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PubMed2005

Molecular methods for the identification of Aspergillus species.

Invasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. In some institutions, species of Aspergillus less susceptible to amphotericin B than Aspergillus fumigatus are becoming more common, making an accurate identification of species important. However, species identification has traditionally relied on macroscopic colony characteristics and microscopic morphology, which may require several days of culture. Additional sub-culturing on specialized media may be required to induce conidia formation; in some cases conidia may never form, confounding identification. Therefore, rapid, nucleic acid-based methods that identify species of Aspergillus independent of morphology are now being developed to augment or replace phenotypic identification methods. The most successful methods to date have employed polymerase chain reaction (PCR) amplification of target sequences within the ribosomal RNA gene complex, including the 28S ribosomal subunit (D1-D2 region) and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). We therefore developed a PCR-based assay to differentiate medically important species of Aspergillus from one another, and from other opportunistic moulds and yeasts, by employing universal, pan-fungal primers directed to conserved ribosomal genes and species-specific DNA probes directed to the highly variable ITS2 region. Amplicons were then detected in a simple, colorimetric enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 and ITS2 regions and of the D1-D2 region was also conducted for the differentiation of species by comparative GenBank sequence analysis. The PCR-EIA method was found to be rapid, sensitive, and specific for the identification and differentiation of the most medically important species of Aspergillus. In addition, methods to identify species of Aspergillus by comparative GenBank sequence analysis were found to be more reliable using the ITS1 and ITS2 regions than the D1-D2 region.

Aspergillosis↗

PubMedDate not supplied

Cooling towers--a potential environmental source of slow-growing mycobacterial species.

Over the last decade a rise in the frequency of disease caused by nontuberculous mycobacteria (NTM) has occurred, especially among AIDS patients. The lack of evidence for person-to-person transmission indicates the environment is a source of infection. The ecology and environmental sources of NTMs are poorly understood, and many pathogenic strains have not been observed outside of clinical cases. Several species of NTMs have been reported from treated water distribution systems; however, one type of manmade environment that has not been examined for mycobacteria is that of cooling towers of air-conditioning systems. Such environments not only harbor a variety of microbial species, they also disseminate them in aerosols. The present investigation examined nine cooling towers from various locations in the United States. Cooling tower water was concentrated, treated with cetylpyridinium chloride, and plated onto Middlebrook 7H10 agar supplemented with OADC and cycloheximide. Colonies presumed to be mycobacterial species were isolated and acid-fast stained. Identification was made by amplifying and sequencing 1450 bp fragments of the 16S rRNA gene in both directions, and comparing resulting sequences with those in GenBank. Results showed that at least 75% of tower samples contained NTMs, and most of the isolates closely matched known mycobacterial pathogens. Isolates most closely matched the following GenBank sequences: Mycobacterium intracellulare, M. szulgai, M. bohemicum, M. gordonae, M. nonchromogenicum, and M. n. sp. "Fuerth 1999." This is the first report of specific NTMs in cooling tower water, and the first report of M. n. sp. "Fuerth 1999" from any environmental sample. Although cooling towers have a relatively high pH, they may favor the growth and dissemination of such potential pathogens, and future epidemiologic investigations should consider cooling towers as possible environmental sources of mycobacteria.

Air Conditioning↗

PubMed1997

Isoforms of ankyrin-3 that lack the NH2-terminal repeats associate with mouse macrophage lysosomes.

We have recently cloned and characterized ankyrin-3 (also called ankyrin(G)), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313-330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29-amino acid segment of the membrane ("repeat") domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrin(R)), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3-positive vesicles appear dark on phase-contrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran-containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3-positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.

Amino Acid Sequence↗

PubMed2001

Genomic divergences between humans and other hominoids and the effective population size of the common ancestor of humans and chimpanzees.

