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PubMed1998

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

Blotting, Southern↗
PubMed1991

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.

Base Sequence↗
PubMed2001

Structural and phylogenetic analysis of TRAS, telomeric repeat-specific non-LTR retrotransposon families in Lepidopteran insects.

TRAS1 is a non-LTR retrotransposon inserted specifically into the telomeric repeat (TTAGG)(n) in the silkworm, Bombyx mori. To characterize the evolutionary origin of TRAS-like elements, we identified seven TRAS families (TRAS3, TRAS4, TRAS5, TRAS6, TRASY, TRASZ, and TRASW) from B. mori and four elements from two Lepidoptera, Dictyoploca japonica (TRASDJ) and Samia cynthia ricini (TRASSC3, TRASSC4, and TRASSC9). More than 2,000 copies of various Bombyx TRAS elements accumulated within (TTAGG)(n) sequences as unusual but orderly tandem repeats. The 5' and 3' regions were highly conserved within each class of Bombyx TRAS elements without truncation. This suggests that distinct classes of TRAS have been maintained independently by retrotransposition into (TTAGG)(n). The phylogenetic tree of site-specific retroelements showed that nine TRAS families in Lepidoptera constitute a single phylogenetic group that is closely related to the R1 family that inserts specifically into arthropod 28S rDNA. The higher amino acid sequence identity from endonuclease (EN) to reverse transcriptase (RT) domains between TRAS groups (about 37%-70%) than among TRAS elements and R1Bm (about 25%-30%), may reflect the presence of some DNA structure responsible for their target specificity. Sequence comparison from EN to RT domains among non-LTR elements revealed several regions conserved only within TRAS elements. We found a highly conserved region that resembles the Myb-like DNA-binding structure, between the EN and RT domains. These regions may be involved in site-specific integration of TRAS elements into the (TTAGG)(n) telomeric repeats.

Amino Acid Sequence↗
PubMed1995

RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element.

R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects. The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities. Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3' hydroxyl group to prime the reverse transcription of the R2 RNA transcript. We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3' untranslated region of the R2 transcript were necessary and sufficient for the reaction. To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3' end of the RNA were tested. The R2 templates used most efficiently had 3' ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo. Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently. R2 RNAs that were truncated at their 3' ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA. The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3' end of most non-long terminal repeat-retrotransposable elements.

Animals↗
PubMed1996

Basal and vitamin D-responsive activity of the rat osteocalcin promoter in stably transfected osteosarcoma cells: requirement of upstream sequences for control by the proximal regulatory domain.

Osteocalcin (OC) is a bone-specific vitamin D- responsive protein that is developmentally expressed during osteoblast differentiation. In transient transfection assays, as little as approximately 0.1 kilobase (kb) of the OC proximal promoter is sufficient for basal expression. Because eukaryotic genes are packaged as nucleosomes that contribute to both chromatin organization and transcriptional control, we functionally examined the activity of OC promoter constructs within a chromatin context. ROS 17/2.8 osteosarcoma cells were stably transfected with a series of rat OC promoter-reporter constructs, containing progressive 5'-deletions. The results demonstrate that in contrast to transient transfection assays, the proximal 0.11-kb promoter is no longer active when integrated in the genome. Progressive gain of basal expression with 0.35-, 0.53-, and 0.72-kb promoters suggests that upstream sequences facilitate the formation of an appropriate higher order nuclear structure, thereby potentiating the activity of the chromosomally integrated proximal promoter elements. This is consistent with location of both deoxyribonuclease I (DNase I)-hypersensitive sites and nuclear matrix protein-DNA interaction sites in the osteocalcin promoter. Vitamin D responsiveness in the stably transfected cells is obtained with the inclusion of 0.53 kb or additional upstream promoter sequences. Therefore, these sequences satisfy the requirements for binding of basal and enhancer transcription factors as well as interactions between them within a chromatin context. Both maximal basal expression and maximal vitamin D responsiveness are obtained with cells carrying either the 0.72-kb or the 1.1-kb promoter fragment. Cells carrying the 1.1-kb promoter show DNase I hypersensitivity at both the basal promoter and the vitamin D response element-containing domains, locations that also exhibit DNase I hypersensitivity in the endogenous OC promoter. In addition, we have documented changes in the basal activity and vitamin D responsiveness of the stably integrated 1.1 kb promoter as a function of cell density-mediated growth inhibition, which is accompanied by up-regulation of bone phenotypic genes. Thus, important aspects of OC gene transcriptional regulation that cannot be investigated in transient transfection assays can be addressed using ROS 17/2.8 cells stably transfected with OC promoter-reporter constructs.

