cells of the innate immune system sense host invasion by detecting structural determinants that are broadly conserved among pathogens of a given phylogenetic group .
the lipopolysaccharides ( lps or endotoxin ) that decorate the outer membrane of gram - negative bacteria are excellent examples of such determinants . in response to minute concentrations of lps derived from certain gram - negative bacteria ,
macrophages secrete cytokines such as tumor necrosis factor ( tnf ) , interleukin-1 ( il-1 ) and il-6 , which contribute to the containment of infection and help to initiate a specific immune response . on the other hand , overstimulation of the innate immune system through this channel
dramatic differences in lps responses are apparent between closely related species , and there are substantial differences even among genetically heterogeneous members of the same species .
for example , whereas humans and chimpanzees are generally considered to be very sensitive to lps , baboons and most other primates are highly resistant .
it is likely that part of the difference in sensitivity may be explained at a very proximal level , although differences in responses to cytokines ( for example tnf ) may also have a role .
neither interspecific differences nor interindividual differences in lps responses have , until recently , been accessible to systematic genetic analysis .
although lps was once thought to exert its effects through intercalation into biological membranes , or to bind to many different receptors on the cell surface , it has been clear for nearly three decades that there is , in fact , a single biochemical pathway for lps detection .
this was indicated by the observation that allelic   mutations of a single gene ( lps ) in mice could entirely abrogate the response to lps , and did so with great specificity .
mice of the strains c3h / hej   and c57bl/10sccr   are highly resistant to lps , showing none of the usual biological effects , yet respond normally to other bacterial products and to most cytokines induced by lps .
we recently identified the lps locus through positional cloning   and showed that the lps - resistance phenotype was caused by defects in the toll - like receptor 4 gene ( tlr4 ) . in c3h
/ hej mice , a point mutation ( p712h ; single - letter amino - acid notation ) modifies the protein within the cytoplasmic domain , creating a codominant inhibitory effect on lps signal transduction . in c57bl/10sccr mice the gene is deleted , yielding a recessive abolition of the lps response .
subsequently , overexpression of the wild - type tlr4 protein was found to enhance lps signal transduction in wild - type macrophages , lowering the effective concentration ( ec50 ) for lps by a factor of 30 , whereas overexpression of the tlr4 isoform represented in c3h / hej mice almost completely suppresses signaling .
furthermore , genetic complementation studies have demonstrated that lps and tlr4 enter into close physical proximity in the course of signal transduction - tlr4 appears to bind directly to lps .
hence , the species origin of tlr4 is the sole determinant of species preference for a given lps structure .  
mice of the c3h / hej and c57bl/10sccr strains are abnormally susceptible to infection by certain gram - negative bacteria , suggesting that timely recognition of lps is essential for successful containment of infection . because deleterious mutations of tlr4 have become fixed spontaneously in two strains of mice , we considered it possible that other functionally important mutations might be identified in mice and humans .
moreover , information on the degree of tlr4 polymorphism in these and other species might allow inferences about the importance of different tlr4 domains . accordingly , we decided to sequence the entire tlr4 gene of both humans and mice , and to survey genetic variation at the locus in each species .
we also examined the tlr4 sequence from two species of subhuman primates that have dramatically different responses to lps .
the mouse tlr4 gene is somewhat longer than its human counterpart tlr4 , owing to the greater length of intronic sequence - 15,337 base pairs ( bp ) from beginning to end of the transcribed sequence in the mouse , compared with 11,467 bp in the human . there are three exons in tlr4 , and each corresponds to a homologous sequence in the human gene .
rock et al .   reported a human cdna sequence ( genbank accession number u88880 ) that includes a fourth exon , positioned between the ' normal ' first and second introns .
when included in the processed transcript , however , this exon specifies early termination of the polypeptide chain . although it is possible that translation is initiated distal to the added stop codon , and that a shorter product results in the human than in the mouse , this would be unusual , given the length of the 5 ' untranslated region ( utr ) that would then exist and the presence of multiple upstream initiation codons .
