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Insulin induces the expression of the SHARP-2/Stra13/DEC1 gene via a phosphoinositide 3-kinase pathway.

Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high carbohydrate diet. A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified. Using multiple copies of the ChoRE as the bait in a yeast one-hybrid system, a rat liver cDNA library was screened, and the cDNA of ChoRE-binding proteins was cloned. A positive clone that encodes a basic helix-loop-helix protein, enhancer of split- and hairy-related protein-2 (SHARP-2), was obtained. Northern blot analysis revealed that the levels of SHARP-2 mRNA increase when a high carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats. In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA even in the absence of glucose. A time course for the increase in SHARP-2 mRNA levels indicated that it followed by those of FAS and L-type pyruvate kinase mRNAs and that the initial time course of SHARP-2 mRNA was similar to changes in the levels of glucokinase mRNA and phosphoenolpyruvate carboxykinase mRNA. Although wortmannin, LY294002, and actinomycin D blocked the increase in SHARP-2 mRNA levels by insulin, rapamycin, staurosporine, PD98059, okadaic acid, and 8-bromocyclic AMP had no effect. In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin. Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway.

Animals↗

Production of anti-fibroblast growth factor receptor monoclonal antibodies by in vitro immunization.

Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes involved in FGF binding and signal transduction which mediate coronary and tumor angiogenesis (the development of blood vessels). Monoclonal antibodies against the FGF receptor were made by in vitro immunization. Receptor was PAGE-purified and transblotted to nitrocellulose. The nitrocellulose was dissolved with acetonitrile, and the receptor used as antigen in an in vitro immunization system. Screening for FGF receptor positive clones was done using membrane preparations from Coronary Venular Endothelial Cells (CVEC) expressing the FGF receptor, both by Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. Culture supernatants of several clones tested positive for IgM or IgG monoclonal antibodies against the FGF receptor. Antibodies were affinity purified. Ascites fluid was produced in Balb/c mice primed with Incomplete Freund's adjuvant (IFA). The monoclonal antibodies were also found to be suitable for receptor immunoprecipitation. Immunocytochemistry was done on sections from a variety of species. Further characterization of these antibodies, as well as the production of the remainder of a panel of anti-FGF receptor monoclonal antibodies, is underway.

Animals↗

Production and characterization of monoclonal antibodies to mesenchymal stem cells derived from human bone marrow.

The bone marrow (BM) serves as a reservoir for different classes of stem cells. In addition to haematopoietic stem cells, bone marrow contains a population of mesenchymal stem cells (MSCs). These cells have a multilineage differentiation capacity and are able to generate progenitors with restricted developmental potential, which include fibroblasts, osteoblasts, adipocytes, and chondrocytes. Characteristic markers have been reported for expanded MSCs, but none of these markers are specific for MSCs. Thus, the objective of this study was to produce monoclonal antibodies against MSCs. MSCs derived from human bone marrow were cultured, expanded, and immunized into mice, and spleen cells subsequently harvested were used to generate hybridoma cell lines secreting antibodies against MSCs. Hybridoma culture supernatants were screened for antibodies against MSCs by enzyme-linked immunosorbent assay (ELISA), and 33 positive clones were then screened against cell suspensions of MSCs by immunofluorescence staining and flow cytometry. Ten clones were positive in immunofluorescence staining. Among these, three hybridoma cell lines, namely, YS08, YS14, and YS18 were found to be reactive with MSC by flow cytometry, but non-reactive with human tumor cell lines and hematopoietic stem cells. YS08 and YS14 showed specific bands in Western blotting. In conclusion, we developed three monoclonal antibodies, YS08, YS14, and YS18, that recognize human MSC cell surface antigen.

Antibodies, Monoclonal↗

GeneMachine: gene prediction and sequence annotation.

MOTIVATION: A number of free-standing programs have been developed in order to help researchers find potential coding regions and deduce gene structure for long stretches of what is essentially 'anonymous DNA'. As these programs apply inherently different criteria to the question of what is and is not a coding region, multiple algorithms should be used in the course of positional cloning and positional candidate projects to assure that all potential coding regions within a previously-identified critical region are identified. RESULTS: We have developed a gene identification tool called GeneMachine which allows users to query multiple exon and gene prediction programs in an automated fashion. BLAST searches are also performed in order to see whether a previously-characterized coding region corresponds to a region in the query sequence. A suite of Perl programs and modules are used to run MZEF, GENSCAN, GRAIL 2, FGENES, RepeatMasker, Sputnik, and BLAST. The results of these runs are then parsed and written into ASN.1 format. Output files can be opened using NCBI Sequin, in essence using Sequin as both a workbench and as a graphical viewer. The main feature of GeneMachine is that the process is fully automated; the user is only required to launch GeneMachine and then open the resulting file with Sequin. Annotations can then be made to these results prior to submission to GenBank, thereby increasing the intrinsic value of these data. AVAILABILITY: GeneMachine is freely-available for download at http://genome.nhgri.nih.gov/genemachine. A public Web interface to the GeneMachine server for academic and not-for-profit users is available at http://genemachine.nhgri.nih.gov. The Web supplement to this paper may be found at http://genome.nhgri.nih.gov/genemachine/supplement/.