Animals↗
PubMed1999

Floricultural traits and transposable elements in the Japanese and common morning glories.

The Japanese morning glory has an extensive history of genetic studies and over 200 different spontaneous mutant lines have been described. Of these, we identified that two mutable alleles, flecked and speckled, for flower variegations are caused by integration of transposable elements, belonging to the En/Spm family, into the DFR-B and CHI genes for flower pigmentation, respectively. The mutable flaked allele of the common morning glory bearing variegated flowers is caused by insertion of a new transposable element, Tip100, into one of the CHS genes for pigmentation and that Tip100 belongs to the Ac/Ds family. These results are discussed with regard to spontaneous transposon mutagenesis and generation of floricultural traits of morning glories.

Alleles↗
PubMed2005

Pulsed field gel electrophoresis to rapidly detect the presence of IncJ conjugative transposon-like elements.

AIMS: To develop a screening method to detect the presence of the IncJ group of integrating conjugative transposon-like elements upon transfer to Escherichia coli. METHODS AND RESULTS: The unique insertion site of known IncJ elements, the prfC gene, is located in a region of the E. coli chromosome between 98.5 and 100 min on the E. coli genetic map. Using pulsed field gel electrophoresis and the rare cutting restriction enzymes SfiI and XbaI insertions of IncJ elements and an estimate of their size could be determined physically. CONCLUSIONS: This method allows initial screening of putative IncJ conjugative transposon-like elements by physical determination of their integration. SIGNIFICANCE AND IMPACT OF THE STUDY: IncJ-like elements, which appear to be highly homologous to the prototype IncJ element R391, have been found associated with recent epidemic outbreaks of cholera in a number of locations worldwide. Because of their integrative biology this method provides the first initial screening method to physically determine their presence upon transfer to E. coli.

Conjugation, Genetic↗
PubMed1985

Extrachromosomal DNA forms of copia-like transposable elements, F elements and middle repetitive DNA sequences in Drosophila melanogaster. Variation in cultured cells and embryos.

Drosophila melanogaster embryos and cells in culture were screened for the presence of unintegrated covalently closed circular DNA forms that hybridize to copia-like transposable elements, the F element and uncharacterized dispersed middle repetitive DNA elements. Our results indicate that the majority of copia-like elements (including copia, 297, 412, mdg1, mdg3 and gypsy), the F elements, and 9 of 12 middle repetitive DNA elements are present as free DNA forms in cultured cells and embryos. An 18 base-pair inverted repeat has been reported to flank the long direct repeat of mdg3, implying that mdg3 is not an orthodox copia-like element; however, we have sequenced two independently isolated mdg3 clones and shown that the inverted repeat is not part of the element. The relative abundance with which free DNA forms are found varies between the cultured cells used, and between cultured cells and embryos. This variation, which can be up to 20-fold for some elements, does not correlate well with either the amount of element-specific poly(A)+ RNA present per cell or the number of element-specific sequences integrated in the genome.

Animals↗
PubMed2000

Repair of gaps in retroviral DNA integration intermediates.

Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.

Base Sequence↗
PubMed2000

Sperm-mediated transformation of the honey bee, Apis mellifera.

Our primary objective was to identify techniques to transform the genome of the honey bee (Apis mellifera) with foreign DNA constructs. The strategy we adopted was to linearize foreign DNA and introduce it with sperm during the instrumental insemination of virgin queen honey bees. We analysed extracts from larvae within the same cohort and isolated the predicted fragment by means of PCR amplification of genomic DNA. Larvae that carried the construct also expressed the introduced DNA. We propagated several transgenic lines for up to three generations, which demonstrates its heritability. Once carried by a queen, the construct can be detected in that queen's larvae over several months. However, there was no evidence of integration of the construct, at least as determined by genomic Southern analysis. Nevertheless, this demonstrates the general viability of the technique for introduction of DNA, and it should be augmented by further use of transposable elements that enhance integration.

Animals↗
PubMed1991

[Development of integrative vectors for Actinomycetes--producers of antibiotics].

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.

Anti-Bacterial Agents↗
PubMed1995

A transgenic neuroanatomical marker identifies cranial neural crest deficiencies associated with the Pax3 mutant Splotch.

