Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a pre–MSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration.Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish ( erbb3b gene is required to establish melanocyte stem cells in the embryo that are responsible for regenerating melanocytes after melanocytes are ablated in the larval zebrafish. Because this adult stem cell is not required for the development of embryonic melanocytes, we conclude that adult melanocyte stem cells develop in parallel to the embryonic tissues that they regulate. We also show that overexpressing kit ligand a will result in over-recruiting these adult stem cells to produce excess melanocytes. Further exploration into the mechanisms by which the zebrafish melanocyte stem cells are maintained and recruited will inform how adult stem cells might be manipulated for medical applications.The promise of regenerative medicine lies in the ability to find or create stem cells that can be manipulated to replace damaged tissues and organs. Such ability requires an understanding of how adult stem cells are established and then later recruited to regenerate different tissues. Here, we study the zebrafish's ability to regenerate melanocytes, a pigment cell shared with humans, to understand these mechanisms. We show that the  As with other teleosts, the zebrafish has a remarkable ability to regenerate a wide range of tissues and organs including fins The zebrafish embryonic pigment pattern is an excellent genetic model for investigating developmental processes kitkit acts cell autonomously in the melanocyte lineage kitla in zebrafish) expressed in the skin or in cells adjacent to the notochord kit signaling have been demonstrated for both larval kit is in the MSC itself, to promote recruitment of these quiescent stem cells, or for subsequent differentiation of regeneration melanoblasts, or both.We have developed a model for cell-specific regeneration of melanocytes during the period of stasis between 3 and 14 dpf puma and picasso each show deficits in forming new melanocytes at the onset of metamorphosis. The positional cloning of picasso revealed that it was caused by a mutation in erbb3b, an epidermal growth factor receptor (EGFR)-like tyrosine kinase. Furthermore, drug inhibitors of the ErbB family of receptors, such as AG1478, administered to WT larvae as early as 14–22 hpf phenocopied the picasso metamorphic defect. This finding strongly suggested a role for ErbB signaling during early embryonic development in establishing the stem cells involved in adult melanocyte development. An attractive possibility is that ErbB-dependent stem cells responsible for metamorphosis are also responsible for surveilling and regulating the larval melanocyte population.Genes identified by mutant phenotypes expressed during metamorphosis of the embryonic or early larval pigment pattern to the adult pigment pattern may also be involved in processes of adult stem cell regulation picasso mutation and the ErbB inhibitor, AG1478, abolish melanocyte regeneration following embryonic melanocyte ablation by MoTP. Our results indicate a requirement of ErbB activity concurrent with embryonic melanocyte development and prior to melanocyte ablation by MoTP, suggesting that ErbB signaling is required during the establishment of larval MSCs. That both larval regeneration and metamorphosis require early ErbB signaling raises the possibility that the same stem cell population is involved in both larval regeneration and metamorphosis.In this study, we explore the role of ErbB signaling in establishing MSC populations that are drawn on for larval melanocyte regeneration. We show that both the picasso or ErbB inhibitor treated embryos have normal embryonic pigment pattern but lack functional stem cells involved in regeneration or metamorphosis indicates there are two types of melanocytes: secondary larval regeneration melanocytes that develop from an ErbB-dependent stem cell, and primary ontogenetic, or embryonic, melanocytes that develop independent of ErbB function.Furthermore, the finding that kit ligand a, Tg(cmv:kitla), which causes an increase in the number of ontogenetic melanocytes. We investigated which melanocyte lineage is responsible for increased melanocytes by examining the effects of drugs that specifically affect the stem-cell derived melanocytes. Thus, we show that the drug ICI-118,551, which specifically blocks regeneration melanocyte development at the dct+ melanoblast stage but has no effect on the development of embryonic or ontogenetic melanocytes, ablates the kitla-mediated melanocyte increase in Tg(cmv:kitla) larvae. Moreover, AG1478, that blocks the establishment of functional MSCs, when given to Tg(cmv:kitla) larvae results in an additional increase in ectopic melanocytes. This additional increase is also blocked by the regeneration specific drug, ICI-118,551. That both regeneration-specific drugs have an effect on the number of ectopic melanocytes in Tg(cmv:kitla) larvae is evidence that overexpression of kitla is acting through the MSC lineage to produce excess melanocytes. That AG1478 does not reduce the number of excess melanocytes in Tg(cmv:kitla) larvea suggests that AG1478 arrests the stem cell lineage in a pre-stem cell stage (pre-MSC) and that these pre-MSC cells can be activated by kitla overexpression.We also examined how AG1478 affects the establishment of MSCs. We took advantage of a transgenic line with constitutive overexpression of the We were interested in testing whether ErbB signaling is required for larval melanocyte regeneration and ontogeny. Thus, we treated fish with the ErbB inhibitors AG1478 and PD158780 erbb3b was specifically required for regeneration, we treated erbb3b (picasso) mutants with DMSO or MoTP from 24–72 hpf and assayed for melanocyte ontogeny and regeneration at 7 dpf . Following melanocyte ablation with MoTP, erbb3b larvae also showed a defect in regeneration  compared to those that regenerate in WT larvae (12.4±7.8 melanocytes) is that partial suppressors of the regeneration defect have accumulated in the mutant stocks, that act equally on the mutant and the pharmacological inhibitor. Whatever is the cause of these increased residual melanocytes, the finding that the deficit in regeneration is not increased by combining the mutant and the pharmacological inhibitor  is consistent both with reports that AG1478 is highly specific to the ErbB family of receptor tyrosine kinases erbb2- and erbb3b-dependent signaling in zebrafish In both AG1478-treated larvae and The early requirement for ErbB activity in promoting metamorphosis suggested that it was required to establish a population of precursors for adult melanocytes. To determine whether ErbB signaling has a similarly early role in establishing stem cells important for larval regeneration, we treated fish with AG1478 at various time intervals both before and after MoTP treatment and examined the number of dorsal trunk melanocytes (somites 5–12) that regenerated . We founIn the foregoing analysis, the ErbB inhibitor AG1478 was applied in a series of applications overlapping with the embryo's exposure to the melanocyte ablation agent, MoTP. In those experiments, melanocytes or melanoblasts likely begin to die early in the MoTP treatment, between 24 and 48 hpf Although the above experiment positively demonstrates the effect of AG1478 on blocking MSC establishment, it does not exclude an additional effect of AG1478 on preventing recruitment of the MSC following melanocyte ablation. To rule out such a role, we performed a reciprocal experiment, where MoTP was applied first, from 24–72 hpf, followed by AG1478 treatment from 72 to 144 hpf. In these larvae, melanocytes regenerated in identical numbers to control larvae that were only treated with MoTP . This reThe preceding analyses, which show that AG1478 acts on the MSC before embryonic melanocytes and melanoblasts are ablated, argue that ErbB signaling acts to promote the establishment of the melanocyte stem cell population rather than on recruitment or differentiation of melanocytes from the stem cell population during larval melanocyte regeneration. This conclusion is consistent with previous studies that showed that AG1478 treatment during early embryonic stages was sufficient to block melanocyte development during pigment pattern metamorphosis nearly two weeks later Because many aspects of zebrafish embryonic development and neural crest development proceed in rostrocaudal progression We were also interested in testing whether the ErbB inhibitor AG1478 blocked the mechanism of stem cell self-renewal that we postulate to occur during melanocyte regeneration. We have shown that following multiple rounds of MoTP-induced melanocyte ablation kit receptor tyrosine kinase in stem cell function. Previous work with conditional mutations in kit have shown that, although kit activity is required for regeneration, kit does not play an essential role in establishing MSCs, but is required for early stages in the development of melanocytes from stem cell precursors kit ligand, kitla in embryos results in 50% more melanocytes than wild type kitla overexpression might be recruiting the MSC to overproliferate or generate excess melanocytes.We were interested in the role of the kitla overexpression is acting on a transient population of melanoblasts during the ontogenetic development of melanocytes in the embryo. Wild-type animals develop nearly all of their embryonic melanocytes by 3 dpf. These differentiated melanocytes are post-mitotic, and there is little or no turnover of melanocytes between 3 dpf and the onset of metamorphosis at 14 dpf kitla overexpression is acting on a transient population of melanoblasts that are present only from neural crest appearance at 14 hpf to, at the latest, completion of embryonic melanocyte patterning at 72 hpf, we induced kitla expression using the heat shock promoter (pT2hsp70:kitla) in transiently injected embryos after 3 dpf. When injected embryos are heatshocked at 4 and 5 dpf, we observed an increase of 30% in the number of melanocytes by 8 dpf  melanocyte nuclei examined had BrdU label. This fraction is nearly identical to the fraction of excess cells we observe for this heatshock treatment (∼30%), indicating that cell division accounts for all of the ectopic melanocytes observed in pT2hsp70:kitla heatshocked embryos , after 3 dpf could inhibit the appearance of these excess melanocytes. For this experiment we used a stable transgenic line of fish that contain kitla under the control of the cmv promoter (Tg(cmv:kitla)). Tg(cmv:kitla) animals have ubiquitous kitla mRNA expression (not shown) and, similar to the previously reported transient experiments, have ∼50% more melanocytes than wild type (see Tg(cmv:kilta) larva by 6 dpf (data not shown), indicating that Tg(cmv:kitla)-induced melanocyte increase is not a consequence of cell division of melanized melanocytes. We therefore conclude that kitla recruits undifferentiated, or nonpigmented, cells to divide and differentiate into melanocytes. That these cells can be recruited by overexpression of kitla at 4 or 5 dpf, when in situ expression analysis reveals no dct+, melanin- melanoblasts present, suggests that excess kitla is acting on an earlier stage of melanocyte development. A candidate for a cell capable of recruitment by kitla is the MSC.To rule out the possibility that the above demonstrated cell division events involve the cell division of differentiated melanocytes in response to overexpression of type see . In the kitla increases the production of melanocytes through recruitment of the MSC lineage, rather than the embryonic melanocyte lineage, we asked whether this increase was sensitive to the MSC lineage-specific drug, ICI-118,551. We identified ICI-118,551 in a screen for small molecules that specifically blocked melanocyte regeneration following MoTP induced ablation . Ontogenetically developing embryonic melanocytes develop normally, albeit