Download clustalw software

In , a new release was made, called ClustalV 7 , 8 , which incorporated profile alignments alignments of existing alignments and the facility to generate trees from the multiple alignment using the Neighbour-Joining NJ method 9.

The third generation of the series, ClustalW 10 , released in , incorporated a number of improvements to the alignment algorithm, including sequence weighting, position-specific gap penalties and the automatic choice of a suitable residue comparison matrix at each stage in the multiple alignment.

In addition, the approximate word search used for the pre-comparison step was replaced by a more sensitive dynamic programming algorithm, and the dendogram construction by UPGMA was replaced by NJ.

The ClustalW program looked very similar to ClustalV, with simple text menus for interactive use and the possibility of running the program in batch mode by specifying the input file and the parameter options on the command line.

The rationale behind the development of the Clustal series has been to provide robust, portable programs that are capable of providing good, biologically accurate alignments within a reasonable time limit. A close collaboration between biologists and computer scientists is probably one of the main reasons for the success and continued widespread use of the Clustal programs. ClustalW has given rise to a number of developments, including the latest member of the family, ClustalX Although the alignments produced are the same as those produced by the current release of ClustalW, the user can better evaluate alignments in ClustalX.

The program displays the multiple alignment in a scrollable window and all parameters are available using pull-down menus. Within alignments, conserved columns are highlighted using a customizable colour scheme and quality analysis tools are available to highlight potentially misaligned regions. Numerous options are provided, such as the realignment of selected sequences or selected blocks of the alignment and the possibility of building up difficult alignments piecemeal, making ClustalX an ideal tool for working interactively on alignments.

A number of other significant developments have been based on the ClustalW program. For example, ClustalNet 12 is a Clustal alignment CORBA server and DbClustal 13 is a program for aligning sequences detected by database searches, which uses local alignment information to anchor the global multiple alignment.

The EBI ClustalWWW interface provides extensive help, ranging from an introduction to multiple alignments for new users to detailed descriptions of each alignment option. An important factor in obtaining a high-quality alignment is the ability to change the numerous alignment parameters available in ClustalW.

While the default values of the parameters have been optimised to work in the majority of cases, they are not necessarily optimal for any given alignment problem.

In the ClustalWWW interface, all the options are easily accessible on the top page. Sequences can be entered by either pasting them or by uploading a file from the user's local computer. Although users are encouraged to submit large numbers of sequences, there is no guarantee that the alignment will be completed within the job run limits. Therefore, users who experience problems when attempting to make very large alignments are advised to download the software and run it locally.

In addition to the input format, the user can also specify the preferred output format for the multiple sequence alignment. It is also possible to configure the browser to automatically load the results files from ClustalW into a suitable external application.

Many commercial packages, e. The resulting multiple alignments can be displayed as either black and white or colour coded text. The alignment consists of four oxidoreductase NAD binding domains. The colouring of residues takes place according to physicochemical criteria highlighting conserved positions in the sequences. A multiple alignment of four oxidoreductase NAD binding domain protein sequences. The residue range for each sequence is shown after the sequence name.

JalView is a fully featured multiple sequence alignment editor which allows the user to perform further alignment analysis. Special features include the definition of sequence sub-groups, links to the SRS server at the EBI and an option to output the alignment as a colour postscript file for printing purposes.

As well as constructing multiple alignments, ClustalWWW can also calculate trees from a multiple alignment using the NJ method, a widely used and relatively fast algorithm that clusters sequences by minimising the sum of branch lengths. Both ClustalW and ClustalX are being actively maintained and updated.

Recent enhancements have included the possibility of saving both alignments and phylogenetic trees in the NEXUS format 14 for compatibility with a number of phylogeny programs. Some work has also been done to optimise the alignment parameters, for example the Gonnet series of residue comparison matrices 15 is now used by default for protein sequence alignments. Also, collinearity modules explained the difference in the position of the Wnt gene family in cattle relative to the other five species in Bovinae.

For instance, the position variation of Wnt2B , Wnt11 , Wnt1 and Wnt10B between Bos taurus and Bos grunniens might have been caused by complex intra-chromosomal translocation events Fig.

Wnt3 and Wnt9B are distributed on different chromosomes between cattle and buffalo bovine Chr 19 and buffalo Chr 3. This may be caused by inter-chromosomal rupture or fusion during the evolution Supplementary Info File 6. Collinearity analysis of Wnt genes between cattle and other organisms. Syntenic genes pairs are linked by grey lines whereas syntenic Wnt genes are shown as red lines.

