JP2002501761A - Methods for identifying polynucleotide and polypeptide sequences for physiological and medical conditions - Google Patents

Abstract

  (57) [Summary] The present invention identifies polynucleotide and polypeptide sequences in humans and / or non-human primates that are associated with a physiological condition, such as a disease (including susceptibility (human) or resistance (chimpanzee) to the development of AIDS). To provide a way to: This method uses a comparison of human and non-human primate sequences using statistical methods. The sequences thus identified are useful as therapeutic targets for the host and / or in screening assays.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention is a human disease or specific or intensified human brain function, longer it became human life, sensitivity or resistance to the development of (AIDS or C type, such as hepatitis) infections Polynucleotides and polypeptides that represent the evolved features associated with human conditions, such as susceptibility or resistance to cancer development, and aesthetic features such as hair growth, susceptibility or resistance to acne, or increased muscle mass It relates to using molecular and evolutionary techniques to identify sequences.

BACKGROUND Human invention, physiological and functional related to key areas in health and welfare of humans, in certain aspects, the closest evolutionary related, i.e. non-human primates such as chimpanzees Is distinguished from For example, (1) humans have unique or enhanced brain function (eg, cognitive abilities, etc.) compared to chimpanzees; (2) humans have a longer lifespan than non-human primates; (3) chimpanzees Are resistant to certain infectious diseases such as AIDS and hepatitis C that afflict humans; (4) chimpanzees appear to have a lower incidence of certain cancers than humans; (5) chimpanzees Does not suffer from acne or alopecia (baldness); (6) chimpanzees have a higher ratio of muscle to fat; (7) chimpanzees are resistant to malaria; (8) chimpanzees are susceptible to Alzheimer's disease Small; and (9) chimpanzees have a low incidence of atherosclerosis. At present, the genes underlying the human / chimpanzee differences described above are unknown, and more importantly, the genes are specific changes that have evolved in the genes that confer these capabilities. Understanding these differences between humans and evolutionary relatives closer to us provides useful information for developing effective treatments for the relevant human conditions and diseases.

[0003] Classical evolutionary analysis, which primarily compares animal anatomical features, has shown dramatic morphological and functional differences between humans and non-human primates ;
However, the human genome is known to share significant sequence similarity with the genomes of other primates. For example, it has been generally concluded that human DNA sequences are approximately 98.5% identical to chimpanzee DNA, and only slightly similar to gorilla DNA (McConkey and Goodman, TIG 13: 350-). 351, 1997). Although the genomic differences between humans and closely related primates are relatively small, perhaps, if not relatively, relatively few changes in the genomic sequence indicate that human health or human health as described above It can cause welfare features. Therefore, it is desirable to collect information from evolved changes in the protein encoded by that gene in order to identify the genes underlying these features and develop therapeutics that can benefit human health and well-being, This is also possible. Identifying and further characterizing these sequence changes is crucial to benefiting from the evolutionary solution of eliminating or minimizing disease or conferring unique or enhanced functions.

[0004] Recent developments in the Human Genome Project have provided a great deal of information on human gene sequences. In addition, the structures and activities of many human genes and protein products have been directly studied in human cultured cells and several animal model systems such as nematodes, Drosophila, zebrafish and mice. These model systems have many advantages: they are relatively simple, easy to operate, and have short generation times. Because the basic structure and biological activity of many important genes are preserved throughout evolution, homologous genes can be identified in many species by comparing the sequences of the macromolecules. Information about important gene products and functional domains from lower species can be used to help identify homologous genes or functional domains in humans. For example, the homeodomain with DNA binding activity first discovered in Drosophila was used to identify human homologous chromosomes with similar activity.

[0005] Comparing homologous genes or proteins between humans and lower model organisms provides useful information about evolutionarily conserved molecular sequences and functional characteristics, but this approach involves natural selection of the sequences. In identifying genes altered by
Its use is restricted. As sophisticated algorithms and analytical methods were developed, it was possible to extract more information about DNA sequence changes. The most powerful of these methods, "KA / KS", is a line of ratio of (non-synonymous nucleotide substitution per non-synonymous site (KA) / synonymous substitution per synonymous site (KS)). It also relates to pairwise comparisons between nucleotide sequences that encode proteins (where non-synonymous means substitutions in which the encoded amino acid changes, and synonyms denote substitutions in which the encoded amino acid does not change). ). "
The "KA / KS type method" includes this method and similar methods. These methods were used to demonstrate the emergence of a positive selection at the molecular level of the Darwinian theory, namely amino acid differences in homologous proteins. Some groups have used such methods to prove that certain proteins have evolved more rapidly than neutral replacement rates, that is, that they support the existence of a positive choice at the molecular level in Darwin's theory. used. For example, McDonald and Kreitman
(Nature 351: 652-654, 1992) proposes to statistically test the neutral protein evolution hypothesis based on a comparison of the number of amino acid substitutions and synonymous substitutions within the coding region of one locus. are doing. They tested this test with three Drosophila Ad
As an explanation for most protein evolution, rather than applying to the h locus, where the locus undergoes adaptive fixation of a selective dominant mutation, selective fixation of adaptive mutations is a key to the correct accumulation of neutral mutations. He concluded that it could be a viable option. Jenkins et al. (Proc. R. Soc. Lond. B261: 203-207, 1995) have reported that McDonald & Kreitman have examined adaptive evolution in sequences that control transcription (non-coding sequences). Tests are used.

[0006] Nakashima et al. (Proc. Natl. Acad. Sci USA 92: 5606-5609, 1995) reported that Miyata and Miyata used a pairwise comparison of the nucleotide sequences of 10 PLA2 isozyme genes from two snake species. The method of Yasunaga is used; this method uses the number of nucleotide substitutions per site for non-coding regions containing introns (
KN) and comparing KA with KS. They conclude that protein-coding regions evolve at a faster rate than non-coding regions, including introns. Highly accelerated displacement rates provide Darwin's theory at the molecular level of the PLA2 isozyme gene that seeks to produce new bioactivity that must have provided a strong selective advantage for catching prey or protecting against predators Due to evolution. Endo et al. (Mol. Biol. Evol. 13 (5): 685-690, 1996).
Nei and Gojob, with the aim of identifying candidate genes manipulated by positive selection
The ori method was used. Here, dN means the number of non-synonymous substitutions, and dS means the number of synonymous substitutions. Mets and Palumbi (Mol. Biol. Evol. 13 (2): 397-406, 1996)
And Gojobori, Nei and Jin, and similar to the method attributed to Kumar, Tamura and Nei
The McDonald & Kreitman test was used to determine if the sea urchin binding gene evolved rapidly in preparation for speciation, and to look for evidence of positive selection for this gene, use pN; The number of substitution alternatives and pS; the average ratio of the number of silent substitutions per silent site is examined. Goodwin et al. (Mol. Biol. Evol. 13
(2): 346-358, 1996) used a similar method to study the evolution of a particular mouse gene family, a system that was experimentally operable, and showed how the selection He concludes that it provides important fundamental insight into whether to take a branch. Edwards et al. (1995) used degenerate primers to derive MHC loci from a variety of birds and alligators, and then used the method of Nei and Gojobori (dN: (dS ratio). Whitfield et al. (Nature 364: 713-715, 1993) conducted a Ka / Ks analysis to look for directional selection within regions flanking the conserved region in the SRY gene (which determines male sex). I'm using They suggest that the rapid evolution of SRY is an important cause of reproductive isolation leading to new species. Wettsetin et al. (Mol. Bio.
Evol. 13 (1): 56-66, 1996) describe the Kumar, Tamua and Nei MEGA program and phylogenetic analysis to examine the diversity of MHC class I genes in squirrels and related rodents. I use. Parham and Ohta (Science 272: 67-74, 1996) describe a population biologic approach, including testing for selection, as well as genetic recombination, and the vague flow of human MHC class I polymorphisms. States that it is necessary to analyze and maintain. Hughes (Mol. Biol. Evol. 14 (1): 1-5, 1997)
To examine the hypothesis that proteins expressed in the cells of the vertebrate immune system evolve abnormally rapidly, we use the method of Nei and Gojobori (dN: dS ratio) to compare between humans and rodents. More than a hundred orthologous immunoglobulin C2 domains were compared. Swanson and Vacquier (Science 281: 710-712, 1998
) Was used to demonstrate the coordinated evolution between lysin and oocytes for lysin
The role of such coordinated evolution is being studied for new species formation (speciation) using the: S ratio.

[0007] According to the distant evolutionary relationship between humans and lower-human animals, adaptive beneficial genetic changes fixed by natural selection often accumulate neutral or arbitrary mutations over time. Will be hidden by. Furthermore, while some proteins evolve by accident, if the two compared genomes are not close enough,
Such accidental changes are masked and result in no point (Messier and Stewart, Nature 385: 151-154, 1997). Indeed, studies have shown that the occurrence of adaptive selection in protein evolution is often neglected when comparing significantly distantly related sequences (Endo et al., Mol. Biol. Evol. 37: 441-456). , 1996; Messi
er and Stewart, Nature 385: 151-154, 1997).

[0008] Studies of molecular evolution within primates have been reported, primarily to assess the rate and pattern of molecular evolution and to explore the evolutionary mechanisms responsible for such changes. Focusing on comparing gene products with a small number of known individual genes. In general, Li, Molecular Evolution, Sinauer Associates, S
See underland, MA, 1997. In addition, the sequence comparison data is used for phylogenetic analysis, where the primate evolutionary history is reconstructed based on relevant ranges of sequence similarity among molecules examined from different primates. For example, DNA and amino acid sequence data for the enzyme lysosome from different primates have been used to study protein evolution in primates and to study the emergence of adaptive selection within specific lineages (Malcom
Et al., Nature 345: 86-89, 1990; Messier and Stewart, 1997). Other genes devoted to molecular evolution studies in primates include hemoglobulin, cytochrome C oxidase, and major histocompatibility complex (MHC) (Nei and Hughes in: Evolutio
n at the Molecular Level, Sinauer Associates, Sunderland, MA 222-247, 19
91; Lienert and Parham, Immunol. Cell Biol. 74: 349-356, 1996; Wu et al., J. Mo.
l. Evol. 44: 477-491, 1997). Many uncoded sequences have also been used in phylogenetic analysis of primates (Li, Molecular Evolution, Sinauer Associate).
s, Sunderland, MA 1997). For example, gene distance in primate lineage is determined from the beta-type globin locus and the orthologous, non-coding nucleotide sequence of the flanking region, and the evolution constructed for nucleotide sequence orthologs. The tree depicted a branching pattern that roughly matched the figure obtained from phylogenetic analysis of morphological features (Goodman et al., J. Mol. Evol. 30: 260-266,
1990).

[0009] Zhou and Li (Mol. Biol. Evol. 13 (6): 780-783, 1996) applied KA / KS analysis to primate genes. It was previously reported that gene conversion events appeared to occur in introns 2 and 4 between the red and green retinal pigments during human evolution. However, the intron 4 sequences for the red and green retinal pigments from one European were completely identical, suggesting that a recent gene conversion has occurred. The authors obtained from male Asians, male chimpanzees, and baboons to determine whether the gene conversion event occurred in individuals, in common ancestors of Europeans, or in an earlier human ancestor. The sequence of intron 4 of the red and green retinal pigment genes was examined and KA / KS analysis was applied. They observed that the difference between the two genes was much lower in intron 4 than in the surrounding exons, suggesting that strong natural selection was performed for sequence homogenization.

[0010] Wolinsky et al. (Science 272: 537-542, 1996) have shown that the HIV virus itself (ie, not the host class) is susceptible to adaptive evolution in individual human patients, and has been reported to use nonsynonymous bases. A comparison of substitution and synonymous base substitution was used. Their purpose is simply to prove that a positive selection occurs in a short time frame (the time frame for progression of the human patient to the disease). Niewiesk and Bangham (J. Mol. Evol. 42: 452-458, 1996) raised a related question about the HTLV-1 virus, namely, what is the selectivity of the virus itself? A / Ds approach was used. They could not determine the nature of the selectivity, probably due to insufficient sample size. In these cases, KA / KS-type methods are used for human viruses, but no attempt has been made to use such methods for therapeutic purposes (as in the present application), but rather to pursue narrow academic purposes. Was made.

As can be seen from the above-mentioned reports, molecular evolution analysis methods (KA / KS type methods) for identifying rapidly evolving genes are used to achieve a number of different purposes. And most commonly can be used to confirm the existence of a positive choice at the molecular level of Darwin's theory. In addition, assess the frequency of positive selection at the molecular level in Darwin's theory, understand phylogenetic relationships, elucidate the mechanisms by which new species are formed, or identify single or multiple It can also be used to establish origin. It is clear from the above reports and the literature that none of the authors believe that evolutionary solutions, i.e., further prevent or treat human conditions or diseases, or have unique or enhanced human functions. No one has applied the KA / KS-type method to identify specific evolutionary changes that mimic or use in the development of modulating therapies. The authors identify genes in human or nonhuman primates that contain evolutionarily significant sequence changes and use such genes and the identified changes in the development of treatments for human conditions and diseases KA / KS type analysis was not used as a systematic approach for

The identification of human genes that have evolved to confer a unique or enhanced human function relative to a homologous chimpanzee gene is a reagent that modulates such a unique human function or restores function when the gene is defective. Could be used to develop. Identification of the genes and specific nucleotide changes in the evolving underlying chimpanzees (or other non-human primates), as well as characterizing the physiological and biochemical changes in the proteins encoded by these evolved genes, For example, what determines infectivity or resistance to infectious diseases such as AIDS, what determines infectivity or resistance to the development of certain cancers, what determines infectivity or resistance to acne. It could provide valuable information, such as making decisions, how hair growth is controlled, and how muscle and fat formation is controlled. This valuable information could be adapted to the development of reagents that cause human proteins to behave as homologous chromosomes in chimpanzees.

[0013] All references cited herein are expressly incorporated by reference. SUMMARY OF THE INVENTION The present invention relates to methods for identifying polynucleotide and polypeptide sequences that have evolutionarily significant changes, wherein the changes are related to physiological conditions, including medical conditions. The present invention relates to physiological conditions, such as the evolution of features that are medically or commercially relevant, i.e., the primate genome by comparison, to identify particular genetic changes that are responsible for such features. And use the information obtained from such evolved genes in the development of human therapies. The sequence of a non-human primate used in the methods described herein may be any non-human primate, preferably a member of the primate human superfamily, more preferably a chimpanzee, a cockpit chimpanzee , Gorillas and / or orangutans, most preferably chimpanzees.