To study the genomic divergences among hominoids and to estimate the effective population size of the common ancestor of humans and chimpanzees, we selected 53 autosomal intergenic nonrepetitive DNA segments from the human genome and sequenced them in a human, a chimpanzee, a gorilla, and an orangutan. The average sequence divergence was only 1.24% +/- 0.07% for the human-chimpanzee pair, 1.62% +/- 0.08% for the human-gorilla pair, and 1.63% +/- 0.08% for the chimpanzee-gorilla pair. These estimates, which were confirmed by additional data from GenBank, are substantially lower than previous ones, which included repetitive sequences and might have been based on less-accurate sequence data. The average sequence divergences between orangutans and humans, chimpanzees, and gorillas were 3.08% +/- 0.11%, 3.12% +/- 0.11%, and 3.09% +/- 0.11%, respectively, which also are substantially lower than previous estimates. The sequence divergences in other regions between hominoids were estimated from extensive data in GenBank and the literature, and Alus showed the highest divergence, followed in order by Y-linked noncoding regions, pseudogenes, autosomal intergenic regions, X-linked noncoding regions, synonymous sites, introns, and nonsynonymous sites. The neighbor-joining tree derived from the concatenated sequence of the 53 segments--24,234 bp in length--supports the Homo-Pan clade with a 100% bootstrap value. However, when each segment is analyzed separately, 22 of the 53 segments (approximately 42%) give a tree that is incongruent with the species tree, suggesting a large effective population size (N(e)) of the common ancestor of Homo and Pan. Indeed, a parsimony analysis of the 53 segments and 37 protein-coding genes leads to an estimate of N(e) = 52,000 to 96,000. As this estimate is 5 to 9 times larger than the long-term effective population size of humans (approximately 10,000) estimated from various genetic polymorphism data, the human lineage apparently had experienced a large reduction in effective population size after its separation from the chimpanzee lineage. Our analysis assumes a molecular clock, which is in fact supported by the sequence data used. Taking the orangutan speciation date as 12 to 16 million years ago, we obtain an estimate of 4.6 to 6.2 million years for the Homo-Pan divergence and an estimate of 6.2 to 8.4 million years for the gorilla speciation date, suggesting that the gorilla lineage branched off 1.6 to 2.2 million years earlier than did the human-chimpanzee divergence.

Alu Elements↗

PubMed2001

Enteropathogenic Escherichia coli and ulcerative colitis in cotton-top tamarins (Saguinus oedipus).

The cotton-top tamarin (CTT; Saguinus oedipus) is an endangered New World primate that develops a highly prevalent idiopathic colitis resembling human ulcerative colitis. This study found that enteropathogenic Escherichia coli (EPEC) caused acute colitis in CTTs, which was associated with ulcerative colitis. EPEC clinical isolates revealed localized adherence patterns by HEp-2 assay and were devoid of Shiga-toxin production. Sequencing of the eae gene (GenBank accession no. AF319597) revealed 99.2% identity to sequences of human isolates (GenBank AF116899) and corresponded to the epsilon intimin gene subtype. Detection of intimin sequences by polymerase chain reaction on primary fecal cultures indicated widespread EPEC infection in the CTT colony. Prospective analysis revealed that animals with fecal cultures positive for intimin sequences had a higher frequency of active colitis (75.0% vs. 27.2%; P<.005, chi(2) test) and higher histological scores of colonic inflammation (0.875 vs. 0.455, respectively; P<.05, Mann-Whitney rank sum test).

Adhesins, Bacterial↗

PubMed2000

Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region.

A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized. This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids). An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene. Sequence analysis revealed three ORFs upstream of the gene. The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A. tumefaciens. In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions. These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B. henselae and other gram-negative bacteria such as A. tumefaciens.

Agrobacterium tumefaciens↗

PubMed2003

Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea.

A total of 1,467 tick (1,463 of Haemaphysalis longicornis, three of Ixodes persulcatus and one of I. turdus) collected from nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia and Anaplasma species. One set of primers and a probe were designed for detection of all of the Ehrlichia and Anaplasma species. Template DNAs (total 803) were prepared either from pools of larvae, nymphs, adult males and females, or from the salivary gland and midgut of adult ticks. Only DNAs positive in TaqMan PCR were examined for A. phagocytophilum with nested PCR and for E. chaffeensis with PCR. Four A. phagocytophilum 16S rRNA gene PCR products were sequenced for comparison with sequences previously reported. Amplification of a 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (45.3% of the total). Of these 364 positive ticks, species-specific PCRs confirmed that 35 H. longicornis and one I. persulcatus were positive for A. phagocytophilum and one I. persulcatus was positive in E. chaffeensis. Except for one (AB-GGHL, GenBank accession number [GAN] AF470698), three of the four 16S rRNA gene fragment sequences of the A. phagocytophilum-positive samples were similar or identical to the sequences of variants of A. phagocytophilum deposited in GenBank. The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma (Ehrlichia) bovis 16S rRNA (GAN U03775). The identities of the Anaplasmataceae genus and species DNA in the 327 ticks that could not be confirmed infected with either E. chaffeensis, A. phagocytophilum, or A. bovis are not known. This study is the first to demonstrate the presence of E. chaffeensis, A. phagocytophilum and A. bovis in Korean ticks.

