|Coordinate||41,670,961 bp (GRCm38)|
|Base Change||A ⇒ T (forward strand)|
|Gene Name||transient receptor potential cation channel, subfamily V, member 5|
|Chromosomal Location||41,652,173-41,680,769 bp (-)|
|MGI Phenotype||Homozygous mutant mice exhibit increased calcium excretion and reduced bone thickenss.|
|Amino Acid Change||Valine changed to Glutamic Acid|
|Institutional Source||Beutler Lab|
V306E in Ensembl: ENSMUSP00000031901 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||Autosomal Recessive|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Sperm, gDNA|
|Last Updated||2016-11-10 11:53 AM by Anne Murray|
|Record Created||2010-08-10 7:26 PM by Wataru Tomisato|
The gingame phenotype was identified among ENU-mutagenized G3 mice in a screen for mutants with susceptibility to dextran sulfate sodium (DSS)-induced colitis (Figure 1). The screen uses weight-loss as an indication of colitis. The gingame mouse were susceptible to low doses of DSS (1%) and showed bleeding, diarrhea, and severe weight loss. Further observation determined that the gingame mice are polyuric, subsequently leading to polydipsia, an increase in uptake of the water with the DSS, and the phenotype observed in the screen.
|Nature of Mutation|
The gingame mutation was mapped using bulk segregation analysis (BSA) of F2 backcross offspring using C57BL/10J as the mapping strain. The mutation showed strongest linkage with marker B10SNPSG0061 at position 67742189 bp on Chromsome 6 (synthetic LOD=5.6). Whole genome SOLiD sequencing of a homozygous gingame mouse and validation by capillary sequencing identified an T to A transversion at base pair 426047032, 27.1 Mb from the marker of peak linkage, on Chromosome 6 in the Genbank genomic region NC_000072 encoding Trpv5. The mutation corresponds to residue 1031 in exon 8 of 15.
The mutated nucleotide is indicated in red lettering, and results in substitution of valine (V) 306 with a glutamic acid (E) in the encoded protein.
Transient receptor potential (TRP) cation channel, subfamily V, member 5 (Trpv5) belongs to the transient receptor (TC 1.A.4) family, the TRPV subfamily and the TRPV5 sub-subfamily. Like the other transient receptor potential (TRP) family members (e.g. TRPV6), the 723 amino acid Trpv5 protein (TRPV5) has large intracellular N- and C-terminal tails as well as six transmembrane (TM)-spanning domains, with a putative hydrophobic, pore-forming region between the fifth and sixth domains (aa 517-539) (1-3) (Figure 4 & 5). Permeation of the channel by Ca2+ is essential for the proper functioning of TRPV5 in Ca2+ (re)absorption in the kidney, intestine and placenta (4). The first 38 residues of the N-terminus are essential for TRPV5 activity as well as for efficient trafficking out of the endoplasmic reticulum (ER) and golgi (5). Residues 64-77 in the N-terminal tail are essential for channel assembly and tetramerization (6;7). In addition, the protein has six putative protein kinase C (PKC) phosphorylation sites as well as protein kinase A (PKA) and Ca2+/calmodulin-activating kinase II phosphorylation sites (8-11). PKC has been shown to upregulate TRPV5 through an alteration of channel properties (i.e. single channel open probability) and/or channel trafficking (12). Activation of TRPV5 by PKC can inhibit caveolin-dependent endocytosis of TRPV5, causing accumulation of TRPV5 at the cell surface (12). PKC-mediated phosphorylation (Ser299, Ser654) is essential not only for hormone-triggered phosphorylation of TRPV5, but is also necessary for tissue kallikrein-mediated TRPV5 stimulation (5;10). Kallikrein is a kidney protease that releases kinin to stimulate Ca2+ reabsorption through a PKC-dependent plasma membrane accumulation of TRPV5 (10). Activation of PKC following kallikrein application to cells regulates the balance between constitutive exocytosis and endocytosis, favoring exocytosis (13). PKC-dependent phosphorylation of TRPV5 may also lead to an activation of motor proteins that transport TRPV5 to the plasma membrane (13). These effects lead to an accumulation of TRPV5 at the cell surface.
