Phenotypic Mutation 'L1' (pdf version)
AlleleL1
Mutation Type missense
Chromosome3
Coordinate96,827,513 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Gja8
Gene Name gap junction protein, alpha 8
Synonym(s) Cnx50, connexin 50, dcm, Cx50, Lop10, alpha 8 connexin, Aey5
Chromosomal Location 96,820,882-96,833,336 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a transmembrane connexin protein that is necessary for lens growth and maturation of lens fiber cells. The encoded protein is a component of gap junction channels and functions in a calcium and pH-dependent manner. Mutations in this gene have been associated with zonular pulverulent cataracts, nuclear progressive cataracts, and cataract-microcornea syndrome. [provided by RefSeq, Dec 2009]
PHENOTYPE: Homozygous mutants exhibit microphthalmia, with small lenses and nuclear or total cataracts. Heterozygotes may be equally or less affected, depending on the particular mutation and the genetic background. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_008123; MGI: 99953

MappedYes 
Amino Acid Change Serine changed to Proline
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold P28236
SMART Domains Protein: ENSMUSP00000049532
Gene: ENSMUSG00000049908
AA Change: S50P

DomainStartEndE-ValueType
CNX 43 76 1.76e-20 SMART
low complexity region 134 147 N/A INTRINSIC
Connexin_CCC 168 234 2.8e-41 SMART
Pfam:Connexin50 267 333 7.3e-35 PFAM
low complexity region 337 355 N/A INTRINSIC
low complexity region 423 438 N/A INTRINSIC
Predicted Effect probably damaging

PolyPhen 2 Score 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000062944)
Meta Mutation Damage Score 0.7408 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Dominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(8) : Targeted, knock-out(2) Targeted, other(2) Spontaneous(1) Chemically induced(3)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01335:Gja8 APN 3 96826558 missense probably benign
IGL02114:Gja8 APN 3 96827341 missense probably benign 0.00
IGL02237:Gja8 APN 3 96827249 missense probably benign 0.00
IGL03204:Gja8 APN 3 96827408 missense probably damaging 1.00
guidance UTSW 3 96826740 missense probably benign 0.00
prediction UTSW 3 96826664 missense possibly damaging 0.64
R1024:Gja8 UTSW 3 96826740 missense probably benign 0.00
R2215:Gja8 UTSW 3 96827218 missense probably damaging 0.98
R2240:Gja8 UTSW 3 96827618 missense probably benign 0.05
R2510:Gja8 UTSW 3 96827033 missense probably damaging 1.00
R2511:Gja8 UTSW 3 96827033 missense probably damaging 1.00
R2926:Gja8 UTSW 3 96826469 missense probably benign 0.00
R3725:Gja8 UTSW 3 96827161 missense probably damaging 1.00
R4090:Gja8 UTSW 3 96826468 missense probably benign 0.00
R4933:Gja8 UTSW 3 96826351 intron probably benign
R5010:Gja8 UTSW 3 96827165 missense probably benign 0.24
R5497:Gja8 UTSW 3 96827513 missense probably damaging 1.00
R5532:Gja8 UTSW 3 96827648 missense probably benign 0.39
R6997:Gja8 UTSW 3 96826657 missense probably benign
R7381:Gja8 UTSW 3 96827338 missense probably benign
R7576:Gja8 UTSW 3 96827209 missense probably benign 0.05
R7792:Gja8 UTSW 3 96827092 missense probably damaging 1.00
R7827:Gja8 UTSW 3 96827635 missense possibly damaging 0.52
R8444:Gja8 UTSW 3 96826990 missense probably damaging 1.00
R9016:Gja8 UTSW 3 96827521 missense probably damaging 1.00
R9081:Gja8 UTSW 3 96826676 missense probably damaging 1.00
R9230:Gja8 UTSW 3 96826664 missense possibly damaging 0.64
Z1177:Gja8 UTSW 3 96827552 missense probably damaging 1.00
Mode of Inheritance Autosomal Dominant
Local Stock None
MMRRC Submission 030332-UCD
Last Updated 2016-05-13 3:09 PM by Stephen Lyon
Record Created unknown
Record Posted 2012-02-02
Phenotypic Description

The L1 mutant was identified among G1 mice born to ENU-mutagenized sires by slit-lamp examination, which detects cataracts (1).  Heterozygous and homozygous animals develop cataracts that affect the entire lens and also have small eyes (30% the size of wild type at the age of 3 weeks; Figure 1).  