Anaplasma↗

PubMed1994

Biological evaluation of d2, an algorithm for high-performance sequence comparison.

A number of algorithms exist for searching sequence databases for biologically significant similarities based on the primary sequence similarity of aligned sequences. We have determined the biological sensitivity and selectivity of d2, a high-performance comparison algorithm that rapidly determines the relative dissimilarity of large datasets of genetic sequences. d2 uses sequence-word multiplicity as a simple measure of dissimilarity. It is not constrained by the comparison of direct sequence alignments and so can use word contexts to yield new information on relationships. It is extremely efficient, comparing a query of length 884 bases (INS1ECLAC) with 19,540,603 bases of the bacterial division of GenBank (release 76.0) in 51.77 CPU seconds on a Cray Y/MP-48 supercomputer. It is unique in that subsequences (words) of biological interest can be weighted to improve the sensitivity and selectivity of a search over existing methods. We have determined the ability of d2 to detect biologically significant matches between a query and large datasets of DNA sequences while varying parameters such as word-length and window size. We have also determined the distribution of dissimilarity scores within eukaryotic and prokaryotic divisions of GenBank. We have optimized parameters of the d2 program using Cray hardware and present an analysis of the sensitivity and selectivity of the algorithm. A theoretical analysis of the expectation for scores is presented. This work demonstrates that d2 is a unique, sensitive, and selective method of rapid sequence comparison that can detect novel sequence relationships which remain undetected by alternate methodologies.

Algorithms↗

PubMed1993

An intracisternal A-particle DNA sequence is closely linked to the thrombomodulin gene in some strains of laboratory mice.

Sequence analysis of the 5' end of the thrombomodulin (TM) gene has identified a difference in two strains of laboratory mice, BALB/cBD (maintained at the Oak Ridge National Laboratory) and the strain used for the sequence listed in the GenBank data base. An intracisternal A-particle (IAP) provirus was present in the BALB/cBD sequence but absent from the GenBank sequence. Thus, there are two different 5' regions for the TM gene in mice. Southern blot and polymerase chain reaction analyses on other mouse strains demonstrated the presence of this proviral sequence in five out of ten strains tested. Northern blot analyses demonstrated that there were differences (up to three-fold) in the steady-state levels of TM mRNA among the strains. However, TM mRNA levels did not correlate directly with the presence of the IAP DNA upstream from the TM gene. This is in contrast to other loci (the interleukin-3 gene, c-mos, and the immunoglobulin Ck gene), in which the IAP element is known to affect levels of RNA transcription. While the IAP provirus does not appear to have an effect on TM transcription under normal conditions, it is possible that transcription levels altered by factors like thrombin and tumor necrosis factor are affected by the IAP provirus.

Animals↗

PubMed2004

Molecular cloning of TSARG6 gene related to apoptosis in human spermatogenic cells.

Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accession No. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan program and PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns is located in human chromosome 11q13.3. Its protein containing 316 amino acid residues is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6 protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result of RT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and the transcript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene which probably inhibited human testis spermatogenesis apoptosis.

Amino Acid Sequence↗

PubMed2000

IE-Kb: intron exon knowledge base.

SUMMARY: IE-Kb (Intron Exon-Knowledge base) illustrates the intron-exon dynamics in eukaryotic genes. We have developed three different knowledge sets, namely 'Non-redundant ExInt', 'Non-redundant Pfam-ExInt complement' and 'Non-redundant GenBank eukaryotic subdivisional sets' to understand this phenomenon. Statistical analysis is performed on each knowledge set and the results are made available online. The entries in knowledge sets are ranked based on their intron length, exon length and protein length with relational hyper-links to the corresponding intron phase, intron position, intron sequence, gene definition and parent GenBank entry.