Animals↗

Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization.

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.

Chromosomes, Bacterial↗

Cloning and expression of Treponema pallidum (Nichols) antigen genes in Escherichia coli.

Hybrid pBR322 plasmid clone banks comprised of more than 125,000 recombinant DNA clones and representing the entire Treponema pallidum Nichols genome were constructed in Escherichia coli K-12 RR1. The two clone banks individually contain over 53,000 and 72,000 recombinant clones. The number average and mass average sizes of the cloned DNA inserts were found to be approximately 12 and 13 kilobase pairs, respectively, indicating the presence of large treponemal DNA inserts in a majority of recombinant clones. To detect E. coli clones synthesizing T. pallidum antigens as hybrid plasmid gene translation products in the clone bank, a simplified, direct, solid-phase radioimmuno-colony blot (RICB) assay was developed employing immunoglobulin G antibody isolated from anti-T. pallidum immune rabbit serum. Clones with positive reactivities in the RICB assay were isolated at frequencies of 0.1 to 0.2%. One isolated RICB-positive clone, designated RICB2-1, produced a very strong signal in the RICB assay and was subsequently found through E. coli cell-free in vitro transcription/translation analysis to encode the synthesis of two gene translation products with apparent molecular weights of 77,000 and 44,000. The 44,000-dalton protein was effectively immunoprecipitated from [35S]methionine-labeled E. coli clone cells by using either immune rabbit serum (preabsorbed with Treponema phagedenis biotype Reiter antigens) or selected human syphilitic serum, whereas the 77,000-dalton protein was never immunoprecipitable by similar methods. Purified plasmid DNA from clone RICB2-1 contained a treponemal DNA insert of 3.70 kilobase pairs, which was of suitable size to code for the 121-dalton (44 + 77) protein. The insert was also flanked on each end by PstI sites and possessed three internal PstI sites with fragment sizes of 2.15, 1.18, 0.20, and 0.17 kilobase pairs. Purified clone RICB2-1 plasmid DNA was capable of transforming recipient E. coli cells to virtually 100% RICB reactivity, thus substantiating the plasmid-encoded characteristic. Further experiments employing various antisera in radioimmunoprecipitation systems utilizing cell-free in vitro synthesized gene translation products from clone RICB2-1 also provided the first evidence that E. coli may be capable of using endogenous T. pallidum DNA promotors for genetic expression. These studies, amplified by the isolation of a potentially significant immunoprecipitable 44,000-dalton recombinant protein antigen, point to the importance of the "cloned antigen gene" approach for the eventual eludication of specific antigens or immunogens operative in the pathogenesis, immunology, and serodiagnosis of T. pallidum infection.

Antigens, Bacterial↗

Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line.

A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.

Amino Acid Sequence↗

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

Adipose Tissue↗

A large set of Finnish affected sibling pair families with type 2 diabetes suggests susceptibility loci on chromosomes 6, 11, and 14.

The aim of the Finland-United States Investigation of NIDDM Genetics (FUSION) study is to identify genes that predispose to type 2 diabetes or are responsible for variability in diabetes-related traits via a positional cloning and positional candidate gene approach. In a previously published genome-wide scan of 478 Finnish affected sibling pair (ASP) families (FUSION 1), the strongest linkage results were on chromosomes 20 and 11. We now report a second genome-wide scan using an independent set of 242 Finnish ASP families (FUSION 2), a detailed analysis of the combined set of 737 FUSION 1 + 2 families (495 updated FUSION 1 families), and fine mapping of the regions of chromosomes 11 and 20. The strongest FUSION 2 linkage results were on chromosomes 6 (maximum logarithm of odds score [MLS] = 2.30 at 95 cM) and 14 (MLS = 1.80 at 57 cM). For the combined FUSION 1 + 2 families, three results were particularly notable: chromosome 11 (MLS = 2.98 at 82 cM), chromosome 14 (MLS = 2.74 at 58 cM), and chromosome 6 (MLS = 2.66 at 96 cM). We obtained smaller FUSION 1 + 2 MLSs on chromosomes X (MLS = 1.27 at 152 cM) and 20p (MLS = 1.21 at 20 cM). Among the 10 regions that showed nominally significant evidence for linkage in FUSION 1, four (on chromosomes 6, 11, 14, and X) also showed evidence for linkage in FUSION 2 and stronger evidence for linkage in the combined FUSION 1 + 2 sample.

Age of Onset↗

[Molecular cloning of EXT2 and EXT4 gene].

Hereditary multiple exostose(EXT) is an autosomal dominant disorder of skeletal system. Three genetic loci have been identified at 8q24.1(EXT1), 11p11(EXT2) and 19p(EXT3) respectively. In this paper, EXT2 gene was cloned with positional cloning and homologous screening. SSCP and sequencing analysis have been done in 37 EXT patients who came from 20 EXT families, 2 mutations of insertion were tested in 2 patients. This confirmed that the gene cloned in this paper was EXT2 gene which locus at 11p11. Additionally EXT4 gene was cloned with homologous screening and located at 1p36.1 with FISH in this paper.