moreover , there is no murine sequence homologous to the alternative second exon of the human gene .
the biological significance of this exon is therefore unclear and , in all likelihood , its inclusion in the mrna leads to the formation of a nonfunctional protein .
neither the human nor the mouse gene has a tata element or caat box in the proximal promoter region .
a number of conserved promoter and enhancer motifs are apparent on alignment of the murine and human 5 ' flanking sequences , and are described in detail elsewhere .
both tlr4 and tlr4 lie amid repetitive sequences of retroviral origin , and no other genes are detected close to either of them using homology searches or the gene prediction algorithm grail . in figure 1 , the grayscale images of the human and mouse genes call attention to the repetitive elements in the region and illustrate the relationship between exons and spacing in the two species . the landscape of genomic dna in the region of human and mouse tlr4 genes .
exons are numbered 1 to 3 . in the human gene model , an added exon (  ) is also portrayed , as found in the alternative sequence of rock et al . , in which early truncation of the protein is predicted .
the grayscale image was generated using x - grail , version 1.3c , and depicts gc content as well as repetitive elements ( both complex and simple ) identified by repeatmasker ( which appear as unbroken stretches of white ) .
grail exons are shown in green ( highest quality ) and blue ( intermediate quality ) above each grayscale image .
restriction sites indicate enzymes that cut at single sites within the interval . among 35 strains of mus musculus
, 10 different alleles were identified on the basis of mutations at 22 sites with respect to the reference sequence . of these
the ancestry of different tlr4 alleles can be traced by haplotype analysis , as many deviations from the reference allele occur in conjunction with one another .
for example , the p / j strain tlr4 gene exhibits 11 mutations the distinguish it from the most common haplotype , six of them specifying changes in the tlr4 amino - acid sequence ; the sea / gnj strain differs by nine mutations , and the strains nzw / j and vm / dk , which are identical to one another , differ from the most common haplotype at six sites .
shared mutations suggest that introgression took place after mutational separation had occurred , leading to the introduction of groups of mutations by genetic recombination .
hence , mice of the p / j , nzw / j , and vm / dk strains have several mutations that are observed in the a / j and balb / c strains , but also lack some of the mutations of the latter strains and have unique mutations of their own .
polymorphisms of tlr4 in mice * strains are as follows : i , nzo / hilt ; 2 , si / col ; 3 , dba / ij ; 4 , a / j ; 5 , el / suz ; 6 , cba / j ; 7 , akr / j ; 8 , balb / cj ; 9 , ddy / jci ; 10 , p / j ; i i , mrl / mpj ; 12 , sjl / j ; 13 , nod / ltj ; 14 , 129/j ; 15 , fl / ire ; 16 , ma / myj ; 17 , swr / j ; 18 , lp / j ; 19 , pro / irej ; 20 , sodi / ei ; 21 , sea / gnj ; 22 , sm / j ; 23 , kk / hij ; 24 , st / bj ; 25 , wb / re ; 26 , ybr / ei ; 27 , fvb / nj ; 28 , pl / j ; 29 , lt / chresv ; 30 , rills / j ; 31 , rf / j ; 32 , nzb / binj ; 33 , au / ssj ; 34 , nzw / lacj ; 35 , vm / dk .   
exon i , 26041 - 26424 ; exon 2 , 32397 - 32563 ; exon 3 , 37732 - 41297 .
ecto , extracellular domain ; cyto , cytoplasmic domain ;   tm , transmembrane domain .  
yes implies one or two forms ; no implies three to five forms among the six mammalian species examined .
mutations occurring at sites that are relatively conserved among species ( only one or two forms among six species ) are shown in blue ; those occurring at sites that are less conserved ( three to five forms among six species ) are shown in black .