Although both heterozygous and homozygous L1 animals develop similar postnatal phenotypes, examination of lens development revealed significant differences in lens defects.  Mature fiber cells are severely altered in both genotypes, but the elongation of primary fiber cells is significantly perturbed only in heterozygous animals.  At embryonic day (E) 15.5, a large cystic lumen was observed in the lens between posterior primary fiber cells and anterior epithelium with 100% penetrance in heterozygote L1 mice.  No such defect was observed in wild type or homozygous animals.

Nature of Mutation
The L1 mutation was mapped to Chromosome 3, and corresponds to a T to C transition at position 219 of the Gja8 transcript (Figure 2), in exon 2 of 2 total exons.  The first exon encodes the majority of the 5’ untranslated region of Gja8.
 
204 TGGGGCGATGAGCAATCTGATTTTGTATGCAAC
43  -W--G--D--E--Q--S--D--F--V--C--N-
 
The mutated nucleotide is indicated in red lettering.
Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain structure of mouse Cx50. Connexin 50 (Cx50) is one of a large family of connexin proteins which form gap junction channels important in intracellular communication and transport. The domain contains four transmembrane regions (TM), shown in gold. Two extracellular loops (EC) are shown in purple.  The cytoplasmic domains,aa 2-23, 100-157, and 230-440, are labeled Cyto.  The L1 mutation (S50P) is denoted by a red asterisk.

Figure 4. Membrane topology of Cx50. The N- and C-termini of Cx50 are cytoplasmic.  The four α-helix transmembrane domains of Cx50 are labeled TM1-4. The extracellular loops are labeled E1 and E2. The L1 mutation at S50 is indicated in red in E1. Cx50 forms a single connexin, which joins with other connexins to form a connexon (see Figure 5). Adapted from reference (3).

FIgure 5. Structure of gap junction intracellular channels. The protein Cx50 forms one connexin unit. Six connexins combine to form a connexon. Several members of the connexin family may combine to form homomeric or heteromeric intracellular channels (gap junctions) between adjacent cells. Gap junctions provide a mircrocirculatory system between cells by allowing passage of a variety of small cellular metabolites, ions, and second messangers. Adapted from (4).

Gja8 encodes mouse Connexin 50 (Cx50), a 440 amino acid protein member of a large family of connexin proteins (over 20 different connexins exist).  These proteins are the subunits of gap junction channels which are an important means of cellular communication and allow the exchange of ions (e.g. Na+, K+, Ca2+, and Cl-), second messengers (e.g. cAMP, cGMP, and inositol trisphosphate (IP3)), nutrients and small metabolites (e.g. glucose and amino acids) between cells (2-4).   Human Cx50 contains 433 residues and shares 89% identity with the mouse protein (5;6).

The absence of introns in the coding sequence is a common feature for all connexins (2;5).  Connexin proteins have four transmembrane domains with three intracellular regions (the N-terminus, a cytoplasmic loop and the C-terminus) and two extracellular loops (E1 and E2) (Figure 3 & 4) (4).  Both the transmembrane and extracellular domains of Cx50 are highly homologous to those of other connexins.  Each extracellular domain contains three characteristically spaced cysteine residues.  The central and carboxy terminal cytoplasmic domains are not conserved (3;5;7;8).  

Connexins form hexamers known as connexons (Figure 5).  Homomeric connexons are composed of six monomers of the same type of connexin whereas heteromeric connexons contain different subunit types (4;9).  A gap junction channel is produced when two connexons from adjacent cells align in the extracellular space via interactions between their extracellular domains.  Hundreds of gap junction channels come together to form gap junctions, which are morphologically defined as punctuate “plaques” of cell-cell contacts.  In addition to the homomeric and heteromeric composition of individual connexons, gap junction channels are classified as homotypic (the docking of two identical connexons), or heterotypic (the docking of two different types of connexons) (10).  Crystallography, electron microscopy and x-ray scattering analysis of various gap junction channels [reviewed in (8) and (3;7)], suggest an α-helical conformation for the four transmembrane domains of each connexin subunit.  The third transmembrane domain of each subunit is thought to be the major pore-lining helix with the E1 domain contributing to the aqueous pore at the extracellular end (11).  The two connexons composing the channel are rotationally staggered by approximately 30° relative to each other so that the α-helices of each connexin monomer are axially aligned with the α-helices of two adjacent monomers in the apposed connexon.  The conserved cysteines in the extracellular loops of each connexin subunit form intra-monomer disulfide bonds between the two loops.  These bonds stabilize the β structures formed by the extracellular domains and permit docking between connexons.  The second extracellular loop primarily determines the specificity of connexon interaction (3;7;8).  In some connexins, including Cx50, the C-terminal domain affects channel sensitivity to pH (12).  The C-terminal domain of Cx50 has also been shown to be important in conductance of gap junction channels (13), while the N-terminal half of the protein, probably due to the E1 domain, appears to interact with monovalent cations (14).  The cytoplasmic tail and loop are proposed to undergo regulatory posttranslational modifications (4).