In one preferred embodiment, the non-human primate polynucleotide or polypeptide undergoes natural selection that is responsible for a positive, evolutionarily significant change (ie,
Non-human primate polynucleotides or polypeptides have positive properties not found in humans). In this aspect, the positively selected polynucleotide or polypeptide is associated with susceptibility or resistance to certain diseases, or other commercially relevant features. Examples of this embodiment include, but are not limited to, a polynucleotide or polypeptide that is positively selected for a non-human primate, preferably a chimpanzee, and that is associated with susceptibility or resistance to infection or cancer. Examples of commercially relevant features include aesthetic features such as hair growth, muscle mass, susceptibility or resistance to acne. Examples of this embodiment include HIV seeding, spreading,
And / or polynucleotides and polypeptides associated with susceptibility or resistance to the development of AIDS. Thus, the present invention is effective in adding insight into the molecular mechanisms underlying resistance to HIV seeding, spread, and / or the development of AIDS, and the use of agents to prevent and / or delay the development of AIDS. It provides information that is also useful for discovering and / or designing such reagents. Specific genes positively selected in chimpanzees involved in AIDS or other infectious diseases are ICAM-1, ICAM-2, ICAM-3 and MIP-
1-α. 17-β-hydroxysteroid dehydrogenase type IV is a specific gene involved in positively selected cancers in chimpanzees.

[0015] In another preferred embodiment, the human polynucleotide or polypeptide has undergone natural selection that has caused a truly evolutionary significant change (ie, the human polynucleotide or polypeptide is a non-human primate). With unparalleled positive properties). One example of this aspect is that the polynucleotide or polypeptide is associated with a unique or enhanced functional capacity of the human brain as compared to a non-human primate. Others have a longer human life span compared to non-human primates. That is, the present invention is useful for adding insight into the molecular mechanisms underlying specific or enhanced human function, and designing reagents such as agents that modulate such specific or enhanced human function. And provide information that may be useful in designing treatments for diseases or conditions involving humans.
By way of example, the present invention is useful in adding insight to the molecular mechanisms underlying human cognitive function, designing reagents such as drugs that enhance human brain function, and It is to provide information that may be useful in designing treatments for brain-related diseases. A specific example of a human gene that has a truly evolutionary significant change when compared to a non-human primate is the tyrosine kinase gene KIAA641.

Accordingly, in one aspect, the present invention provides a method of identifying a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises (e.g., a medically or commercially involved Associated with a physiological condition (such as a significant change). Just evolutionarily significant changes can be found in human or non-human primates.

In one aspect of the invention, there is provided a method for identifying a polypeptide sequence of a non-human primate that encodes a polypeptide, wherein the polypeptide is physiologically non-human in a non-human primate. Related to the condition, such as a physiological condition listed (through the specification), for example, susceptibility or resistance to the development of a medically relevant disease state, such as an infectious disease (a viral disease such as AIDS). including)
Alternatively, it includes, but is not limited to, cancer. In some aspects,
a) comparing a polynucleotide sequence encoding a protein of a non-human primate, preferably a chimpanzee, with a polynucleotide sequence encoding a human protein, wherein the human includes one that is not in a physiological state; A method is provided that comprises selecting a non-human primate polynucleotide sequence that includes a nucleotide change relative to the matching sequence, wherein the change includes being evolutionarily significant. In some embodiments, the sequence encoding the non-human protein corresponds to a cDNA. In some embodiments, the sequence to be compared is obtained from the brain. The methods used to assess nucleotide changes and the nature of the nucleotide changes are described herein and utilized in some or all of the embodiments. In these methods (as well as in other methods similar to those described herein), the sequence encoding the non-human protein (and / or the polypeptide encoded therein) can be used to generate physiological characteristics. And / or related to maintenance.

For any aspect of the present invention, the physiological condition may be any physiological condition, such as those mentioned herein, such as cancer and infectious diseases (viral diseases such as AIDS). (Including susceptibility or resistance to the disease); life span; including brain functions including cognitive function.

In one aspect of the invention, there is provided a method for identifying a polynucleotide sequence encoding a human polypeptide, wherein the polypeptide is associated with a physiological condition present in humans, and A) comparing a polynucleotide sequence encoding a human protein with a polynucleotide sequence encoding a non-human primate protein, wherein the non-human primate has no physiological condition; The sequences corresponding to primates are compared to select a human polynucleotide sequence that contains a single nucleotide change, wherein the change consists of steps that include being evolutionarily significant. In some embodiments, the sequence encoding the human protein (and / or the polypeptide encoding therein) is associated with the development and / or maintenance of a physiological condition. In some embodiments, the sequence encoding the human protein corresponds to a cDNA. In some correspondences, the sequences to be compared are obtained from the brain. In some aspects, the physiological condition is lifespan. In another aspect, the physiological condition is brain function. In another aspect, the brain function is a cognitive function. The methods used to assess nucleotide changes and the nature of the nucleotide changes are described herein and are to be utilized in any or all aspects.

In another embodiment, (a) comparing a nucleotide sequence encoding a human protein with a nucleotide sequence encoding a protein of a non-human primate, preferably a chimpanzee, that is resistant to a particular medically relevant disease state. Wherein the human protein-encoding sequences include those associated with the development of a disease, and (b) selecting a non-human polynucleotide sequence comprising at least one nucleotide change compared to a sequence corresponding to a human. , Wherein the change provides a method comprising steps that include those that are evolutionarily significant. Sequences identified by these methods are further characterized and / or analyzed to ascertain that they are associated with disease development or condition. The most preferred disease states applicable to these methods are cancer, and AIDS, hepatitis C,
Infectious diseases including leprosy.

In another aspect, the invention provides a method for identifying a polynucleotide sequence encoding a polypeptide, wherein the polypeptide is associated with resistance to development of AIDS, and a) comparing a sequence encoding a non-human primate protein that is resistant to AIDS with a sequence encoding a human protein, wherein the sequence encoding the human protein includes one associated with the development of AIDS; and b)
Selecting a non-human primate sequence that is resistant to AIDS containing at least one nucleotide change compared to the corresponding human sequence, wherein the nucleotide change is evolutionarily significant Shall consist of As set forth herein, these methods include, for example, alignment of sequences according to sequence homology, and human polymorphism including at least one unique nucleotide change relative to the corresponding non-human primate polynucleotide sequence. It can be achieved by identifying a nucleotide sequence, wherein the unique nucleotide changes are to be positively selected according to evolutionary analysis (as described herein).

In another aspect, there is provided a method for identifying an evolutionarily significant change in a polynucleotide sequence encoding a human brain protein, and a) providing a polynucleotide sequence encoding a human brain protein; Comparing the corresponding sequence of a non-human primate, and b) selecting a human polynucleotide sequence comprising one nucleotide change compared to the corresponding sequence of a non-human primate; Here, it is assumed that the change includes a stage including evolutionary significance. In some embodiments, the nucleotide sequence encoding a human brain protein corresponds to a human brain cDNA.

Another aspect of the invention includes a method for identifying an evolutionarily significant change in a positively selected human. These methods include: (a) comparing a nucleotide sequence encoding a human protein with a nucleotide sequence encoding a non-human primate protein;
And (b) selecting a human polynucleotide sequence comprising at least one (ie, one or more) nucleotide change compared to the corresponding sequence in a non-human primate, wherein the change is evolutionary , Including the steps that are significant to Sequences identified by this method may be further characterized and / or analyzed for their potential to be associated with a unique or enhanced biological or medically relevant function in humans. I do.

Another aspect of the invention is a method for large-scale sequence comparison between a polynucleotide sequence encoding a human protein and a polynucleotide sequence encoding a protein obtained from a non-human primate, such as a chimpanzee. And (a) aligning the human polynucleotide sequence with the corresponding polynucleotide sequence of a non-human primate according to sequence homology, and (b) corresponding polymorphism obtained from a non-human primate. Any nucleotide changes in the human sequence are identified as compared to the nucleotide sequence, wherein the changes shall comprise steps that include being evolutionarily significant. In some embodiments, the sequence encoding the protein is obtained from the brain.

[0025] In some embodiments, the nucleotide change identified by any of the methods described herein is a non-synonymous substitution. In some aspects, the evolutionary significance of the nucleotide change is determined according to the non-synonymous substitution rate (KA) of the nucleotide sequence.
In some embodiments, the evolutionarily significant changes are assessed by determining the KA / KS ratio between a human gene and a homologous gene obtained from a non-human primate (such as a chimpanzee), preferably The ratio is at least about 0.75, more preferably greater than about 1 (match) (ie, at least 1), more preferably at least about 1.25, and more preferably at least about 1.50.
And more preferably about 2.00. In another embodiment, once a positively selected gene is identified between a human and a non-human primate (such as a chimpanzee), the human or non-human primate (such as a chimpanzee) gene is positively identified. Further comparisons are made with other non-human primates to see if they have been selected.

In another aspect, the invention is a method of correlating an evolutionarily significant human nucleotide change to a physiological condition in humans (or humans), if any, of any of the methods described herein. Analyzing the functional effect (including determining the presence of the functional effect) of the polynucleotide sequence (presence / absence) identified thereby, wherein the presence of the functional effect is evolutionarily significant It shall include those that show a correlation between nucleotide changes and physiological conditions. Alternatively, in these methods, the functional effect (if any) may be based on the polypeptide sequence (or a portion of the polypeptide sequence) encoded by the nucleotide sequence identified by any of the methods described herein. Is evaluated using

[0027] The present invention also provides a method for identifying poly (poly) isolated by physical and biochemical methods widely used in the art to determine the structural or biochemical significance of evolutionarily significant changes. 2 provides a comparison of peptides. The physical method is understood to include a method used to examine structural changes in a protein encoded by a gene likely to have undergone adaptive evolution. Proximity comparisons of the three-dimensional structure of a protein (either human or non-human primate) and an evolved homologous protein (either human or non-human primate, respectively) are related human states And will provide valuable information to develop treatments for the disease. For example, by using the method of the present invention, the chimpanzee ICAM-1 gene has been identified as having a positive evolutionary change compared to human ICAM-1. Human I
In a three-dimensional model of the two functional domains of the CAM-1 protein, five of the six amino acids altered in the chimpanzee were identified as LF, an ICAM-1 counterreceptor.
It is right next to (ie, in physical contact with) an amino acid residue known to be critical for binding to A-1, and in each case, the human amino acid is , Was replaced by a larger amino acid in chimpanzee ICAM-1. Such information provides insight into the design of appropriate therapeutic interventions.

Thus, in another aspect, the invention provides a method for identifying one target site, which may include one or more target sites, that is suitable for therapeutic intervention, comprising any of the methods described herein. Comparing the human polypeptide encoded by the sequence identified above with the corresponding non-human polypeptide (or a portion of that polypeptide), where the location of the molecular difference, if any, is the target site Is shown. In another aspect, the invention provides a method for identifying a target site (including one or more target sites) that is suitable for therapeutic intervention, wherein the method comprises the steps of: Comparing the non-human polypeptide (or part of the polypeptide) encoded by the sequence with the corresponding human polypeptide (or part of the polypeptide), where any differences in amino acids The position of such a molecular difference indicates the target site.

Biochemical methods include those used to determine functional differences in proteins encoded by genes that have undergone adaptive evolution, such as binding specificity, binding strength, or the most desirable binding state. Understand. Proximity comparisons of the biochemical characteristics of a protein (either human or non-human primate) and an evolved homologous protein (either non-human primate or human, respectively) are based on the relevant human Will reveal valuable information for developing treatments for the disease and conditions of the disease.

[0030] In another aspect, the invention provides a method for identifying a reagent that modulates a physiological condition, the method comprising the steps of identifying one reagent (ie, one reagent to be tested). Inoculating cells transformed with a polynucleotide sequence identified by any of the methods described in, wherein the reagent is identified by its ability to modulate the function of the polypeptide sequence. In another aspect, the present invention provides a method for identifying a reagent that modulates a physiological condition, the method comprising the steps of: Contacting a polypeptide (or a composition comprising a polypeptide fragment and / or a polypeptide or polypeptide fragment) encoded within or within a polynucleotide identified by any of The reagent is identified by its ability to modulate the function of the polypeptide. The present invention also provides reagents identified using the screening methods described herein.

[0031] In another aspect, the invention modulates a specific or enhanced human function or modifies a characteristic of a non-human primate, such as susceptibility or resistance to the development of a disease such as AIDS. It provides a method of screening for a reagent that modulates the activity of a mimicking human polynucleotide or polypeptide. These methods involve inoculating cells transformed with the polynucleotide sequence with the reagent to be tested, and identifying the reagent based on its ability to modulate the function of the polynucleotide, or testing the polypeptide preparation. Contacting the reagent to be performed and identifying the reagent based on its ability to modulate the function of the polypeptide.

Detailed Description of the Invention The present invention relates to physiological conditions, such as evolved features that are medically or commercially relevant, that is, specific genes that contribute or contribute to those conditions. It utilizes comparative genomics to identify changes. The present invention relates to the differences in functions and diseases that distinguish humans from non-humans, especially primates, most preferably the two known species, namely the chimpanzees and the chimpanzees, including the chimpanzee chimpanzee (pygmy chimpanzee). It consists of a comparative genomic approach to identify specific genetic alterations that are responsible. For example, chimpanzees and humans are 98.5% identical at the DNA sequence level, and the present invention is capable of identifying adaptive molecular changes that underlie differences between species in many geographical regions, providing unique or Includes enhanced human cognitive ability, including chimpanzee resistance to AIDS and certain cancers. Rather than the typical genomics that merely identify genes, the present invention provides accurate information for evolutionary solutions that eliminate disease and provide unique functions. The present invention identifies genes that have evolved to confer evolutionary benefits and their specifically evolved changes.

The present invention provides that the polynucleotide encoding the human protein can be used in humans, but not in species having close evolutionary relationships to humans, such as non-human primates.
Due to the observation that it involves sequence changes found as a result of adaptive selection during evolution.

Furthermore, the present invention stems from the observation that the genetic information of non-human primates includes changes found in certain non-human primates, but not in humans, as adaptive choices during evolution. are doing. In this embodiment, the non-human primate polynucleotide or polypeptide has undergone natural selection that results in a positively evolutionary significant change (i.e., the non-human primate polynucleotide or polypeptide is Has a non-existent positive property). In this aspect, the positively selected polynucleotide or polypeptide is associated with susceptibility or resistance to certain diseases or other commercially relevant features. Medically relevant examples of this aspect include, but are not limited to, polynucleotides or polypeptides that are positively selected in non-human primates, preferably chimpanzees, and that are associated with susceptibility or resistance to infections and cancers It is not something to try. Examples of this embodiment are HIV
Includes polynucleotides or polypeptides associated with susceptibility or resistance to progression from infection to AIDS. In other words, the present invention provides a method for converting AIDS
Add insight into the molecular mechanisms underlying resistance to progression to onset,
Is useful in finding and / or designing reagents, such as drugs, that prevent and / or delay the onset of. Commercially relevant examples include polynucleotides or polypeptides positively selected in non-human primates associated with aesthetic features such as hair growth, acne, or muscle mass,
It is not intended to limit its scope.