TRPV5 and TRPV6 originate from two genes that are juxtaposed on mouse chromosome 6 and the proteins share a 75% homology; the main differences between the two proteins can be found within the N and C-termini (1;14;15). Similar to other TRPV channels, TRPV5 and TRPV6 have six-repeat ankyrin repeat domains at the N-terminal cytosolic domain (16). The crystal structure of the mouse TRPV6 ankyrin repeats has been solved [PDB #2RFA; illustrated in Figure 5; (16)]. The repeats are anti-parallel inner and outer α-helices and the helices are joined by finger loops (16). In addition there is a twist between repeats ANK1-4 and ANK5-6 caused by conserved TRPV substitutions in the ankyrin repeat consensus (16). Only four of the ankyrin repeats in TRPV5 have been delineated (aa 72-101, 110-149, 156-185, 232-261). The ankyrin repeats maintain the activity of the channel, the interaction of TRPV5 with other proteins or itself, and the targeting of TRPV5 to the plasma membrane (1;9;10;17;18). Ankyrin repeat 1 (aa 72-101) of TRPV5 has been implicated in channel assembly (16). In TRPV6, the ankyrin repeat region(s) within aa 116-191 has been shown to be sufficient to enable self-interaction and functional subunit assembly (6;19). The ankyrin repeat has been proposed to initiate a molecular zippering process that proceeds past the last ankyrin repeat to create an anchor for subunit assembly (7;19). Interestingly, evidence suggests that this region in TRPV6 is involved in TRPV5 channel assembly (6;7). A TRPV5 construct that contains only residues 11-267 (residues surrounding all four ankyrin repeats) binds calmodulin (CaM) (16). CaM is a cytoplasmic protein that regulates the biological activities of proteins and ion transporters, often in a Ca2+-dependent manner, although Ca2+-CaM had no effect on TRPV5 activity in the 11-287 TRPV5 construct (20). Another study found that there are CaM binding sites within the 1-327 region of the N-terminus as well as the 578-730 region of the C-terminus (21).
Within the transmembrane domain of TRPV5 there are two important areas that are essential for the proper functioning of TRPV5. The first is three residues within the intracellular loop between TM2 and TM3 (aa 409, 411, and 412) are essential for the initial inactivation kinetics of TRPV5 (9)(9). In addition to these essential residues, TRPV5 is Asn (N)-linked glycosylated at N358 within the extracellular loop between the first and second TM segments and is necessary for Klotho-mediated stimulation of TRPV5 (17;22). Klotho is a member of the β-glucuronidase family of enzymes that catalyze the breakdown of complex carbohydrates. In cells that had N358 mutated to a glutamine, Ca2+ influx was no different than in cells that expressed the wild-type protein (17;22). However, it was found that glycosylation is necessary for accumulation of TRPV5 at the plasma membrane (17). It is speculated that Klotho-mediated accumulation of TRPV5 is mediated by a hydrolysis of extracellular sugar (i.e. terminal 2,6-sialic acids) from TRPV5 (13;17;23). Once the terminal sialic acids are removed, the underlying disaccharide N-acetyllactosamine (LacNAc) is exposed to galectin-1 (23). It is binding of the galectin-1 that leads to accumulation of TRPV5 at the plasma membrane (23).
The C-terminus of TRPV5 has several important motifs. Similar to other TRP family members, TRPV proteins contain a TRP box (a 6-amino acid motif) and a proline-rich sequence within a TRP domain located after the sixth TM segment (14;24). The TRP domain is a short hydrophobic segment that is necessary for phosphatidylinositol 4,5-bisphosphate (PIP2) binding (a ubiquitously expressed phospholipid that is a regulator of channel function). The TRP box is proposed to be a coiled-coil zipper that regulates TRPV channel gating (keeping it in a closed conformation) (5;14;24;25). There are two regions within the cytoplasmic C-terminus that are involved in Ca2+-dependent inactivation of TRPV5 (aa 643-646, 693-723) (26). These sites may interact with proteins that are necessary for inactivation. C-terminal residues 596-601 are essential for channel assembly and trafficking of TRPV5 (6;19). The C-terminal PDZ (PSD95/Discs-large/ZO-1) binding motif of TRPV5 interacts with the PDZ domain of NHERF2 (Na+/H+ exchanger regulatory factor 2) functioning to link TRPV5 to cytoskeletal proteins as well as targeting and stabilizing TRPV5 at the cell membrane (18). Furthermore, TRVP5 contains a putative serum- and glucocorticoid-inducible kinase (SGK1) phosphorylation site, allowing for SGK1-dependent stimulation of activity (18). SGK1 is transcriptionally upregulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a vitamin D metabolite that also regulates TRPV5 (through its NHERF2 interaction) and is involved in several functions within the cell including regulation of transport, hormone release, neuroexcitability, inflammation, coagulation, cell proliferation and apoptosis (18;27). SGK1 may act to increase the abundance of TRPV5 in the membrane (18).