The L1 mutation substitutes a hydrophobic proline for a highly conserved hydrophilic serine at position 50, in the extracellular loop 1 (E1) of the Cx50 protein (Figure 4).  Proline can disrupt α helixes and β sheets and may disrupt the β structure necessary for normal docking of connexons in the gap junction channel (15).  This change may also affect the ability of Cx50 to interact appropriately with monovalent cations.

Expression/Localization

In vertebrate species, Gja8 mRNA and protein expression has been reported to be specifically expressed in the embryonic and adult lens, particularly lens fiber cells (5;6;16;17).  Localization of the Cx50 protein is on the membranes of cells particularly in areas of cell-cell contact, consistent with its role as a gap junction protein (5;6;16;17).

Other studies have suggested additional areas of expression in the eye such as the corneal epithelium, ciliary body and Müller cells and astrocytes of the retina (18-21).  In the ciliary body, Cx50 protein is found only in the nonpigmented epithelium (NPE) at apical and basolateral membranes while Cx43 is found in the apposing pigmented epithelium (PE) (20).  Retinal expression of Cx50 was confirmed through Western blotting, RT-PCR and immunocytochemistry. Expression in the retina appears restricted to Müller cells and astrocytes of the optic nerve and along retinal projections into the CNS.  In Müller cells, labeling is strongest in the endfeet and in the filamentous processes ensheathing the photoreceptors (21).  Cx50 protein was also found transiently expressed in rat heart valves (22).  However, examination of the antibody used in these studies on Cx50-deficient mice, suggests that it cross-reacts with an epitope other than Cx50 and that Cx50 expression is restricted to the lens fibers of the eye (17).

Gja8 mRNA is observed throughout development and interestingly is expressed by embryonic day 9, preceding the development of the lens and the fiber cells (20;21;23).  The significance of this expression pattern in early lens development is unknown.

Background

Figure 6.  Normal development of the mammalian lens. Letters A through E illustrate key features in lens development. (A) The early surface ectoderm layer and the optic vesicle orginate from the diencephalon (not shown). (B) The ectoderm invaginates, forming the lens placode as it touches the optic vesicle. (C) Further movement of the ectodermal layer continues to form the primitive lens (i.e. the lens pit). The early retina begins as the presumptive neuroretina.  (D) Complete closure of the lens placode results in the formation of the lens vescicle. The optic vescicle has now formed the optic cup. (E) The lens vescicle is closed by elongation of the primary fibers and the neuroretina is formed. The optic stalk will later become the optic nerve. (F) The cellular structure of the lens. The epithelium caps the front of the lens. Cubidal epithelial cells migrate toward the lens equator, where they begin a process of elongation (i.e. differentiation) and eventual loss of intracellular organelles and the cell nucleus to form mature fibers. Once denucleation has occured, the lens fibers remain in place throught the life of the organism.   Abbreviations: SE, surface ectoderm; OV, optic vesicle; LPL, lens placode; LP, lens pit; PNR, presumptive neuroretina; LV, lens vesicle; OC, optic cup; PF, primary fibers; NR, neuroretina; OS, optic stalk. Adapted from (4).