numbers adjacent to arrows indicate the mutational distance ( number of mutations separating each strain from its presumed ancestor ) , with reference to both coding and noncoding substitutions listed in table 1 .
arrows point in the direction of descent , and their lengths are proportional to distance .
most of the murine mutations reside within exon 3 , and only two substitutions are noted that modify the cytoplasmic domain ( figure 2 . ) of these , however , one mutation ( r761h ) is fairly common among the strains surveyed , and the corresponding residue has been reported as an h in the hamster . a single conservative substitution ( v637i )
the human and chimpanzee amino - acid sequences are nearly identical over the interval studied , distinguished by only three substitutions .
the baboon sequence is 93.5% identical to the human in the extracellular domain , differs in the transmembrane domain by one substitution out of 30 residues , and differs in the proximal cytoplasmic domain by only one residue in 155 . at the carboxyl terminus ,
however , homology is badly disrupted , so that 16 of the last 21 human residues are not replicated in the baboon protein , which is 13 amino acids shorter than the human protein .
similarly , among rodents , the carboxyl terminus of the protein is the least conserved .
overall , the order of conservation with respect to domain is : proximal cytoplasmic domain > transmembrane domain > extracellular domain > distal cytoplasmic domain ( table2 , figure 4 ) .
conservation of tlr4 among six mammalian species , calculated according to region with respect to the human sequence , the extracellular domain is amino acids 1 - 624 ; transmembrane domain , amino acids 625 - 658 ; proximal cytoplasmic domain , amino acids 659 - 618 ; distal cytoplasmic domain , amino acids 619 - 638 .
spline curve illustrating interspecific sequence variation across the tlr4 protein . a multiple alignment of tlr4 sequences from three rodent species ( mouse , rat and hamster ) and three primate species ( human , chimpanzee and baboon )
the number of amino acids observed at each residue was plotted using the program prism 3.0 ( a value of 1 was assigned if a single amino acid was observed in the six species ; a value of 5 was assigned if five different amino acids were observed among the six species , and so on ) .
where a deletion was introduced by pileup , a single mismatch was assumed . where the sequence was truncated , each missing residue was tabulated as a separate mismatch .
the mouse tlr4 gene is somewhat longer than its human counterpart tlr4 , owing to the greater length of intronic sequence - 15,337 base pairs ( bp ) from beginning to end of the transcribed sequence in the mouse , compared with 11,467 bp in the human . there are three exons in tlr4 , and each corresponds to a homologous sequence in the human gene .
rock et al .   reported a human cdna sequence ( genbank accession number u88880 ) that includes a fourth exon , positioned between the ' normal ' first and second introns .
when included in the processed transcript , however , this exon specifies early termination of the polypeptide chain . although it is possible that translation is initiated distal to the added stop codon , and that a shorter product results in the human than in the mouse , this would be unusual , given the length of the 5 ' untranslated region ( utr ) that would then exist and the presence of multiple upstream initiation codons .
moreover , there is no murine sequence homologous to the alternative second exon of the human gene .
the biological significance of this exon is therefore unclear and , in all likelihood , its inclusion in the mrna leads to the formation of a nonfunctional protein .
neither the human nor the mouse gene has a tata element or caat box in the proximal promoter region .
a number of conserved promoter and enhancer motifs are apparent on alignment of the murine and human 5 ' flanking sequences , and are described in detail elsewhere .
both tlr4 and tlr4 lie amid repetitive sequences of retroviral origin , and no other genes are detected close to either of them using homology searches or the gene prediction algorithm grail . in figure 1 , the grayscale images of the human and mouse genes call attention to the repetitive elements in the region and illustrate the relationship between exons and spacing in the two species . the landscape of genomic dna in the region of human and mouse tlr4 genes .
exons are numbered 1 to 3 . in the human gene model , an added exon (  ) is also portrayed , as found in the alternative sequence of rock et al . , in which early truncation of the protein is predicted .