TRPV5 can interact with several proteins to regulate its function and/or trafficking. At aa 591-595, TRPV5 interacts with S100A10 as well as the S100A10/annexin 2 heterotetramer (comprised of a S100A10 dimer and two annexin 2 molecules) to facilitate the trafficking of TRPV5 to the plasma membrane (28). S100A10 is a member of the S100 family that is generally localized to the cytoskeleton underlying the plasma membrane and participates in membrane trafficking and/or organization (29). It is unclear whether the association of TRPV5 and S100A10/annexin 2 facilitates the translocation of TRPV5 to the plasma membrane or enhances channel retention (28). Downregulation of annexin 2 in the S100A10-TRPV5 complex leads to inhibition of currents through TRPV5 (28). Other proteins that interact with TRPV5 are BSPRY (B-box and SPRY-domain containing protein) and Rab11a (30). BSPRY associates with the C-terminus of TRPV5 (30). Evaluation of Ca2+ influx via TRPV5 after BSPRY co-expression found that influx was inhibited although TRPV5 expression levels were not changed (30). Therefore, BSPRY is thought to be involved in inhibitory signaling cascades that can control the activity of TRPV5 at the cell surface (30). The TRPV5 C-terminus (aa 595-601) interacts with the GTPase Rab11a to target TRPV5 to the plasma membrane (31). GDP-bound Rab11a initially interacts with TRPV5 in a cytoplasmic compartment. During GDP-GTP exchange, Rab11a does not associate with TRPV5. Finally, the GTP-bound Rab11a mediates transport of TRPV5-containing structures to the plasma membrane (31). TRPV5 and TRPV6 can form homo- and heterotetramers through interactions at both the N- (aa 64-77) and C-termini within the ER before being trafficked to the plasma membrane (1;3;5;32). The tetrameric TRPV5 and TRPV6 structures can also combine to form heteromultimeric channels that may have novel properties (32). The formation of a tetrameric structure between TRPV5 and TRPV6 generates a negatively charged ring of four aspartate residues (D542) that can function as a Ca2+ filter (1;3;4;32;33).
The gingame mutation results in a substitution of valine (V) 306 with a glutamic acid (E). V306 is within the intracellular N-terminal tail of TRPV5, between the last ankyrin repeat and the first TM domain.
RT-PCR, Northern blot, and dot blot analyses detected Trpv5 in kidney, pancreas, testis, prostate, placenta, brain, intestine (including duodenum, jejunum, colon, and rectum), as well as in mammary, sweat, pituitary, and salivary glands (1;15;32;34-37). The most abundant expression was noted in kidney, duodenum, jejunum, and pancreas (pancreatic islets); lower abundance was observed in testis, prostate, placenta, brain, colon, rectum, trachea, esophagus, and stomach; there was no signal in the ileum or the distal segments of the large intestine (15;36). In addition, Trpv5 was detected in the spinal cord, whole brain, cerebral cortex, caudate nucleus, occipital lobe, putamen, substantia nigra, temporal lobe, and hippocampus in the nervous system (36). Trpv5 is also expressed in mouse osteoclasts at the ruffled border membrane, the cellular domain that facilitates removal of bone matrix (3;38;39). In the rat intestine, Trpv5 mRNA is expressed in nodose ganglion neurons that express cholecystokinin (CCK) 1 (i.e. interneurons that mediate feedforward inhibition and behavioral fear responses) (40).
Further analysis determined that in the kidney, the TRPV5 protein is localized at the apical domain of kidney distal convoluted and connecting tubular cells (1;13;32;39). In the intestine, TRPV5 is localized to the apical region of enterocytes (brush border membrane) in the mucosa of the small and large intestine (1;2;41).