Amino Acid Sequence↗

[Construction of a bacterial artificial chromosome (BAC) contig encompassing the bacterial blight resistance gene Xa4 locus in rice].

The gene Xa4 confers dominantly resistance to rice bacterial blight, which has been finely mapped between RFLP markers G181 and L1044, and co-segregated with the resistance gene homologues sequence marker RS13. The three markers were used to screen a rice Bacterial Artificial Chromosome (BAC) library constructed from IRBB56, a Xa4-harborring indica variety, resulting in the detection of totally 128 positive clones. Of the 18 positive clones picked out by RS13, 4 and 6 clones were simultaneously detected by G181 and L1044, respectively. Based on their HindIII restriction patterns, 12 clones were selected out to construct a contig that spanned about 420 kb covering the Xa4 locus, which is a solid base for the isolation of Xa4 gene.

Chromosomes, Artificial, Bacterial↗

[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].

A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.

Base Sequence↗

[Construction of fetal mesenchymal stem cell cDNA subtractive library].

UNLABELLED: To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. CONCLUSION: SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

Cloning, Molecular↗

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.

Amino Acid Sequence↗

[Screening of TNF-alpha mimotopes from c7c phage display peptide library].

AIM: To acquire oligo-peptides mimicking TNF-alpha epitopes. METHODS: The TNF-alpha mimotopes were screened from c7c phage display peptide library by using neutralizing mAb J1D9 against TNF-alpha, and positive clones were identified by sandwich ELISA. RESULTS: 9 out of 20 phage clones were identified as positive clones which can bind to mAb J1D9. The deduced amino acid sequence analysis showed three different sequences: c-RRPAQSG-c, c-NKHNRKI-c and c-RGMSRKI-c. CONCLUSION: All of the three peptides obtained can mimic the epitopes of TNF-alpha and bind to mAb J1D9.

Amino Acid Sequence↗

[Phylogenetic relationship between tufted deer(Elaphodus cephalophus) and Muntiacus deer is revealed by the exon and intron of K+ channel gene].

In this study, partial fragments of potassium ion channel gene were amplified using the genomic DNA of muntjak, reevesi, crinifrons, and Elaphodus cephalophus. The PCR products were ligated to the plasmid of pMD18-T Vector by the method of direct T-A cloning. The positive clones were identified by colony PCR. The sequences of the recombinant clones were determined using M13-47/RV-M universal primers and aligned by the software CLUSTALW. The nucleotide divergences of exon were 0.90%-1.44% among three species of Muntiacus, 0.90%-1.26% between E. cephalophus and each of Muntiacus deer. In the nucleotide of intron there is 0%-1.22% difference among these muntjac deers, and the divergene reached about 1.83% between E. cephalophus and the three species of Muntiacus. Using the software of MEGA to analyse molecular phylogeny, Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. The result showed Muntiacus, crinifrons is most closely related to muntjak, with reevesi as their sister species. E. cephalophus is in the other genus.

Animals↗

[Identification of up-regulated genes induced by angiotensin II in cardiac fibroblasts].

To identify up-regulated genes in adult rat cardiac fibroblasts (CF) induced by angiotensin II (Ang II), suppression subtractive hybridization (SSH) was performed between the CF stimulated by Ang II (tester) and unstimulated CF (driver) to generate subtractive cDNA library. The library was screened with dot blots hybridization to further verify the differentially expressed cDNA clones. Partial positive clones (19 up-regulated genes) were sequenced and BLAST analyzed. Twelve up-regulated genes related to extracellular matrix, cell cycle, intracellular signal transduction, cell cytoskeleton, cell metabolism and 7 new expressed sequence tags (EST) were acquired (GenBank accession number: CN382808, CN382809, CN382810, CN382811, CN382812, CN382813, CN382814). Our data reveal that SSH is a powerful technique of high sensitivity for the detection and cloning of up-regulated genes expressed in CF induced by Ang II, which may be helpful to clarify the mechanism of cardiac remodeling.

Angiotensin II↗

[Screening and preliminary identification of mimetic peptides of Plasmodium falciparum-infected erythrocyte membrane surface protein 1].

OBJECTIVE: To screen and identify mimetic peptides of Plasmodium falciparun-infected erythrocyte membrane surface protein 1 in order to explore anti-adhesive agent against cerebral malaria. METHODS: Phage-borne peptide KLYLIAEGSVAA was used as panning targets to select target binders in a disulfide-constrained heptapeptides library. Three rounds of biopanning were carried out and then ELISA and competitive ELISA were used to evaluate the binding character between phage-borne peptides and ICAM-1. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. RESULTS: After three-round panning, 22 clones were randomly chosen from the third panning and analyzed. Three clones showed positive interaction with ICAM-1, and two of them possessed the amino acid sequence C-ITAVPVR-C, the other one was C-DIMGGYN-C. These peptides specifically inhibited the binding of 15.2 antibody to ICAM-1 detected by competitive ELISA. CONCLUSION: Two kinds of mimetic peptides of PfEMP-1 have been obtained, which can bind with ICAM-1 specifically.

Amino Acid Sequence↗