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At least 19 recordsLinked to original sources

Cloning of the novel isoform of the estrogen receptor beta cDNA (ERbeta isoform M cDNA) from the human testicular cDNA library.

Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.

Alternative Splicing↗

[Screening and identification of apolipoprotein CIII cDNA clones from human liver cDNA library and preparation of apolipoprotein CIII cDNA probe from the clones].

Four of positive clones were obtained by three runs of screening the cDNA library of human liver with rabbit antihuman apolipoprotein (apo) CIII IgG. One of them was identified to be the positive clone of apoC III cDNA by restriction endonuclease map studies of the cDNA insert fragment and by Western blot analysis of the expression products. The apoCIII cDNA-gamma gt11 recombinant DNA prepared from the positive clone can be used in RNA-dot blot analysis as apoCIII cDNA probe. And the apoCIII cDNA clone can be also used to produced apoCIII peptide under the induction of isopropyl-beta-D-thiogalactoside. The apoCIII peptide, after purification, has a lot of use in further studies.

Apolipoprotein C-III↗

Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells.

AIM: To elucidate molecular mechanism of chemopreventive efficacies of garlic against human gastric cancer (HGC). METHODS: HGC cell line BGC823 was treated with Allitridi (a kind of garlic extract) and Allitridi-treated and parental BGC823 cDNA libraries were constructed respectively by using lambdaZAP II vector. cDNA Representational Difference Analysis (cDNA RDA) was performed using Bam H I cutting-site and abundant cDNA messages provided by the libraries. Northern blot analysis was applied to identify the obtained difference products. RESULTS: Two specific cDNA fragments were obtained and characterized to be derived from homo sapiens folate receptor alpha (FRalpha) gene and calcyclin gene respectively. Northern blot results showed a 4-fold increase in FRalpha gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: The method of cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with abundant cDNA messages in cDNA library; this expands the application of cDNA library and increases the specificity of cDNA RDA. Up-regulation of FRalpha gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.

Allyl Compounds↗

Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.

Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Isolation of wound-specific cDNA clones from a cDNA library prepared with mRNAs of alkali-burned rabbit corneas.

Alkali burn is one of the most severe corneal injuries. In order to gain a better understanding of the healing of alkali-burned corneas, it is necessary to identify and characterize proteins that are specifically synthesized by the injured corneal tissues. In this study, we developed a useful procedure to identify and isolate cDNA clones that encode messenger ribonucleic acids (mRNAs) that are specific and/or abundant in alkali-burned rabbit corneas (ARCs), but absent in normal rabbit corneas (NRCs). At first, a cDNA library was prepared by cloning cDNA of mRNA isolated from ARCs into the lambda ORF-8 vector. A differential plaque hybridization was used to screen 2.5 x 10(4) plaque-forming units (pfu) from an ARC cDNA library using 32P-labeled cDNAs prepared from mRNA of ARCs and NRCs. Thirty-seven cDNA clones of mRNAs specific for ARCs were identified and isolated in their pureform. The cDNA inserts of these lambda ORF-8 phages were subcloned into the pSM216 vector by in vivo recombination. The cDNA inserts then were characterized by restriction enzyme digestion, i.e., BamHI, HindIII, and EcoRI. The size of the cDNA inserts ranged from 210 to 5,000 base pairs. Using Northern blot hybridization of total RNA prepared from polymorphonuclear neutrophils, mononuclear leukocytes, alkali-burned corneas, and normal corneas, the cDNA clones were divided into three groups. Five cDNA clones encoded mRNA of corneal cells in ARCs. Twenty-four cDNA clones derived from mRNA of inflammatory cells were present in alkali-burned corneas, but Northern blot hybridization failed to identify mRNA of discrete sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkalies↗

Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to Latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrifugation was hybridized repeatedly to the cDNA-oligo(dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned into pBluescript II KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA inserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.

Acetamides↗

A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

Animals↗

PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L micron3 myeloma cells, total cDNA was synthesized and amplified approximately 10(5) fold. A library containing 10(6) clones was established from 1/6 of the amplified cDNA. Screening of the library with probes for three genes expressed in these cells revealed a number of corresponding clones in each case. The longest obtained clones contained inserts of 1.5 kb length. No sequences originating from carriers or from rRNA was found in 14 randomly picked clones.

Cloning, Molecular↗

cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesis with polymerase chain reaction amplification.