[0035] Positively selected human evolutionary significant changes in polynucleotide and polypeptide sequences are characteristic of or enhanced human brain function, especially when compared to the closest evolutionary relative, chimpanzees. And the human ability to provide such competitive advantages to humans. The present invention identifies human genes that have evolved to provide unique or enhanced human cognitive abilities, and the actual protein changes that confer functional differences enhance cognitive abilities for the general population, It will be quite effective for therapeutic approaches to treating cognitive deficits.

The practice of the present invention will employ, unless otherwise indicated, typical techniques of molecular biology, genetics and molecular evolution that are within the skill of the ordinary art. Such techniques are explained fully in the literature, for example: "Molecular Cloning: A Laboratory Manual" 2nd edition (Sambrock et al., 1989); "Oligonucleotide Synthesis" (edited by MJ Gait,
1984); "Current Protocols in Molecular Biology" (edited by FM Ausubel et al.,
1987); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994);
Molecular Evolution ”(Li, 1997).

Definitions As used herein, “polynucleotide” refers to a nucleotide of any length,
Or, reference is made to polymeric forms of ribonucleotides or deoxynucleotides, or analogs. The term refers to the primary structure of the molecule, ie, includes double- and single-stranded DNA, as well as double- and single-stranded RNA. Also includes modified polynucleotides, such as methylated and / or capped polynucleotides. "Polynucleotide" and "nucleotide sequence" are used interchangeably.

As used herein, “gene” refers to a polynucleotide or a portion of a polynucleotide comprising a sequence that encodes a protein. The gene is a sequence that is not coded, a sequence located on the 5 ′ and 3 ′ side like the intron (promoter,
It is also well understood in the art that they are also comprised of enhancers, repressors, and other control sequences.

“Polypeptide,” “peptide” and “protein” are used interchangeably herein and refer to a macromolecule of amino acids of any length. These terms also include proteins that have been post-translationally modified through reactions involving glycosylation, acetylation, and phosphorylation.

“Physiological condition” is a term well understood in the art and means any condition or aspect measured and / or observed. "Physiological condition" includes the degree of body fat, alopecia (baldness), physical condition such as acne, life expectancy, disease aspects (including susceptibility or resistance to disease) such as cancer and infections, It is not intended to limit its scope. Examples of physiological conditions are set forth below (e.g., "human medically relevant medical conditions", "human commercially relevant conditions", "
(See definitions of "medically relevant evolved features" and "commercially relevant evolved features"), and throughout the specification, and these terms and examples may refer to physiological states. Understood. Certain physiological conditions are not necessarily necessary, but are the result of multiple factors, any of which are subsequently considered to be one physiological condition. Physiological conditions "present" in humans or non-human primates occur within certain populations, are unique to certain populations when compared to other populations, and / or are enhanced Including physiological conditions.

The terms “human medically relevant condition” or “human commercially relevant condition” refer to a human condition for which medical or non-medical (respectively) intervention is desired. Used here.

The term “medically relevant evolved feature” is used herein to refer to a feature that has evolved in a human or non-human primate, the analysis of which has led to the development of medical treatments in humans. (E.g., physical or biochemical data).

The term “commercially relevant evolved features” is used herein to refer to evolved features in humans or non-human primates, the analysis of which comprises the use of non-medical products used by humans. Or it may provide information (eg, physical or biochemical data) relevant to the development of a therapy.

The term “KA / KS-type method” is the difference between the number of non-synonymous and the number of synonymous substitutions in a homologous gene, often (but not always) expressed as a ratio, to evaluate this difference. Means a method (including more accurate methods of determining non-synonymous and synonymous sites). These methods are not limited to KA / KS, but dN / dS, DN
Designed using some terminology, including / DS.

The term “evolutionary significant change” or “change in applied evolution” refers to a change in one or more nucleotide or peptide sequences between two species attributed to positive selective pressure. I do. One method for determining the existence of an evolutionarily significant change is
The use of a KA / KS type analysis method such as measuring the KA / KS ratio. Typically, the KA / KS ratio is at least about 0.75, more preferably at least about 1.0, more preferably at least about 1.25, more preferably at least about 1.5, and more preferably at least about 2 A value of 0.0 indicates a positive selection effect and is considered to be an evolutionarily significant change.

The term “positive evolutionary significant change” means an evolutionarily significant change in a particular species that results in an application change that is positive compared to other related species. Examples of positive evolutionarily significant changes are changes resulting in enhanced cognitive abilities in humans and also application changes in chimpanzees resulting in the ability of HIV-infected chimpanzees to resist progression to terminal AIDS. is there.

The term “resistant” refers to the ability of an organism, such as a chimpanzee, to avoid or reduce the development of a disease state and / or disease, and is preferably a non-resistant organism, typically Depends on comparison with human. For example, chimpanzee is H
It is resistant to certain effects of IV and infection with other viruses, and / or chimpanzees do not develop AIDS, the ultimate disease.

The term “susceptible” is known to mean that an organism, such as a human, cannot avoid or reduce the development of a disease state and / or disease state, and is preferably resistant. As compared to a living organism, eg, a non-human primate, such as a chimpanzee. For example, humans are susceptible to certain effects of HIV and other viral infections, and / or AIDS, the ultimate disease.

[0049] Tolerance and susceptibility vary among individuals, and for the purposes of the present invention, these terms apply to populations within a species, and intra-specific comparisons are used, but generally a comparison of resistance and susceptibility Refers to the overall or average difference between species.

The terms “homology”, “homologous” or “ortholog” are known in the art, are well understood, have a common ancestor, and are based on the degree of sequence identity. Reference to a related sequence determined by: These terms describe the relationship between genes found in one species and corresponding or equivalent in other species. For the purposes of the present invention, homologous sequences are compared. "Homologous sequence",
"Homology" or "orthologs" are considered, believed, or known to be functionally related. Functional relationships may be indicated in any one of a number of ways, including, but not limited to, (a) degree of sequence identity; (b) indicated by the same or similar biological functions. It is not done. Preferably, (a
) And (b) are both shown. The degree of sequence identity varies, and is preferably at least 50% (using a standard sequence alignment program known in the art), more preferably at least 60%, more preferably at least 60%. 75%, more preferably at least 85%. Homology can be determined using software programs readily available in the art, for example, Current Protocols in Molecular Biology (FM Ausubel).
Eds., 1987) Some of these were reviewed in special issue No. 30, Chapter 7.718, and Table 7.71. Preferred alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK) and ALIGN
Plus (Scientific and Educational Software, Pennsylvania). Another preferred alignment program is Seqencher (G) using default parameters.
ene Codes, Ann Arbor, Michigan).

The term “nucleotide change”, as is well understood in the art,
Reference is made to nucleotide substitutions, deletions, and / or additions. As used herein, the term "nucleotide sequence encoding a human protein", which is "associated with susceptibility to AIDS", is associated with HIV dissemination (infectious in vivo, i.e., in vivo), transmission and / or development of AIDS. Reference is made to a human nucleotide sequence that encodes a protein of interest. Extensive studies of the mechanisms underlying the progression from HIV infection to AIDS development indicate that many human candidate genes are believed or known to be associated with one or more of these phenomena. Polynucleotide sequences (including any polypeptide encoded therein) that are associated with susceptibility to AIDS may include HIV seeding, replication, and / or subsequent terminal AI.
One that is known or involved in playing a role in DS progression. Examples of such candidate genes are shown below.

“AIDS resistance” refers to the ability of an organism, such as a chimpanzee, to avoid or reduce the extent of HIV infection (such as transmission or dissemination) and / or avoid the development of AIDS, Is compared with a person susceptible to AIDS.

“Susceptibility” to AIDS means that an organism, such as a human, cannot avoid or reduce the consequences of HIV infection (such as transmission or dissemination) and / or the development of AIDS. Preferably, when compared to an organism known to be resistant to AIDS, such as a non-human primate, such as a chimpanzee.

As used herein, the term “nucleotide sequence encoding a brain protein”
Reference is made to the nucleotide sequence expressed in the brain that encodes the protein. An example of a "nucleotide sequence encoding a brain protein" is a brain cDNA sequence.

As used herein, the term “unique or enhanced brain function in humans”, “unique functional ability of the human brain” or “unique or enhanced brain functional ability in humans” refers to Reference is made to any brain function identified and / or observed to be enhanced in humans, as compared to other non-human primates, in kind or degree. Such brain functions include, but are not limited to, advanced information processing, storage and repair, creativity, memory, language, emotional responses through the brain, movement, pain / pleasure, smell, and temper It does not mean that.

“Housekeeping gene” is a well understood term in the art that includes, but is not limited to, growth, differentiation, quiescence, metabolism, and / or death, and Genes associated with various cell functions. "housekeeping"
Genes generally perform functions found in one or more cell types. In contrast, cell-specific genes generally function in a particular cell type (such as the nervous system) and / or class (such as a nerve cell).

“Reagent” as used herein refers to a single or mixed organic or inorganic molecule,
A biological or chemical compound, such as a peptide, protein, or oligonucleotide. A vast number of compounds have been synthesized, for example oligomers such as oligopeptides and oligonucleotides, and synthetic organic and inorganic compounds based on a variety of core structures, which are also included in the term "reagent." In addition, various natural resources, such as plant and animal extracts, provide compounds for screening. Compounds can be tested alone or in combination with other compounds.

The term “modulating the function” of a polynucleotide or polypeptide means that the function of the polynucleotide or polypeptide is changed as compared to the case where no reagent is added. Regulation can occur at any level that affects function. Polynucleotide or polypeptide function is direct or indirect, and is measured directly or indirectly.

“Polynucleotide function” includes, but is not limited to, replication, translation, and expression patterns. Polynucleotide function also includes functions associated with the polypeptide encoded within the polynucleotide. For example, it acts on a polynucleotide to express, conform, or fold a protein (
Or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), control and / or reagents that affect other aspects of protein structure or function are polynucleotides It is considered that the function was adjusted.

“Polypeptide function” refers to conformation, folding (or other physical characteristic), binding to another moiety (such as a ligand), activity (or other functional characteristic), control and / or Or other aspects of protein structure or function,
It is not intended to be limited to them. For example, acting on a polypeptide, conformation, folding (or other physical characteristic), binding to another molecule (such as a ligand), activity (or other functional characteristic), control and / or protein structure Alternatively, reagents that affect other aspects of function may modulate polypeptide function. An indication that an effective reagent acts to modulate the function of a polypeptide is 1
) Altering the conformation, folding or other physical characteristics; 2) altering the avidity of the native ligand or altering the specificity of binding to the ligand; and 3) altering the activity of the polypeptide. But not intended to be limited thereto.

As used herein, “modulates susceptibility to the development of AIDS” and “A
The term "modulates resistance to the development of IDS" refers to the intercellular H
Modulating IV transmission or infection. Further, these terms include reducing the onset of AIDS and / or susceptibility to HIV cell-to-cell transmission or infection. Further, these terms include increasing the onset of AIDS and / or the resistance to cell-to-cell transmission or infection of HIV. One means of assessing whether a reagent modulates susceptibility or resistance to the development of AIDS is to use the cell-based systems described herein in comparison to appropriate controls,
Measuring whether at least one indicator of HIV susceptibility is affected. The characteristics of HIV susceptibility include, but are not limited to, the intercellular transmission of the virus, as measured by the total number of cells infected with HIV and the formation of fused cells.

The term “target site” refers to a polypeptide that is a single amino acid of a structural and / or functional center, such as, for example, a binding site, a dimerization domain, or a catalytically active site, and / or a portion thereof. Means a certain position. The target site is effective for direct or indirect interaction with the reagent, such as a therapeutic agent.

The term “molecular difference” includes any structural and / or functional differences.
Methods for detecting such differences are described herein, as are examples of such differences.

“Functional effect” is a term well-known in the art and means any effect, whether direct or indirect, exhibited at any level of activity. General Methods Known in the Art For the purposes of the present invention, sources of human and non-human polynucleotides include:
It can be any suitable source, such as a gene or cDNA sequence. Preferably, cDNA sequences obtained from human and non-human primates are compared. Sequences encoding human proteins are available from Genome Sequence Data Bank and GenBank.
From public databases such as These databases serve as a repository for molecular sequence data generated by ongoing research.
Alternatively, the sequence encoding the human protein can be obtained, for example, from a sequence of cDNA reverse transcribed from mRNA expressed in human cells, or a sequence after PCR amplification, according to methods well known in the art. Alternatively, sequences of the human genome can be used for sequence comparison. The sequence of the human genome can be obtained from public databases, sequences of commercially available human genomic DNA libraries, or from genomic DNA after PCR.

A sequence encoding a non-human primate protein can be obtained, for example, by sequencing a cDNA clone arbitrarily selected from a non-human primate cDNA library. Non-human primate cDNA libraries can be constructed from total mRNA expressed in primate cells using standard techniques in the art. In some embodiments, cDNA is prepared from mRNA from tissues at a limited stage of development, or obtained after primates have been subjected to certain environmental conditions. The cDNA libraries used in the sequence comparisons of the present invention can be constructed using routine cDNA library construction techniques well described in the technical literature. Total mRNA is used as a template to reverse transcribe the cDNA. The transcribed cDNA is subcloned into an appropriate vector to establish a cDNA library. Although shorter than full length cDNAs can be used, established cDNA libraries are maximized to include full length cDNAs. Furthermore, the sequence frequency can be determined, for example, by Bonaldo et al. (Genome Research 6: 791-806, 1996).
Standardized according to CD arbitrarily selected from the constructed cDNA library
NA clones can be sequenced using standard automated sequencing techniques. Preferably, a full-length cDNA clone is used for sequencing. Although some or all of the cDNA clones obtained from the cDNA library are sequenced, some embodiments of the invention are practiced by sequencing a small number of clones, such as one cDNA clone or several cDNA clones. You can, but.

In one preferred aspect of the invention, the non-human primate cDNAs to be sequenced can be pre-selected according to their expression specificity. To select a cDNA corresponding to a specifically expressed active gene, the cDNA can be subjected to subtractive hybridization using mRNA obtained from other organs, tissues or cells of the same animal. Hybridization with tissue non-specific mRNA under certain conditions of hybridization with the appropriate stringency and concentration will result in a cDNA that represents a "housekeeping" gene.
Could be removed from the cDNA pool. Thus, the remaining cDNA to be sequenced is the cDNA most likely to be associated with tissue-specific functions.
DNA. For the purpose of subtraction hybridization, tissue-nonspecific mRNA is obtained from one organ, or preferably a mixture of cells and heterologous organs. The amount of non-tissue specific mRNA is used by saturating the tissue-specific cDNA.