with fainter pigment, in the presence of ICI-118,551 (dct+ positive melanoblasts (gfpt1 (eartha) mutation, which also blocks melanocyte regeneration at the dct+ melanoblast stage and has lighter pigmented ontogenetic melanocytes than WT To establish that ectopic overexpression of -118,551 . In contnoblasts , but thenoblasts . This dikitla overexpression is recruiting melanocytes from the stem cell lineage rather than from the ontogenetic melanocyte lineage, we treated Tg(cmv:kitla) animals with ICI-118,551 beginning at 9 hpf (Tg(cmv:kitla) larva  animals develop from the stem cell lineage rather than from the ontogenetic lineage.To test whether at 9 hpf  and askekitkitla. To test whether kitla could induce AG1478-treated larvae to produce excess melanocytes, we treated wild-type and Tg(cmv:kitla) transgenic larvae with AG1478 from 9–30 hpf (Tg(cmv:kitla) larva show an increase in the number of melanocytes in both the trunk  larva are treated with AG1478, they show an additional increase of 52% in the number of melanocytes in the trunk  (P<0.0001). In contrast to the two-melanocyte width of the dorsal stripe typically observed in wt larvae (Tg(cmv:kitla) (Tg(cmv:kitla) (Tg(cmv:kitla) larvae  -induced overproduction of melanocytes primarily in the trunk region where AG1478 prevents the establishment of MSCs reinforces our conclusions developed above that kitla overexpression is acting on the MSC lineage, rather than embryonic melanoblasts and melanocytes, to cause overproduction of melanocytes.We were next interested in how AG1478 is acting to inhibit MSC establishment. Specifically, we wanted to know whether AG1478 killed the stem cell lineage, or whether it blocked the MSC from developing from a pre-stem cell (pre-MSC) state. We suspected that AG1478 prevented cells in the neural crest lineage from achieving melanocyte stem cell specification, and that if these precursors were still alive, they might be expressing 9–30 hpf . This ti9–30 hpf , providi9–30 hpf . Untreatt larvae , dorsal v:kitla) , or AG14v:kitla)  larvae akitla is recruiting cells from the ontogenetic lineage when the MSCs are blocked with AG1478, we treated Tg(cmv:kitla) larvae with both AG1478 and ICI-118,551 (See Tg(cmv:kitla) larva are treated with AG1478 and ICI-118,551 together, we observed even fewer ontogenetic melanocytes (44.6% of that of WT). This result provides further evidence that kitla is capable of recruiting the AG1478 pre-MSC. That fewer ICI-118,551-insensitive melanocytes develop in Tg(cmv:kitla) AG1478-treated larva might suggest that the proliferation of the ontogenetic melanoblast lineage is suppressed when the stem cell lineage is overproliferating to such an extent (230% of wild type).To rule out the possibility that ,551 See . When Tgkitla arise from the MSC lineage, rather than the ontogenetic (ErbB-independent) lineage. These include (1) overexpression of kitla is capable of inducing an excess of melanocytes after 4 dpf when embryonic melanoblasts are not present; (2) all excess melanocytes arise through cell division after kitla overexpression; (3) ICI-118,551, which does not affect embryonic melanocyte number, blocks the increase in kitla overexpressing animals and lastly; (4) AG1478, acts specifically in the MSC lineage, modifies the number of excess melanocytes in kitla overexpressing animals.Taken together, four lines of evidence converge to indicate that the excess melanocytes that develop following overexpression of kitla. This cell does not have the full regulative abilities of a MSC, as evidenced by the fact that it cannot respond to ablation of embryonic melanocytes by making new melanocytes. Its presence, rather than immediate death, is revealed by its responsiveness to excess kitla. A parsimonious interpretation is that AG1478 prevents—and erbb3b is required for—the progression of a precursor to the fully regulative MSC. The second conclusion is that this precursor, that we refer to as a pre-MSC, is responsive to kit signaling. Together with the findings that kitla overexpression after 4 days induces overproduction of new melanocytes through cell division of undifferentiated precursors . Our model for MSC establishment and recruitment is presented in We have both genetic and pharmacological evidence that ErbB signaling is required for the establishment of the adult melanocyte stem cell. Although we lack direct markers for the melanocyte stem cell, we can draw strong inferences about its existence and regulation from the ability of larvae to regenerate melanocytes, the formation of new melanocytes upon pigment pattern metamorphosis, or the regeneration of melanocyte stripes in the regenerating adult fin. We have previously shown that following ablation of embryonic melanocytes, cells with stem cell properties are recruited to replace or regenerate the missing melanocytes erbb3b mutant was also defective for larval melanocyte regeneration following ablation with MoTP. We find that embryos treated with ErbB inhibitor and the erbb3b (picasso) mutant, which are both defective for metamorphic melanocyte development Our finding that larval melanocyte regeneration was sensitive to ErbB signal inhibition led us to explore whether the Our interpretation that larval regeneration melanocytes and metamorphic melanocytes arise from the same adult stem cells sheds some light on how adult stem cells may be regulated by different mechanisms of repression or induction at different stages of development. During larval stages (3–14 days), melanocyte development is nearly static, with few or no new melanocytes developing during this period but see . That aberbb3b or following treatment with ErbB inhibitors that the ErbB-dependent MSC is not a precursor for ontogenetic melanocytes. Moreover, that the ErbB independent melanocyte precursors in the ontogenetic lineage cannot regulate the larval pigment pattern when the ErbB-dependent MSCs are blocked suggests that a precursor for these cells do not persist in the larva through self-renewal. Self-renewal, in contrast to depletion during development, is a hallmark for embryonic or adult stem cells This study now provides strong evidence for two distinct melanocyte lineages that populate the larval zebrafish. One melanocyte population, which we have discussed extensively in this study, is derived from an ErbB-dependent melanocyte stem cell . Embryos and larval fish were reared at 28°C in 2 ppt marine salt  in carbon-filtered tap water. Larvae were fed rotifers after 5 dpf. Wild type embryos photographed were sjA (http://zfin.org). All quantitative analysis for wild-type fish was conducted with fish mutant for melanophilin (mlpha), which contracts melanosomes within the melanocyte but does not effect the number or pattern of melanocytes erbb3b (picasso) mutant, which has normal number of embryonic melanocytes but fails to develop normal adult melanocyte patterning, was previously described Adult fish were maintained at 25–27°C, 14L∶10D standard treatment  and PD158780  were dissolved in dimethyl sulphoxide (DMSO) to make a stock solution (6 mM AG1478 and 600 µM PD158780) and stored at −20°C in a light protected vessel. Dose response testing  revealed a strong effect with limited lethality at 3 µM for both drugs. Unless otherwise noted, embryos were incubated in Petri dishes with 3 µM AG1478 with 0.5% DMSO in 2 ppt marine salt water, with one embryo per ml. Embryos were kept in the dark during incubation to protect drugs from light degradation.The regeneration blocker ICI-118,551 was identified in a small molecule screen for drugs that specifically inhibit larval regeneration. Embryos were placed in MoTP (50 µM) and each of the 1250 drugs (10 µM) in the LOPAC  panel at 12 hpf in 96 well plates. MoTP was washed out of each well at 3 dpf and replaced with additional drug. Drugs that inhibited regeneration by 6 dpf were tested for their effects on ontogenetic melanocyte development. Embryos treated with 30 µM ICI-118,551, had a complete absence of regenerated melanocytes but displayed a normal number of ontogenetic melanocytes.MoTP (4-(4-morpholinobutylthio)phenol) stock solution (50 mM) was made up in DMSO, and stored at room temperature in a light protected vessel. Embryos were incubated in 50 µM MoTP in 2 ppt water for 48 hour incubation periods and assayed for regeneration after at least an additional 48 hours post washout. Embryos were visually inspected for complete melanocyte death 24 hours after washout.To assay for either melanocyte ontogeny or regeneration quantitatively, we counted dorsal melanocytes. Unless otherwise noted, these include melanocytes along the dorsum of the larva from somites 1–26. In each Figure we present the mean number of dorsal melanocytes with error bars indicating standard deviation. P-values for statistical significance were obtained by using Student's t-test in Microsoft Excel.kit ligand a (kitla) driven under the cmv promoter, j901Tg(cmv:kitla), were created. The cmv:kitla construct previously described (pT2cmv:kitla). Capped transposase RNA was synthesized from pCS-TP, provided by K. Kawakami, using the mMessage mMachine SP6 RNA synthesis kit . A transposase injection solution was prepared fresh each day of injecting composed of 25 mg/ml pT2cmv:kitla DNA, 25 mg/ml transposase mRNA, and 0.1% phenol red  in 1× Danieau buffer. Animals were injected with 1–2 nl of pT2cmv:kitla transposase injection solution at the 1 or 2 cell stage. The mosaic F0 animals were scored for kitla induced hyperpigmentation at 5 dpf and bred to sjA to test for germ line transmission. A single F1 male was selected that had robust hyperpigmentation phenotype in 50% of its offspring when crossed with sjA. Expression of kitla was examined with RNA in situ hybridization in these siblings. All embryos with kitla phenotype stained positive for ubiquitous kitla mRNA expression, whereas only ∼5% of animals scored as WT stained for ubiquitous kitla mRNA. Experiments presented here are with j901Tg(cmv:kitla)/+ F4 embryos.Fish transgenic for an overexpression line of the kitla overexpression, kitla was placed under the control of the heatshock promoter, hsp70 Xenopus elongation factor 1 alpha promoter. Animals were injected with between 1–2 nl of 25 µg/ml pT2hsp70:kitla and 25 µg/ml transposase RNA at the 1 or 2 cell stage. Genomic integration of the pT2hsp70:kitla construct was determined by GFP expression at 24 hpf, typically observed in >95% of injected animals. Only GFP+ embryos were used in heatshock experiments. Heatshock activation of kitla expression was performed by placing embryos in 50 ml conical tubes with 25 ml embryo medium and placing the tubes in a 37°C water bath for 30 minutes. All of the pT2hsp70:kitla construct experiments presented here are using F0 mosaic embryos.For heat inducible pT2hsp70:kitla genomic integration were immersed in BrdU (5 mM) for 96 hours beginning at 4 dpf. To induce kitla expression, animals were heatshocked at 4 and 5 dpf. Animals were then harvested at 8 dpf and fixed with 4% paraformaldehyde in phosphate buffered saline buffer (pH 7). More than 20 animals for each treatment were embedded in paraffin wax and sectioned into 5-micron-thick sagittal sections. Sections were deparaffinized and incubated with mouse anti-BrdU monoclonal antibody , followed by a secondary incubation with Alexa Fluor 594-conjugated goat anti-mouse Ig , and 4′,6-diamidino-2-phenylindole  for nuclear staining Cell division events in the melanocyte lineage were detected by retention of 5-bromo-2′-deoxyuridine (BrdU). Animals mosaic for in situ analysis was used to asses the presence of dct-expressing melanoblasts. Larvae were fixed in 4% paraformaldehyde in phosphate buffered saline (pH 7) at 5 dpf. cDNA for dct was amplified using PCR primers 5′-TACCTGTCGGAGGCTCAGTT-3′ and (with T7 sequence in lowercase) 5′-taatacgactcactatagggTACGCTGGTCACAGTTCAGC-3′ from first strand cDNA of 48 hpf embryos. Digoxygenin (DIG) labeled RNA probes from dct cDNA template were made using T7 RNA polymerase using the protocol at http://zfish.wustl.edu. Anti-DIG in situ hybridization was performed as previously described RNA