We describe here a general and simple procedure for cDNA library construction making use of in vitro amplification of cDNA by polymerase chain reaction (PCR). The first-strand cDNA is synthesized from total RNA with a primer EcoRI-(dT)17 and oligo(dG) tailed. An oligonucleotide, EcoRI-BamHI-(dC)13, is used to prime the second-strand synthesis by the thermostable DNA polymerase of Thermus aquaticus. The double-stranded cDNA is then amplified directly by PCR. A study of the effect of the elongation time on the PCR products showed that a long extension time is necessary to overcome the size heterogeneity of the cDNA population. Starting from 1 microgram of total brain RNA, the products obtained ranged from 200 to more than 2000 bp. The presence of the myelin basic protein cDNA sequence was determined. A lambda gt10 library containing 2 x 10(6) clones was established with the amplified cDNA. No sequences originating from rRNA were detected by Southern blot analysis. The ability to produce representative cDNA libraries from minute amounts of total RNA by this protocol should have many applications to studies of gene expression in small amounts of tissues or cells.

Animals↗

The novel isoform of the estrogen receptor-alpha cDNA (ERalpha isoform S cDNA) in the human testis.

In order to clone the novel isoform of the cDNA for the human estrogen receptor-alpha (ERalpha), the human testicular cDNA library was screened by the exons 4-8 corresponding sequence of the human ERalpha cDNA. As a result, a novel isoform of the ERalpha cDNA (termed the ERalpha isoform S cDNA), which consists of a previously unidentified 5'-sequence and the exons 4-8 of the ERalpha gene, has been cloned. The structure of the ERalpha isoform S cDNA is essentially similar to that of the progesterone receptor (PR) isoform S cDNA that was identified in our recent report. Analysis of the genomic DNA revealed that the 5'-sequence of the ERalpha isoform S mRNA originated from a novel exon (termed the exon S). Moreover, the reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the primers specific to the ERalpha isoform S mRNA on the total RNA from the human spermatozoon (Sp), liver (Li), uterine endometrium (Em) and myometrium (Mm). The ERalpha isoform S mRNA was detected in the uterine Em and Sp. Moreover, the molecular size of the ERalpha isoform S encoded by the ERalpha isoform S mRNA, which was analyzed by the transfection of the expression vector with ERalpha isoform S cDNA into the 293T cell, was approximately 39kDa. It was indicated that the one of the ATGs in the exon S could be used as the translation initiation codon. This is the first report on the ERalpha mRNA isoform that is not caused by exon-skipping or alternative utilization of the untranslated 5'-exons.

Base Sequence↗

Discovery of differentially expressed genes in human breast cancer using subtracted cDNA libraries and cDNA microarrays.

Identifying novel and known genes that are differentially expressed in breast cancer has important implications in understanding the biology of breast tumorigenesis and developing new diagnostic and therapeutic agents. In this study we have combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarray, as a high throughput methodology designed to identify cDNA clones that are breast tumor- and tissue-specific and are overexpressed in breast tumors. Approximately 2000 cDNA clones generated from the subtracted breast tumor library were arrayed on the microarray chips. The arrayed target cDNAs were then hybridized with 30 pairs of fluorescent-labeled cDNA probes generated from breast tumors and normal tissues to determine the tissue distribution and tumor specificity. cDNA clones showing overexpression in breast tumors by microarray were further analysed by DNA sequencing, GenBank and EST database searches, and quantitative real time PCR. We identified several known genes, including mammaglobin, cytokeratin 19, fibronectin, and hair-specific type II keratin, which have previously been shown to be overexpressed in breast tumors and may play an important role in the malignance of breast. We also discovered B726P which appears to be an isoform of NY-BR-1, a breast tissue-specific gene. Two additional clones discovered, B709P and GABA(A) receptor pi subunit, were not previously described for their overexpression profile in breast tumors. Thus, combining PCR-based cDNA subtraction and cDNA microarray allowed for an efficient way to identify and validate genes with elevated mRNA expression levels in breast cancer that may potentially be involved in breast cancer progression. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for breast cancer.

Amino Acid Sequence↗

Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells.

A cDNA sequence coding for mouse interleukin 2 (IL-2) has been cloned from a cDNA library prepared from mRNA derived from a concanavalin A-activated mouse T-cell clone. The library was constructed by using the pcD vector system, which permits the expression of cDNA inserts in mammalian cells. Screening of the library was performed by transfecting COS-7 monkey cells with pools of cDNA clones in order to express the products encoded by full-length cDNA inserts. By assaying the supernatant fluid, IL-2 cDNA clones that express T-cell growth-factor (TCGF) activity were identified. The DNA sequence codes for a polypeptide of 169 amino acid residues including a putative signal peptide. The mouse IL-2 amino acid sequence deduced from the nucleotide sequence of its cDNA shares extensive homology with the human IL-2 amino acid sequence reported previously. These results demonstrate that identification of full-length cDNA clones for many lymphokines may be achieved entirely on the basis of detection of the functional polypeptides in mammalian cells.