TRPV5 forms a heterotetramer with TRPV6 and the proteins are co-expressed in the epithelia of organs that mediate transcellular Ca2+ transport such as duodenum, jejunum, colon and kidney (37;42). They are also co-expressed in other tissues such as pancreas, prostate, mammary gland, sweat gland, and salivary gland (37). Although they are co-expressed in these tissues, expression levels vary between the two proteins (43). For example, TRPV5 is the major isoform in the kidney, while TRPV6 is highly expressed in the intestine (1;43).
Ca2+ is essential for several processes throughout the body including muscle contraction, bone formation, and neuronal function. To maintain Ca2+ balance, three organ systems work together: the gastrointestinal tract, bone and kidney. More specifically, Ca2+ is absorbed by the gastrointestinal tract (~90% through the small intestine; ~10% in the colon), filtered and reabsorbed in the kidney, and stored and released from the bone as needed (1;3;44). Changes in the rates of renal and intestinal epithelia transport as well as the storage and release from the bone regulate Ca2+ balance in the body (3;39). The efficiency of intestinal Ca2+ absorption regulates the availability of dietary Ca2+. An alteration in the vitamin D endocrine system as well as the lipid composition and fluidity of intestinal membranes can increase the intestinal Ca2+ absorption rates when dietary Ca2+ levels are low (1). Changes in intestinal Ca2+ absorption rates can be observed in several physiological processes including growth, pregnancy and lactation (an increase); a decrease in Ca2+ absorption is observed with age (1). Impaired neural excitability, arrhythmia and altered bone formation can be observed when there is a failure to maintain Ca2+ levels in the plasma (3).
The Paracellular and Transcellular Pathways
In the intestine, Ca2+ can be absorbed by two mechanisms: a metabolically driven, saturable transport (i.e. the transcellular pathway) or a passive, non-saturable route (i.e. the paracellular pathway) (1). Factors such as hormones and nutrients regulate both mechanisms (1). For example, the transcellular pathway is regulated mainly by a vitamin D metabolite 1,25(OH)2D3 that binds to the nuclear vitamin D receptor, leading to the activation of gene transcription (1). Under normal conditions, ~90% of Ca2+ transport occurs along concentration and electrical gradients via the paracellular pathway through junctional complexes between cells in the jejunum and ileum of the small intestine (1;3;45). When the demand for Ca2+ increases (e.g. during laction, pregnancy, or when dietary Ca2+ is low), Ca2+ absorption occurs predominantly (~80%) via the transcellular route in enterocytes of the duodenum (3;45). In the transcellular pathway, Ca2+ enters the cell through Ca2+ channels (e.g. TRPV5). These channels are involved in regulating several cell functions such as contraction, secretion, gene expression, transepithelial ion transport, osmoregulation, and transduction of painful stimuli (9). Within the cell of the kidney and intestine, Ca2+ is bound to calbindins, carrier proteins that facilitate the diffusion of Ca2+ to the basolateral membrane. Calbindin D is the carrier protein in the enterocyte (calbindin-D9K in the mouse duodenum) that mediates Ca2+ transport either through diffusion or vesicle transport (3;39;45). At the basolateral membrane, the Ca2+ is transported into the interstitial fluid by ion exchangers and pumps such as the plasma membrane ATPase (PMCA1b) and the Na+/ Ca2+ exchanger (NCX1) (3;39;44;45).
The TRP channels
The TRP ion channel family has seven subfamilies: TRP Canonical (TRPC), TRP Melastatin (TRPM), TRP Ankyrin (TRPA), TRP Mucolipin (TRPML), TRP Polycystin (TRPP), TRP NOPMC (TRPN), and TRP Vanilloid (TRPV) (2;3;45;46). Vertebrates have at least one receptor from each subfamily except for TRPN (47). These receptor subfamilies respond to several external stimuli such as light (i.e. phototransduction), chemicals and temperature as well as mechanical and osmotic pressures (2;32;48-50). TRP channels have roles in diverse processes including olfaction, nociception, speech, regulation of blood circulation, pain signal transduction, gut motility, mineral absorption and fluid balance epithelial Ca2+ transport, development of airway and bladder hypersensitivities, and cell survival, growth and death (48). The TRP channels function by facilitating the transmembrane flow of cations (i.e. Na+ and Ca2+) down electrochemical gradients to depolarize the cell as well as to mediate signal transduction (45;47).