Optionally, information from online public databases is used to select or prioritize cDNAs most likely to be associated with specific functions.
For example, non-human primate cDNA candidates for sequencing are selected by PCR using primers designed from the candidate human cDNA sequences.
Candidate human cDNA sequences are, for example, those found only in certain tissues, such as the brain, or corresponding to genes that are likely to be important in certain functions, such as brain function. Such human tissue-specific cDNA sequences can be obtained by searching an online human sequence database, such as GenBank, in which information regarding the expression profile and / or biological activity of the cDNA sequences is specified. I have.

The sequence of a non-human primate (eg, obtained from an AIDS-resistant non-human primate) having homology to a known human gene can be obtained using art-standard methods,
For example, a public database such as GenBank or a PCR method (for example, GeneAmp P
CP System 9700 thermocycler (using Applied Biosystems, Inc.). For example, non-human primate cDNA candidates for sequencing
Can be selected by PCR using primers designed from candidate human cDNA sequences. Primers for PCR can be prepared from human sequences using standard techniques in the art, and include publicly available primer design programs such as PRIMER (Whitehead Institute). The amplified sequence is then sequenced using techniques and equipment standard in the art, for example, an automated sequencer (Applied Biosystems, Inc.).

General Method of the Invention The general method of the present invention is as follows. Briefly, nucleotide sequences are obtained from human and non-human primate sources. The sequences of human and non-human primates are compared to each other to identify sequences of homology. Homologous sequences are analyzed to identify sequences that have nucleic acid sequence differences between the two species. Subsequently, molecular evolution analysis is performed to quantitatively and qualitatively assess the evolutionary significance of the difference. For genes that are positively selected between the two species, eg, human and chimpanzee, it is useful to determine whether the differences occur in other non-human primates. The sequence is then characterized with respect to molecular / genetic identity and biological function. Finally, that information can be used to identify reagents that are effective in diagnosing and treating human medically or commercially relevant conditions.

The general method of the invention involves comparing a nucleotide sequence encoding a human protein with a nucleotide sequence encoding a non-human, preferably primate, most preferably chimpanzee protein. Examples of other non-human primates are Kobito chimpanzees, gorillas, orangutans, gibbon, old world monkeys, and new world monkeys. A phylogenetic tree for primates within the primate human superfamily is shown in FIG. Bioinformatics is applied for comparison to select sequences that contain nucleotide changes that are evolutionarily significant changes. The present invention allows the identification of genes that have evolved to confer certain evolutionary advantages and the identification of specifically evolved changes.

[0071] Sequences encoding human and other non-human primate proteins are compared to compare homologous sequences. Any suitable mechanism for completing this comparison is contemplated by the present invention. Alignment can be performed manually or with software (examples of suitable alignment programs are known in the art). Preferably, sequences encoding proteins obtained from non-human primates are compared to human sequences by database searches, such as BLAST searches. The high score "hits", ie, sequences that show significant similarity after BLAST analysis, will be collected and analyzed. Sequences exhibiting significant similarity have a sequence identity of at least about 60%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%
And more preferably at least about 90%. Preferably,
Sequences showing greater than about 80% identity will be further analyzed. Homologous sequences identified by database searches were identified in the art by available sequence alignment techniques and programs, such as CLUSTAL V, a commonly used simple alignment program (Higgins et al., CABIOS 8; 189 -191) is used to arrange the entire sequence.

Alternatively, the sequencing and homology comparison of protein-encoding sequences between human and non-human primates is performed simultaneously using newly developed sequencing chip technology. See, for example, Rava et al., US Patent No. 5,545,531.

Sequences encoding aligned human and other non-human primate proteins are analyzed to identify nucleotide sequence differences at specific locations. On the other hand, any suitable method for accomplishing this analysis is contemplated by the present invention. If there is no difference in the nucleotide sequences, the sequence encoding the non-human primate protein is usually not further analyzed. The detected sequence change is generally and preferably initially verified for accuracy. Preferably, the initial confirmation involves performing one or more of the following steps, any or all of which are known in the art: (a) non-human primates and human Finding the point where there is a change between sequences; (b) Determining whether bases that appear to be unique to a non-human primate correspond to a specific, strong and well-defined signal for the corresponding base (C) Check the number of human hits to see if there is more than one human sequence corresponding to the sequence change. The registration of diverse human sequences for the same gene with the same nucleotide at the position where a different nucleotide is present in the sequence of a non-human primate provides independent support that the human sequence is accurate and that the changes are significant. provide. Such changes are examined using public database information and the genetic code, to determine if such nucleotide sequence changes are due to changes in the amino acid sequence of the encoded protein. Given the definition of a "nucleotide change", the present invention relates to substitutions, deletions, or additions in a polynucleotide sequence encoding a human protein as compared to the corresponding sequence obtained from a non-human primate. And at least one nucleotide change. Preferably, the change is a nucleotide substitution. More preferably, one or more substitutions are present in the identified human sequence and are subject to molecular evolution analysis.

[0074] Any of several different molecular evolution analyzes, or KA / KS-type methods, quantitatively and quantitatively determine the evolutionary significance of nucleotide changes identified between human and non-human primate gene sequences. It can be used for qualitative evaluation (Kreitman and Akashi, Annu. Rev. Ecol. Syst. 26: 403-422, 1995; Li, Molecular Evoluti
on, Sinauer Associates, Snderland, MA, 1997). For example, a positive selection for a protein (ie, application evolution at the molecular level) can be made by comparing pairs of ratios of non-synonymous nucleotide substitutions per non-synonymous site (KA) to synonymous substitutions per synonymous site (KS). It can be detected in the gene encoding the protein (Li et al., 1985; Li, 1993).
It is particularly convenient and most effective to compare the two variables KA and KS as a ratio, but any comparison of KA and KS is used. Sequences are identified by showing statistically significant differences between KA and KS using standard statistical methods.

Preferably, KA / KS analysis by Li et al. Is used to carry out the invention (Li et al., Mol.), Although other analysis programs capable of detecting interspecies positively selected genes can be used. Biol. Evol. 2: 150-174, 1985; and Li, J. Mol. Evol. 36:96.
-99, 1993; Messier and Stewart, Nature 385: 151-154, 1997; Nei, Mo.
lecular Evolutionary Genetics (New York, Columbia University Press), 198
7). The KA / KS method consists of comparing the non-synonymous substitution rate per non-synonymous site and the synonymous substitution rate per synonymous site between the gene regions encoding the homologous proteins with respect to the ratio. Used to identify sequence substitutions forced by adaptive selection. Synonymous ("static") substitutions are those in which there is no change in the encoded amino acid sequence due to the degeneracy of the genetic code; non-synonymous substitutions result in amino acid substitutions. The degree of change for each type can be evaluated as KA and KS, the number of synonymous substitutions per synonymous site and the number of non-synonymous substitutions per non-synonymous site, respectively. K
The calculation of A / KS can be performed manually or using software. An example of a suitable program is MEGA (Molecular Genetics Institute, Pennsylvania Sta.)
te University).

For the purpose of assessing KA and KS, sequences encoding fully or partially human proteins are often used to calculate the total number of synonymous and non-synonymous substitutions, It is. The length of the analyzed polynucleotide sequence can be any suitable length. Preferably, the entire coding sequence is compared to determine any or all significant changes. Li93 (Li, J. Mol. Evol. 36: 96-99,
A publicly available computer program such as 1993) or INA can be used to calculate KA and KS for all pairwise comparisons. This analysis further
Applies to examine sequences in a "sliding window" fashion so that a few significant changes are not hidden by the entire sequence. A "sliding window" refers to the examination of subsections of a continuous, overlapping gene (subsections can be of any length).

The comparison between the non-synonymous substitution rate and the synonymous substitution rate is represented by the KA / KS ratio. KA
/ KS showed that the applied evolution was a reflection of the extent to which it acted on the sequence under study. The full length or fragment of the encoding sequence can be used for KA / KS analysis. As the KA / KS ratio increases, it is more likely that one sequence undergoes adaptive evolution and that non-synonymous substitutions are evolutionarily significant. For example, Mess
See ier and Stewart (1997). Preferably, the KA / KS ratio is at least about 0.5. 75, more preferably at least about 1.0, more preferably at least about 1.25, more preferably at least about 1.50, and even more preferably at least about 2.00. Preferably, the statistical analysis indicates that all high KA / K
S-ratio was performed and includes standard methods such as student t-test and likelihood ratio test described by Yang (Mol. Biol. Evol. 37: 441-456, 1998), but is not limited to that range. Absent.

A KA / KS ratio sufficiently greater than 1 indicates that the positive selection fixes more amino acid substitutions than could be expected as a result of chance alone, with the commonly observed pattern where the ratio is less than 1. Strongly suggests contrast (Nei, 1987; Hughes and Hei, Nature 335: 167-170, 1998; Messier and Stewart, Current Biol. 4: 9.
11-913, 1994; Kreitman and Akashi, Ann. Rev. Ecol. Syst. 26: 403-422, 1995.
Messier and Stewart, 1997). A ratio of less than one generally means a negative role, or purifying choice: there is a strong force to remain unchanged in the primary structure of a functional and effective protein.

[0079] Any method for calculating the KA / KS ratio is based on the number of non-synonymous substitutions per non-synonymous site and the number of synonymous substitutions per synonymous site for regions of homologous genes encoding proteins from related species. Based on pairwise comparison of numbers. Each method implements a different modification to evaluate the "multiple hit count" (ie, there is one or more nucleotide substitutions at the same position). Each method also uses different models for how DNA sequences change during evolution time. That is, preferably, the combination of the results obtained from the different algorithms is used to increase the level of detection sensitivity of the positively selected genes and the level of confidence in the results.

Preferably, the KA / KS ratio should be calculated as an orthologous gene pair relative to a paralogous gene pair (ie, a gene derived from speciation as opposed to a gene that is the result of gene duplication). (Messier and Stewart, 19
97). This distinction is made by making further comparisons with other non-human primates, such as gorillas and orangutans, allowing for the creation of a phylogenetic tree. The orthologous gene used in phylogenetic tree construction will give a known "seed tree", ie, produce a tree that revives a known biological tree. In contrast, paralogous genes will produce trees that violate known biological trees.

It is understood that the methods described herein have led to the identification of human porin nucleotide sequences functionally related to sequences encoding human proteins. Such sequences include non-coding sequences or coding sequences that do not code for human proteins, and are not intended to limit its scope. For example, these related sequences may include sequences encoding human proteins in the human genome, such as introns, or sequences flanking the 5′- and 3′-sides (including regulatory elements such as promoters and enhancers). Can be physically adjacent. These related sequences are
It can be obtained by searching public human genome databases such as k, or alternatively, by screening and sequencing a human genomic library using a sequence encoding one protein as a probe. Methods and techniques for obtaining non-coding sequences using related coding sequences are well known to those skilled in the art.

Evolutionarily significant nucleotide changes are detected by molecular evolution analysis such as KA / KS analysis, but their unique occurrence in humans (or non-human primates), or human (or non-human) Primates) can be further evaluated for the extent to which those changes are unique. For example, the identified changes can be tested for the presence or absence in other non-human primate sequences. Sequences having at least one evolutionarily significant change between human and non-human primate are used as primers for PCR analysis of sequences encoding other non-human primate proteins and the resulting poly- The nucleotides are sequenced to see if the same change is present in other non-human primates. Furthermore, these comparisons indicate whether changes in adaptive evolution are unique to human lineage compared to other non-human primates, or whether adaptive changes are relative to humans and other non-human primates, Allow distinction as to whether it is unique to non-human primates (ie, chimpanzees). Nucleotide changes detected in humans, but not other primates, likely represent changes in human adaptive evolution. Alternatively, nucleotide changes detected in non-human primates or not detected in humans or other non-human primates are likely indicative of changes in chimpanzee application evolution. Other non-human primates used for comparison may be selected based on their systematic relationship to humans. Closely related primates can be primates within the primate human superfamily sublineage, such as chimpanzees, chimpanzees, gorillas, and orangutans. Non-human primates also fall outside the primate human superfamily, ie, are not as closely related to humans as the Old World monkeys and the New World monkeys. The statistical significance of such comparisons can be determined using established and available programs such as Messier and Stewart (Nature 385: 1
51-154, 1997). Such genes that exhibit a statistically high KA / KS ratio appear to have undergone adaptive evolution.

[0083] Sequences with significant changes can be used as probes in genomes from multiple human populations to determine if the sequence changes are shared by one or more human populations. Gene sequences obtained from different human populations include, for example, Huma
n Genome Project, a database made available by the Human Genome Diversity Project, or, optionally, DNA from a large number of unrelated different human populations, amplified by PCR and obtained from direct sequencing . further,
The presence of an identified change in different human populations will indicate the evolutionary significance of the change. Chimpanzee sequences with significant changes can be obtained and evaluated using similar methods to determine whether sequence changes are shared by many chimpanzees.

Sequences that have significant variation between species are further characterized for their molecular / genetic identity and biological function using methods and techniques known to those of ordinary skill in the art. For example, the sequences are genetically and physically located within the human genome using a bioinformatics program made publicly available. The newly identified significant changes in the nucleotide sequence are indicative of a potential role for the gene in human evolution and a potential link to functional capabilities unique to humans. The putative gene having the identified sequence is further characterized, for example, by a homology search. The shared homology of the putative gene and the known gene may indicate a similar biological role or function. Another exemplary method for characterizing a putative gene sequence is based on known sequence motifs. Certain sequence patterns are known to encode for regions of the protein having specific biological characteristics, such as signal sequences, DNA binding domains, or transmembrane domains.

[0085] Human identified sequences with significant changes can also be further evaluated by examining where the gene is expressed in terms of tissue or cell type specificity. For example, the identified coding sequence can be used as a probe to perform in situ mRNA hybridization indicating the expression pattern of the sequence. Genes that are expressed in certain tissues may be better candidates for being associated with important human functions associated with that tissue, such as brain tissue. The timing of gene expression at each stage of human development can also be determined.

[0086] As another exemplary method of sequence characterization, the functional role of identified nucleotide sequences with significant changes can be assessed using functional assays for different alleles of the identified genes in yeast, nematodes, Drosophila. , And by performing on a model system such as a mouse. Model systems are cell-based systems or in vivo systems such as transgenic animals. Preferably, transgenic mice are used. Methods for making cell-based and / or transgenic animal systems are known in the art and need not be described in detail here.

As another exemplary method of sequence characterization, computer programs are permitted to model and visibly model the three-dimensional structure of homologous proteins from humans and chimpanzees. The particular and accurate understanding that amino acids have been replaced by chimpanzee proteins allows the detection of structural changes associated with functional differences. That is, the use of modeling techniques is closely related to the identification of functional roles discussed in the previous paragraph. The use of one or a combination of these techniques forms part of the present invention. In chimpanzee ICAM-3, human ICAM-3 has a proline residue (P1
01) consists of a glutamine residue (Q101). Human proteins are known to be sharply curved at this point. Replacing glutamine with proline in chimpanzee proteins appears to result, at this point, to a lesser extent, a sharp curve. This converts the chimpanzee protein of ICAM-3 to HIV.
Obviously there is a close connection to packaging into a virus.