Amino Acid Sequence↗

Complete cDNA sequence and cDNA-directed expression of CYP4A11, a fatty acid omega-hydroxylase expressed in human kidney.

A cDNA was isolated from a human kidney lambda gt10 library using the rat CYP4A3 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 519 amino acids that was designated CYP4A11 (Nelson et al., 1993) and exhibited 76%, 72%, 80%, and 53% similarities to rat CYP4A1, rat CYP4A3, rabbit CYP4A6, and human CYP4B1, respectively. The deduced amino-terminal amino acid sequence of this cDNA agreed with the amino-terminal amino acid sequence of a major P450 protein purified from human renal microsomes. A second variant form of CYP4A11 cDNA, designated CYP4A11v, was isolated from the same library and had a deletion of a single adenine residue, thereby extending the reading frame and resulting in a protein of 591 amino acids. CYP4A11v is probably encoded by a rare allelic variant of CYP4A11, since no mutant alleles were uncovered in 15 normal individuals, as determined by a polymerase chain reaction (PCR) diagnostic test. Baculovirus-mediated cDNA expression of CYP4A11 yielded a P450 protein having a lambda max of 452 nm when reduced and complexed with carbon monoxide. The expressed enzyme efficiently catalyzed omega-hydroxylation of lauric acid. No detectable activity was uncovered toward arachidonic acid and prostaglandin E1. The cDNA-expressed variant, CYP4A11v, was found to be unstable and not to efficiently metabolize lauric acid, as assessed by both baculovirus and monkey kidney COS cell cDNA expression systems. These studies indicate that CYP4A11 is a major fatty acid-metabolizing P450 that is expressed in human kidney.

Amino Acid Sequence↗

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.

Adult↗

Evaluation of a cDNA scanning method concerning the fidelity and efficiency of cDNA selection using the YAC CIC3B1-S region of Arabidopsis thaliana chromosome 5.

We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated. We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region. In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones. Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein. We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region. We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning.

Arabidopsis↗

Construction of expression-ready cDNA clones for KIAA genes: manual curation of 330 KIAA cDNA clones.

We have accumulated information on protein-coding sequences of uncharacterized human genes, which are known as KIAA genes, through cDNA sequencing. For comprehensive functional analysis of the KIAA genes, it is necessary to prepare a set of cDNA clones which direct the synthesis of functional KIAA gene products. However, since the KIAA cDNAs were derived from long mRNAs (> 4 kb), it was not expected that all of them were full-length. Thus, as the first step toward preparing these clones, we evaluated the integrity of protein-coding sequences of KIAA cDNA clones through comparison with homologous protein entries in the public database. As a result, 1141 KIAA cDNAs had at least one homologous entry in the database, and 619 of them (54%) were found to be truncated at the 5' and/or 3' ends. In this study, 290 KIAA cDNA clones were tailored to be full-length or have considerably longer sequences than the original clones by isolating additional cDNA clones and/or connected parts of additional cDNAs or PCR products of the missing portion to the original cDNA clone. Consequently, 265, 8, and 17 predicted CDSs of KIAA cDNA clones were increased in the amino-, carboxy-, and both terminal sequences, respectively. In addition, 40 cDNA clones were modified to remove spurious interruption of protein-coding sequences. The total length of the resultant extensions at amino- and carboxy-terminals of KIAA gene products reached 97,000 and 7,216 amino acid residues, respectively, and various protein domains were found in these extended portions.

Cloning, Molecular↗

Analysis of full-length cDNA clones carrying GAL1 of Saccharomyces cerevisiae: a model system for cDNA expression.

A cDNA cloning vector that allows expression in Saccharomyces cerevisiae has been developed using the plasmid primer approach described by Okayama and Berg [Mol. Cell. Biol. 2:161-170(1982)]. The vector contains ARS1 and TRP1 for plasmid maintenance in yeast and the ADC1 or GAL1 promoter and the TRP5 terminator for expression of the cloned cDNA. Using this system, several recombinants with nearly full-length GAL1 cDNA inserts in a cDNA library made with galactose-induced yeast mRNA were identified. By measurement of galactokinase mRNA and its protein, the expression of GAL1 cDNA was shown to be under the control of the promoter placed upstream of the cDNA insert. Nucleotide sequence analysis revealed that the 3'-ends of the GAL1 cDNA inserts were not unique, indicating that polyA tails were added to GAL1 transcripts at multiple sites in the GAL1 gene. Genetic complementation of appropriate yeast mutants permitted the isolation of clones containing the coding sequences for GAL1, HIS3, and LEU2 from the same cDNA library.

Base Sequence↗

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP.

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Animals↗