The ocular lens is a unique organ able to focus light on the retina as a result of unique physiological properties and architecture that eliminates light scattering.  Normal development of the mouse lens begins by embryonic day 9.5-10 (E9.5-10) when the developing optic vesicles have approached the surface ectoderm, which begins to thicken and form the lens placode (Figure 5).  At E10.5, the lens placode begins to invaginate to form the lens vesicle.  At E11-13, the lens is characterized by elongation of cells from the posterior wall of the lens vesicle to form primary lens fibers, leading to obliteration of the vesicle cavity.  These primary fibers are maintained throughout life and constitute the nucleus of the adult lens.  The lens of the adult eye is an avascular tissue surrounded by the lens capsule, an extracellular matrix (ECM) secreted by lens cells.  Beneath the capsule, a single layer of cuboidal epithelial cells covers the anterior surface and is bathed in aqueous humour.  Further enlargement of the lens continues by proliferation and elongation of the epithelial cells at the lens equator to form secondary fibers, a process that continues throughout adult life.  Lens fibers face the posterior chamber of the eye and are bathed in vitreous humour.  Cues, such as fibroblast growth factor (FGF) present in the vitreous humour initiate the change from lens epithelial cell to lens fiber [reviewed in (24)].  The fiber cells adopt a flattened hexagonal profile that facilitates packing into an ordered array that promotes lens transparency.  Additionally, the fiber cells lose their intracellular organelles and undergo dramatic changes in the expression of cytoplasmic and membrane proteins.  An overabundance of soluble cytoplasmic crystallin proteins, creates a high index of refraction.  Some crystallins have a chaperone-like function and maintain the solubility of other proteins in the lens.  The crystallin concentration is highest in the nucleus and creates a radial gradient in refractive index that corrects for the shape of the lens (23-26).

Because of its unique function and anatomy, the mammalian lens is dependent on the proper functioning of gap junction proteins.  Along with Na+-K+ pumps, water channels and glucose transporters, gap junctions are critical components of the lens microcirculatory system, a flow of ionic current that is directed inwards at the poles and outwards at the equator and allows for nutrient and waste transport and maintenance of appropriate membrane potential.  Gap junctions couple the metabolically active lens epithelium with lens fibers that have lost their organelles to help support homeostasis of the lens fibers.  Moreover, gap junctions are differentially distributed on more equatorial fiber cells thus directing the microcirculatory current (2;26;27).  Loss of transparency of the lens due to disruption of this microcirculatory system or the lens architecture causes cataracts, the most common cause of blindness in humans.  Clinical descriptions of cataracts are based on the physical appearance and location of opacities within the lens.  Whole cataracts affect the entire lens while nuclear cataracts affect the center of the lens and the primary lens fibers.  Cortical cataracts originate in the lens cortex and lamellar cataracts are present in only one layer of the lens.  The term “zonular” refers to opacities that are confined to one or more discrete zones of the lens other than the poles.  The term “pulverulent” refers to powdery dustlike opacities that can be either zonular or widely dispersed throughout the lens.  Cataracts are often caused by mutations in crystallins (see L1N, L10, L23), channel proteins involved in the transport of water and metabolites, and connexins, the components of gap junction channels (2;24;28).

The lens expresses three connexins: Cx43, Cx46 and Cx50 (4).  Lens fiber cells are coupled by intercellular gap junction channels formed by Cx46 and Cx50 connexin subunits, while Cx43 is expressed in lens epithelial cells.  Mice with mutations in connexins Cx46 and Cx50 develop cataracts with varying phenotypes.  Cx46-deficient animals display nuclear cataracts while Cx50-null mice exhibit both microphthalmia and nuclear cataracts (16;29;30).  At postnatal day 14, the eyes of Cx50 knockout mice weigh 32% less than those of controls, whereas lens mass is reduced by 46%.  Deletion of Cx50 does not alter the amount or distribution of Cx46 or Cx43.  In addition, intercellular passage of tracers reveals the persistence of communication between all cell types in the Cx50-knockout lens.  However, an increase in insoluble crystallin proteins is detected in these animals (16;29).  Mice lacking both Cx46 and Cx50 display severe cataracts resulting from cell swelling and degeneration of inner fibers while normal peripheral fiber cells continue to form throughout life.  Neither an increase of degraded crystallins nor an increase of water-insoluble crystallins is found in double mutant lenses.  However, a substantial reduction of γ-crystallin proteins, but not α- and β-crystallins, is detected.  These observations suggest that the presence of both Cx46 and Cx50 in fiber cells is critical for maintenance of the lens architecture (31).  Targeted replacement of the Gja8 coding region with the Gja3 (Cx46) coding sequence corrects defects in cellular differentiation and prevents cataracts, but does not restore normal growth (27;32).  A major difference observed between animals that exhibit loss of Cx46 and Cx50 is that in the absence of Cx50 there is a reduction in lens growth (16;29;30).  Interestingly, mitosis was decreased in Cx50-defiecient mice, indicating that there is a role in epithelial mitosis for Cx50-mediated gap junction communication (4).  