The sequences identified by the methods described herein have important applications in the diagnosis and treatment of medically or commercially relevant human conditions. Thus, the present invention provides a method for identifying a reagent that modulates a functional ability unique to humans or enhanced in humans and / or corrects deletions in such sequences by the use of such sequences. I will provide a. These methods use art-known screening techniques such as, for example, in vitro systems, cell-based expression systems, and transgenic animal systems. The approach provided by the present invention not only identifies genes that have evolved immediately, but also points out adjustments made to proteins that are less toxic as the genes are present in other species.

Screening Methods The present invention also provides screening methods using polynucleotides and polypeptides identified and characterized using the methods described above. These screening methods are useful for identifying reagents that modulate the function of a polynucleotide or polypeptide as effective for human therapy. Typically,
The method involves contacting a cell transformed with a polynucleotide sequence identified by the method described above, or at least one reagent tested with a preparation of a polypeptide encoded by such a polynucleotide sequence; Here, the reagent is identified by its ability to modulate the function of the polynucleotide sequence or polypeptide.

As used herein, the term “reagent” refers to a single or complex organic or inorganic molecule, peptide, protein or biological or chemical compound such as an oligonucleotide. I do. Large arrays of compounds can be synthesized, for example, as oligomers, such as oligopeptides and oligonucleotides, and as synthetic organic or inorganic compounds based on a variety of core structures, which are also referred to as the term "reagent". included. In addition, various natural resources, such as plant and animal extracts, provide compounds for screening. The compounds can be tested alone or in combination with one another.

“Modulate function” of a polynucleotide or polypeptide means that the function of the polynucleotide or polypeptide is altered as compared to the case without the addition of a reagent. Regulation can occur at any level that affects function. The function of the polynucleotide or polypeptide is direct or indirect;
It can also be measured directly or indirectly. "Function" of a polynucleotide includes, but is not limited to, substitutions, transcription, and expression patterns.
Polynucleotide function also includes functions related to the polypeptide encoded in the polynucleotide. For example, acting on a polynucleotide, expression, conformation, folding (or other physical characteristic) of a protein, binding to another moiety (such as a ligand), activity (other functional characteristic), protein structure Reagents that affect control and / or other aspects of function may modulate polynucleotide function. The ways in which effective reagents act to regulate the expression of a polynucleotide include: 1) altering the binding of a transcription factor to a transcription factor responsive element in the polynucleotide; Altering the interaction between species transcription factors; 3) altering the ability of the transcription factor to express the polynucleotide to enter the cell nucleus; 4) increasing the activity of transcription factors involved in polynucleotide transcription. Inhibiting; 5) interacting with a ligand, and binding of the ligand usually alters a cell surface receptor resulting from expression of the polynucleotide; 6) a component of the signaling cascade that leads to expression of the polynucleotide. Inhibiting the interaction; and 7) enhancing the activity of transcription factors involved in polynucleotide transcription, including Not intended to be limiting.

The “function” of a polypeptide is conformation, folding (or other physical characteristic), binding to another moiety (such as a ligand), activity (or other functional characteristic), and / or protein. It is not intended to limit the scope of the invention including many aspects of the structure or function of the invention. For example, it can act on a polypeptide to affect its conformation, folding (or other physical characteristic), binding to another moiety (such as a ligand), activity (or other functional characteristic), and / or protein Reagents that affect many aspects of structure or function may modulate polypeptide function. The ways in which effective reagents can act to modulate the function of a polypeptide are: 1) altering conformation, folding, or other physical characteristics; 2) altering the avidity of a normal ligand Or altering the binding specificity of the ligand; and 3) altering the activity of the polypeptide, but is not intended to limit its scope.

Generally, the choice of reagent to be screened depends on several parameters,
For example, a particular polynucleotide or polypeptide target, its perceived function,
Governed by its three-dimensional structure (if known or assumed) and other aspects of rational drug design. Combinatorial chemistry techniques can also be used to generate countless combinations of candidates. One skilled in the art can invent and / or obtain suitable reagents for testing.

The in vitro screening assays described herein have several advantages over typical drug screening assays: 1) if certain reagents do not enter cells to achieve the expected therapeutic effect; If not, the in vivo assay may give instructions as to whether the reagent can enter cells: 2) in v
An ivo screening assay is effective in that once a reagent has been added to an assay system, the reagent is ineffective at eliciting at least one characteristic associated with modulating polynucleotide or polypeptide function, once inside the cell. 3) Most importantly, but in vivo assay systems ultimately require the identification of features associated with polynucleotide or polypeptide function. It allows the identification of reagents that affect any component of the originating pathway.

In general, screening involves adding a reagent to an appropriate cell sample transformed with a polynucleotide identified using the methods of the present invention and effecting the effect, ie, the polynucleotide or polypeptide encoded within the polynucleotide. This can be done by tracking changes in functionality. The experiment preferably includes a control sample that does not receive the candidate reagent. The treated and untreated cells are then contrasted by any suitable phenotypic characteristics, microscopic analysis, viability testing, replication competence, histological testing, the level of specific RNA or polypeptide associated with the cells. Including the level of enzyme activity expressed by the cell or cell lysate, its interaction with the cell when exposed to an infectious reagent such as HIV, and the ability of the cell to interact with other cells or compounds. No attempt is made to limit the scope. For example, the transformed cells are exposed to the reagent to be tested, and before, during, and after the treatment of the reagent, the cells undergo HIV infection.
And is tested for any indication of the susceptibility of the cell to viral infection, such as, for example, the susceptibility of the cell to viral infection between cells, replication of the virus, production of viral proteins, and / or infection by the virus. Followed by the formation of fused cells. The difference between treated and untreated cells is the effect due to the candidate reagent. Optimally, the reagent has a greater effect on experimental cells than on control cells. Suitable host cells are eukaryotic cells, and preferably include mammalian animal cells, but are not intended to limit its scope. The selection of cells is, at least in part,
It will depend on the nature of the assay contemplated. To test for a reagent that upregulates expression of a polynucleotide, the polynucleotide was transformed with a polynucleotide of interest such that the polynucleotide is expressed (expression as used herein includes translation and / or transcription). Appropriate host cells are contacted with the reagent to be tested. Certain reagents will be tested for their ability to result from increased expression of mRNA and / or polypeptide. Methods for preparing and transforming vectors are well known in the art. "Transfection" includes any method of introducing an exogenous sequence, for example, lipofection, introduction, infection, or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector (such as a plasmid) or may be integrated into the host genome.

To identify a reagent that specifically activates transcription, a transcription control region is linked to a reporter gene and the construct is added to a suitable host cell. As used herein, the term "reporter gene" means a gene that encodes a gene product that can be identified (ie, a reporter protein). Reporter genes include, but are not intended to limit the scope of, alkaline phosphatase, chloramphenicol acetyltransferase, β-galactosidase, luciferase, and green fluorescein protein (GFP). Methods for identifying reporter gene products include enzyme assays and fluorometric assays, and are not intended to limit its scope. Reporter genes and assays for detecting these products are well known in the art and are described, for example, in Ausubel et al. (1987) and in periodic revisions. Reporter genes, reporter gene assays, and reagent kits are also readily available from commercial sources. Examples of suitable cells include fungi, yeast, mammals, and other eukaryotic cells, and are not intended to limit its scope. Those skilled in the art will appreciate techniques for transfecting eukaryotic cells, including the preparation of suitable vectors such as viral vectors, and introducing the vectors into cells by, for example, electroporation, e.g., a reporter. Or they may be well versed in selecting cells transformed with drug susceptibility factors. The effect of the reagent on transcription from the control region in such constructs will be assessed through the activity of the reporter gene product.

[0097] Except for an increase in expression under conditions that are normally suppressed as described above, normal expression results in reduced expression. Certain reagents accomplish this through a reduction in transcription rate, and the reporter gene system described above would provide an assay tool for this. Host cells for assaying such reagents will need to be tolerant of expression.

Cells that transcribe mRNA (obtained from a polynucleotide of interest) have the mRN
It can be used to identify reagents that specifically regulate the half-life of A and / or translation of mRNA. Such cells are also used to assay the effect of the reagent on ongoing and / or post-translational modification of the polypeptide. Reagents can modulate the amount of polypeptide in a cell by altering the turnover (ie, increasing or decreasing the half-life) of the polypeptide. The specificity of a reagent for mRNA and polypeptide is determined by examining the product in the absence of the reagent and examining the products of unrelated mRNA and polypeptide. Methods for determining mRNA half-life, protein processing, and protein turnover are well known to those skilled in the art.

[0099] In vivo screening methods are also useful in identifying reagents that modulate polypeptide function through direct interaction with the polypeptide. Such reagents could block normal polypeptide-ligand interactions, and enhance or stabilize such interactions, if any. Such reagents may also alter the conformation of the polypeptide. The effect of the reagent is
It can be determined using an immunoprecipitation reaction. Suitable antibodies are used to precipitate the polypeptide and any proteins closely related thereto. By comparing polypeptides immunoprecipitated from treated and untreated cells, if any,
Reagents that increase or inhibit the polypeptide-ligand interaction are identified. Polypeptide-ligand interactions can also be assayed using a cross-linking agent that converts close but non-covalent interactions between the polypeptides into covalent interactions. Techniques for examining protein-protein interactions are well known to those skilled in the art.
Techniques for assaying protein conformation are also well known to those skilled in the art.

Screening methods include in vitro methods such as transcription and translation systems in cell-free systems.
It is also understood to be relevant to the method. In such systems, transcription or translation is allowed to occur, and the reagents are tested for their ability to modulate function.
In vitro transcription / translation systems can be used in assays to determine whether a reagent modulates mRNA or polynucleotide. Such systems are commercially available and provide an in vitro approach to producing mRNA that matches a polynucleotide sequence of interest. After production of the mRNA, it is translated in vitro and the translation products are compared. Contains no reagent (negative control) in
Comparison of translation products between in vitro expression systems and in vitro expression systems containing reagents
Indicates whether the reagent affects translation. A comparison of the translation product between the control and the test polynucleotide was made if the reagents were to act at this level (general,
Indicates whether it selectively affects translation (as opposed to affecting non-selective or non-specific translation). Modulation of polypeptide function can be achieved in a number of ways, including in vivo and in vitro assays listed above, as well as in vitro assays using protein preparations, and is not intended to limit its scope. . Polypeptides can be extracted and / or purified from natural resources or recombinant to make protein preparations. A reagent is added to a protein preparation sample and whether and how the reagent acts on the polypeptide, and whether the reagent conforms, folds (or other physical characteristics) of the polypeptide, etc. Binding to a moiety (such as a ligand), activity (or other functional characteristic), and / or whether or not it affects other aspects of protein structure or function. The effect is thought to be modulation of polypeptide function.

In the example of an assay for a reagent that binds to a polypeptide encoded by a polynucleotide identified by the methods described herein, the polypeptide (encoded by the polynucleotide identified as described above) is well characterized. The polypeptide is initially expressed recombinantly in a prokaryotic or eukaryotic expression system, either as a natural or fusion protein complexed with the assigned epitope or protein. Thereafter, the recombinant polypeptide is purified, for example, by immunoprecipitation using a suitable antibody or anti-epitope antibody, or by binding the complex to an immobilized ligand. Thereafter, an affinity column consisting of a polypeptide or fusion protein is used to screen a mixture of appropriately labeled compounds. Suitable labeling reagents include fluorophores, radioisotopes, enzymes and chemiluminescent compounds, and are not intended to limit its scope. Unbound and bound compounds can be separated by washing with various conditions as commonly used by those skilled in the art (eg, high salts, surfactants). Non-specific binding to the affinity column can be minimized by pre-washing the mixture of compounds using an affinity column consisting of only one of the complex or epitope. Similar methods can be used as a screen for reagents that compete for binding to the polypeptide. In addition to affinity chromatography, changes in the melting temperature of proteins that will alter the binding to other molecules,
Or there are other techniques such as measuring the change in fluorescence anisotropy. For example, a BIAcore assay (Pharmacia Bio) using a sensor chip covalently bound to a polypeptide
Provided by sensors, Stit et al., Cell 80: 661-670, 1995) are performed to determine the binding activity of different reagents.

The in vitro screening methods of the present invention include structural or rational drug design, wherein the amino acid sequence, three-dimensional atomic structure, or other property of the protein is expected to bind to the polypeptide. Provides the basis for the design of the reagents used. In general, the design and / or selection of reagents in this context is a side-by-side comparison of the human and homologous non-human primate polypeptide structures, their three-dimensional structure (if known or putative). ), And other aspects of rational drug design. Combinatorial chemistry techniques are also used to generate countless combinations of candidate reagents.

[0103] Transgenic animal systems are also contemplated in the screening methods of the invention and are known in the art. The screening methods described above represent a primary screen and are designed to detect any agent that modulates the function of a polynucleotide or polypeptide. One skilled in the art will recognize that a secondary test will be required to further evaluate the reagent. For example, secondary screening involves testing the reagents for infection assays using mice and other animal models (such as rats), which are known in the art. In addition, toxicity tests will be performed as further confirmation that reagents that yield a positive result in the primary screen will be suitable for use in organisms. Any toxicity test is suitable for this purpose, including for example the MTT assay (Promega).

[0104] The invention also includes reagents identified by the screening methods described herein. Efficient Methods for Identifying Positively Selected Non-Human Features In one aspect of the invention, a non-human primate polynucleotide or polypeptide undergoes natural selection due to a positive, evolutionarily significant change. (Ie, a non-human primate polynucleotide or polypeptide has positive properties and is absent in humans). In this aspect of the invention, a positively selected polynucleotide or polypeptide may be involved in susceptibility or resistance to a disease, or may be associated with other commercially relevant features. Examples of this embodiment include polynucleotides and polypeptides positively selected in non-human primates, preferably chimpanzees, which are associated with susceptibility or resistance to infections, cancer, or acne, or It is not intended to limit its scope, including those associated with an aesthetic condition of interest to humans, such as growth or muscle mass.
Examples of this aspect include polynucleotides and polypeptides associated with susceptibility or resistance to progression from HIV to AIDS. Therefore, the present invention
It is useful to add insight into the molecular mechanisms underlying resistance to progression from V infection to AIDS development, discover agents such as agents that prevent and / or delay the development of AIDS, and / or It provides information that is also useful for designing. Commercially relevant examples include, and limit the scope of, positively selected polynucleotides and polypeptides in non-human primates that are associated with aesthetic features such as hair growth, acne, or muscle mass It is not something to try.

