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Drosophila P-element transposase is a novel site-specific endonuclease.

We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5' and 3' P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3' cleavage site is at the end of the P-element, whereas the 5' cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo.

Animals↗

Potential gene conversion and source genes for recently integrated Alu elements.

Alu elements comprise >10% of the human genome. We have used a computational biology approach to analyze the human genomic DNA sequence databases to determine the impact of gene conversion on the sequence diversity of recently integrated Alu elements and to identify Alu elements that were potentially retroposition competent. We analyzed 269 Alu Ya5 elements and identified 23 members of a new Alu subfamily termed Ya5a2 with an estimated copy number of 35 members, including the de novo Alu insertion in the NF1 gene. Our analysis of Alu elements containing one to four (Ya1-Ya4) of the Ya5 subfamily-specific mutations suggests that gene conversion contributed as much as 10%-20% of the variation between recently integrated Alu elements. In addition, analysis of the middle A-rich region of the different Alu Ya5 members indicates a tendency toward expansion of this region and subsequent generation of simple sequence repeats. Mining the databases for putative retroposition-competent elements that share 100% nucleotide identity to the previously reported de novo Alu insertions linked to human diseases resulted in the retrieval of 13 exact matches to the NF1 Alu repeat, three to the Alu element in BRCA2, and one to the Alu element in FGFR2 (Apert syndrome). Transient transfections of the potential source gene for the Apert's Alu with its endogenous flanking genomic sequences demonstrated the transcriptional and presumptive transpositional competency of the element.

Alleles↗

A rice Tc1/mariner-like element transposes in yeast.

The Tc1/mariner transposable element superfamily is widely distributed in animal and plant genomes. However, no active plant element has been previously identified. Nearly identical copies of a rice (Oryza sativa) Tc1/mariner element called Osmar5 in the genome suggested potential activity. Previous studies revealed that Osmar5 encoded a protein that bound specifically to its own ends. In this report, we show that Osmar5 is an active transposable element by demonstrating that expression of its coding sequence in yeast promotes the excision of a nonautonomous Osmar5 element located in a reporter construct. Element excision produces transposon footprints, whereas element reinsertion occurs at TA dinucleotides that were either tightly linked or unlinked to the excision site. Several site-directed mutations in the transposase abolished activity, whereas mutations in the transposase binding site prevented transposition of the nonautonomous element from the reporter construct. This report of an active plant Tc1/mariner in yeast will provide a foundation for future comparative analyses of animal and plant elements in addition to making a new wide host range transposable element available for plant gene tagging.

Base Sequence↗

Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility.

The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.

Ampicillin Resistance↗

Repetition patterns in Weddell seal (Leptonychotes weddellii) underwater multiple element calls.

Many vocalizations produced by Weddell seals (Leptonychotes weddellii) are made up of repeated individual distinct sounds (elements). Patterning of multiple element calls was examined during the breeding season at Casey and Davis, Antarctica. Element and interval durations were measured from 405 calls all > 3 elements in length. The duration of the calls (22+/-16.6 s) did not seem to vary with an increasing number of elements (F4,404=1.83,p = 0.122) because element and interval durations decreased as the number of elements within a call increased. Underwater vocalizations showed seven distinct timing patterns of increasing, decreasing, or constant element and interval durations throughout the calls. One call type occurred with six rhythm patterns, although the majority exhibited only two rhythms. Some call types also displayed steady frequency changes as they progressed. Weddell seal multiple element calls are rhythmically repeated and thus the durations of the elements and intervals within a call occur in a regular manner. Rhythmical repetition used during vocal communication likely enhances the probability of a call being detected and has important implications for the extent to which the seals can successfully transmit information over long distances and during times of high level background noise.

Acoustics↗

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.

Bacteroides↗

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.

Amino Acid Sequence↗

Excision, transfer, and integration of NBU1, a mobilizable site-selective insertion element.

The Bacteroides species harbor a family of conjugative transposons called tetracycline resistance elements (Tcr elements) that transfer themselves from the chromosome of a donor to the chromosome of a recipient, mobilize coresident plasmids, and also mediate the excision and circularization of members of a family of 10- to 12-kbp insertion elements which share a small region of DNA homology and are called NBUs (for nonreplicating Bacteroides units). The NBUs are sometimes cotransferred with Tcr elements, and it was postulated previously that the excised circular forms of the NBUs were plasmidlike forms and were transferred like plasmids and then integrated into the recipient chromosome. We used chimeric plasmids containing one of the NBUs, NBU1, and a Bacteroides-Escherichia coli shuttle vector to show that this hypothesis is probably correct. NBU1 contained a region that allowed mobilization by both the Tcr elements and IncP plasmids, and we used these conjugal elements to allow us to estimate the frequencies of excision, mobilization, and integration of NBU1 in Bacteroides hosts to be approximately 10(-2), 10(-5) to 10(-4), and 10(-2), respectively. Although functions on the Tcr elements were required for the excision-circularization and mobilization of NBU1, no Tcr element functions were required for integration into the recipient chromosome. Analysis of the DNA sequences at the integration region of the circular form of NBU1, the primary insertion site in the Bacteroides thetaiotaomicron 5482 chromosome, and the resultant NBU1-chromosome junctions showed that NBU1 appeared to integrate into the primary insertion site by recombining within an identical 14-bp sequence present on both NBU1 and the target, thus leaving a copy of the 14-bp sequence at both junctions. The apparent integration mechanism and the target selection of NBU1 were different from those of both XBU4422, the only member of the conjugal Tcr elements for which these sequences are known, and Tn4399, a mobilizable Bacteroides transposon. The NBUs appear to be a distinct type of mobilizable insertion element.