the grayscale image was generated using x - grail , version 1.3c , and depicts gc content as well as repetitive elements ( both complex and simple ) identified by repeatmasker ( which appear as unbroken stretches of white ) .
grail exons are shown in green ( highest quality ) and blue ( intermediate quality ) above each grayscale image .
among 35 strains of mus musculus , 10 different alleles were identified on the basis of mutations at 22 sites with respect to the reference sequence . of these
the ancestry of different tlr4 alleles can be traced by haplotype analysis , as many deviations from the reference allele occur in conjunction with one another .
for example , the p / j strain tlr4 gene exhibits 11 mutations the distinguish it from the most common haplotype , six of them specifying changes in the tlr4 amino - acid sequence ; the sea / gnj strain differs by nine mutations , and the strains nzw / j and vm / dk , which are identical to one another , differ from the most common haplotype at six sites .
shared mutations suggest that introgression took place after mutational separation had occurred , leading to the introduction of groups of mutations by genetic recombination .
hence , mice of the p / j , nzw / j , and vm / dk strains have several mutations that are observed in the a / j and balb / c strains , but also lack some of the mutations of the latter strains and have unique mutations of their own .
polymorphisms of tlr4 in mice * strains are as follows : i , nzo / hilt ; 2 , si / col ; 3 , dba / ij ; 4 , a / j ; 5 , el / suz ; 6 , cba / j ; 7 , akr / j ; 8 , balb / cj ; 9 , ddy / jci ; 10 , p / j ; i i , mrl / mpj ; 12 , sjl / j ; 13 , nod / ltj ; 14 , 129/j ; 15 , fl / ire ; 16 , ma / myj ; 17 , swr / j ; 18 , lp / j ; 19 , pro / irej ; 20 , sodi / ei ; 21 , sea / gnj ; 22 , sm / j ; 23 , kk / hij ; 24 , st / bj ; 25 , wb / re ; 26 , ybr / ei ; 27 , fvb / nj ; 28 , pl / j ; 29 , lt / chresv ; 30 , rills / j ; 31 , rf / j ; 32 , nzb / binj ; 33 , au / ssj ; 34 , nzw / lacj ; 35 , vm / dk .   
exon i , 26041 - 26424 ; exon 2 , 32397 - 32563 ; exon 3 , 37732 - 41297 .
ecto , extracellular domain ; cyto , cytoplasmic domain ;   tm , transmembrane domain .  
yes implies one or two forms ; no implies three to five forms among the six mammalian species examined .
mutations occurring at sites that are relatively conserved among species ( only one or two forms among six species ) are shown in blue ; those occurring at sites that are less conserved ( three to five forms among six species ) are shown in black . genetic distance and probable ancestral relationships among tlr4 genes of 35 mus musculus strains .
numbers adjacent to arrows indicate the mutational distance ( number of mutations separating each strain from its presumed ancestor ) , with reference to both coding and noncoding substitutions listed in table 1 .
arrows point in the direction of descent , and their lengths are proportional to distance .
most of the murine mutations reside within exon 3 , and only two substitutions are noted that modify the cytoplasmic domain ( figure 2 . ) of these , however , one mutation ( r761h ) is fairly common among the strains surveyed , and the corresponding residue has been reported as an h in the hamster . a single conservative substitution ( v637i )
the human and chimpanzee amino - acid sequences are nearly identical over the interval studied , distinguished by only three substitutions .
the baboon sequence is 93.5% identical to the human in the extracellular domain , differs in the transmembrane domain by one substitution out of 30 residues , and differs in the proximal cytoplasmic domain by only one residue in 155 . at the carboxyl terminus ,
however , homology is badly disrupted , so that 16 of the last 21 human residues are not replicated in the baboon protein , which is 13 amino acids shorter than the human protein .
similarly , among rodents , the carboxyl terminus of the protein is the least conserved .
overall , the order of conservation with respect to domain is : proximal cytoplasmic domain > transmembrane domain > extracellular domain > distal cytoplasmic domain ( table2 , figure 4 ) .