Thus, in one aspect, the invention provides a method for identifying a polynucleotide sequence that encodes a polypeptide, wherein the polypeptide has a medically relevant positive evolutionally significant change. Related. Positive evolutionary significant changes are found in human or non-human primates, but evolutionarily significant changes in positively selected non-human primates will be described here for the first time. Would. The method comprises: (a) comparing a nucleotide sequence encoding a human protein with a nucleotide sequence encoding a non-human primate protein; A human polynucleotide sequence comprising at least one nucleotide change is selected, wherein the change comprises steps that include being evolutionarily significant. Sequences identified by this method have been further characterized and / or
Alternatively, it is analyzed for possible association with human-specific or enhanced biologically or medically relevant functions.

Within the present invention, there is also provided a method for identifying positive evolutionarily significant changes in a nucleotide sequence encoding a human protein, comprising: (a) a sequence encoding a human protein; Comparing with a non-human primate sequence, wherein (b) selecting a human nucleotide sequence comprising at least one nucleotide change relative to the non-human primate identity sequence, wherein The change consists of stages that include being evolutionarily significant.

Efficient Methods for Identifying Positively Selected Human Features The present invention relates to human polymorphisms that are associated with human specific or enhanced functional abilities, such as prolonged brain function or longevity. Specifically provided are methods for identifying nucleotide and polypeptide sequences. More specifically, such methods identify gene sequences associated with unique or enhanced abilities in humans and include brain functions such as advanced information processing, storage and repair, creativity, and language. It is not intended to limit its scope. In addition, such methods identify sequences that are associated with other brain function characteristics with respect to the functioning of the human brain at enhanced levels compared to other non-human primates: Including prolonged emotional reactions, exercise, sensations of pain / joy, olfaction, temper and longevity. In this method, the general method of the present invention is utilized as described above. In general, the methods described herein include (a) comparing a polynucleotide sequence encoding a human protein to a non-human primate sequence; and (b) comparing a human sequence to a non-human primate. It requires selecting polynucleotide sequences encoding human proteins that have evolutionarily significant changes associated with unique or enhanced functional capabilities.

In this aspect, human sequences include sequences that are evolutionarily significant (ie, wherein the human sequence has one or more of: It differs from the sequence of non-human primates). The identity and functionality of the protein encoded by the gene including the evolutionarily significant changes are characterized and a determination is made whether the protein is involved in a particular or enhanced human function. if,
If the protein is associated with a unique or enhanced human function, the information may include:
It is useful as a means to identify reagents that supplement or otherwise modulate unique or enhanced human functions.

As an example, without limiting the scope of the invention, identifying genetic (ie, nucleotide sequence) differences that are functionally underlying human brain modulates human brain function and / or functions. It provides a basis for designing reagents that help to compensate for failure. Such sequences can also be used in the development of diagnostics and / or biomedical research tools. The present invention also provides a method for large-scale comparison of sequences encoding human brain proteins with sequences of non-human primates.

[0110] The identified human sequence changes can be used to establish a database of candidate human genes involved in human brain function. Candidates are ordered for those whose genes may be responsible for the unique or enhanced functional abilities found in the human brain compared to chimpanzees or other non-human primates. In addition, the database not only provides a regular collection of candidate genes,
It also provides the exact molecular sequence differences that exist between humans and chimpanzees (and other non-human primates), thus defining the changes that underlie functional differences. This information can be useful in identifying possible sites on the protein that serve as effective targets for the drug.

Thus, the present invention also provides a method for determining the correlation between an evolutionarily significant nucleotide change and the ability of a brain to be unique or enhanced in a human, comprising: Identifying the human nucleotide sequence, (b)
It consists of analyzing the functional effects of the presence or absence of the sequence identified by the model system.

Further studies can be performed to confirm the putative function. For example, putative functions may be in transfected or cultured cells of animals transfected with stably or stably transfected animals, or animals transfected with an antisense clone that inhibits expression of a polynucleotide identified to assess the effect of non-expression of the encoded polypeptide. The cells are tested in an appropriate iv in vitro assay. Such as one or two hybrid studies can be performed, for example, to determine what other macromolecules a polypeptide interacts with. Transgenic nematodes or Drosophila can be used for various functional assays, including ethology. Proper study depends on the nature of the identified polynucleotide and the polypeptide encoded within the polynucleotide, as will be apparent to those of skill in the art.

Details of AIDS Specific Examples (Examples of Positively Selected Non-Human Features) It is estimated that the AIDS (Acquired Immune Deficiency Syndrome) epidemic threatens 30 million people worldwide. (UNAIDS / WHO, 1998. Report on HIV / AIDS pandemic worldwide). Just over a million people in developed countries are infected, and in sub-Saharan Africa, one in four adults is now the carrier of the virus (UNAIDS
/ WHO, 1998). Efforts to develop vaccines are ongoing, but prospects for vaccine success in the near future are severe (Balter and Cohen, Science 281: 159-160, 1988;
Baltimore and Heilman, Scientific Am. 279: 98-103, 1998). Further complicating the development of therapies is the rapid mutation rate of HIV, the human immunodeficiency virus that causes AIDS, which results in rapid changes in viral proteins. These changes eventually escape the virus from current therapies targeting viral proteins (Dobkin, Inf. Med. 15 (3): 159, 1998). Even drug cocktails, which initially showed great promise, are subject to the emergence of drug-resistant mutants (Balter and Cohen, 1998; Dobkin, 1998). Thus, there is still a significant shortfall in developing treatments that delay or prevent the spread of AIDS to people infected with HIV. Chun et al., Proc. Natl. Acad. Sci. USA 94: 13193-13197, 1997; Dobkin, 199.
8).

The human analog chimpanzee (Pan troglodytes) has been proven to be an unfavorable model for disease progression following infection with HIV-1 (Novembre et al., J. Virol). 71 (5): 4086-4091, 1997). Once infected with HIV-1, chimpanzees are resistant to disease transmission. To date, more than 100 captured chimpanzees have been infected, but only one chimpanzee is known to develop terminal AIDS (Novembre et al., 1997; Villinger et al., J. Med. Primatol 26 (1-2): 11-18, 1997). Clearly, understanding the mechanisms that confer resistance to disease transmission in chimpanzees proves to be invaluable for efforts to develop therapeutics for humans infected with HIV.

It is generally believed that wild chimpanzee populations harbored the HIV-1 virus (perhaps for thousands of years) prior to recent cross-species transmission to humans (Du
be et al., Virology 202: 379-389, 1994; Zhu and Ho, Nature 373: 503-504, 1995; Z
Hu et al., 1998; Quinn, Proc. Natl. Acad. Sci. USA 91: 2407-2414, 1994). This long-term virus / host coevolution is apparently due to adaptation and explains chimpanzee resistance to AIDS transmission (Burnet and White, Natural History
of Infectious Disease (Cambridge, Cambridge Univ. Press), 1972; Ewald, Hu
m, Nat. 2 (i): 1-30, 1991). All documents mentioned herein are hereby incorporated by reference.

One aspect of the invention is that (a) chimpanzees (Pan troglodytes) are susceptible to HIV infection but resistant to the development of AIDS (Alter et al., Science 226).
: 549-552, 1984; Fultz, J. Virol. 58: 116-124, 1986; Novembre et al., J. Virol.
71 (5): 4086-4091, 1997) while humans are susceptible to the development of this catastrophic disease, so genes in chimpanzees contribute to this resistance; (b) other species ( When comparing homologous genes (such as chimpanzees), the change in human genes results from the observation that they are evolutionarily significant (ie, positive selective pressure). That is, the protein encoding the polynucleotide is not human,
It contains plausible sequence changes as a result of positive adaptive selection during evolution found in chimpanzees (similar to other AIDS-resistant primates). Furthermore, such evolutionary significant changes in polynucleotide and polypeptide sequences may result in the ability of AIDS-resistant non-human primates (such as chimpanzees) to resist the development of AIDS. The method of the present invention uses selective comparative analysis to identify candidate genes associated with susceptibility or resistance to AIDS, as well as specific information on changes that have evolved to confer resistance, as well as new information for therapeutic intervention. A host target may be provided. The development of therapeutic approaches involving host proteins (as opposed to viral proteins and / or mechanisms) may even be to delay and / or avoid the emergence of resistant virus mutants. The invention also provides screening methods using the identified sequences and structural differences.

The present invention provides methods for identifying human polynucleotide and polypeptide sequences that are associated with susceptibility to developing AIDS after infection. Conversely, the present invention relates to AIDS-resistant non-human primates that are associated with resistance to the development of AIDS (
Also provided are methods for identifying polynucleotide and polypeptide sequences obtained from such a chimpanzee. Identifying the genetic (ie, nucleotide sequence) and consequent protein structure and biochemical differences that underlie susceptibility and resistance to the development of AIDS may be useful in preventing and / or treating the progression of HIV infection to AIDS. It will probably provide a basis for the discovery and / or design of reagents that can be provided. These differences can also be used in the development of diagnostics and / or biomedical research techniques. For example, identification of a protein that confers resistance can lead to the development of diagnostics or biomedical research techniques based on disruption of disease pathways involving the resistance protein.

In general, the methods described herein comprise (a) comparing a polynucleotide sequence encoding a human protein to a sequence of a non-human primate (such as a chimpanzee) that is resistant to AIDS, wherein The polynucleotide sequence encoding the human protein comprises A
It requires selecting polynucleotide sequences encoding human proteins that are associated with the development of IDS and (b) have evolutionarily significant changes associated with susceptibility to the development of AIDS. In another embodiment, the method comprises: (a) comparing a polynucleotide sequence encoding a human protein to a sequence of a non-human primate (such as a chimpanzee) resistant to AIDS, wherein the human protein is encoded. The polynucleotide sequence is associated with the development of AIDS; and (b
2.) It requires selecting polynucleotide sequences encoding non-human primate proteins that have evolutionarily significant changes associated with resistance to the development of AIDS.

[0119] Clearly, the methods described therein are applicable to other infectious diseases. For example, the method is known or believed to have latent infections for a significant period of time (i.e., sufficient time to allow positive selection) by non-human primates, And in situations such as resisting the onset of disease. Thus, in another aspect, the invention provides a method for identifying a polynucleotide sequence encoding a polypeptide, wherein the polypeptide is associated with resistance to the development of an infectious disease, and further comprising: a) comparing a sequence encoding a protein of a non-human primate resistant to infection with a sequence encoding a human protein, wherein the sequence encoding the human protein comprises being associated with the development of an infection; and (B) selecting a non-human primate sequence that is resistant to an infectious disease comprising at least one nucleotide change compared to the matching human sequence, wherein the nucleotide change is evolutionarily significant Are included. In another embodiment, the invention is directed to
A method for identifying a polynucleotide sequence encoding a human polypeptide is provided, wherein the polypeptide is associated with susceptibility to the development of an infectious disease, and further comprising: (a) a sequence encoding the human protein; Comparing a polynucleotide sequence encoding a protein of a non-human primate resistant to infection, wherein the sequence encoding the human protein is associated with the development of the infection, and (b)
At least one non-human primate matched sequence that is resistant to infection;
Selecting a human polynucleotide sequence containing one nucleotide change, wherein the nucleotide change is evolutionarily significant.

In the present invention, human sequences to be compared to homologous sequences obtained from AIDS-resistant non-human primates are associated with HIV transmission (ie, replication), seeding, and / or subsequent progression to AIDS. Selected based on known or implicit gender. Such knowledge can be obtained, for example, from publications and / or public databases (GenB
including sequence databases such as ank). Since the pathways involved in the development of AIDS, including viral replication, are associated with many genes, a large number of suitable candidates can be tested using the methods of the present invention. Table 1 contains an illustration of the genes tested.
In general, sequences are known in the art.

[0121]

Table 1: Sample list of human genes tested / tested Gene Function eIF-5A Initiator hPC6A Protease hPC6B Protease p56lek Signaling FK506 Binding Protein Immunophilin Calnexin? Bax PCD promoter Bcl-2 apoptosis inhibitory factor lck tyrosine kinase MAPK (mitogen activated protein) protein kinase CD43 sialoglycoprotein CCR2B chemokine receptor CCR3 chemokine receptor Bonzo chemokine receptor BOB chemokine receptor BOB chemokine receptor BOB chemokine receptor BOB chemokine receptor BOB chemokine receptor BOB chemokine receptor BOB chemokine receptor (SDF-1) Chemokine Tumor necrosis factor-α (TNF-α) PCD promoter TNF receptor II (TNFRII) receptor Interferon γ (IFN-γ) site Cain interleukin 1α (IL-1α) site Cain interleukin 1β (IL -1β) Site Caine Interleukin 2 (IL-2) Site Caine Interleukin 4 (IL- 4) Site Cain Interleukin 6 (IL-6) Site Cain Interleukin 10 (IL-10) Site Cain Interleukin 13 (IL-13) Site Cain B7 Signaling Protein Macrophage Colony Stimulating Factor (M-CSF) Site Cain Granulocyte Macrophage colony stimulating factor site Cain phosphatidylinositol 3-kinase (PI3-kinase) kinase phosphatidylinositol 4-kinase (PI4-kinase) kinase HLA class I α chain histocompatibility antigen β2 microglobulin lympho site antigen CD55 decay accelerating factor CD56 complement protein CD63 glycoprotein antigen CD71? Interferon α (IFN-α) site cytokine CD44 cell adhesion CD8 glycoprotein Genes already tested (12) ICAM-1 immune system ICAM-2 immune system ICAM-3 immune system Leukocyte-related factor 1 molecule α (LFA-1) immunity System Leukocyte-related factor 1 molecule β (LFA-1) immune system Mac-1α Immune system Mac-1β (same as LFA-1β) Immune system CXCR4 Chemokine receptor CCR5 Chemokine receptor MIP-1α Chemokine MIP-1β Chemokine RANTES Chemokine Human Aligned sequences encoding AIDS-resistant non-human primate proteins such as and chimpanzees are analyzed to identify nucleotide sequence differences at specific sites. Detected sequence changes are generally and preferably initially confirmed for accuracy, as described above. Evolutionarily significant nucleotide changes are KA / K
It can be detected by molecular evolution analysis, such as S-analysis, and further evaluated to determine whether a non-human primate gene or a human gene has undergone a positive selection.
For example, the identified changes are examined for the presence in other AIDS-resistant non-human primate sequences. Sequences having at least one evolutionarily significant change between human and AIDS-resistant non-human primates are used as primers for PCR analysis of sequences encoding other non-human primate proteins The resulting polynucleotide is then sequenced to see if the same change is present in other non-human primates. Such a comparison allows further distinction as to whether adaptive evolutionary changes are unique to AIDS-resistant non-human primates (such as chimpanzees) as compared to other non-human primates. For example, nucleotide changes detected in chimpanzees but not in other primates probably indicate a positive selection in the chimpanzee gene. Other non-human primates used for comparison may be selected based on phylogenetic relationships with humans. Closely related primates may be present in sublineage of primates, such as chimpanzees, chimpanzees, gorillas, and orangutans. Non-human primates are located outside the primate human superfamily, meaning they are not very closely related to humans, such as the Old World monkey or the New World monkey. Such statistical significance can be determined using established and available programs, for example, t-tests as used by Messier and Stewart (Nature 385: 151-154, 1997).