Bacteroides↗

IS1630 of Mycoplasma fermentans, a novel IS30-type insertion element that targets and duplicates inverted repeats of variable length and sequence during insertion.

A new insertion sequence (IS) of Mycoplasma fermentans is described. This element, designated IS1630, is 1,377 bp long and has 27-bp inverted repeats at the termini. A single open reading frame (ORF), predicted to encode a basic protein of either 366 or 387 amino acids (depending on the start codon utilized), occupies most of this compact element. The predicted translation product of this ORF has homology to transposases of the IS30 family of IS elements and is most closely related (27% identical amino acid residues) to the product of the prototype of the group, IS30. Multiple copies of IS1630 are present in the genomes of at least two M. fermentans strains. Characterization and comparison of nine copies of the element revealed that IS1630 exhibits unusual target site specificity and, upon insertion, duplicates target sequences in a manner unlike that of any other IS element. IS1630 was shown to have the striking ability to target and duplicate inverted repeats of variable length and sequence during transposition. IS30-type elements typically generate 2- or 3-bp target site duplications, whereas those created by IS1630 vary between 19 and 26 bp. With the exception of two recently reported IS4-type elements which have the ability to generate variable large duplications (B. B. Plikaytis, J. T. Crawford, and T. M. Shinnick, J. Bacteriol. 180:1037-1043, 1998; E. M. Vilei, J. Nicolet, and J. Frey, J. Bacteriol. 181:1319-1323, 1999), such large direct repeats had not been observed for other IS elements. Interestingly, the IS1630-generated duplications are all symmetrical inverted repeat sequences that are apparently derived from rho-independent transcription terminators of neighboring genes. Although the consensus target site for IS30 is almost palindromic, individual target sites possess considerably less inverted symmetry. In contrast, IS1630 appears to exhibit an increased stringency for inverted repeat recognition, since the majority of target sites had no mismatches in the inverted repeat sequences. In the course of this study, an additional copy of the previously identified insertion sequence ISMi1 was cloned. Analysis of the sequence of this element revealed that the transposase encoded by this element is more than 200 amino acid residues longer and is more closely related to the products of other IS3 family members than had previously been recognized. A potential site for programmed translational frameshifting in ISMi1 was also identified.

Amino Acid Sequence↗

The genome of hepatitis B virus contains a second enhancer: cooperation of two elements within this enhancer is required for its function.

Previous studies have identified an enhancer (enhancer I) at nucleotides (nt) 1074 to 1234 in the genome of the human hepatitis B virus (HBV), which locates immediately upstream from the X gene. By analysis of the expression of the chloramphenicol acetyltransferase gene driven by a heterologous simian virus 40 early promoter, we describe the identification of a second enhancer (enhancer II) at nt 1636 to 1741, which locates downstream of enhancer I and immediately upstream of the core gene. With various deletions at the 5' end of enhancer II, a positive regulatory element was identified at nt 1636 to 1690 (the II-A element), with the 5' boundary between nt 1636 and 1671. The II-A element alone did not have an enhancer function, but the enhancer activity was achieved by the concomitant presence of the sequence from nt 1704 to 1741 (the II-B element). The II-B element alone did not have enhancer activity. These results indicate that cooperation between the II-A and II-B elements is required to exhibit the enhancer activity of enhancer II. We also show that enhancer II stimulates the transcriptional activity of both the SPI and SPII promoters of the surface gene. Therefore, the SPI promoter activity is regulated by the proximal HNF-1 binding element and the distal enhancers I and II. These results indicate that multiple regulatory elements scattered over the whole viral genome are involved in the regulation of expression of each individual HBV gene and that the same regulatory element controls the expression of different HBV genes. The relative positions of these regulatory elements in the HBV genome suggest that they may control the expression of HBV genes in a coordinate and cooperative manner.

Carcinoma, Hepatocellular↗

Composite transposable elements in the Xenopus laevis genome.