To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering.Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies.An interactive We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies. Large-scale projects in genomic sequencing and protein structure determination are producing enormous quantities of data on the relationships between 2D gene sequence and 3D protein structure. Moreover, such efforts are providing experimental data on success factors at every step in the gene to structure research endeavor. Ideally, this wealth of information should be used in a feedback cycle to facilitate the design and production of genes and protein constructs that are optimized for the successful production of functional protein samples for structural studies. Fundamentally, this goal represents a bioinformatics software challenge. With the goal of improving yield and success rates of heterologous protein production for structural studies, we have developed Gene Composer, a database software package which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences.100 (~5 × 1047) different possible coding sequences. The degeneracy of the genetic code therefore allows the pressures of natural selection to simultaneously influence both DNA and RNA sequence features in addition to protein coding function. DNA sequence elements and folded RNA structures are known to play significant roles in gene expression. As such, the overlapping information contained in a gene sequence can be significantly more complex than coding for a linear amino acid sequence. For example in the tryptophan operon of E. coli, the mRNA can fold into one of two mutually exclusive conformations that are a direct consequence of tryptophan availability [The redundancy of the genetic code allows any given protein to be encoded by a very large number of possible synonymous gene sequences. On average, each amino acid can be encoded by approximately three different codons (61 amino acid codons/20 amino acids). For a typical 100 amino acid protein there would be 30 ~5 × 107 differe® operating system and utilizes a network based SQL server or Access® database that is populated by users as they design genes. This arrangement makes it possible for multiple users to go back after time to design new construct variants that improve on existing designs by inclusion of new sequence or structural information from international genome sequencing and structural genomics efforts. In this report we describe how the synthetic gene design modules of Gene Composer facilitate protein construct engineering for structural studies, codon engineering for heterologous protein production, and oligonucleotide planning for PCR-based gene assembly with mismatch endonuclease error correction.Given the overlapping nature of information content in gene sequences  we endeavored to create a database and software tool called Gene Composer which facilitates the design of protein constructs, guided by 2D and 3D information, while the corresponding nucleic acid sequences are engineered for both codon usage and other desired sequence features. Gene Composer also enables the virtual cloning of the designed gene constructs which, depending on user preferences, can be parsed into data files for online ordering of complete genes or overlapping oligonucleotides that can be used for PCR-based gene assembly in any standard molecular biology lab. Gene Composer operates within the WindowsAlignment Viewer) that facilitates interactive protein construct design with virtual cloning (Construct Design Module), followed by codon engineering of novel synthetic gene sequences that are optimized for protein expression in various recombinant systems (Gene Design Module). Gene Composer is written in C++ for Windows® operating systems, and runs together with either an Access® or SQL® database.Gene Composer has a modular design to facilitate the work of protein engineers and structural biologists. It combines, within a single database software product, the ability to carry out comparative sequence alignments . From this imported information, Gene Composer uses the popular ClustalW algorithm [Alignment Viewer  which Gene Composer uses to generate a simple distance matrix between non-bonded, non-hydrogen atom centers in PDB files. The resulting matrix is used to flag residues in the protein model that participate in ligand contacts, water contacts, and/or crystal contacts. Each contact type is annotated within the Alignment Viewer with special visual symbols displayed below the residue of interest Figure . CrystalConstruct Design Module works in concert with the Alignment Viewer allowing the researcher to interactively define novel protein constructs with altered amino- and carboxy-termini, internal insertions or deletions, point mutations, and added affinity tags. The construct design tools are connected to the Alignment Viewer by a cursor that shows the user exactly where in the sequence alignment the desired changes are being made. For example, the user can set the cursor within the Alignment Viewer at a domain boundary as visualized in the comparative sequence alignment and then truncate the construct at that site. The desired modifications can be virtually combined and permuted in silico to arrive at multiple desired protein constructs  and mutagenic oligonucleotides needed to produce the gene constructs of interest by PCR from a defined template. The template sequence can either be a native cDNA sequence or a codon engineered synthetic DNA sequence crafted by the Gene Design Module of Gene Composer. The software creates a spreadsheet list of the required oligonucleotides with defined quantity and location within a bar coded 96 well plate that conforms to Society of Biomolecular Screening (SBS) standards. The spreadsheet is sent directly to oligonucleotide vendors  who will typically prepare and ship the oligo-plates within a week. Once SBS plates containing the amplimers  and mutagenic oligonucleotides have been obtained, they can then be placed on the deck of the Tecan liquid handler together with plates containing template DNA (either cDNA or synthetic DNA), vector DNA, buffer mixes, polymerase, and other enzymes at defined locations. With this information, the Construct Design Module can compute a script of pipetting instructions for the Tecan liquid handler to perform all the necessary liquid handling events needed to pool template DNA with amplimers, polymerase, and buffers within individual wells of a 96-well PCR plate. Alternatively, the same script of pipetting, PCR, and cloning events can be executed manually by a researcher with a handheld pipettor. After the PCR plates are filled with the necessary reagents, either the user or the robotic arm of the Tecan robot places them onto thermal cycler which carries out the necessary PCR reactions to produce "adapted" inserts. In the case where point mutations, deletions, or insertions are required at a single site within the insert sequence, the Tecan platform (or the researcher with a hand held pipette) is scripted to add mutagenic oligos to plasmid constructs with inserts, wherein the QuikChange® site directed mutagenesis method is then used to obtain the desired mutated constructs [In order to automate the physical cloning of designed constructs using either the Tecan Freedom Evo2 liquid handling system nstructs .®), restriction enzyme (RE) cloning, ligase independent cloning (LIC) [polymerase incomplete primer extension (PIPE) cloning [® cloning, Gene Composer designs terminal amplimers (forward and reverse) with the required recombination sequences recognized by recombinase enzyme to affect recombination with the vector DNA to form the final constructs. For restriction enzyme cloning, Gene Composer allows the user to create custom terminal amplimers with defined terminal restriction enzyme recognition sites and any extra bases needed to ensure efficient restriction enzyme cutting at ends. For LIC and PIPE methods, Gene Composer allows the user to define standard terminal amplimers with the necessary terminal LIC or PIPE cloning sequences.Gene Composer supports recombinational cloning (ie. Gatewayng (LIC) , and pol cloning . For Gat®, RE, and LIC methods for cloning each require that the PCR insert samples be subjected to an extra step of treatment or purification prior to mixing with the vector sample for final cloning. In the case of Gateway® cloning, the PCR products must be purified away from amplimers that could otherwise interfere with vector recombination. For RE cloning, the inserts must be digested with restriction enzymes and then purified away from the enzymes and terminal bits of DNA released during digestion. For LIC cloning the inserts must be purified away from nucleotides and primers, so that they can be treated with T4 DNA polymerase to affect a specific 3' to 5' single strand digestion of DNA termini in the presence of a specific a defined deoxynucleotide triphosphate mix [®, RE, and LIC procedures produces open reading frames with "extra" non-native amino acids encoded by the terminal cloning sequences used to adapt the insert to the vector.The Gatewayhate mix . Each ofIn contrast, PIPE cloning enables the direct enzyme-free cloning of PCR products without any unwanted "extra" amino acid sequences in the final cloned open reading frame. The PIPE method relies on the observation that a significant quantity of PCR products have incompletely polymerized strands, leaving single-stranded 5' overhangs of various length defined by the variable 3' end points of the complementary strands . Since tGene Design Module uses a protein-to-DNA algorithm to automate the "back-translation" of a protein amino acid sequence into a codon engineered nucleic acid sequence. This can be done for a variety of heterologous expression systems based on their known codon bias represented in pre-installed codon usage tables constructed from the calculated codon frequencies from genomic sequence information [e.g., bacteria or insect cells. Users can also generate their own preferred codon usage tables as more genomic sequence data is published.The ormation . In addiPrior to running the back-translation algorithm, users are asked by Gene Composer to select a codon usage table (CUT), define a minimal codon usage frequency threshold to eliminate the use of "rare" codons and set a target guanine:cytosine percentage (G:C%) content for the intended expression system. Codons with fractional usage above the minimal threshold setting are re-normalized so that their fractional usage settings reflect their relative proportional usage when rare codons are eliminated. This allows the back-translation algorithm to plan synthetic "codon engineered" gene sequences that lack rare codons, but otherwise have codon usage which mimics natural codon usage. This is achieved algorithmically by random selection of available codons in a weighted fashion according to their re-normalized fractional usage. Based on these user defined parameters, Gene Composer generates thousands of possible new synonymous gene sequences for any given protein sequence. The software then selects a single synthetic gene sequence that most closely matches the target G:C%. to calculate local RNA secondary structure and minimum ΔG in a step-wise fashion across the entire gene sequence [NP_620581, 360 amino acids, 1080 bp of DNA) in approximately 45 sec. This step is only the first step of our process. Detailed below are additional modifications that the user may select to further enhance the gene design and arrive at a highly optimized sequence. We also want the reader to note that the user can elect to skip the back-translation step and begin instead with a native mRNA sequence. When modifying a native mRNA the user still selects a codon usage table and codon frequency cutoff. Within this framework, various