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Nested insertions of short mobile sequences in Drosophila P elements.

A potentially full-sized P element isolated from the genome of Drosophila ambigua by polymerase chain reaction amplification was completely sequenced. It has a length of 3329 bp and the termini are formed by 33 bp inverted repeats. Sequence comparisons show that it can be classified as a member of the T-type P element subfamily. The translational reading frames of all four exons are interrupted by stop codons and frameshift mutations. At the 3' end of exon 3 a 687 bp insertion sequence (IS-amb-P) is found that also occurs in the form of dispersed copies (IS-amb) in the genome in D. ambigua. At the interspecific level it shows homology to mobile sequences of other species of the obscura group. Although variable in length, these IS elements are characterized by conserved sections without coding function and by 14 bp inverted repeats, one at a terminal, the other at a subterminal position. In situ hybridization revealed that P elements in D. ambigua are restricted to only two euchromatic sites on chromosome elements A and E. This situation resembles that found in Drosophila guanche and Drosophila subobscura where P homologs are clustered at a single site on chromosome element E and where the section corresponding to exon 3 of P elements carries an IS element. The gene sik-hom, which is located at the 5' side of the D. guanche cluster of P homologs, was used as a marker to examine whether the P element sites on chromosome element E of D. guanche and D. ambigua are homologous. The results suggest that the nested insertions of IS elements into P elements must have occurred independently in the two different lineages.

Amino Acid Sequence↗

Haloperidol-induced Fos expression in striatum is dependent upon transcription factor cyclic AMP response element binding protein.

Haloperidol has been shown to induce rapid and transient expression of c-fos messenger RNA and Fos protein in striatal neurons via dopamine D2 receptors. Regulation of the c-fos gene by cyclic AMP and Ca2+ has been shown to be dependent on a DNA regulatory element within its promoter that binds the constitutively expressed transcription factor cyclic AMP response element binding protein. Cyclic AMP response element binding protein binds to an oligonucleotide containing the calcium/cyclic AMP response element of the c-fos promoter sequence in striatal cell extracts; the amount of binding is not regulated by haloperidol treatment. We have previously shown that haloperidol induces cyclic AMP response element binding protein phosphorylation in the striatum. Here we show by intrastriatal injection of antisense oligonucleotides that haloperidol-induced Fos expression is dependent on cyclic AMP response element binding protein. Intrastriatal injections of phosphorothioate oligonucleotides, in antisense orientation to cyclic AMP response element binding protein messenger RNA, reduce levels of cyclic AMP response element binding protein and completely prevent haloperidol-mediated induction of Fos. Oligonucleotides in sense orientation have no such effect. We observed a markedly different time course of the Fos protein inhibition by cyclic AMP response element binding protein antisense oligonucleotides compared to c-fos antisense oligonucleotides. This most likely reflects the different half-lives of c-fos and cyclic AMP response element binding protein messenger RNA and proteins. Neither cyclic AMP response element binding protein nor c-fos antisense oligonucleotide injection reduced c-Jun immunostaining in the striatum. We conclude that haloperidol induces Fos via transcription factor cyclic AMP response element binding protein.

Activating Transcription Factor 2↗

Geldanamycin induction of grp78 requires activation of reactive oxygen species via ER stress responsive elements in 9L rat brain tumour cells.

The molecular mechanism whereby anticancer agent geldanamycin (GA) impacts endoplasmic reticulum (ER) stress pathway is largely unknown. Here, we investigate the effect of GA on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signalling to ER stress. GA induces the expression of mRNA and protein of grp78 by Northern blot analysis and metabolic labelling experiment in cultured rat brain tumour 9L cells. The induced grp78 expression is sensitive to antioxidant N-acetylcysteine (NAC) addition, indicating the involvement of reactive oxygen species (ROS) in GA-induced ER stress. Results from direct determination of oxidation status using dichlorodihydrofluorescein diacetate (H(2)DCFDA) showed that accumulation of ROS elicited GA was quenched by addition of NAC. Reporter genes harbouring deletions of transcription elements from grp78 promoter demonstrated that controlling elements of ERSE1, ERSE2 and CRE are required in GA treatment. The critical ROS-dependent elements in grp78 promoter can be confined within ER stress responsive element (ERSE) region, since reporter constructs loss of ERSE elements that lost the susceptibility to be modulated by NAC after GA treatment. Hence, ER stress elements correlate well with ROS-mediated elements in grp78 promoter. Reporter construct loss of ERSE element retains the susceptibility by NAC after GA treatment, indicating that CRE element might represent a ROS-independent, GA-inductive element. Conclusively, we show that ROS is required for GA to launch the transactivation of grp78, and a firm link was established between the ROS signalling pathway to specific promoter elements-ERSE1 and ERSE2 elements in ER stress marker gene grp78 promoter.