In addition, sequences that have significant changes can be used as probes to genomes from different humans to determine if the sequence changes are shared by one or more individuals. For example, in certain populations, progression to AIDS is slow ("slow progressors"), (a) a comparison between a chimpanzee sequence and a homologous sequence obtained from a slow progressor group, and / or (b) A comparison between the AIDS susceptible group and the slow progressor group would be of interest. Gene sequences obtained from different human populations may be obtained, for example, from databases made available by the Human Genome Diversity Project, or alternatively, may be obtained from many unrelated and different human populations.
Obtained from direct sequencing of CR amplified DNA. The presence of the changes identified in human slow progressors further indicates the evolutionary significance of the changes.

The following examples are provided to further assist those of ordinary skill. Such examples are intended to be illustrative and therefore should not be considered as limiting the invention. Many illustrative modifications and variations are described in this application, and others will be apparent to those skilled in the art. Such changes are
It is considered to be included within the scope of the described invention and the claims herein.

[0124] EXAMPLES Example 1: Construction chimpanzee cDNA library cDNA library was constructed using the chimp tissues.

[0125] Total mRNA was extracted from the tissue (RNeasy kit, Quiage; RNAse-free R
apid Total RNA kit, 5 Prime-3 Prime, Inc.), RNA retention and purification were determined according to typical molecular cloning procedures. PolyA + RNA was isolated (Min
i-Oligo (dT) Cellulose Spin Columns, 5 Prime--3 Prime, Inc.), oligo (d
It is used as a template for reverse transcription of cDNA with T) as a primer. The synthesized cDNA is processed and modified for cloning using commercially available kits. The recombinant is then packaged and propagated in a host cell line. Amplify part of the packaging mixture and retain the rest before amplifying. The library was standardized and the number of independent recombinants in the library was determined.

Example 2: Sequence Comparison [0126] PCR of chimpanzee cDNA was performed by preparing appropriate primers based on a candidate human gene and using cDNA prepared from a cDNA library or mRNA.
Used for amplification. Selected chimpanzee cDN obtained from cDNA library
The A clone is sequenced using an automated sequencer such as ABI377. Commonly used primers and reverse primers for cloning vectors such as M13 Universal are used to screen. For inserts that have not been completely sequenced by end sequencing, dye-labeled terminators are used to fill in any remaining gaps.

For example, finding places where there is a difference between the chimpanzee and human sequences; determining whether a base that seems to be human-specific corresponds to a specifically strong and distinct signal for the corresponding base To determine the number of human hits to see if there is one or more human sequences that correspond to a sequence change; and By other known methods, the detected sequence differences are first checked for accuracy. The invasion of various human sequences into the same gene with the same nucleotides where different chimpanzee nucleotides are present provides independent support that human sequences are correct and that chimpanzee / human differences exist. Such changes are examined using public database information and the genetic code that determines whether these DNA sequence changes are due to changes in amino acids in the encoded protein. The sequence can also be determined by direct sequencing of the encoded protein.

Example 3 Molecular Evolution Analysis Chimpanzee and human sequences subjected to comparison undergo KA / KS analysis.
In this analysis, commonly available computer programs such as Li93 and INA were used, and for each sequence submitted to the study described above, the number of synonymous changes per site (KS) / non-synonymous changes per site Determine the number (KA). Full length coding regions or fragments of coding regions can be used. The higher the KA / KS ratio, the more likely the sequence was undergoing adaptive evolution. Statistical significance of KA / KS values was determined using established statistical methods and available programs such as t-test.

To further support the significance of high KA / KS ratios, the sequences delegated to the study are compared with a diverse population of chimpanzees and other non-human primates such as gorillas, orangutans, and chimpanzees be able to. Such a comparison further allows a distinction as to whether changes in adaptive evolution are specific to human lineage compared to other non-human primates. The sequence is also examined by directly sequencing the gene of interest from a sample of several different human populations to assess to what extent the sequence is conserved in humans.

Example 4 Identification of Positively Selected ICAM-1, ICAM-2 and ICAM-3 Using the method of the invention described herein, the intercellular adhesion molecule, ICAM-1
, ICAM-2 and ICAM-3 were shown to be strongly positively selected. I
CAM molecules are known to be involved in several immune response interactions and play a role in progression to AIDS in HIV-infected humans. The ICAM protein is a member of the immunoglobulin (Ig) superfamily and a ligand for the integrin leukocyte associated factor-1 molecule (LFA-1) (Makg
oba et al., Nature 331: 86-88, 1988). LFA-1 is expressed on the surface of most leukocytes, whereas ICAM is expressed on the surface of leukocytes and other cell types (Larson et al., J. Cell Biol. 108: 703-712, 1989). ICAM and LFA-1 proteins are involved in several immune response interactions, include T cell function, and direct leukocytes to areas of inflammation (Larson et al., 1989).

Cells taken from primate tissue (chimpanzee brain and blood, gorilla blood and kidney, orangutan blood) or the following B white blood cell lines: CARL (chimpanzee), ROK (gorilla), and PUTI (orangutan) From total RNA
RNese kit (Quagen) or RNAse-free Rapid Total RNA kit (5 Prime--3 Pr
ime, Inc.). mRNA was isolated from total RNA using a Mini-Oligo (dT) cellulose spin column (5 Prime--3 Prime, Inc.). cD
AN was synthesized oligo dT and / or random priming from mRNA using a cDNA synthesis kit (Stratagene). The protein coding region of the primate ICAM-1 gene was amplified from cDNA using primers (concentration = 100 nmol / μl) manually designed from the published human sequence. PC for ICAM-1 amplification
The R conditions were first pre-melted at 94 ° C (4 minutes), followed by 35 cycles of 94 ° C (15 seconds), 58 ° C (1 minute 15 seconds), 72 ° C (1 minute 15 seconds) and finally Extended for 10 minutes at 72 ° C. The PCR is ready-ready with a total reaction volume of 50 μl.
It was completed using to-Go RCR beads (Amersham Pharmacia Biotech). Qia Quick
The appropriate sized product was purified from agarose gel using the Gel Extraction kit (Qiagen). The strand of the amplification product was directly sequenced using the Big Dye Cycle Sequencing Kit, and analyzed using a 373A DNA sequencer (ABI BioSystems).

Comparison of the portion encoding the protein of the ICAM-1 gene of human, gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) with the corresponding portion of the chimpanzee showed a statistically significant KA / KS ratio. (Table 2). The portions encoding the human and chimpanzee ICAM-1 gene proteins have been previously published and the portions encoding the gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) ICAM-1 gene proteins are shown in FIG. Each is shown in FIG.

In this example, the paired KA / KS ratio was calculated for the mature protein using the algorithm of Li (1985; 1993). Statistical significance (determined by t-test)
Is shown in bold. Although a comparison of gorillas and humans is sufficient to show that chimpanzee ICAM-1 has been positively selected, the presumed historical scope of African gorillas is that gorillas are exposed to the HIV-1 virus. Suggest that you got
Orangutans were similarly compared (Nowak and Paradiso, Walker's Mammals of t
he World (Baltimore, MD. The Johns Hopkins University Press), 1983). However, orangutans are always restricted to Southeast Asia, which means that they will not be exposed to HIV throughout evolutionary time (Nowak and Paradis
o, 1983) (gorillas are closest to humans and chimpanzees, while orangutans are farther away).

[0134]

Table 2: KA / KS ratio: ICAM-1 total protein comparison Species compared KA / KS ratio Chimpanzee and human 2.1 (P <0.01) Chimpanzee and gorilla 1.9 (P <0.05) ) Chimpanzees and orangutans 1.4 Humans and gorillas 1.0 Humans and orangutans 0.87 Gorillas and orangutans 0.95 Even among the proteins that showed positive selection, KA / K as high as ICAM-1 comparison
Few exhibit S ratios (Lee and Vacquier, Biol. Bull. 182: 97-104, 1992; S
wanson and Vacquier, Proc. Natl. Acad. Sci. USA 92: 4957-4961, 1995; Messi.
er and Stewart, 1997; Sharp, Nature 385: 111-112, 1997). The results are consistent with strong selection pressure due to adaptive changes with the chimpanzee ICAM-1 molecule.
The domains of the ICAM-1 molecule (D1 and D2) that bind to LFA-1 have been demonstrated (Staunton et al., Cell 61: 243-254, 1990). The paired KA / KS comparison between the primate ICAM-1 genes, ie, the KA / KS ratio, was calculated only for domains D1 and D2 using the algorithm of Li (1985; 1993) (Table 3). Statistical significance comparison is (t
(Determined by inspection), shown in bold. A rather high, statistically significant KA / KA ratio for domains D1 and D2 suggests that these regions of the protein were very strongly positively selected. These regions of chimpanzee ICAM-1
Showing a significant KA / KS ratio (Table 3) than that seen for the whole protein comparison;
This suggests that the ICAM-1 / LFA-1 interaction was under abnormally high selection pressure.

[0135]

Table 3: KA / KS ratio: domain D1 + D2 of ICAM-1 Species compared KA / KS ratio Chimpanzee and human 3.1 (P <0.01) Chimpanzee and gorilla 2.5 (P <0 .05) Chimpanzee and orangutan 1.5 (P <0.05) Human and gorilla 1.0 Human and orangutan 0.90 Gorilla and orangutan 1.0 Basically, ICAM-2 of positively selected chimpanzee The same procedure was used to identify as ICAM-3 (see Table 4). The ligand binding domain of ICAM-1, in contrast to ICAM-2 and ICAM-3, is positioned as presenting a particularly strong positive selection, which results from amino acid substitutions throughout the protein. That is, this comparative genomic analysis was performed using chimpanzee ICAM-1.
Indicates that the positive selection in has altered the primary structure of the protein, for example, a critical binding domain. These changes may have conferred resistance to AIDS progression in chimpanzees.

[0136]

Table 4 KA / KS ratio: total protein comparison between ICAM-2 and 3 Species compared KA / KS ratio ICAM-2 in chimpanzee and human 2.1 (P <0.01) ICAM- in chimpanzee and human 33.7 (P <0.01) Binding of ICAM-1, 2, and 3 plays an essential role in the formation of fused cells (ie, giant, multinucleated cells) in HIV infected cells in vitro. (Pantaleo et al., J. Ex. Med. 173: 511-514, 1991). Fusion cell formation is performed in vitro
o followed CD-cell depletion (Pantaleo et al., 1991; Levy, Microbiol. Rev. 57: 183).
-189, 1993; Butini et al., Eur. J. Immunol. 24: 2191-2195, 1994; Finkel and Ban.
ad, Curr. Opin. Immunol. 6: 605-615, 1994). Fusion cell formation is difficult to detect in vivo, but clumps of infected cells are found in the lymph nodes of infected individuals (Pantaleo et al., N. Eng. J. Med. 328: 327- 335, 1993; Finkel and Banda
Embretson et al., Nature 362: 359-362, 1993; Pantaleo et al., Nature 362: 35.
5-358, 1993). The fused cells are rapidly removed from the body and can only be detected (Fouchier et al., Virology 219: 87-95, 1996). It is speculated that in vivo fusion cell-mediated deletion of CD44 cells occurs, which is directly linked to immune system impairment, leading to opportunistic infections and terminal AIDS (Sodeosky et al., Nature 322: 4).
70-474, 1986; Hildreth and Orentas, Science 244: 1075-1078, 1989; Finkel and Banda, 1994). Thus, definitive changes in chimpanzee ICAM-1, ICAM-2, or ICAM-3 may prevent fusion cell formation in chimpanzees and may help explain chimpanzee resistance to AIDS. Because of the multifunctional nature of ICAM-1, these positively selected changes in the ICAM gene may confer additional resistance to other infectious diseases and may be of value in developing therapeutics in humans. Can play a role in the inflammatory process. The alignment of the polypeptide sequences of ICAM-1, -2 and -3 is shown in FIGS. 5, 6 and 7, respectively.

Example 6 Characterization of ICAM-1, ICAM-2, and ICAM-3 Positively Selected Sequences The sequences identified by the method of the present invention were further tested by cell transfection assays and characterized. Can be attached. For example, cultured human cells can be transfected with the chimpanzee nucleotides identified by the methods described herein (ICAM-1 (or ICAM-2 or ICAM-3, see below)) in a technically standard assay. Can be tested for reduced virus seeding and / or transmission using a control cell and compared to control cells.Depending on the observed or apparent functional properties of the polynucleotide / polypeptide to be tested, Other characteristics can also be measured, for example, ICAM-1 (or ICAM-2 or ICAM
In case of -3), the formation of fused cells can be measured and compared with control (untransfected) cells. This will test whether resistance results from inhibition of the formation of fused cells by infected cells.

Cells effective to characterize the sequences identified by the methods of the invention and their effects on cell-to-cell infection by HIV-1 are human T cell lines that are tolerant to HIV-1 infection, eg, ATCC Includes H9 and HUT78 cell lines available from.

For the cell transfection assay, ICAM-1 (or ICAM
-2 or ICAM-3) cDNA (or any cDNA identified by the methods described herein) can be cloned into a suitable expression vector. To obtain maximum expression, cloned ICAM-1 (or ICAM-2)
Alternatively, the region encoding ICAM-3) is operably linked to a promoter that is active on human T cells, such as the IL-2 promoter. Alternatively, the ICAM-1 (or ICAM-2 or ICAM-3) cDNA can be placed under the transcriptional control of a strong constitutive or inducible promoter. Expression systems are well known in the art and are a method for introducing an expression vector into cells. For example, ICAM-1 (or ICAM-2 or ICAM-3)
The expression vector making up the cDNA can be introduced into cells by DEAE dextran, electroporation, or any other known method. Thereafter, the cloned ICAM-1 (or ICAM-2 or ICAM-3) molecule is expressed on the cell surface. ICAM-1 (or ICAM-2 or ICAM-3) c
The determination of whether the DNA has been expressed on the cell surface is completed using an antibody specific for ICAM-1 (or ICAM-2 or ICAM-3). Chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) expressed on the surface of human T cells
In the case of chimpanzee and human ICAM-1 (or ICAM-2 or ICAM-2)
Antibodies that distinguish AM-3) can be used. The antibody comprises a detectable labeling reagent,
For example, it can be labeled with a fluorescent dye. Chimpanzee ICAM-1 (or I)
Cells expressing CAM-2 or ICAM-3) are identified using fluorescence-activated cell sorting or appropriately labeled antibody ICAM-1 (or ICAM-2 or ICAM-3). Or using well-established techniques.