Artificial Intelligence↗

PubMed2000

Object-oriented parsing of biological databases with Python.

MOTIVATION: While database activities in the biological area are increasing rapidly, rather little is done in the area of parsing them in a simple and object-oriented way. RESULTS: We present here an elegant, simple yet powerful way of parsing biological flat-file databases. We have taken EMBL, SWISSPROT and GENBANK as examples. EMBL and SWISS-PROT do not differ much in the format structure. GENBANK has a very different format structure than EMBL and SWISS-PROT. Extracting the desired fields in an entry (for example a sub-sequence with an associated feature) for later analysis is a constant need in the biological sequence-analysis community: this is illustrated with tools to make new splice-site databases. The interface to the parser is abstract in the sense that the access to all the databases is independent from their different formats, since parsing instructions are hidden.

Databases, Factual↗

PubMed2001

Extending traditional query-based integration approaches for functional characterization of post-genomic data.

MOTIVATION: To identify and characterize regions of functional interest in genomic sequence requires full, flexible query access to an integrated, up-to-date view of all related information, irrespective of where it is stored (within an organization or across the Internet) and its format (traditional database, flat file, web site, results of runtime analysis). Wide-ranging multi-source queries often return unmanageably large result sets, requiring non-traditional approaches to exclude extraneous data. RESULTS: Target Informatics Net (TINet) is a readily extensible data integration system developed at GlaxoSmith- Kline (GSK), based on the Object-Protocol Model (OPM) multidatabase middleware system of Gene Logic Inc. Data sources currently integrated include: the Mouse Genome Database (MGD) and Gene Expression Database (GXD), GenBank, SwissProt, PubMed, GeneCards, the results of runtime BLAST and PROSITE searches, and GSK proprietary relational databases. Special-purpose class methods used to filter and augment query results include regular expression pattern-matching over BLAST HSP alignments and retrieving partial sequences derived from primary structure annotations. All data sources and methods are accessible through an SQL-like query language or a GUI, so that when new investigations arise no additional programming beyond query specification is required. The power and flexibility of this approach are illustrated in such integrated queries as: (1) 'find homologs in genomic sequence to all novel genes cloned and reported in the scientific literature within the past three months that are linked to the MeSH term 'neoplasms"; (2) 'using a neuropeptide precursor query sequence, return only HSPs where the target genomic sequences conserve the G[KR][KR] motif at the appropriate points in the HSP alignment'; and (3) 'of the human genomic sequences annotated with exon boundaries in GenBank, return only those with valid putative donor/acceptor sites and start/stop codons'.

Animals↗

PubMed2002

A kinase sequence database: sequence alignments and family assignment.

UNLABELLED: The Kinase Sequence Database (KSD) located at http://kinase.ucsf.edu/ksd contains information on 290 protein kinase families derived by profile-based clustering of the non-redundant list of sequences obtained from a GenBank-wide search. Included in the database are a total of 5,041 protein kinases from over 100 organisms. Clustering into families is based on the extent of homology within the kinase catalytic domain (250-300 residues in length). Alignments of the families are viewed by interactive Excel-based sequence spreadsheets. In addition, KSD features evolutionary trees derived for each family and detailed information on each sequence as well as links to the corresponding GenBank entries. Sequence manipulation tools, such as evolutionary tree generation, novel sequence assignment, and statistical analysis, are also provided. AVAILABILITY: The kinase sequence database is a web-based service accessible at http://kinase.ucsf.edu/ksd CONTACT: buzko@cmp.ucsf.edu; shokat@cmp.ucsf.edu/ksd

Cluster Analysis↗

PubMed1988

The Annotator's Assistant: an expert system for direct submission of genetic sequence data.

As DNA sequencing technology improves and more rapid techniques become routine in molecular biology labs, researchers need to expedite the incorporation of information into genetic sequence databases, such as GenBank, by directly submitting sequence data. The Annotator's Assistant is an expert system that runs on an IBM PC and helps the molecular biologist, who may have little knowledge of the structure or content of a GenBank entry, to construct a complete and valid sequence submission file. This expert system uses a simple molecular biology knowledge base and a selection of customized screen entry forms to guide the user through the entry and annotation of a sequence and its biological features. The system compiles information about the contributor, journal references, physical and functional characteristics of the nucleic acid, source organism and features, and checks it to eliminate incomplete answers and simple errors. Users supply input by answering direct and multiple-choice questions, selecting menu items and completing entry forms; on-line help is available. Users may also enter new or unusual information using generic forms. Several modules of the expert system were converted into Prolog programs and compiled, decreasing the running time significantly. The expert system rules and the data entry forms are easy to modify, update and customize for specific sequence classes.

