolutionary studies of these disjunct species.

Cloning, Molecular↗
PubMed2006

Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata).

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated.

Base Sequence↗