engineering tools (listed below) can be applied to modify a native gene sequence. Moreover, the software allows users to visualize graphically the location of rare codons across a gene sequence relative to a selected codon usage table and defined codon frequency threshold. This allows the user to identify rare codons within native gene sequences so that they may be appropriately modified.If users wish to eliminate stable mRNA hairpins from their synthetic gene sequences, the back-translation algorithm can be run in "delta G" mode wherein the standard back-translation is carried out together with a user defined threshold for an allowable calculated change in Gibbs free energy (ΔG) for local RNA folding. In this mode, the standard back-translation algorithm runs the Vienna RNA Package sequence . The usesequence ,19. The Beyond simple back-translation, there are at least three general classes of synonymous codon engineering features that synthetic gene engineers may desire: (i) those that facilitate DNA cloning operations such as the introduction or elimination of restriction enzyme cleavage sites; (ii) those that improve the "translation quality" of mRNA such as the elimination of nucleotide repeats, codon repeats, cryptic Shine-Dalgarno translation start sites, or the introduction of alternate reading frame "ambush" stop codons ; and ii. Many ofMany expression vector plasmids have multiple cloning sites (MCS) with several unique restriction enzyme sequences in a defined order which facilitates unidirectional cloning of gene sequences. In order to take full advantage of the utility of multiple cloning sites, careful synthetic gene design allows one to use synonymous nucleotide changes to "silently" eliminate undesired MCS restriction endonuclease sites from the body of the open reading frame Figure . In thisThe degeneracy of the genetic code, even when constrained to the preferred codons of highly expressed proteins, usually allows sufficient flexibility to enable the "silent" introduction of unique restriction sites located at strategic positions within the open reading frame of the gene of interest. The introduction of unique restriction sites throughout the body of a gene enables it to be cleaved into several defined pieces, which may be designed to encode alternate mutations within each gene segment. In this way, re-ligation of the pools of mutant gene segments allows for the rapid generation of several combined mutations. This strategy can be used to select for protein variants with desired properties and can be a powerful combinatorial protein engineering strategy. It should be noted that within the within the restriction site database manager, users can specify any DNA sequence, including wild card sequences that they wish to consider for silent introduction or removal without altering the amino acid coding sequence.When designing synthetic gene sequences for optimal protein expression it is important to consider that simple back-translation process may inadvertently design open reading frame sequences with one or more cryptic Shine-Dalgarno  or KozakIn eukaryotic organisms, the Kozak consensus sequence for translation initiation is loosely defined as 5'GCCACCAUGG3' wherein translation initiation usually starts at a methionine AUG codon within the sequence. Given the relatively small size of the Kozak consensus sequence, there are relatively few possible alternative synonymous sequence choices that would eliminate an unwanted cryptic Kozak site. As such, Gene Composer has the flexibility to allow the user to define any unwanted sequence of any size. The software will choose any synonymous change that effectively eliminates the defined sequence, even if its use would be counter to previously defined codon usage parameters.Sequence repeats of greater than five nucleotides in length are a source of ribosomal frameshifting, wherein the mRNA can "slip" out of frame . Therefond or 3rd frame translation and thereby promote the termination of any mistranslated proteins.As noted above, the ribosomal machinery is not perfect and can suffer a reading frame shift under certain circumstances, such as when it encounters a stable RNA hairpin, pseudoknot structure, or a "slippery" repeated nucleotide sequence . Once a E. coli that carry an inducible MazF gene. This gene encodes the MazF endoribonuclease which cleaves any unstructured single-stranded RNA molecules that contain the "ACA" sequence [E. coli cells into a "quasi-dormant" state, lacking all mRNA except for the mRNA encoded by the synthetic "ACA-less" gene. The cells in this growth arrested state can be concentrated and added to medium containing spin-labeled organic salts, which results in the NMR-ready labeling of only the protein of interest that is encoded by the synthetic "ACA-less" gene. This MazF procedure for producing only one protein of interest in vivo requires that the gene of interest be engineered to silently eliminate all "ACA" nucleotide combinations. Conveniently, the degeneracy of the genetic code allows all possible ACA coding sequences to be changed to alternative synonymous coding sequences. Gene Composer is capable of designing ACA-less genes through synonymous codon engineering which eliminates the ACA triplet in all three reading frames.Protein production levels from synthetic genes can sometimes be improved by synonymous codon engineering of mRNAs to resist destruction through the silent elimination of RNase cleavage sites. This is handled by Gene Composer using the same procedure described above for the elimination of cryptic Kozak sequences. Similarly, synonymous codon engineering by Gene Composer can be used to create "ACA-less" mRNAs for "single protein" production in strains of sequence . Since m®), traditional restriction enzyme cloning, and the polymerase incomplete primer extension cloning method (PIPE) [Once a codon engineered gene sequence has been formulated with the aid of the Gene Composer protein-to-DNA algorithm, the gene can either be purchased from a synthetic gene vendor, or be manufactured by PCR in a standard molecular biology lab from the Oligo-sets designed by the Gene Composer Gene Design Module. This module includes a gene-to-oligonucleotide algorithm that is used to plan all required overlapping oligonucleotides needed to synthesize a desired gene sequence by PCR -34, and d (PIPE) .Gene Composer has the ability to design overlapping oligonucleotide sets (Oligo-sets) for a variety of reported PCR gene assembly methods that use a range of different oligonucleotide building block sizes Figure . As suchWith these parameters defined, the gene-to-oligo algorithm computationally cuts the top and bottom strand sequences at random positions that are otherwise constrained only by the aforementioned oligo size and overlap constraints. The algorithm starts on the top strand and generates the first cut by choosing a random integer value between the maximum and minimum sizes (inclusive). The algorithm continues marching down the top strand in the 5' to 3' direction generating cuts until the remaining length of the sequence is less than the minimum oligo length. Once this point is reached, the algorithm iteratively shifts all upstream cut sites by -1 base until the terminal oligo length is the size of the target overlap length. This process adjusts the cuts across the top strand such that the final ensemble of cuts produces oligos that meet all of the user defined size parameters. Once the top strand has been cut up, the algorithm then generates bottom strand cuts by making calculated cuts at nucleotide positions approximately half way between the ends of the top strand oligos. This ensures that each bottom strand cut falls in the approximate middle of two top-strand oligos, which is validated in a final step that checks that the top and bottom strand oligo sizes and overlaps conform to the user defined parameters.m) for the overlapping segments of each possible top and bottom strand pair [m value and the lowest overall variance in Tm, which together help ensure fidelity in oligonucleotide pairing during pooled PCR reactions for gene assembly. Moreover, an Oligo-set should not contain any oligonucleotides that can form stable hairpins , or which have the same or similar sequence as other oligonucleotide members. Therefore, the gene-to-oligo algorithm selects only the best candidate Oligo-set which has the highest average Tm, lowest variance in Tm, and have a calculated ΔG for intramolecular folding which is above the value set during the backtranslation step [The gene-to-oligo algorithm is run thousands of times to generate a large number of candidate Oligo-sets from which to choose. The Oligo-sets are then ranked according to their calculated average melting temperature . Once identified, the algorithm determines how it can shift the oligonucleotide endpoints to either raise the overlap Tm and/or bring it closer to the mean. With each shift, a check is done to ensure that the Oligo-set variables  are not violated, and that the Tm is closer to the mean and the overall Tm variance has been reduced. The final Oligo-set is checked again to ensure that no oligonucleotides violate the minimal allowable ΔG for intramolecular folding.Once the top candidate Oligo-set has been selected, the gene-to-oligo algorithm proceeds to make further improvements to the Oligo-set by adjusting the oligonucleotide end points for any top and bottom strand oligo pairs whose overlap Tm of overlaps between top and bottom strands increases. The variance in overlap Tm is influenced by both the number of oligos in the pool and their average overlap length. Therefore, in order to improve the successful outcome of gene synthesis, our Gene Composer PCA protocol starts with the PCR production of gene fragments from sub-pools of oligos. Once the successful production of each overlapping gene fragment has been confirmed by agarose gel electrophoresis or capillary electrophoresis, all sub-gene fragments are pooled for PCR amplification of the full length gene using an excess of flanking primers covering the extreme 5' and 3' ends of the desired gene. While it is certainly possible to obtain complete genes by PCA from a large pool of oligos, we have found that the failure rate for PCA from large pools is too high to justify this as a standard approach to gene synthesis, and have therefore adopted the sub-fragment/pool approach to PCA for synthetic genes.The Gene Composer protocol for PCR assembly (PCA) of synthetic genes from designed oligonucleotides (Oligo-sets) is illustrated in Figure Most PCA-based gene synthesis methods can suffer from a mutation rate of ~2 to 4 point mutations per 1–2 Kbp of synthetic gene product -38. The The use of mismatch specific endonuclease surveillance in conjunction with PCA greatly reduces the error rate synthetic gene production. Nevertheless, in order to obtain a final sequence validated synthetic gene of ~1–2 Kbp, one may have to examine several (~5) independent gene clones to identify one which is 100% accurate. Single base mistakes can also be repaired by site-directed mutagenesis, and/or by reconstructing the final gene from sub-cloned gene fragments that are free of mutations. Finally, many researchers may simply wish to purchase a fully sequence validated synthetic gene of their own design from a number of synthetic gene vendors.Bacillus subtilis, and the Hepatitis C viral polymerase NS5B. The sequences of these genes were re-designed by codon engineering using Gene Composer as described in the Materials and Methods. In Figure We chose three genes as examples of our methods from design to final synthetic gene product. These are the human protein kinase P38α, the bacterial protein FtsZ from The sequence analysis results for several clone isolates of the six synthetic genes  are presented in Table Gene Composer™ that facilitates the information-rich design of protein constructs and their nucleic acid coding sequences. This user friendly software