Base Sequence↗

PubMed2004

A software program combining sequence motif searches with keywords for finding repeats containing DNA sequences.

MOTIVATION: One of the most interesting features of genomes (both coding and non-coding regions) is the presence of relatively short tandemly repeated DNA sequences known as tandem repeats (TRs). We developed a new PC-based stand-alone software analysis program, combining sequence motif searches with keywords such as organs, tissues, cell lines or development stages for finding exact, inexact and compound, TRs. Tandem Repeats Analyzer 1.5 (TRA) has several advanced repeat search parameters/options over other repeat finder programs as it does not only accept GenBank, FASTA and expressed sequence tag (EST) sequence files but also does analysis of multifiles with multisequences. Advanced user-defined parameters/options let the researchers use different motif lengths search criteria for varying motif lengths simultaneously. The outputs show statistical results to be evaluated by the user. The discovery of TRs in ESTs could be useful for both gene mapping and association studies and discovering TRs located in coding regions of important genes that are expressed under various conditions of environment, stress, organ, tissue and development stage. RESULTS: In this paper, we demonstrated applications of TRA using 175 899 ESTs sequences for three Arabidopsis spp. downloaded from GenBank. The EST-SSRs/ESTs ratios were found 43.1%, 15.3% and 2.34% in A.lyrata, A.thaliana and A.halleri, respectively. Analysis revealed that organs, tissues and development stages possessed different amounts of repeats and repeat compositions. This indicated that the distribution of TRs among the tissues or organs may not be random differing from the untranscribed repeats found in genomes. AVAILABILITY: The program can be obtained free by anonymous FTP from ftp.akdeniz.edu.tr/Araclar/TRA.

Arabidopsis↗

PubMed2005

DNA polymorphism detector: an automated tool that searches for allelic matches in public databases for discrepancies found in clone or cDNA sequences.

SUMMARY: DNA polymorphism detector (DPD) is a new web application developed to help automate the process of cDNA clone validation. DPD identifies and highlights discrepancies between any cDNA clone sequence and its expected reference sequence. To determine if these differences correspond to natural genetic polymorphisms (versus artifacts introduced during clone production or evaluation), DPD uses the discrepancies, along with flanking sequences, to search GenBank for identical matching strings. If matching DNA sequences are found, DPD verifies that they are from the same gene. The application then reports the discrepancy as a polymorphism along with the corresponding GenBank reference information. AVAILABILITY: DPD is currently hosted by the Harvard Institute of Proteomics at http://www.hip.harvard.edu

Cloning, Molecular↗

PubMed2005

Large-scale prokaryotic gene prediction and comparison to genome annotation.

MOTIVATION: Prokaryotic genomes are sequenced and annotated at an increasing rate. The methods of annotation vary between sequencing groups. It makes genome comparison difficult and may lead to propagation of errors when questionable assignments are adapted from one genome to another. Genome comparison either on a large or small scale would be facilitated by using a single standard for annotation, which incorporates a transparency of why an open reading frame (ORF) is considered to be a gene. RESULTS: A total of 143 prokaryotic genomes were scored with an updated version of the prokaryotic genefinder EasyGene. Comparison of the GenBank and RefSeq annotations with the EasyGene predictions reveals that in some genomes up to approximately 60% of the genes may have been annotated with a wrong start codon, especially in the GC-rich genomes. The fractional difference between annotated and predicted confirms that too many short genes are annotated in numerous organisms. Furthermore, genes might be missing in the annotation of some of the genomes. We predict 41 of 143 genomes to be over-annotated by >5%, meaning that too many ORFs are annotated as genes. We also predict that 12 of 143 genomes are under-annotated. These results are based on the difference between the number of annotated genes not found by EasyGene and the number of predicted genes that are not annotated in GenBank. We argue that the average performance of our standardized and fully automated method is slightly better than the annotation.

Base Composition↗