Acetylcysteine↗

Localization of P elements, copy number regulation, and cytotype determination in Drosophila melanogaster.

Seventeen highly-inbred lines of Drosophila melanogaster extracted from an M' strain (in the P/M system of hybrid dysgenesis) were studied for their cytotype and the number and chromosomal location of complete and defective P elements. While most lines were of M cytotype, three presented a P cytotype (the condition that represses P-element activity) and one was intermediate between M and P. All lines were found to possess KP elements and only eight to bear full-sized P elements. Only the lines with full-sized P elements showed detectable changes in their P-insertion pattern over generations; their rates of gain and of loss of P-element sites were equal to 0.12 and 0.09 per genome, per generation, respectively. There was no correlation between these two rates within lines, suggesting independent transpositions and excisions in the inbred genomes. The results of both Southern blot analysis and in situ hybridization of probes made from left and right sides of the P element strongly suggested the presence of a putative complete P element in region 1A of the X chromosome in the three lines with a P cytotype; the absence of P copy in this 1A region in lines with an M cytotype, favours the hypothesis that the P element inserted in 1A could play a major role in the P-cytotype determination. Insertion of a defective 2 kb P element was also observed in region 93F in 9 of the 13 M lines. The regulation of the P-element copy number in our lines appeared not to be associated with the ratio of full-length and defective P elements.

Animals↗

Transposable element sequences involved in the enhancement of yeast gene expression.

The his4-917 mutation of yeast results from the insertion of a Ty element, Ty917, into the 5' regulatory region of the HIS4 gene. Ty917 prevents HIS4 transcription, thus rendering the cell histidine requiring. Recombination between Ty917 and a Ty element elsewhere in the yeast genome can result in the replacement of part or all of the Ty917 element by sequences from the Ty element. Recombinant derivatives display a variety of phenotypes including His-, weakly His+, and strongly His+. In most of the His+ derivatives, the expression of HIS4 is controlled by genes at the mating type locus. To identify the Ty sequences important in controlling the expression of an adjacent gene, we used Ty elements that have different effects on gene expression to construct hybrid Ty elements in vitro. The effects of these hybrid elements on HIS4 expression were examined. These experiments indicate that the critical sequence differences between Ty elements that permit HIS4 expression and those that prevent its expression lie in the rightmost (HIS4-proximal) 730 base pairs of the element. The DNA sequence of this region was determined for three elements: Ty917, which prevents HIS4 expression; Ty917(467), which confers a weak His+ phenotype; and Ty917(480), which confers a strong His+ phenotype. Within this region, Ty917(467) differs from Ty917 by a single base-pair change that is in the internal (epsilon) region of the Ty element. Ty917(480) differs from Ty917 by this same base-pair change and by 10 changes in the terminal delta sequence. The sequence change common to Ty917(467) and Ty917(480) lies in a region of the Ty element that is homologous to the simian virus 40 enhancer of transcription.

Base Sequence↗

A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes.

Several recent reports indicate that mobile elements are frequently found in and flanking many wild-type plant genes. To determine the extent of this association, we performed computer-based systematic searches to identify mobile elements in the genes of two "model" plants, Oryza sativa (domesticated rice) and Arabidopsis thaliana. Whereas 32 common sequences belonging to nine putative mobile element families were found in the noncoding regions of rice genes, none were found in Arabidopsis genes. Five of the nine families (Gaijin, Castaway, Ditto, Wanderer, and Explorer) are first described in this report, while the other four were described previously (Tourist, Stowaway, p-SINE1, and Amy/LTP). Sequence similarity, structural similarity, and documentation of past mobility strongly suggests that many of the rice common sequences are bona fide mobile elements. Members of four of the new rice mobile element families are similar in some respects to members of the previously identified inverted-repeat element families, Tourist and Stowaway. Together these elements are the most prevalent type of transposons found in the rice genes surveyed and form a unique collection of inverted-repeat transposons we refer to as miniature inverted-repeat transposable elements or MITEs. The sequence and structure of MITEs are clearly distinct from short or long interspersed nuclear elements (SINEs or LINEs), the most common transposable elements associated with mammalian nuclear genes. Mobile elements, therefore, are associated with both animal and plant genes, but the identity of these elements is strikingly different.