conservation of tlr4 among six mammalian species , calculated according to region with respect to the human sequence , the extracellular domain is amino acids 1 - 624 ; transmembrane domain , amino acids 625 - 658 ; proximal cytoplasmic domain , amino acids 659 - 618 ; distal cytoplasmic domain , amino acids 619 - 638 .
a multiple alignment of tlr4 sequences from three rodent species ( mouse , rat and hamster ) and three primate species ( human , chimpanzee and baboon ) was generated using the gcg program pileup .
the number of amino acids observed at each residue was plotted using the program prism 3.0 ( a value of 1 was assigned if a single amino acid was observed in the six species ; a value of 5 was assigned if five different amino acids were observed among the six species , and so on ) .
where a deletion was introduced by pileup , a single mismatch was assumed . where the sequence was truncated , each missing residue was tabulated as a separate mismatch .
the pathway by which lps activates host innate immune defenses has been illuminated by the positional cloning of lps and establishment of its identity with tlr4 , a mammalian representative of an ancient family of receptors   that serve both developmental and defensive functions .
the function of tlr4 as the lps signal transducer became clear when mapping   and sequencing   data revealed tlr4 as the only gene in a critical 2.6 megabase ( mb ) region and , furthermore , showed that two strains of lps - resistant mice are homozygous for mutations of tlr4 that are absent in closely related lps - sensitive strains . in drosophila ,
the prototypic homolog of the mammalian toll - like receptors ( toll ) defends against fungal infection , whereas the protein 18-wheeler defends against bacterial infection . in the case of toll , there is no evidence of direct contact between the receptor and the microbial pathogen or its components .
rather , infection activates a proteolytic cascade that leads to the production of an endogenous ligand ( sptzle ) , which in turn engages toll .
the situation is apparently different in mammals , in which tlr4 is clearly a direct interface with the microbial world . as such
, the primary structure of a tlr4 molecule determines ligand specificity , accounting for the well - known observation that mouse cells can recognize tetra - acyl lipid a as an agonist   whereas human cells recognize it as an antagonist . as mice of the c3h / hej strain
are highly susceptible to infection by gram - negative bacteria , it would seem plausible that among human patients with gram - negative sepsis , some individuals may have been at risk by virtue of mutations in tlr4 . the first step in determining whether different isoforms of tlr4 confer added risk of , or protection against
, sepsis is the assessment of genetic variability at this locus in the normal population .
whereas ablative mutations of tlr4 abolish the capacity to detect lps , the question arises as to whether less severe mutation might alter either the specificity of lps detection , as discussed above , or the magnitude of the lps signal .
our present knowledge of tlr4 structural variation in mice may permit an answer to this question , insofar as the mutations might easily be recreated , and measurements of signal - transducing activity through the modified receptors carried out in immortalized c3h / hej macrophages .
animals of the a / j and p / j strains have defective tumoricidal capacity , although in neither case is the lps locus implicated in the defect . deleterious mutations of tlr4 might reasonably be sought in individuals who have developed serious gram - negative infections , on the premise that mice with deleterious mutations of tlr4 are rendered susceptible to such infections . similarly , in birds , a polymorphism at the tenascin locus ( which lies a few megabases proximal to tlr4 in mice ) predicts susceptibility to gram - negative infection , suggesting that it may lie in linkage disequilibrium with a particular form of the avian tlr4 gene . beyond this , it may be assumed , given the powerful pro - inflammatory activity of the receptor , that germline or somatic mutations of tlr4 could in some instances cause constitutive signal transduction ( as observed with the drosophila toll locus ) .
in fact , in the lpr model of systemic lupus erythematosus , the lps locus was believed a likely modulator of phenotype .
this would seem still more likely at present , given the paucity of other candidate genes in the immediate vicinity of tlr4 .