Members of two related families of transposable elements, Tx1 and Tx2, were isolated from the genome of Xenopus laevis and characterized. In both families, two versions of the elements were found. The smaller version in each family (Tx1d and Tx2d) consisted largely of two types of 400-base-pair tandem internal repeats. These elements had discrete ends and short inverted terminal repeats characteristic of mobile DNAs that are presumed to move via DNA intermediates, e.g., Drosophila P and maize Ac elements. The longer versions (Tx1c and Tx2c) differed from Tx1d and Tx2d by the presence of a 6.9-kilobase-pair internal segment that included two long open reading frames (ORFs). ORF1 had one cysteine-plus-histidine-rich sequence of the type found in retroviral gag proteins. ORF2 showed more substantial homology to retroviral pol genes and particularly to the analogs of pol found in a subclass of mobile DNAs that are supposed retrotransposons, such as mammalian long interspersed repetitive sequences, Drosophila I factors, silkworm R1 elements, and trypanosome Ingi elements. Thus, the Tx1 elements present a paradox by exhibiting features of two classes of mobile DNAs that are thought to have very different modes of transposition. Two possible resolutions are considered: (i) the composite versions are actually made up of two independent elements, one of the retrotransposon class, which has a high degree of specificity for insertion into a target within the other, P-like element; and (ii) the composite elements are intact, autonomous mobile DNAs, in which the pol-like gene product collaborates with the terminal inverted repeats to cause transposition of the entire unit.

Amino Acid Sequence↗

Characterization of Stowaway MITEs in pea (Pisum sativum L.) and identification of their potential master elements.

We have investigated miniature inverted-repeat transposable elements (MITEs) of the Stowaway family and corresponding Mariner-like master elements that could potentially facilitate their mobilization in the genome of the garden pea (Pisum sativum L.). The population of pea Stowaway MITEs consists of 103-104 copies dispersed in the genome. Judging from a sequence analysis of 17 isolated Stowaway elements and their flanking genomic regions, the elements are relatively uniform in size and sequence and occur in the vicinity of genes as well as within repetitive sequences. Insertional polymorphism of several elements was detected among various Pisum accessions, suggesting they were still transpositionally active during diversification of these taxa. The identification of several Mariner-like elements (MLEs) harboring intact open reading frames, capable of encoding a transposase, further supports a recent mobilization of the Stowaway elements. Using transposase-coding sequences as a hybridization probe, we estimated that there are about 50 MLE sequences in the pea genome. Among the 5 elements sequenced, 3 distinct subfamilies showing mutual similarities within their transposase-coding regions, but otherwise diverged in sequence, were distinguished and designated as Psmar-1 to Psmar-3. The terminal inverted repeats (TIRs) of these MLE subfamilies differed in their homology to the TIRs of Stowaway MITEs. The homlogy ranged from 9 bp in Psmar-3 to 30 bp in Psmar-1, which corresponds to the complete Stowaway TIR sequence. Based on this feature, the Psmar-1 elements are believed to be the most likely candidates for the master elements of the Stowaway MITEs in pea.

Amino Acid Sequence↗

Species-specific differences in positive and negative regulatory elements in the renin gene enhancer.

A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4. 1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.

Animals↗

Genomic comparisons among Escherichia coli strains B, K-12, and O157:H7 using IS elements as molecular markers.

BACKGROUND: Insertion Sequence (IS) elements are mobile genetic elements widely distributed among bacteria. Their activities cause mutations, promoting genetic diversity and sometimes adaptation. Previous studies have examined their copy number and distribution in Escherichia coli K-12 and natural isolates. Here, we map most of the IS elements in E. coli B and compare their locations with the published genomes of K-12 and O157:H7. RESULTS: The genomic locations of IS elements reveal numerous differences between B, K-12, and O157:H7. IS elements occur in hok-sok loci (homologous to plasmid stabilization systems) in both B and K-12, whereas these same loci lack IS elements in O157:H7. IS elements in B and K-12 are often found in locations corresponding to O157:H7-specific sequences, which suggests IS involvement in chromosomal rearrangements including the incorporation of foreign DNA. Some sequences specific to B are identified, as reported previously for O157:H7. The extent of nucleotide sequence divergence between B and K-12 is < 2% for most sequences adjacent to IS elements. By contrast, B and K-12 share only a few IS locations besides those in hok-sok loci. Several phenotypic features of B are explained by IS elements, including differential porin expression from K-12. CONCLUSIONS: These data reveal a high level of IS activity since E. coli B, K-12, and O157:H7 diverged from a common ancestor, including IS association with deletions and incorporation of horizontally acquired genes as well as transpositions. These findings indicate the important role of IS elements in genome plasticity and divergence.

Chromosome Mapping↗

Element concentrations of dry-grind corn-processing streams.