Although the Gja8 knockout has a recessive phenotype, point mutations in the mouse Gja8 gene can produce dominant and semi-dominant cataract phenotypes with distinct characteristics (33-35).  For instance, Gja8Aey5 results in a nuclear cataract caused by lack of degradation of fiber cell nuclei deep within the lens (35).  Like the L1 mutation, some of these mutations perturb the first extracellular domain of Cx50 (1;34-36). The dominant nature of such mutations suggests that mutant Cx50 proteins form atypical and/or dysfunctional gap junction channels in the eye.  This hypothesis is strengthened by the finding that transgenic overexpression of Cx50 in the lens also leads to cataracts perhaps by disrupting connexons containing Cx46 (37).  Studies of Cx50 and other connexin mutants in mice suggest a strict requirement for certain types of gap junction channels (homotypic or heterotypic) exists in the development and homeostasis of the lens.  Each of the approximately 20 connexin isoforms produces channels with distinct unitary conductances, molecular permeabilities, and electrical and chemical gating sensitivities (2;27).  Channels formed from Cx50, in particular, exhibit a unique sensitivity to extracellular monovalent cations (14), which may be critical for its role in lens homeostasis.

In humans, GJA8 is located on chromosome 1q21.1, and mutations are commonly linked to congenital cataracts.  Cataract-microcornea syndrome (CCMC, OMIM #116150) has a distinct set of phenotypes within the group of autosomal dominant congenital cataracts. Three human mutations of GJA8 are associated with CCMC while six others are associated with zonular pulverulent cataract 1 (CZP1, OMIM #116200).  These mutations are located throughout the Cx50 protein, although the majority of the mutations occur in the highly conserved transmembrane and extracellular domains that form functional critical structures in gap junction channels (38).

Putative Mechanism

Figure 7. Putative mechanisms involved in the L1 mutant (3; 4) It is proposed that the L1 connexin subunits are able to interact with wild type Cx46 and Cx50 proteins to form atypical connexons that have altered functions.  These atypical channels would interfere with normal lens development and maintenance through altered electrochemical properties of the gap junctions, the loss of a putative signal to promote proper crystallin distribution (marked by ? on the figure, (41)) and/or a loss of transport of factors essential for cell elongation.  Any of these disturbances would subsequently lead to disruption in lens development and/or the aggregation of proteins to impair lens transparency.

Cx50 knockout phenotypes suggest that Cx50 plays a major role in the normal growth of the lens, thus affecting overall eye size.  Lens cell division is significantly retarded during the first postnatal week and additional studies suggest other connexins cannot replace the critical role that Cx50 has in postnatal lens cell division (16;17;29;30;32;39;40).  While knockout animals have a mild nuclear cataract phenotype, this phenotype is significantly affected by genetic modifiers and the lenses of knockout animals still retain functional gap junctions (16;29;39).  

The L1 mutation affects the highly conserved E1 domain of Cx50 which plays an important function in connexon interaction between cells and affects the properties of the resulting gap junction channel (3;7;8;14) (Figure 7).  Similar to Cx50-null animals, L1 animals also develop cataracts (whole instead of nuclear) and microphthalmia.  The L1 mutation is dominant and heterozygote animals display a different phenotype than do homozygous animals.  Although mature fiber cells are severely altered in both homozygous and heterozygous animals, the elongation of primary fiber cells during embryonic development is altered only in heterozygous animals.  Further examination of the L1 mutation in Cx46 and Cx50-deficient backgrounds revealed that mutant Cx50 subunits interact with wild type Cx50 subunits to form connexons and gap junctions that impair primary fiber cell elongation.  Only heterozygous L1 animals displayed defects in primary fibers while Gja8L1/-animals did not.  The presence or absence of Cx46 protein did not affect primary fiber formation in L1 heterozygotes, but the absence of Cx46 protein in these animals surprisingly resulted in normal secondary fibers.  This result suggests that mutant Cx50 subunits interact with Cx46 to disrupt the formation of postnatal secondary fibers.  It is likely that Cx50-S50P connexin subunits are able to interact with wild type Cx46 and Cx50 proteins to form atypical connexons that have altered functions and interfere with normal lens development and maintenance (1).