the evolutionary conservation of tlr4 is of particular interest , in that different species show preferential responses to some lps forms and not others , and have particular ' set points ' for responses to toxic lps molecules .
an assessment of variability may be made through comparison of different species , but is complemented by the study of a large number of individuals within species .
both approaches reveal that the extracellular domain of tlr4 is highly variable compared with the transmembrane domain and proximal cytoplasmic domain of the protein .
pooling the number of mutable sites in the extracellular domain and transmembrane domain of humans and mice , 17 coding changes are observed , compared with two in the proximal cytoplasmic domain . moreover , variability does not seem confined to any particular region of the extracellular domain , but is spread uniformly across its length .
given that the ligand is an endogenous protein , extracellular domain conservation tends to be strict , insofar as mutations affecting extracellular domain structure are likely to diminish specificity or affinity of binding .
the presumed ligand for tlr4 is lps itself , presented alone or in conjunction with another protein ( for example cd14 ) , and the relatively high frequency of polymorphism observed in tlr4 may be viewed as a consequence of the protective effect rendered by lps recognition and the variability of lps structure .
this is probably due to the fact that the cytoplasmic domain of tlr4 is not required to cope with a ligand of variable structure .
rather , when called upon to signal , it must do so , utilizing transducing molecules with conserved structures . on the other hand ,
the intensity of the lps signal has apparently been optimized for each species , perhaps ensuring that the response to lps is appropriately integrated into the immune response as a whole .
humans , ungulates and rabbits , for example , exhibit an intense response to low concentrations of lps , whereas the lower primates and most rodents are relatively more resistant .
the highly variable carboxy - terminal end of the tlr4 cytoplasmic domain may be seen as the embodiment of interspecific differences in lps sensitivity , although the poor conservation of this portion of the molecule might alternatively be taken to indicate a neutral effect of mutation .
it is also possible that this region of the molecule is subject to a higher rate of mutation than that applying to the rest of the protein .
although most such mutations might be removed by selection , some might be discovered in populations defined by the occurrence of gram - negative sepsis .
the mouse bacterial artificial chromosome ( bac ) 152c16 ( from the 129/j strain , research genetics ) was previously shown by us to contain the tlr4 gene in its entirety , and a small fraction of tlr4 was also found in the overlapping bac 309i17 .
human tlr4 was identified in bac 110p15 ( genome systems ) by hybridization screening using a pcr - amplified human tlr4 cdna sequence as a probe .
all three bacs were fragmented by ultrasound , shotgun cloned into the vector pbluescript - ks , and extensively sequenced using abi model 373 and 377 sequencers , using big dye terminators ; 959 reads were obtained from 390i17 , 1503 reads from 110p15 , and 2731 reads from 152c16 .
the average read length was approximately 700 nucleotides . to concentrate data acquisition efforts on the tlr4 and tlr4 genes themselves , pcr primers were fashioned to match regions flanking each gene .
a 16 kb fragment was amplified from the mouse bac 152c16 , and a 12 kb fragment was amplified from the human bac 110p15 , each containing all exons of the respective gene .
these fragments were also shotgun cloned and extensively sequenced , so that the depth of sequence reached an average of 12 reads over the area of greatest interest .
sequence assembly used the programs phred and phrap ( obtained from brent ewing and phil green , university of washington genome center ) .
interpretation of repetitive elements was achieved with the program repeatmasker ( obtained from arian smit , university of washington genome center ) .
a contiguous high - quality sequence 18,974 bp in length , containing tlr4 , was obtained from the human bac , and a contig 91,748 bp in length , containing tlr4 , was obtained from the mouse bac . over these intervals ,
these sequences have been posted to genbank in annotated form ( accession number af177767 for the murine sequence and af177765 for the human sequence ) .
mouse dna , obtained from animals of 35 mus musculus strains , was ordered from the jackson laboratories .
chimpanzee and baboon dna were obtained from kurt benirschke ( university of california , san diego ) and gregory delzoppo ( scripps research institute ) , respectively .