Arabidopsis↗

A comprehensive analysis of recently integrated human Ta L1 elements.

The Ta (transcribed, subset a) subfamily of L1 LINEs (long interspersed elements) is characterized by a 3-bp ACA sequence in the 3' untranslated region and contains approximately 520 members in the human genome. Here, we have extracted 468 Ta L1Hs (L1 human specific) elements from the draft human genomic sequence and screened individual elements using polymerase-chain-reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. One hundred twenty-four of the elements amenable to complete sequence analysis were full length ( approximately 6 kb) and have apparently escaped any 5' truncation. Forty-four of these full-length elements have two intact open reading frames and may be capable of retrotransposition. Sequence analysis of the Ta L1 elements showed a low level of nucleotide divergence with an estimated age of 1.99 million years, suggesting that expansion of the L1 Ta subfamily occurred after the divergence of humans and African apes. A total of 262 Ta L1 elements were screened with PCR-based assays to determine their phylogenetic origin and the level of human genomic variation associated with each element. All of the Ta L1 elements analyzed by PCR were absent from the orthologous positions in nonhuman primate genomes, except for a single element (L1HS72) that was also present in the common (Pan troglodytes) and pygmy (P. paniscus) chimpanzee genomes. Sequence analysis revealed that this single exception is the product of a gene conversion event involving an older preexisting L1 element. One hundred fifteen (45%) of the Ta L1 elements were polymorphic with respect to insertion presence or absence and will serve as identical-by-descent markers for the study of human evolution.

Animals↗

Quantitative effects of P elements on hybrid dysgenesis in Drosophila melanogaster.

Genetic analyses involving chromosomes from seven inbred lines derived from a single M' strain were used to study the quantitative relationships between the incidence and severity of P-M hybrid dysgenesis and the number of genomic P elements. In four separate analyses, the mutability of snw, a P element-insertion mutation of the X-linked singed locus, was found to be inversely related to the number of autosomal P elements. Since snw mutability is caused by the action of the P transposase, this finding supports the hypothesis that genomic P elements titrate the transposase present within a cell. Other analyses demonstrated that autosomal transmission ratios were distorted by P element action. In these analyses, the amount of distortion against an autosome increased more or less linearly with the number of P elements carried by the autosome. Additional analyses showed that the magnitude of this distortion was reduced when a second P element-containing autosome was present in the genome. This reduction could adequately be explained by transposase titration; there was no evidence that it was due to repressor molecules binding to P elements and inhibiting their movement. The influence of genomic P elements on the incidence of gonadal dysgenesis was also investigated. Although no simple relationship between the number of P elements and the incidence of the trait could be discerned, it was clear that even a small number of elements could increase the incidence markedly. The failure to find a quantitative relationship between P element number and the incidence of gonadal dysgenesis probably reflects the complex etiology of this trait.

Animals↗

Sequence analysis of active mariner elements in natural populations of Drosophila simulans.

Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.

Animals↗

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group.

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.

Animals↗

Sequence analysis of transposable elements in the sea squirt, Ciona intestinalis.

A systematic search of 1 Mb of genomic sequences from the sea squirt, Ciona intestinalis, revealed the presence of six families of transposable elements. The Cigr-1 retrotransposon contains identical 245-bp long terminal repeats (LTRs) and a 3,630-bp open reading frame (ORF) encoding translation products in the same order as the domains characteristic of gypsy/Ty3-type LTR retrotransposons. The closest homologs of the reverse transcriptase domain were in gypsy elements from Drosophila and the sushi element from the pufferfish. However, the capsid-nucleocapsid region shows the clearest homology to an echinoderm element, Tgr1. Database searches also indicated two classes of non-LTR retrotransposon, named Cili-1 and Cili-2. The Cili-1 sequences show matches to regions of the ORF2 product of mammalian L1 elements. The Cili-2 sequences possess similarity to the RNaseH domain of Lian-Aa1, a mosquito non-LTR retrotransposon. The most abundant element was a short interspersed nucleotide element named Cics-1 with a copy number estimated at 40,000. Cics-1 consists of two conserved domains separated by an A-rich stretch. The 172-bp 5' domain is related to tRNA sequences, whereas the 110-bp 3' domain is unique. Cics-1 is unusual, not just in its modular structure, but also in its lack of a 3' poly(A) tail or direct flanking repeats. A second abundant element, Cimi-1, has an A+T-rich 193-bp consensus sequence and 30-bp terminal inverted repeats (TIRs) and is usually flanked by A+T-rich 2-4-bp putative target site duplications-characteristics of miniature inverted-repeat transposable elements found in plants and insects. A single 2,444-bp foldback element was found, possessing long TIRs containing an A+T-rich internal domain, an array of subrepeats, and a flanking domain at the TIR ends; this is the first example of a chordate foldback element. This study provides the first systematic characterization of the families of transposable elements in a lower chordate.