TRP channels can be activated through the activation of phospholipase C (PLC) by G protein-coupled receptors and receptor tyrosine kinases. Activation of PLC leads to hydrolysis of PIP2, producing diacylglycerol (DAG) and inositol (1,4,5) triphosphate (IP3). Strong evidence supports roles for PIP2 hydrolysis and DAG production in modulating TRP channel activity. On the other hand, the function of TRP channels as “store-operated calcium entry” channels activated by IP3-mediated release of intracellular Ca2+ stores remains controversial (47). The TRP channels can also be activated by ligands that range from exogenous small organic molecules (e.g. capsaicin), endogenous lipids or products of lipid metabolism (e.g. DAG), purine nucleotides and their metabolites (e.g. adenosine diphosphoribose (ADP-ribose)), to inorganic ions (e.g. Ca2+ and Mg2+) (47). A third mechanism is one of direct TRP channel activation by temperature changes, mechanical stimuli, coupling to IP3 receptors, cell swelling, channel phosphorylation through protein kinases A, C, and G (PKA, PKC, and PKG, respectively), and Ca2+/Calmodulin signaling (47).
The TRPV subfamily has six receptors in mammals (TRPV1—TRPV6), one receptor in C. elegans (Osm-9) and one in Drosophila (Nan) (48;51). The TRPV subfamily members are all Ca2+ entry channels that are stimulated by different physical and chemical stimuli (e.g. TRPV1 is gated by capsaicin and heat; TRPV2, heat; TRPV3, heat; TRPV4, cell swelling) (52). TRPV5 and TRPV6 are homologous to each other and are the most Ca2+ sensitive of the TRP channels (3;45). TRPV5 and TRPV6 are unique among the other TRP channels in that they are constitutively open, they are more selective for Ca2+ than Na+, and the current-voltage relationship of the channels shows inward rectification as opposed to outward rectification (1;9;10).
TRPV5 is predominantly involved in the transcellular transport of Ca2+ in the distal tubules and loops of Henle in the kidney (3;38;45;53;54). Once within the cell, the transfer of the Ca2+ to the basolateral membrane occurs through an association with calbindin D28K, and PMCA1b and NCX1 extrude the Ca2+ from the tubule cells [Figure 6; (10;45)]. TRPV5, along with TRPV6, is also involved in transcellular intestinal Ca2+ reabsorption (2;38;41;53-55).
The regulation of TRPV5 can occur at several levels including: alterations at the gene level, trafficking to the cell membrane, the protein abundance at the cell surface, and the channel activity [Table 1; (31)]. TRPV5 can be regulated by dietary conditions (i.e. by Ca2+ levels) as well as by hormones (2;41;55;56). Klotho is a β-glucuronidase that can hydrolyze the extracellular glycan residues on TRPV5 (as mentioned above in Protein Prediction). The modification of the sugar moiety on TRPV5 leads to trapping of the channel in the plasma membrane, leading to maintenance of channel activity and Ca2+ permeability (3;10;17;23). Other hormones such as 1,25(OH)2D3, parathyroid hormone (PTH), testosterone, and estrogen lead to changes in transcription of Trpv5 (1;3;10;57). PTH can also stimulate the activation of the protein kinase A (PKA) pathway and subsequent TRPV5-mediated Ca2+ transport upon PTH-dependent phosphorylation of TRPV5 (3). Another role of PTH in TRPV5 regulation is that of inhibiting caveolin-mediated endocytosis of TRPV5, leading to an increase in TRPV5 at the cell surface (3).
Accessory proteins can regulate TRPV5 activity by either directing TRPV5 trafficking to the plasma membrane (e.g. NHERF2 (Na+/H+ exchanger regulatory factor 2), NHERF4, Rab11a, WNK4 (with no lysine kinase 4)) or by altering the conformation of the channel at the plasma membrane (10). BSPRY (B-box and SPRY-domain containing protein), calbindin-D28K, and FK506-binding protein 52 modulate TRPV5 activity at the plasma membrane (10).
In experimental settings, TRPV5 can be regulated by the dye ruthenium red, which prevents PTH-stimulated Ca2+ transport as well as leads to decreased expression of calbindin-D28K and NCX1; TRVP5 expression was not changed (9;39).