The dry-grind corn process is one of two technologies used to convert corn into ethanol. In this process, all kernel components are processed through several sequential steps, including fermentation. Only one coproduct (distillers' dried grains with solubles [DDGS]) is available for marketing. DDGS provide income to offset costs of processing; issues that affect marketing have implications in the economic viability of dry-grind plants. Two issues relate to elements in DDGS: high concentrations and excessive variation. Data on element concentrations in dry-grind processing streams could be helpful in addressing these concerns. The objective of this study was to determine element concentrations in primary process streams from dry-grind plants. Samples of corn, ground corn, beer, wet grains, syrup, and DDGS were obtained from nine dry-grind plants, and element concentrations were determined. The concentrations of most elements in corn were not different among processing plants and were similar to published data. However, for the processing streams, there were differences in several element concentrations among processing plants. The concentrations of most elements in beer were about three times those of corn, due to the disappearance of starch during fermentation. Syrup had the highest element concentrations. Variations in element contents of DDGS and parent streams were due to processing conditions and not corn. Appropriate processing of thin stillage (the parent stream of syrup) could reduce the element content of DDGS.

Beer↗

Establishment and maintenance of the P cytotype associated with telomeric P elements in Drosophila melanogaster.

P elements inserted near the left telomere of the X chromosome are associated with the P cytotype, a maternally transmitted condition that strongly regulates the activity of the P transposon family in some strains of Drosophila. The regulatory abilities of two such elements, TP5 and TP6, are stable in homozygous stocks over many generations. However, these regulatory abilities are attenuated when the telomeric P elements are transmitted through heterozygous females, and they are utterly lost when the elements are transmitted through males. Paternally transmitted telomeric P elements reacquire regulatory ability when they pass through a female germ line. This reacquisition is enhanced if the females in which it occurs came from mothers who carried a telomeric P element. The enhancement has two components: (1). a strictly maternal effect that is transmitted to the females independently of the mother's telomeric P element ("presetting" or the "pre-P cytotype") and (2). a zygotic effect associated with inheritance of the mother's telomeric P element. One telomeric P element can enhance the reacquisition of another's regulatory ability. When X chromosomes that carry telomeric P elements are extracted through males and made homozygous by using a balancer chromosome, most of the resulting stocks develop strong regulatory abilities in a few generations. However, some of the stocks do not attain the regulatory ability of the original population.

Animals↗

Evidence for an inactive transposable mariner-like element in Anopheles albimanus.

Transposable elements may eventually be used as transformation vectors of anti-Plasmodium genes in natural populations of Anopheles mosquitoes. The mariner transposable element is widespread in most animal phyla and we wished to test for its presence in the important Latin American malaria vector, Anopheles albimanus. Degenerate primers were designed from the consensus of insect mariner elements and used in a polymerase chain reaction to amplify a fragment of the predicted size from An. albimanus. The fragment was cloned, sequenced, and determined to be a mariner-like element through multiple alignment with known insect mariner elements. Dot blot and Southern blot analyses showed only one or a few elements per haploid genome. The cloned fragment was used as a probe to isolate similar sequences from an An. albimanus genomic DNA library. A 659-base-pair clone was 57% similar at the DNA level with mariner elements from Anopheles gambiae, Chrysoperla ptorapunda, and Haematobia irritans. This low sequence similarity is comparable to that found among mariner-like elements in all insects. However, conserved amino acid motifs were not discovered nor was a single open reading frame found. These aspects suggest that the An. albimanus mariner-like element may represent an ancient transposition event but that the element is no longer active, the typical open reading frame having been disrupted through nucleotide insertions and deletions.

Animals↗

[Distribution of chemical elements in whole blood and plasma].

The distribution factor (Fd) of 35 elements of plasma and whole blood in 26 healthy men and women was detected by ICP-OES. Usilig this parameter the elements were subdivided in 3 pools. 9 of them have Fd higher than 1.5 ("elements of plasma"-Ag, Ca, Cu, In, Li, Na, Se, Si, Sr); 6 have lower than 0.5 ("elements of blood cells"-Fe, K, Mn, Ni, V, Zn), other 20-about 1 ("blood elements"). Fd of all elements depends on ionic radius. Elements of 2nd sub-groups of all groups of Mendeleev's periodic table ("heavy metals") depend on the similar law: "with growing of ionic radius the concentration of elements in plasma enhances". In alkaline metals Fd depends on the opposite law:" with growing of ionic radius of alkaline metal the quantity of elements in blood cells enhance". Dependence of Fd on the value of atomic mass in periods or in exterior electronic cloud (s-, p-, d-, f-) was not established. The table of distribution of all detected elements in whole blood in relation to 8 macroelements (Ca, Mg, K, Na, S, P, Fe, Zn,) is presented, as a basic diagnostic criteria in metal-ligand homeostasis disturbance.

Adult↗