Functionally related genes tend to show a co-expression patterns and often regulate biological processes collaboratively. To explore the expression patterns of the Wnt gene family, we investigated their expression levels in samples of 60 tissue types. The Wnt genes along with other 13 closely related genes can be classified into four groups I to IV Fig. Accordingly, the 60 bovine tissue types also clustered into four main clades a—d based on the expression patterns of all the 31 genes including Wnt family.

The members of the Wnt family and their receptors, the FZD gene family, displayed overlapping expression patterns, suggesting a coordinated regulatory role. Expression analysis of the Wnt gene family in different bovine tissue types. A Expression analysis of the Wnt gene family in 60 bovine tissues. The tissues were classified into 4 groups a to d and the 31 genes were also classified into 4 groups I—IV according to their expression pattern. B Expression analysis of the Wnt gene family in 5 bovine fat tissues.

The horizontal and vertical axis represent genes and bovine tissues, respectively. Knowledge of these patterns will provide useful information in bovine fat research. Meanwhile, the clustering analysis of tissue expression pattern revealed that intramuscular fat and subcutaneous fat of adult cattle got together firstly, indicating that they were the most similar among the five types of fat. This also suggests that primary adipocytes isolated from subcutaneous fat can be used for preliminary expression pattern validation of the Wnt gene family.

Meat tenderness and juiciness are affected by IMF content, whereas it is too limited to be sampled. Subcutaneous fat is significantly associated with IMF 28 , which is consistent with our clustering results Fig. To explore the expression patterns of the Wnt gene family during adipocyte differentiation, primary adipocytes collected from subcutaneous adipose tissue of cattle were induced.

Ten days after induction, Oil red O staining showed a greater extent of lipid droplet accumulation in adipocytes than in preadipocytes Fig. The absorbance at nm was significantly higher in differentiated adipocytes than in preadipocytes Fig.

These results indicate that the induced differentiation of primary adipocytes was successful and could be used in the subsequent gene expression analysis.

Induced differentiation of primary adipocytes. A Oil Red O staining of bovine adipocytes induced at day 0 left and day 10 right of adipogenic differentiation. B Absorbance at nm. Control: isopropanol, 0d: substance extracted from adipocytes before induction 0 day , 10d: substance extracted from adipocytes 10 days after induction..

C The expression of adipogenic marker genes during adipogenic differentiation. These levels were reduced after induction, suggesting a collective involvement in keeping adipocytes undifferentiated.

Wnt2 , Wnt6 , Wnt9B , Wnt10A and their receptors Fzd9 , Fzd10 were significantly up-regulated, indicating a regulatory role during adipocyte differentiation.

Furthermore, Wnt2B , Wnt4 , Wnt8A and Fzd5 , Fzd8 reached the lowest expression at the second day and displayed a similar overall trend of expression. A Expression of 19 Wnt genes. The core motifs and domains of a protein determine its function and activity Gene families usually encode proteins that share similar motifs and act synergistically All the 19 bovine Wnt members have six conserved amino acid sequences Motifs 1, 2, 4, 5, 6 and 7 , pointing to a common functional site.

Thus, it is likely that these four motifs Motifs 3, 8, 9 or 10 are not located at the core of the Wnt protein domain. Their sequences and conserved motifs were similar, and all possessed the WNT conserved domain.

This might play a role in maintaining their three-dimensional structure and binding function. Phylogenetic analysis provides a credible way to explore the relationship between amino acid sequence similarity and function of proteins in the same family In multicellular eukaryotes, the Wnt family proteins is divided into 13 subfamilies.

For instance, a total of 11 Zhikong scallop , 12 Yesso scallop , Pacific oyster and 13 Lingula anatine , Plathynereis dumerlii , Lottia gigantean , Crassostrea gigas , etc. In the Bovinae, Wnt proteins were classified into 12 subfamilies but lacked WntA. This was consistent with previous studies reporting that vertebrates all have reserved subfamilies except for WntA 32 , Although the function of the Wnt family is highly conserved, several members have been lost in many species after the complete set of Wnt genes emerged in cnidarians 34 , The Wnt9 subfamily specific to Bilateria was also found to be absent from Chlamys farreri It remains unclear whether these genes were not identified due to limitations in genome assembly or whether they were lost during evolution.

In the phylogenetic relationship, two genes from distinct species that are located in the same clade are defined as orthologs The orthologous gene pairs among cattle and the other five bovine species were identified based on homologous relationships. Orthologous Wnt members first clustered in a single clade, indicating that they were conserved among different species. Genome-wide collinearity analysis of Wnt genes provided key information on the function and evolution in the Bovidae.