The murine Pax3 gene encodes a transcription factor containing a paired domain as well as a paired-type homeodomain. Its expression during embryonic development is temporally and spatially restricted, including mainly the dorsal part of the neural tube, the mesencephalon, the neural crest derivatives, and the dermomyotome. Development in the absence of Pax3 can be studied in Splotch mutant mice, which bear mutations within the Pax3 gene. Various alleles have been phenotypically and molecularly characterized. Abnormalities have been observed in the brain, the neural tube, the trunk neural crest derivatives and in muscles of these mutants. The importance of PAX3 during human embryonal development is readily seen in Waardenburg patients, who present a dominant inherited syndrome consisting mainly of craniofacial abnormalities, pigmentation deficiencies, and deafness, consecutive to PAX3 mutations. In order to analyze the nervous system of Splotch embryos in more detail, we employed the transgenic mouse line L17. These transgenic mice harbor a beta-galactosidase marker gene under the control of Hoxa-7 promoter elements. Probably in combination with cis-elements adjacent to the integration site of the L17 transgene, the Hoxa-7 elements drive the expression of the marker gene in major parts of the peripheral nervous system, as well as in more restricted parts of the central nervous system. These structures can be visualized during embryonic development, allowing detailed neuroanatomical studies in midgestation embryos. We describe the beta-galactosidase expression in wild-type L17 mice and demonstrate the applicability of L17 mice to the study of the nervous system. We then apply this experimental system to the analysis of Splotch embryos. Our findings underline the importance of Pax3 in the development of neural crest-derived structures, especially of cranial ganglia and nerves. We suggest the use of L17 mice as a valuable tool to perform similar analysis for other embryonal mutant phenotypes.

Animals↗
PubMed1995

Alu repeats: a source for the genesis of primate microsatellites.

As a result of their abundance, relatively uniform distribution, and high degree of polymorphism, microsatellites and minisatellites have become valuable tools in genetic mapping, forensic identity testing, and population studies. In recent years, a number of microsatellite repeats have been found to be associated with Alu interspersed repeated DNA elements. The association of an Alu element with a microsatellite repeat could result from the integration of an Alu element within a preexisting microsatellite repeat. Alternatively, Alu elements could have a direct role in the origin of microsatellite repeats. Errors introduced during reverse transcription of the primary transcript derived from an Alu "master" gene or the accumulation of random mutations in the middle A-rich regions and oligo(dA)-rich tails of Alu elements after insertion and subsequent expansion and contraction of these sequences could result in the genesis of a microsatellite repeat. We have tested these hypotheses by a direct evolutionary comparison of the sequences of some recent Alu elements that are found only in humans and are absent from nonhuman primates, as well as some older Alu elements that are present at orthologous positions in a number of nonhuman primates. The origin of "young" Alu insertions, absence of sequences that resemble microsatellite repeats at the orthologous loci in chimpanzees, and the gradual expansion of microsatellite repeats in some old Alu repeats at orthologous positions within the genomes of a number of nonhuman primates suggest that Alu elements are a source for the genesis of primate microsatellite repeats.

Animals↗
PubMed2005

Alu insertion loci and platyrrhine primate phylogeny.

Short INterspersed Elements (SINEs) make very useful phylogenetic markers because the integration of a particular element at a location in the genome is irreversible and of known polarity. These attributes make analysis of SINEs as phylogenetic characters an essentially homoplasy-free affair. Alu elements are primate-specific SINEs that make up a large portion of the human genome and are also widespread in other primates. Using a combination wet-bench and computational approach we recovered 190 Alu insertions, 183 of which are specific to the genomes of nine New World primates. We used these loci to investigate branching order and have produced a cladogram that supports a sister relationship between Atelidae (spider, woolly, and howler monkeys) and Cebidae (marmosets, tamarins, and owl monkeys) and then the joining of this two family clade to Pitheciidae (titi and saki monkeys). The data support these relationships with a homoplasy index of 0.00. In this study, we report one of the largest applications of SINE elements to phylogenetic analysis to date, and the results provide a robust molecular phylogeny for platyrrhine primates.

Alu Elements↗
PubMed1989

Rearrangement of the cytoskeleton and nuclear transfer in Tetrahymena thermophila cells fused by electric field.