package enables the facile design of totally novel nucleic acid sequences which are codon engineered for improved protein production in heterologous expression systems. We believe that computer aided design software will allow researchers to efficiently design protein constructs and synthetic genes which they can order according to their own design (not just the design provided by any given synthetic gene vendor). The prospect of increasingly affordable synthetic genes opens the opportunity to explore the effects of synonymous gene sequence engineering for improved protein production, or in some cases reduced protein production [Whole gene synthesis is rapidly becoming a powerful and cost effective technology for creating novel proteins and improving protein expression. To capitalize on the availability of low cost synthetic genes, we have created a database software package called oduction . Our effAnother goal in developing Gene Composer is to aid the dissemination of synthetic DNA technology to even very small labs with modest investments in recombinant DNA equipment . We have shown that our PCA method for synthetic gene assembly from Oligo-sets defined by Gene Composer works reliably for both highly engineered synthetic gene sequences and native cDNA sequences, with reasonably low error rates of 1–2 mutations per 2 Kbp. Thus, Gene Composer can aid even the smallest lab to produce synthetic genes in any location where oligonucleotides can be made or ordered for delivery.; Codon Devices, ; Blue Heron, ; Geneart, ; BioBasic, ; GenScript, ; Top Gene Technologies, , and others) and competitive technology development is expected to continually drive prices lower. In general, most synthetic gene production companies also offer limited gene design services, for example to design genes with preferred codons of a selected expression system. However, we anticipate that the design parameters for improved protein production through synonymous gene sequence engineering of open reading frames will ultimately require a more sophisticated user driven approach. For this reason, we have intentionally enabled Gene Composer to accept numerous user defined settings for gene design parameters.Several companies now offer synthetic gene production services  as standard, desalted oligos and were used without further purification. KOD polymerase was obtained from Novagen , and used for all PCR reactions following the manufacturer's instructions. PCR fragments were gel purified from agarose gels using a Qiagen Gel Extraction Kit . Plasmid DNA was prepared using a Qiagen Plasmid Purification Kit . Restriction enzymes were obtained from Fermentas  or New England BioLabs . T4 DNA ligase was purchased from New England Biolabs. CEL1 nuclease was purchased from Transgenomic, Inc .NP_389412, amino acids 1–382), P38α  and Hepatitis C viral NS5B  were engineered for expression in E. coli using Gene Composer software . Back-translation of the amino acid sequence was accomplished using an E. coli highly expressed protein codon usage table . No sequence optimization or alteration was carried out for the cDNAs except to exclude certain restriction enzyme sites from the body of the open reading frame by synonymous codon engineering so as to facilitate cloning into the expression vectors.Native cDNAs encoding these proteins were also assembled  in the third reading frame. Cryptic Shine-Dalgarno sequences upstream from any AUG in all three reading frames were also removed to prevent improper cryptic translation initiation. Restriction enzyme recognition sites for BamHI and HindIII were also silently removed by synonymous codon changes from both the synthetic genes and native cDNAs to facilitate cloning.Synthetic genes were designed using the Gene Composer protein-to-DNA back-translation algorithm using the m (65.84 to 87.84°C), ΔG of oligo folding , and length (50–70 bases). The "Create Primers" algorithm was used to organize the overlapping oligonucleotides into pools of 5 or 6 with combined segment lengths of 300–500 bp for each pool. The flanking amplification primers were set to a Tm of 65°C to 80°C and a length of 20 to 30 bases. Restriction sites were added as 5' and 3' flanking sequences to the ends of the primers in the primer design tool of Gene Composer.The designed genes were then converted into Oligo-sets using the Gene Composer gene-to-oligo algorithm. The overlapping oligonucleotides on top and bottom strands of the NS5B gene were optimized for overlapTm and low Tm variance for overlapping segments of the oligonucleotides. The autoimprove Tm and improve ΔG features were omitted from the Oligo-set optimization for cDNA samples to prevent the introduction of synonymous changes. Primers were generated in the same manner and with the same parameters as for synthetic genes. The Oligo-sets for cDNA gene synthesis were ordered from the same vendors and assembled by PCA using the same methods as for the synthetic codon engineered genes.The native gene sequences were divided into Oligo-sets using the manual Oligo-set creation feature in Gene Composer. The oligo size range was set to 50–70 bases and the overlap to 23–40 bases. The specific Oligo-set used was chosen from a calculated list of more than 100 possible Oligo-sets and was based on high minimum TA two-stage PCR method is used to assemble synthetic and cDNA coding sequences with an intervening mismatch-dependent-endonuclease treatment of PCR products to reduce errors arising from PCR or from oligonucleotides containing synthesis mistakes.4)2SO4, 0.1% Triton X-100, 0.001% BSA. Four cycles of PCR were performed at 94°C for 30 seconds, 72°C for 90 seconds, and 72°C for 45 seconds. After 4 cycles, forward and reverse amplification primers were added to a final concentration of 200 nM each, and the reactions were amplified for 26 cycles of 94°C for 30 seconds, 68°C for 90 seconds, and 72°C for 45 seconds, followed by a final extension of 10 minutes at 72°C. Reaction products were then separated on an agarose gel and the excised bands of appropriate size were purified using a standard gel extraction kit (Qiagen) into a volume of 50 μl of buffer .In Step 1, defined pools of overlapping oligonucleotides and PCR primers (Oligo-sets) were used in individual PCR reactions to produce individual gene sub-fragments of 400–500 bp. Together, each of the gene sub-fragments span the entire gene coding sequence, with overlaps between sub-fragment ends of ~20–30 base pairs. Target genes were parsed by Gene Composer into pools (Oligo-sets) of 10–12 overlapping oligonucleotides (5–6 oligo pairs) for PCR amplification. Oligos in a solution containing 2.8 mM Tris-HCl, pH 7.6, 0.4 mM KCl, 0.04 mM dithiothreitol, and 50 mM NaCl were mixed in an equimolar ratio at a final concentration of 4 μM each. The mixture was then diluted 16-fold with water to create a working stock for PCR, of which 2 μl of each pool were individually amplified in a 50 μl PCR reaction using KOD polymerase (2.5 Units) without amplification primers in the manufacturer's buffer containing 0.12 M Tris-HCl, pH 8.0, 10 mM KCl, 6 mM (NH4). The digested products were separated by agarose gel electrophoresis and the "error-free" CEL1 resistant bands were purified using a standard gel extraction kit (Qiagen) producing a final volume of 50 μl of purified CEL1 nuclease resistant sub-fragment in 10 mM Tris-HCl, pH8.5.In Step 2, the gel purified sub-fragments from Step 1 were heat denatured at 95°C for 10 minutes (20 μl at 50 ng/μl), and then reannealed to form heteroduplexes by decreasing the temperature of the solution at a rate of 0.1 degree per second for 10 minutes in a thermocycler in a buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA and 1 M NaCl. The heteroduplexes were then treated with the mis-match specific endonuclease CEL1 nuclease  in a 50 μl reaction at 40°C for 60 minutes 2SO4, 0.1% Triton X-100, 0.001% BSA containing 200 nM of the extreme 5' and 3'amplification primers (designed by Gene Composer) containing the terminal restriction enzyme recognition sites (BamHI and HindIII) or other sequences necessary for final construct cloning. PCR was performed for 26 cycles of 94°C for 30 seconds, 68°C for 90 seconds, and 72°C for 45 seconds, followed by a final extension of 10 minutes at 72°C. The final PCR products were then gel purified (with or without prior restriction enzyme digestion), and cloned into the desired expression vectors. Alternatively, the final PCR products can be subjected directly to LIC or PIPE cloning methods.In Step 3, the full length coding sequences were assembled using PCR by pooling 2 μl (~1 μg) of each of the gel purified CEL1 resistant sub-fragments in a 50 μl reaction using KOD polymerase (2.5 Units)  in the manufacturers buffer  were transformed with 4.5 μl of the cloning reaction. Aliquots of 50 μl and 200 μl of transformed bacteria culture were spread on 2xYT agar plates containing 50 μg/ml kanamycin and incubated overnight at 37°C. Single colonies of each of the constructs were grown overnight in 2xYT media containing kanamycin (30 μg/ml) and plasmid DNA was isolated by alkaline lysis using the Qiagen plasmid purification kit according to manufacturer specifications. Clones were sequenced with vector primers M13F(-21) (5'-TGTAAAACGACGGCCAGT-3') and M13R(-29) (5'CAGGAAACAGCTATGACC-3') by dideoxy termination using an Applied BioSystems, Inc.  3730 capillary electrophoresis instrument. Sequence analysis was carried out using Sequencher 4.7 DNA Sequence Analysis Software . One correct clone for each of the constructs was selected and digested from the pCR-Blunt II-TOPO vector via double digest using BamHI and HindIII restriction enzymes (Fermentas) according to manufacturer specifications. Digested products were gel purified and the DNA was quantified by absorbance at 260 nm using a NanoDrop ND-1000 spectrophotometer. A 3:1 molar ratio of the insert to vector (15 ng/μl) is used to subclone into the final expression vector using T4 DNA ligase (New England Biolabs) following the manufacturer guidelines, but increasing the suggested ligation time from 10 minutes to 30 minutes in a 10 μl reaction at 22°C in buffer containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP. One Shot TOP10 Chemically Competent E. coli (Invitrogen) are transformed with 6 μl of the ligation reaction. Aliquots of 50 μl and 200 μl of transformed bacteria were spread on 2xYT plates containing 100 μg/ml ampicillin and incubated overnight at 37°C. Resulting colonies were screened for proper insert using PCR and vector specific primers and verified by DNA sequencing as described above.The concentration of gel purified synthetic genes from PCA was determined by absorbance at 260 nm using a NanoDrop ND-1000 spectrophotometer . DNA concentrations were adjusted to 50 ng/μl or greater in 10 mM Tris-HCl, pH 8.5 and then used for cloning into the pCR-Blunt II-TOPO vector (Invitrogen) according to the manufacturer's instructions. One Shot™ TOP10 chemically competent ccdB negative selection cassette. PCR reactions are performed using pfuTurbo (Invitrogen). The extension time for the iPCR is 3 minutes, while the extension time for the vPCR is adjusted to 14 min. to allow sufficient time for amplification to occur. The vector for PIPE cloning is a derivative of pET28a into which the coding sequence of Smt3 has been cloned followed by a ccdB cassette. Equal parts crude iPCR product and crude vPCR product are combined and used to transform competent cells.The gene of interest is amplified in the insert PCR reaction (iPCR) with chimeric primers, with homology to both the gene terminus and the vector junction  and RG designed the gene sequences, designed the oligo-sets, performed the PCA gene assemblies and analyzed DNA sequences. AB(2) and LS conceptualized experimental strategy and guided the manuscript preparation. All authors read and approved the final manuscript.DL contributed to software design, experimental strategy, analyzed data, and drafted the manuscript. AR contributed to experimental strategy, analyzed data, and drafted the manuscript. JW and MM wrote the