Gene duplication events can cause gene family expansion during genome evolution Indeed, both tandem and segmental duplications are responsible for the expansion of the Wnt family in Bovinae Figs.

The members of the Wnt gene family were distributed across nine chromosomes in the six selected species. This deletion may be due to the absence of a tandem duplication event or the loss of WNT9B after the tandem repeats occurred in Bos indicus during evolution.

However, the causes, processes, and outcomes of this evolutionary event are still unclear and need more research; such work may further help to clarify the function of WNT9B.

Due to the different starting points of chromosome annotation among species, the arrangement of genes might be totally reversed. Intra-chromosomal translocation and rearrangement during species evolution also lead to the changes in gene arrangement 38 , For example, the position of Wnt2B was altered in Bos taurus and Bos grunniens due to the inversion of large segments within the chromosome Fig.

Furthermore, the locations of Wnt genes change from collinear conserved in the same order to syntenic not necessarily in the same order This may be caused by inter-chromosomal rupture or fusion during evolution.

It is commonly known that functionally related genes usually exhibit similar expression patterns. Furthermore, gene expression clustering analysis can group genes of similar function Wnt is selective in recognizing its Fzd receptors The observed overlaps in the expression of 19 Wnt and 10 Fzd members in 60 tissue types suggest a coordinated and selective regulatory role Fig.

Meanwhile, previous studies, carried out in humans, showed that Wnt2B and FZD5 exhibited physical interactions and co-expression relationships Table 2 and displayed similar expression patterns during the differentiation of bovine adipocytes Fig. Adipogenic differentiation is a well-organized and complicated process regulated by various genes. Analysis of the interactions between the Wnt and the Fzd family is essential to explore their roles.

To ensure the accuracy of this interaction network, only sources from literature mining and experimental verification were selected. Analysis showed there were extensive and complex direct or indirect relationships between the Wnt and Fzd gene family.

We observed clear bias in the Wnt family members in terms of their ability to recognize their Fzd receptors Table 2 16 , The results provide a foundation for further study Wnt genes and the regulation of adipocyte differentiation in cattle. The cattle used in the experiments was electric shocked before being released. Primary adipocytes were isolated immediately, making all efforts to minimize its suffering.

Candidate sequences were manually checked to confirm Wnt homology. Conserved motifs were detected in MEME 5. Exon—intron structures and motif patterns of the Wnt family were visualized using TBtools v1. Protein sequences were aligned using ClustalW to investigate the phylogenetic relationship of Wnt genes.

FigTree software version 1. The chromosomal locations of Wnt genes in cattle and the five other species of Bovinae were obtained from general feature format GFF3 files.

Collinearity analysis for orthologous genes between Bos taurus and the five other bovine species was performed using the MCScanX toolkit The sequencing quality was checked using FastQC Quality control of the raw sequence data was performed using the trimmomatic The heatmap was constructed using the TBtools software Primary adipocytes were isolated and cultured from subcutaneous adipose tissue of the cattle in the Zerui Ecological Breeding Farm using the Type I collagenase digestion method.

Induction of primary adipocytes differentiation 62 and Oil red O staining 63 were performed as previous described. The absorbance of the substance extracted from adipocytes at 0 day and 10 day after induction was also measured at nm with isopropanol as a control. Primers for the Wnt genes were designed using Primer Premier 5. Then, a total of ng RNA was reverse transcribed using random primers with Moloney murine leukemia virus reverse transcriptase Takara Bio, Kyoto, Japan.

Three replicates were performed for each test. One-way analysis of variance ANOVA was applied to test the statistical significance among groups at 0. All the animal experiments were conducted according to the guidelines of the Regulations for the Administration of Affairs Concerning Experimental Animals Ministry of Science and Technology, China, All of the data generated or analyzed during this study are included in this published article and its supplementary information files.

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Ross, S. Inhibition of adipogenesis by Wnt signaling. Science , Liu, R. Glucagon like peptide-1 promotes adipocyte differentiation via the Wnt4 mediated sequestering of beta-catenin. Google Scholar. Guo, L. Qiu, W. Patients with high bone mass phenotype exhibit enhanced osteoblast differentiation and inhibition of adipogenesis of human mesenchymal stem cells. In addition Biopython includes wrapper code for calling a number of third party command line tools including:.

Recent releases of Biopython require NumPy and not Numeric. Version 1. Please note that Biopython 1. Windows installers for Python 2. A Windows installer for Numeric