Table 1. Regulation of TRPV5 [adapted from (57)]
TRPV5 animal models
The Trpv5 knockout mouse (Trpv5-/-) had no obvious phenotype, the pups thrived to adulthood and were the same size and weight as wild-type mice (76). Analysis of urine and serum samples collected from the knockout mice found that the loss of Trpv5 led to polyuria and hypercalciuria as a result of a diminished rate of renal Ca2+ reabsorption (especially along the distal convulution, the primary expression site of TRPV5); serum Ca2+ levels were normal (3;45;61;76). Polyuria is a mechanism to reduce the possibility of Ca2+ precipitation (76). Also, there were increased levels of 1,25(OH)2D3 in the serum and urine acidification (restrains formation of Ca2+ oxalate stones in the kidneys) (3;61;76). As a result of the hypercalciuria and/or prolonged elevated 1,25(OH)2D3 levels, the mice have disturbances in bone structure including reduced trabecular and cortical bone thickness (45;76). The Trpv5-/- mice show a compensatory upregulation of TRPV6 protein expression in the intestine (but not in the kidney), leading to Ca2+ hyperabsorption to alleviate the diminished Ca2+ levels caused by impaired Ca2+ reabsorption in the kidney (2;13;39;56;61;76). The TRPV5 knockout mice also displayed a decrease in calbindin-D28K and NCX1 mRNA levels, indicating that TRPV5 and/or Ca2+ influx via TRPV5 can control the transcription of other Ca2+ transport genes (76).
The gingame mutation is a T1031A mutation in Trpv5 that results in a V306E amino acid change at the N-terminus of TRPV5. The N-terminus of TRPV5 is essential for channel assembly, activity, targeting to the plasma membrane and interactions with other TRPV5 molecules to form a homotetrameric complex. Residue 306 is within a region of the N-terminus that interacts with CaM. It is possible that a change from the nonpolar valine to a negatively charged glutamic acid residue could alter the ability of CaM to bind to the N-terminus. However, studies have shown that mutations within the N-terminal CaM-binding motif do not alter the activity of TRPV5 (21). In addition to being within a CaM-binding motif, a mutation at V306 may lead to misfolding of the protein. The polyuria and polydipsia phenotype mimics that of the Trpv5-/- mouse (76).
|Primers||Primers cannot be located by automatic search.|
Gingame genotyping is performed by amplifying the region containing the mutation using PCR followed by sequencing of the amplified region to detect the nucleotide change. The following primers were used for PCR amplification and sequencing:
Primers for PCR amplification
Gingame (F): 5’-GCAAATGAGCAATGGCTTCTCGAATC-3'
Gingame (R): 5'-TGGAGACAACATCATCATCCC-3'
Primers for sequencing
Gingame_seq (F): 5’-GAATCTTTCTACAGTAGTTCAGTCC-3’
Gingame_seq (R): 5’-GAATTACCTGTAGTGATTTCTGTTCC-3'
1) 94°C 2:00
2) 94°C 0:30
3) 57°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29x
6) 72°C 7:00
7) 4°C ∞
The following sequence of 560 nucleotides (from Genbank genomic region: NC_000072.5 of the linear genomic sequence of Trpv5) is amplified; sense strand shown:
9448 gca aatgagcaat ggcttctcga atctttctac
9481 agtagttcag tcccctaact actactcatc tattatagac tcaatgttgt tgtatgcgct
9541 gtgattactt ctgtaatact tctatagcac taccacaaag cctaccatgt aatagaaatg
9601 aataatttct tatcaaggaa ataagtgctt ggatgatggt gatgatagtt gctgccctgt
9661 cacctggaat gcagttcaaa cctcaacact gcaaattgtg ttgttcattc tatattgctc
9721 tttatttctg acaggctcga cagattctag aacagacccc agtgaaggag ctggtgagcc
9781 tgaagtggaa gaaatatgga cagccttatt tctgcctcct gggtgctctg tacatcttct
9841 acatggtctg cttcaccaca tgctgtgttt accgccccct caagttccga gatgccaacc
9901 gtacacatgt tcgagataac accatcatgg aacagaaatc actacaggta attcttcttc
9961 aaagggatga aagggaatgg tgtcatggga tgatgatgtt gtctcca
PCR primer binding sites are underlined. Sequencing primer binding sites are highlighted; overlapping sequences with the PCR primers are highlighted and underlined; the mutated nucleotide is highlighted in red (T>A on sense strand; A>T on Chr. + strand).
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|Science Writers||Anne Murray|
|Illustrators||Diantha La Vine, Victoria Webster|
|Authors||Wataru Tomisato, Katharina Brandl, Bruce Beutler|