the three principal exons of tlr4 were amplified independently from mouse genomic dna samples , leaving a margin of approximately 50 bp to each side of the exons so as to indentify intronic mutations that might alter splicing .
all exons of the chimpanzee were amplified and sequenced using the same primers used to amplify and sequence the human exons . for the baboon , the first two exons were also amplified using these same primers ; however , the third exon of the baboon was amplified with a substituted primer at the 5 ' end .
exons 1 and 2 were sequenced using the same primers that were used for amplification .
exon 3 was sequenced using the flanking primers as well as a collection of eight internal primers . in this manner ,
the entire coding region and all splice junctions of tlr4 could be covered with a total of 14 sequencing reads .
all primers used for amplification and sequencing are presented in table 3 ; separate sets were used to amplify and sequence mouse and the primate samples .
oligonucleotide primers used to amplify and sequence mouse and human tlr4 genes  primer matches + strand ; primer matches - strand .
independent assembly of each sample was required as a condition for further analysis , and if such assembly failed , additional reads were executed using a secondary collection of primers .
thereafter , mutations were identified en masse , by pooling all of the reads from 25 to 30 samples at a time and reassembling with the program polyphred , using the phred - phrappoly script ( obtained from natalie kolker , university of washington genome center ) .
consed - alpha ( obtained from david gordon , university of washington genome center ) was used to visualize reads and mutations .
the annotated chimpanzee exon sequences have been submitted to genbank with the accession numbers af179218 , af179219 and af179220 .
the baboon sequences have been submitted with the accession numbers af180962 , af180963 and af180694 . for genetic comparisons , rat ( genbank af057025 ) and hamster ( af153676 ) tlr4 sequences were also used .
a 500 mhz dec - alpha system equipped with 256 mbytes of memory was used for direct analysis of sequence data as described above .
in addition to the programs already mentioned , the gcg software ( version 9.0 ) was used for alignment analysis , with the program pileup used in multiple alignments of protein sequences .
the windows - based program generunner 3.0 ( hastings software ) was used for the design of oligonucleotide primers . a spline curve describing heterogeneity of the tlr4 polypeptide sequence from different species
sequences were prepared for submission with the use of the program sequin 2.90 ( obtained from the national center for biotechnological information ) .
the mouse bacterial artificial chromosome ( bac ) 152c16 ( from the 129/j strain , research genetics ) was previously shown by us to contain the tlr4 gene in its entirety , and a small fraction of tlr4 was also found in the overlapping bac 309i17 .
human tlr4 was identified in bac 110p15 ( genome systems ) by hybridization screening using a pcr - amplified human tlr4 cdna sequence as a probe .
all three bacs were fragmented by ultrasound , shotgun cloned into the vector pbluescript - ks , and extensively sequenced using abi model 373 and 377 sequencers , using big dye terminators ; 959 reads were obtained from 390i17 , 1503 reads from 110p15 , and 2731 reads from 152c16 .
the average read length was approximately 700 nucleotides . to concentrate data acquisition efforts on the tlr4 and tlr4 genes themselves , pcr primers were fashioned to match regions flanking each gene .
a 16 kb fragment was amplified from the mouse bac 152c16 , and a 12 kb fragment was amplified from the human bac 110p15 , each containing all exons of the respective gene .
these fragments were also shotgun cloned and extensively sequenced , so that the depth of sequence reached an average of 12 reads over the area of greatest interest .
sequence assembly used the programs phred and phrap ( obtained from brent ewing and phil green , university of washington genome center ) .
interpretation of repetitive elements was achieved with the program repeatmasker ( obtained from arian smit , university of washington genome center ) .
a contiguous high - quality sequence 18,974 bp in length , containing tlr4 , was obtained from the human bac , and a contig 91,748 bp in length , containing tlr4 , was obtained from the mouse bac . over these intervals ,
these sequences have been posted to genbank in annotated form ( accession number af177767 for the murine sequence and af177765 for the human sequence ) .