Amino Acid Sequence↗

Distribution and conservation of mobile elements in the genus Drosophila.

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.

Animals↗

Trace elements in animals.

Trace element deficiency and toxicity in animals induces a wide variety of clinical effects although few are sufficiently specific to permit diagnosis without supporting investigation of changes in tissue trace element content or of the activity of metabolic processes influenced by trace element supply. Study of such trace element dependent processes has shown that extensive changes often arise before overt signs of disease appear. Some of these subclinical effects have pathological consequences and thus cannot be ignored when seeking correlations between geochemical anomalies and disease incidence. Many past estimates of the quantitative requirements of animals for the essential trace elements are imprecise. Although recent work is providing clearer definition of requirements, many common dietary components have a marked influence upon the efficiency with which such elements can be utilized from the diet. Recent evidence indicates that such antagonists influence both the absorption and the subsequent fate of essential and toxic elements in body tissues and these processes have to be taken into account when investigating the aetiology of disorders believed to be attributable to anomalies in trace element supply. Their existence is not always detectable if attention is confined to the trace element analysis of body tissues or to the nature of clinical lesions. Provided the complexity of soil-plant-animal relations with respect to trace element supply is fully recognized in the interpretation of data, the geochemical approach to the initial recognition of areas associated with a high risk of anomalies in trace element supply to animals and man has considerable potential value. This is already apparent from investigations upon the incidence of trace element problems in animals. As yet, its validity for similar purposes in man is less fully established.

Animals↗

Cooperation between elements of an organ-specific transcriptional enhancer in animals.

The elastase I gene enhancer that specifies high levels of pancreatic transcription comprises three functional elements (A, B, and C). When assayed individually in transgenic mice, homomultimers of A are acinar cell specific, those of B are islet specific, and those of C are inactive. To determine how the elements interact in the elastase I enhancer and to investigate further the role of the C element, we have examined the activity of the three possible combinations of synthetic double elements in transgenic animals. Combining the A and B elements reconstitutes the exocrine plus endocrine specificity of the intact enhancer with an increased activity in acinar cells compared with that in the A homomultimer. The B element therefore plays a dual role: in islet cells it is capable of activating transcription, whereas in acinar cells it is inactive alone but greatly augments the activity specified by the A element. The C element augments the activity of either the A or B element without affecting their pancreatic cell type specificity. The roles of each element were verified by examining the effects of mutational inactivation of each element within the context of the elastase I enhancer. These results demonstrated that when tested in animals, the individual enhancer elements can perform discrete, separable functions that combine additively for cell type specificity and cooperatively for the overall strength of a multielement stage- and site-specific transcriptional enhancer.

Animals↗

The distal enhancer region of the rat prolactin gene contains elements conferring response to multiple hormones.

The ability of an upstream element of the rat PRL gene to permit transcriptional regulation in response to several different hormones has been examined. To test the ability of specific DNA sequences to mediate hormone responsiveness, DNA fragments were subcloned upstream of a thymidine kinase-chloramphenicol acetyltransferase fusion gene and transferred into GH3 pituitary tumor cells. Initially, fragments representing a distal enhancer element (positions -1713 to -1495) and a more proximal element (positions -292 to -38) were tested. The results demonstrate that the distal enhancer permits cAMP, TRH, epidermal growth factor (EGF), and estradiol to stimulate expression of the thymidine kinase-chloramphenicol acetyltransferase gene. The proximal element permitted fusion gene regulation in response to cAMP, TRH, EGF, and phorbol esters. For the cAMP, TRH, and EGF responses, the distal element permitted responses approximately equal to or greater than responses conferred by the proximal PRL gene fragment. The response of the distal element to cAMP and TRH was more than additive with the response to estradiol, suggesting that the estrogen response element is distinct but may interact cooperatively with the other hormone response elements. Mutation of the estrogen-responsive element abolished both the response to estrogen and the cooperative response with cAMP, but not the response to cAMP itself. Mutation of a sequence involved in basal enhancer activity of the distal element reduced both basal transcription and the response to cAMP. These results suggest that the distal enhancer sequence of the PRL gene contains, in addition to an estrogen response element, elements that confer responsiveness to cAMP, TRH, and EGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The P cytotype in Drosophila melanogaster: a maternally transmitted regulatory state of the germ line associated with telomeric P elements.