Single transgene expressing mammary epithelial cells were shown to reconstitute mammary glands in vivo while immunohistochemical staining identified MaSCs in basal and luminal locations, with preponderance towards the basal position. By quantifying luciferase expression using bioluminescent imaging, we were able to track MaSCs non-invasively in individual mice over time. Using this model to monitor MaSC dynamics throughout pregnancy, we found that MaSCs expand in both total number and percentage during pregnancy and then drop down to or below baseline levels after weaning. However, in a second round of pregnancy, this expansion was not as extensive. These findings validate a powerful system for the analysis of MaSC dynamics in vivo, which will facilitate future characterization of MaSCs during mammary gland development and breast cancer.Mammary stem cells (MaSCs) play essential roles for the development of the mammary gland and its remodeling during pregnancy. However, the precise localization of MaSCs in the mammary gland and their regulation during pregnancy is unknown. Here we report a transgenic mouse model for luciferase-based single marker detection of MaSCs  The mammary gland is a highly dynamic organ, undergoing well choreographed growth and involution during development, estrous cycles, and pregnancy in vivo is critical for understanding stem cell-niche interactions in the mammary gland and investigating the potential link between MaSC activity and breast cancer susceptibility. Unfortunately, the high degree of precision in detecting protein level variations using FACS cannot be achieved in immunostaining and thus has made it difficult to localize MaSCs by immunostaining of surface markers in combination. Furthermore, even after definitive identification of MaSCs, the only currently available method to monitor their dynamics was to use flow cytometry after dissecting mammary glands, making it impossible to monitor MaSCs in individual mice over time.Using fluorescence activated cell sorting (FACS) and mammary fat pad transplantation experiments, MaSC-enriched populations were identified by the expression of CD24 along with high expression of either CD29 or CD49f in vivo MaSC tracking based on our discovery that MaSC-enriched cells from a luciferase/GFP-transgenic mouse strain in vivo. By monitoring luciferase expression in recipient mice, we were able to quantitatively track the regulation of MaSCs in individual mice non-invasively using bioluminescence imaging (BLI). Additionally, the restricted expression of luciferase allowed us to precisely identify the location of MaSC-enriched epithelial populations in the mammary gland using immunohistochemistry.In order to overcome these difficulties, we have characterized a novel mouse model for CMV enhancer/β-actin promoter +CD29hi or CD24+Cd49fhi) luciferase mRNA  and other mammary epithelial cells (Lin−CD24−) were sorted from luciferase transgenic mice and transplanted into non-transgenic mammary fat pads cleared of their endogenous epithelium. Luciferase expression, which expanded quickly within the first two weeks after transplantation before reaching a relative steady state, was detected solely in the mice receiving CD24+CD29hi cells  and other mammary epithelial cells (Lin−CD24−). Intriguingly, the percentage of CD24+CD29lo cells both rose and fell with a delayed kinetics following the expansion and regression of CD24+CD29hi cells, indicating that these cells may be direct progenies of CD24+CD29hi cells as has been suggested elsewhere +CD29lo cells also dropped precipitously when mothers did not nurse their pups  by sacrificing mice at each individual time point throughout the pregnancy cycle. Comparing the two methods allowed us to asses the possibility that the expansion and regression in the number of MaSCs observed rcentage , suggesteir pups . As CD24To better observe the morphological changes associated with the massive expansion of MaSC activity, we performed immunostaining of luciferase transgenic mammary glands throughout pregnancy . While i+CD29hi cells, this did not prove that the expansion was the result of an actual increase of MaSCs, since it could also be due to the expansion of another luciferase expressing cell population within the CD24+CD29hi population. To address this, we first performed luciferase assays on sorted mammary epithelial cells from the luciferase transgenic mice just after giving birth to pups (near the peak in both number and percentage of MaSCs) and observed a restricted pattern of luciferase expression in CD24+CD29hi MaSCs similar to virgin controls (+CD29hi cells from pregnant and virgin mice at a limited dilution (500 cells) where we expected not every mouse would show reconstitution. If the parity related rise in CD24+CD29hi luciferase expressing cells were the result of an expansion of a non-MaSC population, we would expect to see less reconstitution when transplanting the same number of cells from pregnant and virgin mice. However, we observed similar levels of reconstitution  suggesting that the rise in luciferase expressing CD24+CD29hicells was indeed a rise in MaSCs.While we clearly observed an expansion of luciferase positive cells, and in the number of CD24controls . Additioin vivo model  and the Wnt receptor Lrp5 are found to be important for MaSC function We have also used this model to monitor MaSC dynamics throughout pregnancy, where we have shown that MaSCs rise in both total number and percentage during pregnancy and then decline to or below baseline levels after weaning. This effect is dampened when mothers do not nurse their young. Additionally, the magnitude of MaSC expansion is decreased during a second pregnancy. These results are particularly interesting when considered in the context of breast cancer, as it is known that in both mice and humans, full-term and multiple pregnancy results in a short term increase, but ultimately a long term decrease in breast cancer susceptibility In summary, our model serves as a unique platform in which MaSC activity can be non-invasively, sensitively and quantitatively monitored by bioluminescence imaging. This system will not only allow for the monitoring of MaSC activity in normal physiologically relevant processes but will also permit more direct evaluation of the susceptibility of MaSCs to oncogenic transformation and the regulation of their growth throughout tumorigenesis. If MaSCs play a critical role in the initiation or progression of breast cancer, this model will serve as an ideal system to develop and test future therapeutic applications that target MaSCs, including potentially novel prophylactic breast cancer treatments for high risk groups.All experiments involving mice were performed in accordance with approved protocols by the Institutional Animal Care and Use Committee of Princeton University. FVB/N-J mice were purchased from The Jackson Laboratory, FVB/N-Luciferase transgenic mouse strain L2G85 6 cells, including single color controls and combination staining. Control samples for triple color FACS included no-staining, propidium iodide  only, CD24-PE only, CD29-FITC only, Biotin-lineage only as well as corresponding PE, FITC and APC single color fluorochrome conjugated antibody isotype controls. Both primary and secondary staining was conducted for 30 minutes at room temperature in FACS buffer. Between staining, cells were washed with 5ml FACS buffer. Cell sorting for transplantation experiments was done using a FACSVantage SE w/DiVa (BD Biosciences), whereas flow cytometric analysis of mammary epithelial cell populations during pregnancy was performed on a four color, dual-laser FACSort instrument (BD Biosciences). All cells were sorted into 1∶1 Fetal Bovine Serum∶MEGM.Single cell suspensions were prepared from the mammary glands of luciferase transgenic animals after mechanical and enzymatic dissociation based on published protocols Sorted cells were centrifuged for 5 minutes at 1000g and then resuspended in 1× lysis buffer and incubated for 1hr at room temperature. Substrate was added and luciferase was detected using a GLOMAX microplate reader and a firefly luciferase assay kit (Promega). All samples were assayed in triplicate.Tubulin Forward: CCTTCATTGGAAACAGCACA, Tubulin Reverse: CCTCCTCTCCGAAATCCTCT; Actin Forward: GTATCCATGAAATAAGTGGTTACAGG, Actin Reverse: GCAGTACATAATTTACACAGAAGCAAT, Luciferase Forward: ATCACAGAATCGTCGTATGC, Luciferase Reverse: GAAATCCCTGGTAATCCGTTRNA was isolated from sorted mammary epithelial populations using the Trizol (Invitrogen) and contaminating DNA was removed using the Ambion DNA-free kit. cDNA synthesis was performed using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). qRT-PCR was run on an ABI 7900 96 well machine using the PowerSYBR Green PCR Master Mix from Applied Biosystems. Samples were run in duplicate using the following primers: in vivo using the Xenogen IVIS 200 imaging system.After FACS, cells were centrifuged for 5 minutes at 1000g and then resuspended in 50% Matrigel/50% PBS. Mice were anesthetized by intraperitoneal injection of ketamine (100mg/Kg) and xylazine (10mg/Kg). Unless otherwise noted, all injections involved placing 1000 sorted mammary epithelial cells into cleared inguinal (#4) mammary fat pads according to standard injection procedures Excised mammary glands were fixed for 1 hour in 3∶1 glacial acetic acid: 100% ethanol, washed in 70% ethanol for 15 minutes and stained in alum carmine overnight at room temperature. Glands were then washed for 15 minutes in 70%, 95% and 100% ethanol and transferred for long term storage into HistocClear II clearing agent .2O. Antigen retrieval was performed using a pressure cooker  at peak temperature of 125°C with EDTA, pH 8. Slides were washed with Tris buffer with Tween-20 before applying peroxidase block (Dako) for 5 minutes, rinsing with 1× Tris, and bathing in serum-free protein block (Dako) for 20 minutes. Primary staining against luciferase was done with 1∶5000 to 1∶10000 diluted Novus goat polyclonal anti-firefly luciferase antibody (NB100-677) in Dako Antibody diluent for 1 hour followed by secondary staining with 1∶500 diluted rabbit anti-goat (Dako) for 30 min. Slides were then treated with Dako envision anti-rabbit for 30 min, and then treated with DAB before counterstaining with hematoxylin.Histology services were performed by the Histopathology Core Facility at Brigham and Women's Hospital. Formalin fixed paraffin embedded mammary glands were sectioned (4µm), incubated at 58°C for 1 hour and then deparaffinized and rehydrated by washing in twice in xylene, 100% ethanol, 95% ethanol and dHFigure S1hi cells were injected into 16 recipient mice while 4000 or 10,000 GFPlo cells were injected into 8 mice per group. BLI activity was subsequently measured over six weeks and normalized to the signal at the time of injection. (B) Reconstitution ability was highly enriched in the GFPhi fraction, with successful reconstitution (green dots) in 9 out of 16 recipients, compared to 3/8 mice receiving 10,000 GFPlo cells and 1/8 mice receiving 4000 GFPlo cells. Overall, the 14 cases of reconstitution that were confirmed by alum carmine staining also represented the top14 final BLI readings.Enriched MaSC activity in GFPhi cells. (A) 500 GFP(9.30 MB TIF)Click here for additional data file.Figure S2In vitro luciferase assay for sorted mammary epithelial cells from virgin and parous mice. Mammary epithelial cell populations were collected based on CD24 and CD29 staining and lysed for in vitro quantification of luciferase activity. Data shown is normalized to P4 cells (+/− SEM). The same pattern of luciferase activity is seen in virgin and pregnant mice, with the higher expression of luciferase in unsorted populations as the result of the higher percentage of luciferase expressing MaSCs in the parous mice.(3.76 MB TIF)Click here for additional data file.


Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events. Furthermore, we decided to exploit the possibility to use peptide arrays to predict kinase consensus sequences for deducing the preferential substrate peptide sequence of MAP3K8, a kinase which is homologous to c-Raf, but has a completely different function in cellular physiology, prompting the question whether both kinases share the same substrate preference or whether, despite the similarity in sequences both kinases have sufficient substrate specificity to account for the differences in biological function. MAP3K8 has been shown to participate in the transcriptional regulation of several important genes, including those for tumour necrosis factor alpha and IL-2 (Interleukin 2) c-Raf is a kinase important in human pathology, for instance, as a mediator of oncogenic Ras 33P-γ-ATP was purchased from Amersham Biosciences . MEKSer218/222/MEK2Ser222/226 antibodies were purchased from Upstate Biotechnology . Lysis buffer was purchased from Cell Signaling Technology. Lysis buffer was supplemented with protease and phosphatase inhibitors, including 1 mM sodium fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF (Phenylmethanesulfonyl fluoride).Truncated constitutively active human MAP3K8 kinase and c-Raf were purchased from Upstate Biotechnology . et alhttp://www.pepscanpresto.com/index.php?id=27. We used a second array consisting of 1024 peptides based on known phosphorylated motifs in human proteins found in the Human Protein Reference Database (HPRD) The trial arrays consisting of 192 peptides were kindly provided by Pepscan Systems . The full list of these substrates is listed elsewhere by Diks Trial peptide arrays consisting of 192 peptide substrates were used to test enzyme kinetics. 5 µg/ml of purified active MAP3K8 was incubated with trial peptide arrays for 1, 5, 10, 20, 30, 45, 60 and 120 minutes.In vitro kinase assays were used according to the instructions of the manufacturer . Active MAP3Ks were diluted in an Mg/ATP mixture and recombinant inactive MEK was added and in vitro kinase assays were performed at 30°C for 20 minutes. Samples were dissolved in sample buffer, incubated at 95°C for 5 minutes, and analyzed on Western blot using an anti-phospho-MEKSer218/222/MEK2Ser222/226 Antibody.2, 0.05% v/v Brij-35, 25 µg/ml BSA (Bovine Serum Albumin)) and 30 µCi 33P-γ-ATP, at 37°C for 60 minutes in a humidified oven. The slide was then washed twice with PBS (+0.1% Triton X-100), 2M NaCl and demineralised H2O and dried with N2 gas. Only active c-Raf was incubated with the peptide array consisting of 1024 pseudo-substrates and the same washing steps were carried out.Peptide arrays with 1176 different kinase pseudo-substrates were incubated with active c-Raf and MAP3K8 incubation mix  and exported to a spreadsheet program . The relative contribution of each individual amino acid at each individual position was calculated and corrected for the relative abundance of that amino acid at that position relative to the central serine, threonine or tyrosine residues and the respective consensus sequences were generated.After drying, the glass slides were exposed to a phosphor imager plate for 72 hours. Acquisition of the peptide array was performed using a phosphor-imager . The level of incorporated radioactivity, which corresponds to the phosphorylation status, was quantified by Scanalyze. . A set of hundred random peptides from the 1176 array design were generated using Microsoft Excel.  and each of these peptides were aligned individually against the single sequence obtained from the 1024 array design using MultAlin We set out to evaluate the usefulness of peptide arrays for deriving consensus substrate sequences for kinases. To extract useful information from the phosphorylation of peptide arrays by kinases, it is important to ensure that such phosphorylation conforms to the Michaelis-Menten laws of enzyme kinetics. Hence, we decided to perform a series of initial experiments using active MAP3K8 on trial arrays consisting of 192 peptide substrates investigating array phosphorylation in the temporal domain. in vitro phosphorylation studies was examined by incubating it with MEK (Mitogen Activated Protein Kinase Kinase), a well established substrate. As evident from 33P, demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation  and the relative contribution of each individual amino acid at each individual position was calculated and corrected for the relative abundance of that amino acid at that position relative to the central serine, threonine or tyrosine. The resulting putative c-Raf consensus sequence is shown in If analysis of the contribution of each amino acid at each position in peptide array phosphorylation patterns yields meaningful results, it should follow that the analysis of phosphorylation of an array containing totally different substrate peptides, which on determination showed only five amino acids to be common, on average, between the two designs, should give a similar result. To test this hypothesis, we analyzed c-Raf-dependent phosphorylation of an array: consisting of 1024 peptides . SimilarSubsequently, we addressed the question whether the various MAP3Ks, which share substantial sequence homology  in theirSubsequent analysis, however, of the importance of the individual amino acids relative to the central residue shows that the substrate preference of both kinases also displays substantial similarities, with preferences for arginine, isoleucine, lysine and alanine at −3, −2, −1 and +1 positions which are also preferred by c-Raf. However, a major difference between the two is the strong preference for phenylalanine at positions −1, −2, −3, +1 in MAP3K8. Another important difference is the absence of proline at the −2 and +1 positions in MAP3K8. .et alThe predominance of phosphorylation as a regulator of cellular metabolism makes it of utmost importance to know kinase substrates for proper understanding of cellular physiology. Unfortunately, our understanding of kinase action does not yet permit the determination of kinase substrates based on the primary sequence of proteins. Indeed, if kinases with highly similar amino acid composition have similar or distinct substrate preferences remain unknown. Hence, empirical methods for determining kinase substrate sequences remain essential. We describe here a novel methodology for predicting kinase substrates, which makes use of a library of peptides, known to serve as phosphorylation motifs to determine a kinase substrate consensus phosphorylation sequence and employ this methodology for comparing substrates for c-Raf and MAP3K8 enzymatic activity respectively. c-Raf and MAP3K8 are two serine/threonine kinases which are associated with cellular transformation, but which are suggested to have divergent functions in cellular physiology despite their high sequence homology. Employing peptide arrays we derived consensus sequences for substrate phosphorylation based on the relative importance of multiple amino acids  carried out at each position and this sequence could then be used to search databases and predict possible substrates. We considered only peptides which had a single phosphorylation site as one of the concerns we had was that some peptides could also be phosphorylated on more than one spot and would thus add to the intensity generated from that peptide. However, as suggested by Diks Confidence in our results was bolstered by the observation that two peptide arrays displaying different peptides yielded similar results. On comparing the consensus sequence derived from the 1176 array design:ST]-[ANPQRILV]-[AGKLPR]-[AGIKR] with that derived from the 1024 array design:[AIKRV]-[ALPKQR]-[AGIKPR]-[ST]-[AGLMPR]-[AKPR]-[DEKLMPR]-[ADLNRV]-[FKLQR] one can see that practically the same group of amino acids are seen at positions −3 to −1, except at positions +1 and +3 with methionine appearing instead of aspargine and glycine, and methionine in place of glycine respectively. Surprisingly, with the 1176 array, two peptide motifs with a central tyrosine residue are among the substrates favoured by c-Raf, showing that serine/threonine restriction of this kinase is not necessarily absolute. Maybe this observation has physiological relevance. Phosphorylation of Y340 in c-Raf itself is important for association to MEK, it's most important substrate, thus auto-phosphorylation of c-Raf at this residue may contribute to its physiological function, but obviously further studies are essential for answering this question. We feel, however, that weighing individual amino acids in their contribution to overall peptide array phosphorylation seems a valid tool for determining consensus sequences.[GKLMR]-[AGKLR]-[AKLQR]-[AGIKLPQR]-[AGKLPR]-[ST]-[AFGKLV]-[AGIKR]-[AGHKLW]) shows the same set of amino acids dominating except for the strong presence of phenylalanine at all 3 positions N terminal and the +1 position C terminal to the centrally fixed serine/threonine residue. Another difference observed is the dominance of lysine over arginine at all positions where basic residues are seen. Although these two kinases show very similar consensus/scaffold sequences, they seem to phosphorylate completely different sets of substrates as seen in Comparison of c-Raf with MAP3K8 ([AFIKR]-[AFHKQR]-[AFGIKPV]-[c-Raf seems to have a strong preference for basic residues at the −3 position and hydrophobic residues at the +1 position relative the phosphorylated serine/threonine residue. The Protein kinase C (PKC), AKT kinase (Protein Kinase B), mammalian AMP-activated protein kinase, SNF1 (sucrose non-fermenting kinase 1), calcium/calmodulin-dependant kinase, phosphorylase kinase have similar preferences for basic residues at the −3 position and hydrophobic residues at the +1 position in vivo. Moreover, these substrates also had phenylalanine at the +1 position C terminal to the centrally fixed serine residue (FKKSFKL for MARCKS and KKFSFKK for MARCKS-related protein), which was one of the important differences seen between the c-Raf and MAP3K8 consensus sequences as described above. It is also interesting to note that BLAST in vivo influence the extent to which a motif is really subject to phosphorylation by c-Raf or MAP3K8. In this sense, the consensus sequence determination is more suitable for determining the possible upstream regulatory kinases when an amino acid is known to undergo phosphorylation, rather than providing insights in the downstream targets of a given kinase. Nevertheless, analysis of possible downstream targets may be useful for hypothesis generation. In conclusion, we have developed a new method to generate consensus sequences using peptide arrays based on the relative intensity of amino acids seen at all the positions N and C terminal to the centrally fixed serine/threonine/tyrosine residue. The general utility of this method would be identification of new substrates and it also has an edge over the oriented and combinatorial peptide microarrays as it has peptides with known phosphorylation sites for different kinases.With the consensus sequences determined, a broad choice of possible substrates remains for both c-Raf as well as MAP3K8 in the human genome. To test this, we performed kinase restricted pattern searches using Scanprosite Data S1Peptides with single phosphorylatable residues considered for 1176 array analysis(0.34 MB DOC)Click here for additional data file.Data S2Peptides on 1024 array with single phosphorylation sites that were used for analysis(0.13 MB DOC)Click here for additional data file.