mouse dna , obtained from animals of 35 mus musculus strains , was ordered from the jackson laboratories .
chimpanzee and baboon dna were obtained from kurt benirschke ( university of california , san diego ) and gregory delzoppo ( scripps research institute ) , respectively .
the three principal exons of tlr4 were amplified independently from mouse genomic dna samples , leaving a margin of approximately 50 bp to each side of the exons so as to indentify intronic mutations that might alter splicing .
all exons of the chimpanzee were amplified and sequenced using the same primers used to amplify and sequence the human exons . for the baboon , the first two exons were also amplified using these same primers ; however , the third exon of the baboon was amplified with a substituted primer at the 5 ' end .
exons 1 and 2 were sequenced using the same primers that were used for amplification .
exon 3 was sequenced using the flanking primers as well as a collection of eight internal primers . in this manner , the entire coding region and all splice junctions of tlr4 could be covered with a total of 14 sequencing reads .
all primers used for amplification and sequencing are presented in table 3 ; separate sets were used to amplify and sequence mouse and the primate samples .
oligonucleotide primers used to amplify and sequence mouse and human tlr4 genes  primer matches + strand ; primer matches - strand .
independent assembly of each sample was required as a condition for further analysis , and if such assembly failed , additional reads were executed using a secondary collection of primers .
thereafter , mutations were identified en masse , by pooling all of the reads from 25 to 30 samples at a time and reassembling with the program polyphred , using the phred - phrappoly script ( obtained from natalie kolker , university of washington genome center ) .
consed - alpha ( obtained from david gordon , university of washington genome center ) was used to visualize reads and mutations .
the annotated chimpanzee exon sequences have been submitted to genbank with the accession numbers af179218 , af179219 and af179220 .
the baboon sequences have been submitted with the accession numbers af180962 , af180963 and af180694 . for genetic comparisons , rat
a 500 mhz dec - alpha system equipped with 256 mbytes of memory was used for direct analysis of sequence data as described above .
in addition to the programs already mentioned , the gcg software ( version 9.0 ) was used for alignment analysis , with the program pileup used in multiple alignments of protein sequences .
the windows - based program generunner 3.0 ( hastings software ) was used for the design of oligonucleotide primers . a spline curve describing heterogeneity of the tlr4 polypeptide sequence from different species
sequences were prepared for submission with the use of the program sequin 2.90 ( obtained from the national center for biotechnological information ) .
we thank the zoologic society of san diego , which was the source of the chimpanzee genomic dna used in these studies .
 background : differences in responses to bacterial surface lipopolysaccharides ( lpss ) are apparent between and within mammalian species . it has been shown in mice that resistance to lps is caused by defects in the toll - like receptor 4 gene ( tlr4 ) , the product of which is thought to bind lps and mediate lps signal transduction in immune system cells.results:we have sequenced the toll - like receptor 4 gene of humans ( tlr4 ; 19.0 kilobases , kb ) and mice ( tlr4 ; 91.7 kb ) , as well as the coding region and splice junctions of tlr4 from 35 mouse ( mus musculus ) strains , from the chimpanzee and from the baboon . 
 no other discernible genes or regions of interspecies conservation lies close to tlr4 and , in both humans and mice , flanking sequences and introns are rich in repeats of retroviral origin . 
 interstrain analyses reveal that tlr4 is a polymorphic protein and that the extracellular domain is far more variable than the cytoplasmic domain , both among strains and among species . 
 the cytoplasmic domain of the tlr4 protein is highly variable at the carboxy - terminal end.conclusions:we suggest that selective evolutionary pressure exerted by microbes expressing structurally distinguishable lps molecules has produced the high level of variability in the tlr4 extracellular domain . 
 the highly variable carboxy - terminal region of the cytoplasmic domain is likely to determine the magnitude of the response to lps within a species . 