This paper reports on electrofusion of Tetrahymena thermophila and on the reorganization of the cytoskeleton in fused cells. Important factors influencing the fusion yield are the number of electric pulses, the strength of alternate current field and cell density. The process of cell fusion consists of a mutual intermingling of cell membranes following their deformation at the contact zone, followed by the formation of cytoplasmic bridges and simultaneous disruption of portions of the submembranous cytoskeleton (epiplasm) and alveolar sacs. The course of further changes in cell organization depends on the polarity of fused cells. Homopolar fusion partners integrate by gradual translocation of portions of cortical cytoskeletal elements. In contrast, cortical integration of heteropolar fused cells is limited. Cytoskeletal integration is particularly promoted if the cells are incubated in non-growing conditions. Cortical integration leads to a high frequency of micronuclear transfer when a micronucleate strain is used as a donor and an amicronucleate strain is used as a recipient in the fusion experiments.

Animals↗
PubMed2003

Correlated discharge among cell pairs within the oculomotor horizontal velocity-to-position integrator.

In the oculomotor system, temporal integration of velocity commands into position signals may depend on synaptic feedback among neurons of a bilateral brainstem cell assembly known as the "neural integrator." Both ipsilateral excitatory and contralateral inhibitory projections between eye position-related integrator cells are hypothesized as a substrate for positive feedback supporting integration. Presence of feedback interactions should be evident in cross-correlation functions of neuron pairs. Here, unilateral and bilateral paired recordings were obtained during fixation behavior from neurons in goldfish brainstem area I, a key element of the integrator. During fixations, discharge of most unilateral pairs, composed of cells with eye position sensitivities of the same sign, was positively correlated with lag of 0-10 msec (n = 11 of 14 significant). Typically, a very narrow peak (mean half-width <4 msec) near zero lag was observed. Discharge of bilateral pairs, composed of cells with position sensitivities of the opposite sign, was either negatively correlated with lag of 0-10 msec (n = 5 of 13 significant) or not correlated. Troughs in negative correlations always had minima between 3 and 5 msec lag. These results are consistent with the feedback hypothesis of temporal integration, highlighting excitation unilaterally and inhibition bilaterally. Absence of visual input did not weaken correlations, but other sources of correlated input extrinsic to area I were not ruled out. Triplet recordings revealed that unilateral pairwise correlations were primarily independent. Correlation between unilateral pairs systematically decreased with increasing eye position, demonstrating that synchrony is not necessary for persistent activity at high firing rates.

Action Potentials↗
PubMed1986

Genomic and structural organization of Drosophila melanogaster G elements.

The properties and the genomic organization of G elements, a moderately repeated DNA family of D. melanogaster, are reported. G elements lack terminal repeats, generate target site duplications at the point of insertion and exhibit at one end a stretch of A residues of variable length. In a large number of recombinant clones analyzed G elements occur in tandem arrays, interspersed with specific ribosomal DNA (rDNA) segments. This arrangement results from the insertion of members of the G family within the nontranscribed spacer (NTS) of rDNA units. Similarity of the site of integration of G elements to that of ribosomal DNA insertions suggests that distinct DNA sequences might have been inserted into rDNA through a partly common pathway.

Animals↗
PubMed1999

A new element within the T-cell receptor alpha locus required for tissue-specific locus control region activity.

Locus control regions (LCRs) are cis-acting regulatory elements thought to provide a tissue-specific open chromatin domain for genes to which they are linked. The gene for T-cell receptor alpha chain (TCRalpha) is exclusively expressed in T cells, and the chromatin at its locus displays differentially open configurations in expressing and nonexpressing tissues. Mouse TCRalpha exists in a complex locus containing three differentially regulated genes. We previously described an LCR in this locus that confers T-lineage-specific expression upon linked transgenes. The 3' portion of this LCR contains an unrestricted chromatin opening activity while the 5' portion contains elements restricting this activity to T cells. This tissue-specificity region contains four known DNase I hypersensitive sites, two located near transcriptional silencers, one at the TCRalpha enhancer, and another located 3' of the enhancer in a 1-kb region of unknown function. Analysis of this region using transgenic mice reveals that the silencer regions contribute negligibly to LCR activity. While the enhancer is required for complete LCR function, its removal has surprisingly little effect on chromatin structure or expression outside the thymus. Rather, the region 3' of the enhancer appears responsible for the tissue-differential chromatin configurations observed at the TCRalpha locus. This region, herein termed the "HS1' element," also increases lymphoid transgene expression while suppressing ectopic transgene activity. Thus, this previously undescribed element is an integral part of the TCRalphaLCR, which influences tissue-specific chromatin structure and gene expression.

Animals↗