We demonstrated that inhibition of neutral endopeptidase (NEP), a protease that degrades vaso- and neuroactive peptides, improves vascular and neural function in diabetic animal models. In this study we explored the role of NEP in neuropathy related to either insulin-deficient diabetes or diet-induced obesity using NEP deficient (−/−) mice. Initial studies showed that streptozotocin, in the absence of subsequent hyperglycemia, did not induce nerve conduction slowing or paw thermal hypoalgesia. Glucose disposal was impaired in both C57Bl/6 and NEP −/− mice fed a high fat diet. Thermal hypoalgesia and nerve conduction slowing were present in both streptozotocin-diabetic and high fat fed C57Bl/6 mice but not in NEP −/− mice exposed to either streptozotocin-induced diabetes or a high fat diet. These studies suggest that streptozotocin does not induce neurotoxicity in mice and that NEP plays a role in regulating nerve function in insulin-deficient diabetes and diet-induced obesity. Peripheral neuropathy affects patients with diabetes as well as patients that have impaired glucose tolerance and are considered to be prediabetic –5. DiabeOur studies have focused on the role of vascular dysfunction in peripheral neuropathy using diabetic animal models as well as animal models of obesity –10. In bUnless stated otherwise all chemicals used in these studies were obtained from Sigma Chemical Co. . μM 7-amido-3-methylcoumarin (AMC)/min/μg protein, respectively (*P < .001 versus C57Bl/6 by unpaired t-test). C57Bl/6 wild type mice were purchased from Jackson Laboratories and Swiss Webster mice were purchased from Harlan laboratories. Breeding pairs of neutral endopeptidase deficient (NEP −/−) mice were provided by Lu et al. and are on the C57Bl/6 background . These mMice were housed in a certified animal care facility and food  and water were provided ad libitum. Adequate measures were taken to minimize pain or discomfort and all of the experiments were conducted in accordance with international standards on animal welfare and were compliant with all institutional and National Research Council's guidelines for use of animals (ACURF protocol 0802033). β cells [An initial study was performed to address whether streptozotocin causes direct neurotoxicity, independent of inducing insulin deficiency and hyperglycemia. This study was performed by two different laboratories  using two different strains of mice. C57Bl/6 and Swiss Webster mice at 12 weeks of age were divided into three groups. The first group was treated with vehicle and served as the control group. The second group was treated with streptozotocin  to induce diabetes. The third group was treated with 3-O-methylglucose  approximately one minute prior to each streptozotocin injection to prevent the effects of the streptozotocin on the pancreatic β cells , 30. DiaFor the study examining the influence of deletion of the NEP gene C57Bl/6 and NEP −/− mice at 12 weeks of age were divided into three groups. One group was fed the standard chow diet. A second group was fed a diet containing 60% kcal as fat . The third group was treated with streptozotocin  to induce diabetes. Mice having blood glucose level of 300 mg/dL (16.7 mM) or greater were considered to be diabetic. The experimental period lasted for 12 weeks. Prior to behavioral and nerve conduction studies control and high fat fed mice were fasted overnight for study of glucose utilization. Mice were injected with a saline solution containing 2 g/kg glucose, i.p. Immediately prior to the glucose injection and for 120 minutes afterwards blood samples were taken to measure circulating glucose levels. The day before the terminal studies thermal nociceptive response in the hindpaw was measured using the Hargreaves method with instrumentation provided by IITC Life Science; Woodland Hills, CA (model 390G) or UARD (San Diego). The test was performed when possible in a blind manner. Thermal nociceptive responses were measured by placing the mouse in the observation chamber on top of the thermal testing apparatus and allowing it to acclimate to the warmed glass surface (30°C) and surroundings for a period of 15 minutes. The mobile heat source was maneuvered so that it was under the heal of the hindpaw and then activated, a process that activates a timer and locally warms the glass surface, when the mouse withdrew its paw, the timer, and the heat source was turned off . Followi Mice were anesthetized with Nembutal  or isoflurane (4% in oxygen) and nonfasting blood glucose levels determined with the use of glucose oxidase reagent strips . Afterwards, motor and sensory nerve conduction velocities were determined as previously described , 13, 32.P-value of less .05 was considered significant. The results are presented as mean ± SE. Comparisons between the groups for body weight, blood glucose, MNCV, SNCV, and thermal nociception latency were conducted using a one-way ANOVA and Bonferroni's test for multiple comparisons . A β cells from streptozotocin toxicity [β cell damage. There has been a considerable amount of discussion about the possible neurotoxic effect of streptozotocin and the suitability of streptozotocin-induced diabetes as an acceptable model for diabetic neuropathy especially in mice when high doses of streptozotocin are used to induce diabetes. To address this issue we used a procedure previously used in rats to protect the pancreatic toxicity , 30. C57Data in Data in After 12 weeks of untreated diabetes or high fat diet motor and sensory nerve conduction velocity was determined in C57Bl/6 and neutral endopeptidase deficient mice and respective controls. Data in Data in Diabetes is the most common cause of peripheral nerve damage rendering both diffuse damage referred to as polyneuropathy and focal damage or mononeuropathy , 34. It My laboratory has focused on the role of microvascular dysfunction in the development and progression of peripheral nerve disorders. Our studies in both type 1 and type 2 diabetic rats and rat models of metabolic syndrome have demonstrated that impaired vascular reactivity precedes the development of nerve dysfunction as identified by reduced nerve conduction velocity , 8–11. RIn order to further investigate the role of neutral endopeptidase in peripheral nerve dysfunction we examined the effect of streptozotocin-induced diabetes and diet-induced obesity on nerve conduction velocity and thermal response latency in the hindpaw of C57Bl/6 mice and mice deficient in neutral endopeptidase. The two major findings of these studies are that streptozotocin itself is not neurotoxic and that neutral endopeptidase deficient mice are protected from the slowing of nerve conduction velocity and thermal hypoalgesia that occur in streptozotocin-induced diabetic- or diet-induced obesity-C57Bl/6 mice.Pabbidi et al.  have repOur findings suggest that neutral endopeptidase activity contributes to peripheral neuropathy observed in type 1 diabetes and diet-induced obesity. Slowing of motor and sensory nerve conduction velocity is a common feature in animal models of streptozotocin-induced diabetes , 50. LikP in the dorsal root ganglion of diabetic mice by nerve growth factor has also been shown to prevent/improve diabetic sensory neuropathy [P it would be expected that mice deficient in neutral endopeptidase would maintain higher levels of both of these neuroactive peptides and perhaps be protected from neuropathy.A reason for the normalcy of nerve conduction velocity in diabetic and obese neutral endopeptidase deficient mice could be the preservation of vascular function. In diabetic and obese rats we have demonstrated that treatment with a vasopeptidase inhibitor prevents vascular dysfunction , 13. Thiuropathy . Since nP neuroactive peptides involved in pain perception. Neutral endopeptidase is expressed in normal skin and is increased in wounds [We observed that diabetes- or obesity-induced thermal hypoalgesia of the hindpaw was prevented in neutral endopeptidase deficient mice. In our studies mice were diabetic or fed a high fat containing diet for 12 weeks prior to experimentation. Beiswenger et al.  reportedn wounds . In normn wounds . Furthern wounds . It is tn wounds . We haven wounds . It remaNormal neutral endopeptidase deficient mice were thermal hyperalgesic compared to control C57Bl/6 mice as determined by latency of withdrawal threshold of the hindpaw. Increased sensitivity to pain stimulus has previously been demonstrated in neutral endopeptidase deficient mice or inhibition of neutral endopeptidase activity –58. It hUsing C57Bl/6 and Swiss Webster mice we found that streptozotocin alone is not neurotoxic or responsible for nerve dysfunction associated with streptozotocin-induced diabetes. These studies also provide additional evidence that expression of neutral endopeptidase plays a role in peripheral neuropathy that accompanies diabetes and/or diet-induced obesity. Continued investigation into therapeutic means to manipulate the activity of neutral endopeptidase and perhaps other metalloendopeptidases may lead to an effective treatment for peripheral polyneuropathy.