The incomplete P elements TP5 and TP6 are inserted in the TAS repeats near the left telomere of the Drosophila melanogaster X chromosome. These telomeric P elements repress P-induced gonadal dysgenesis and germ-line hypermutability in both sexes. However, their capacity to repress hypermutability is lost when they are transmitted patroclinously in a cross. TP5 and TP6 do not repress P-element activity in somatic cells, nor do they alter the somatic or germ-line phenotypes of P-insertion alleles. In the germ line, these elements suppress the phenotype of a P-insertion allele of the singed gene that is evoked by other P elements, presumably because these other elements encode repressor polypeptides. This suppression is more effective when the telomeric P elements are inherited maternally. Regulation by telomeric P elements parallels that of the P cytotype, a state that represses P-element activity in some strains of Drosophila. This state exists only in the germ line and is maternally transmitted along with the P elements themselves. Regulation by known repressor P polypeptides is not restricted to the germ line and does not require maternal transmission of the relevant P elements. Regulation by telomeric P elements appears to be epistatic to regulation by repressor P polypeptides.

Alleles↗

Intravenous solution compatibility and filter-retention characteristics of trace-element preparations.

The compatibility of recently published AMA formulations of the four trace-element injections (zinc, copper, chromium, and manganese) with a representative total parenteral nutrition (TPN) formulation and commonly used intravenous solutions was evaluated in glass and plastic containers. The effect of in-line filtration on trace-element concentration was also studied. Individual metal-ion concentrations, pH, color, and clarity were measured at 0-, 12-, 24-, and 48-hour intervals following mixture in TPN, 5% dextrose injection, 0.9% sodium chloride injection, and 8.5% amino acid injection (Travasol, Travenol Laboratories). Multiple trace elements were studied in one-liter glass bottles of 5% dextrose and 0.9% sodium chloride injection. Trace-element contaminants were measured in the intravenous solutions and trace-element injections. The four trace-element preparations were added individually and in combination to the solutions in plastic or glass containers. The trace-element admixtures were passed through in-line i.v. filters (IVEX-2, Abbott Laboratories) during a three-hour period at controlled-flow rates. Concentrations of trace elements remained within 99% confidence limits in all i.v. solutions, with both individual trace elements and combinations of the four trace elements. Trace-element concentrations did not change when the admixtures were passed through the in-line filter unit. It is concluded that there are no obvious compatibility problems associated with admixture nor any detectable retention by the i.v. filter of trace elements.

Drug Incompatibility↗

Multiple cis-acting elements act cooperatively in directing trochoblast-specific expression of the alpha-tubulin-4 gene in Patella embryos.

During early embryogenesis of the mollusc Patella vulgata the alpha-tubulin-4 gene is specifically expressed in the differentiation process of a particular cell type, the trochoblasts. The 5' region of the gene has been analysed for elements that are required for the regulation of the cell-type-specific activation of the gene. In a functional assay, seven elements that play a role in the spatiotemporal activation of the gene were detected in the promoter sequence. They are not required all together at the same time. A core of two elements, together with two or three auxiliary elements, are sufficient in controlling trochoblast-specific expression of the gene in the Patella embryo. One of the core elements is always required, without it, the gene is not expressed at all. The other core element acts as a positive element in trochoblasts and as a negative element in non-trochoblasts and thus is involved in directing the cell-type specificity of expression. The seven elements act cooperatively, and at least some of them can substitute for each other. Sequence comparison revealed that six of the seven fragments contain the sequence element GTTAA, including one of the core elements. Both core elements seem to play a crucial role in the expression of the alpha-tubulin-4 gene during the differentiation process of trochoblasts in the Patella embryo. A model for the regulation of the cell-type specificity of the gene during trochoblast differentiation is proposed.

Animals↗