An additional study addressing the biophysical properties of L1 mutant Cx50-containing channels showed that channels comprising Cx50-S50Psubunits alone fail to induce electrical coupling, likely because these channels fail to localize to the plasma membrane at cell-cell contacts.  In an in vitro assay, the mixed expression of Cx50-S50P and wild type Cx46 to create heteromeric connexons, resulted in functional intercellular channels with unique voltage-gating properties compared to wild typechannels.  Mutated Cx50-S50P protein was able to colocalize with wild-type Cx46 in both transfected HeLa cells in vitro and mouse lens sections in vivo, further supporting the hypothesis that this altered form of Cx50 is able to form atypical connexons with wild-type connexin proteins perhaps interfering with the formation of appropriate gap junction channels (36).

In order for the lens to remain transparent and to maintain the appropriate refractive index for the transmission and focusing of light on the retina, high concentrations of lens proteins, metabolites, ions need to be arranged among lens fiber cells  (41).  In addition to the role of connexin-mediated gap junction communication in maintaining lens growth and transparency, it has been found that gap junction communication can influence intracellular protein distribution in differentiated lens fiber cells before undergoing cell maturation  (41).  Cx50 was sufficient (in Cx46 knockout lenses) to facilitate the uniform distribution of GFP as well as fiber cell denucleation; Cx46 alone was less efficient (41).  Loss of both Cx50 and Cx46 resulted in an abolishment in GFP exchange in inner differentiated fiber cells, inhibition of fiber cell elongation, disruption of denucleation, and degeneration in the lens core (41).  The mechanism as to how Cx50-mediated gap junction communication mediates GFP transport remains unknown, however, it has been proposed that gap junction communication influences the formation of the macromolecular exchange pathway (41).   The L1 mutation may be leading to an improper distribution of proteins within the cell subsequently leading to cataract formation.

Primers Primers cannot be located by automatic search.
Genotyping
L1 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change. 
 
Primers for PCR amplification
L1(F): 5’-CGGCACAGATGAGGCACTTGATAG -3’
L1(R): 5’-TGTGGCAGACATAGGTCCTTAGCAG -3’
 
PCR program
1) 94°C             2:00
2) 94°C             0:30
3) 56°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 29X
6) 72°C             7:00
7) 4°C               ∞
 
Primers for sequencing
L1_seq(F): 5’- GAGATCATCTCAGAGTTGCACTG -3’
L1_seq(R): 5’- CATAGGTCCTTAGCAGTGTGCC -3’
The following sequence of 638 nucleotides (from Genbank genomic region NC_000069 for linear genomic sequence of Gja8) is amplified:
5433                                    cggcacag atgaggcact tgatagaagc
5461 tgttggatac tatgattgtt ccatcagttc caaaaggaaa gtcactccaa gagctaggaa
5521 agagatcatc tcagagttgc actgtggcca attagatttt gccttctgct tccttggtag
5581 tgagcaatgg gcgactggag tttcctggga aacatcttgg aagaggtgaa tgagcactcc
5641 actgtcatcg gcagagtctg gctcacagtg ctcttcatct tccgcatcct catcctcggg
5701 acagcagcgg agtttgtgtg gggcgatgag caatctgatt ttgtatgcaa cacccagcag
5761 ccaggctgtg agaatgtctg ctacgatgag gcctttccca tctcacacat ccgcctctgg
5821 gtgctgcaga tcatcttcgt ctccactcca tcgctgatgt acgtggggca cgcggtacac
5881 cacgttcgca tggaggagaa gcgaaaggac cgtgaagctg aggagctctg tcagcagtcg
5941 cgcagcaacg ggggtgagag ggtaccaatc gccccagacc aggccagcat ccggaagagc
6001 agcagcagta gcaaaggcac caagaagttc cggctggagg gcacactgct aaggacctat
6061 gtctgccaca
 
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.
References
Science Writers Nora G. Smart, Anne Murray
Illustrators Victoria Webster
AuthorsChun-hong Xia, Xin Du, Xiaohua Gong, Bruce Beutler
Edit History
2011-01-13 2:25 PM (current)
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