The transfected human cells expressing the chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) on the cell surface were then subjected to fusion cell formation, and / or HIV replication, and / or intercellular The number of infected cells is examined using infection as an index. Cells expressing chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) are infected with HIV-1 at an appropriate dose. For example, a tissue culture infection dose of 50, ie, a dose capable of infecting 50% of the cells. Infected cells when seeded at a cell density of about 500,000 cells / ml in an appropriate tissue culture medium and after infection, fusion cells formation and / or virus replication, compared to control uninfected cells. Observe the number. Cells not transfected with chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) are also used as controls. Fusion cell formation generally involves cells infected with HIV-1 about 10 days after infection (chimpanzee ICAM-1 (or I
CAM-2 or ICAM-3).

For observation of HIV replication, cell supernatants are assayed for the presence and amount of p24 antigen. Any assay that detects p24 can be used, for example, using a rabbit anti-p24 antibody as the capture antibody and a biotin-labeled rabbit anti-p as the detection antibody.
Using 24 antibodies, the assay involves an ELISA expressed with horseradish peroxidase conjugated avidin. Any known method can be used to determine the number of infected cells, including indirect immunofluorescence. In indirect immunofluorescence, human HIV positive serum can be used as a source of anti-HIV antibodies to bind to infected cells. Bound antibodies are detected using a FITC-conjugated anti-human IgG antibody, cells are visualized with a fluorescence microscope, and cell numbers are determined.

Another method for assessing the role of a molecule, such as ICAM-1 (or ICAM-2 or ICAM-3), involves the continuous infection of cells with HIV. A human cell line, preferably a human cell line that does not express endogenous ICAM (although cell lines that do not express endogenous ICAM can also be used) are human or chimpanzee ICAM-1 or -2 or-. Transfected in 3. In one series of experiments, HIV was recovered from the supernatant of cells expressing human ICAM-1 (or ICAM-2 or ICAM-3) infected with HIV and chimpanzee ICAM.
It is used to infect cells expressing -1 (or ICAM-2 or ICAM-3) or cells expressing human ICAM-1 (or ICAM-2 or ICAM-3). Measured as described above, but chimpanzee ICA
M-1 (or ICAM-2 or ICAM-3) and human ICAM-1
The initial infectivity of cells expressing (or ICAM-2 or ICAM-3) is expected to be high. After several replications, chimpanzee ICAM-1 (
Or ICAM-2 or ICAM-3) confers resistance, the cell-to-cell infection may be chimpanzee ICAM-1 (or ICAM-2 or ICAM-2).
In cells expressing AM-3), a decrease would be expected. Second
In a series of experiments, HIV was transmitted to the chimpanzee ICAM-1 (
Alternatively, it is recovered from cells expressing ICAM-2 or ICAM-3) and used to infect cells expressing human ICAM-1 (or ICAM-2 or ICAM-3). In this case, ICAM-1 (or ICAM-
2 or ICAM-3) is involved in susceptibility to HIF transmission,
Initial infectivity is expected to be significantly lower than in the first series of experiments.
After several replications, cell-to-cell infection would be expected to increase.

The identified human sequences can be used to establish a database of human candidate genes involved in conferring or contributing AIDS sensitivity or resistance. In addition, the database not only provides an ordered collection of candidate genes, but also reports the exact molecular sequence differences that exist between humans and non-human primates resistant to AIDS (such as chimpanzees). Provide, that is, define clearly the changes that underlie functional differences.

Example 7 Molecular Modeling of ICAM-1 and ICAM-3 The modeling of the three-dimensional structure of ICAM-1 and ICAM-3 demonstrates the role of these proteins in explaining chimpanzee resistance to AIDS progression. Provides additional evidence for

In the case of ICAM-1, five out of six amino acid substitutions that are unique to chimpanzee lineage are immediately adjacent to amino acids identified by mutagenic studies as important for LFA-1 binding (ie, , Physical contact). These five amino acid substitutions result from human L18 to chimpanzee Q18,
No. 9 was replaced with chimpanzee D29, human P45 was replaced with chimpanzee G45, human R49 was replaced with chimpanzee W49, and human E171 was replaced with chimpanzee Q171. This positioning cannot be predicted from the primary structure (ie, the actual sequence of the amino acids). Among the amino acid residues important for binding, chimpanzee ICAM-
Nothing changed in 1.

[0146] Such positioning is based on the basic function of the chimpanzee ICAM-1 protein.
It strongly demonstrates that there is no change between humans and chimpanzees, however,
Evolution has caused fine-tuned changes that help confer resistance to progression of AIDS to chimpanzees. The characteristics of amino acid substitutions are being investigated to allow the use of three-dimensional structural information in developing reagents for therapeutic intervention. Remarkably,
Four out of five chimpanzee residues are adjacent to important binding residues identified as N-linked glycosylation sites. This is human and chimpanzee ICAM-1
Suggest that there is a difference in binding constants (for LFA-1). These binding constants have been measured. Given that binding constants are demonstrated to be low in chimpanzee ICAM-1, it is also possible to invent small molecular reagents that mimic (in a sterically hindered manner) changes in binding constants as a possible therapeutic strategy for HIV infected humans. It is possible. Similarly, stronger binding constants, if observed for chimpanzee ICAM-1, suggest a selective strategy for developing therapeutic interventions for humans infected with HIV.

In the case of ICAM-3, proline (observed in 7 humans) to glutamine (
A key amino acid residue substitution (observed in three chimpanzees) is predicted from our modeling studies to significantly alter the positional angle between domain 2 and domain 3 of human and chimpanzee ICAM-1. You. Human proteins show steep angles at this joint (Klickstein et al., J. Biol. Chem. 27: 239 20-27, 1996). The loss of this sharp angle (bend) is expected to represent chimpanzee ICAM-3, which is not easily packaged in HIV-1 virions (in infected humans, after ICAM was packaged in HIV virions, The intercellular infection increases dramatically (Barbeau, B. et al., J. Virol. 72: 7125-7136, 1998). The inability of this chimpanzee ICAM-3 to be readily packaged into HIV-1 virions would prevent the increase in cell-to-cell infection seen in infected humans. This will explain the resistance of chimpanzees to AIDS progression.

[0148] The binding of appropriately designed small molecules to human proline residues can be attributed to human ICA.
Chimpanzee ICA with larger (ie, not steep) angles of M-1 protein
Enables small molecule therapeutic intervention mimicking M-3 (as a result of steric hindrance). The retention of critical binding residues between the two proteins (and the general similarity of the immune response between humans and chimpanzees) demonstrates that this change in angle does not compromise the basic function of human ICAM-3. I do. However, the human ICAM-3 protein is not resistant to packaging into HIV virions, meaning that infected chimpanzees
Putative pathways that resist progression to DS (in humans infected with HIV)
It may be resistant to imitation.

Example 8: Identifying positive selection of MIP-1α MIP-1α is a site cytokine that has been shown to suppress HIV-1 replication in human cells in vitro (Cocchi, F. et al.). Science 270: 1811-1815, 1995). The chimpanzee homolog of the human MIP-1α gene was amplified by PCR and sequenced. Calculation of the KA / KS ratio (2.1, P <0.05) and comparison with gorilla homology indicate that the chimpanzee gene was positively selected. With respect to the other genes discussed herein, the characteristics of amino acid substitutions in chimpanzees have been examined to determine how to utilize chimpanzee proteins for therapeutic intervention.

Example 9: Identifying Positive Selection of 17-β-Hydroxysteroid Dehydrogenase Using the method of the present invention, positive selection was made in comparison to the human homolog (GenBank Acc. # X87176) ( KA / KS = 1.6) A chimpanzee gene expressed in the brain was identified. The human gene, 17-β-hydroxysteroid dehydrogenase type IV, encodes a protein known to weaken the two most potent estrogens, β-estradiol and 5-diel (Adamski, J. et al., Biochem. . J. 311: 437
-443, 1995). Estrogen-related cancers, including, for example, lung and prostate cancers, account for approximately 40% of human cancers. Interestingly, reports in the literature suggest that chimpanzees are resistant to tumorigenesis, especially estrogen-associated tumorigenesis. This protein may have been positively selected in chimpanzees to allow more efficient depletion of estrogens, ie, confer chimpanzee resistance to such cancers.
If so, the specific amino acid substitutions observed in the chimpanzee protein will provide important information for therapeutic intervention in human cancer.

Example 10: Construction of cDNA Library A cDNA library of chimpanzee brain was constructed using chimpanzee brain tissue. Chimpanzee brain tissue could be obtained after natural death, so there was no need to kill animals for this study. However, mRNA expressed in the brain
The brains were obtained as soon as possible after the animals died in order to increase the chances of obtaining intact. Preferably, the weight and age of the animal are measured before death. The brain tissue used to construct the cDNA library is preferably whole brain, in order to maximize the content of mRNA expressed in whole brain. Following standard surgical procedures, brain tissue was excised from the animals.

[0152] Total RNA was extracted from brain tissue, and RNA retention and purity were determined according to typical molecular cloning techniques. PolyA + RNA was selected and used as a template for reverse transcription of cDNA with oligo (dT) as a primer. Synthesized c
DNA was processed and modified for cloning using commercially available kits. The recombinant was then packaged into a host cell line and propagated. A portion of the packaged mixture was amplified and the rest was retained before amplification. The library was standardized and the number of individual recombinants in the library was determined.

Example 11 Sequence Comparison Chimpanzee brain cDNA clones arbitrarily selected from a cDNA library were sequenced using an automatic sequencer such as ABI377. M
Commonly used primers and reverse primers for cloning vectors such as 13 Universal are used to screen. For inserts that have not been completely sequenced by terminal sequencing, a dye-labeled terminator is used to fill in any remaining gaps.

The resulting chimpanzee sequence was compared to human sequences from a database such as a BLAST search. The "hit count", which is a high score showing significant (eg,> 80%) similarity after BLAST analysis, is collected and analyzed. Thereafter, the two homologous sequences are aligned using the sequence program CLUSTAL V developed by Higgins et al. Any sequence diversity, including nucleotide substitutions, additions and deletions, is detected and recorded by the alignment.

Finding places where there is a difference between chimpanzee and human sequences; to determine whether a base that seems to be unique to humans corresponds to a specifically strong and distinct signal for the corresponding base Examine the sequence fluorogram (chromatogram); examine the number of human hits to see if there is one or more human sequences that correspond to the sequence variation; and By other methods, the detected sequence differences are first checked for accuracy. The invasion of various human sequences into the same gene with the same nucleotides where different chimpanzee nucleotides are present provides independent support that human sequences are correct and that chimpanzee / human differences exist. Such changes are examined using public database information and the genetic code that determines whether these DNA sequence changes are due to changes in amino acids in the encoded protein.

Example 12: Molecular Evolution Analysis Chimpanzee and human sequences subjected to comparison undergo KA / KS analysis.
In this analysis, commonly available computer programs such as Li93 and INA were used, and for each sequence submitted to the study described above, the number of synonymous changes per site (KS) / non-synonymous changes per site Determine the number (KA). This ratio, KA / KS, indicates that it reflects adaptive evolution, ie, the extent to which positive selection has affected the sequence entrusted to the study. Typically, the full length coding region is used in these comparative analyses. However, coding fragments can also be used effectively. The higher the KA / KS ratio, the more likely the sequence was undergoing adaptive evolution. Statistical significance of KA / KS values was determined using established statistical methods and available programs such as t-test. Genes that show a statistically high KA / KS ratio between chimpanzees and humans appear to have undergone the most adaptive evolution. To further support the significance of the high KA / KS ratio, For example, comparisons can be made within other non-human primates, such as gorillas, orangutans, and chimpanzees. Such comparisons further allow a distinction as to whether changes in adaptive evolution are unique to human lineage compared to other non-human primates. The sequence is also examined by directly sequencing the gene of interest from a sample of several different human populations to assess to what extent the sequence is conserved in humans.

Example 13 Further Characterization of Sequences Human brain nucleotide sequences containing evolutionarily significant changes are further characterized in terms of molecular and genetic properties, as well as biological function. The identified coding sequence may represent an expression pattern of the gene in terms of which tissue and cell type the sequence is expressed in and / or when the sequence is expressed during development or during the cell cycle. Used as a probe to perform mRNA hybridization. Sequences expressed in the brain are candidates for being associated with important human brain functions. In addition, putative genes having the identified sequences are subjected to homology searches to determine what class of functions the sequence belongs to.

In addition, for certain proteins, the identified human sequence changes are useful in assessing the functional significance of the change. By using such features, a database of candidate genes is created. Candidates have identified that the gene has unique or enhanced abilities found in the human brain compared to chimpanzees or other non-human primates, for example, as well as advanced information processing, storage and replication It is ranked in terms of likelihood due to proficiency, language ability, etc. In this way, this approach offers a new strategy that such genes can be identified. In conclusion, the database not only provides an orderly collection of candidate genes, but also provides the exact molecular sequence differences that exist between humans and chimpanzees (and other non-human primates); Clarify the change that underlies the differences.

In some cases, functional differences are assessed in a suitable model system, in vitro analyzes such as long-lasting enhancement (LTP) characteristics, and transgenic animals or other suitable model systems. Including but not limited to use. These will be readily apparent to those skilled in the art.

Example 14 Identification of a Positive Selection in the Human Tyrosine Kinase Gene Using the method of the present invention, a human gene (GenBank Acc. # AB014541) expressed in the brain was identified and compared to a chimpanzee homolog. Was exactly selected. This gene encodes a tyrosine kinase and is a well-characterized mouse gene (GenBank
Acc. # AF011908) and its gene product is called AATYK and is known to cause apoptosis (Gaozza, E. et al., Oncogene 15: 3127-3135, 1).
997). Literature suggests that this protein controls apoptosis in the developing mouse brain (ie, "mining" the developing brain). The tyrosine kinase domain of this protein is well conserved among mice, chimpanzees, and humans (mostly tyrosine kinases). Interestingly, however, the region of the protein to which the signaling protein binds is positively selected in humans, but is strongly conserved in chimpanzees and mice. The region of the human protein to which the signaling protein binds is not only positively selected as a result of nucleotide point mutations, but also dimerizes several SH2 binding domains that exist only as a single copy in mice and chimpanzees. Is additionally shown. This suggests that a different set of signaling proteins can bind to human proteins and subsequently trigger different pathways of apoptosis in the developing human brain compared to mice and chimpanzees. Thus, such genes may contribute to a unique or enhanced human cognitive ability.

While the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. . Accordingly, the description and examples are not to be construed as limiting the scope of the claims, but rather are set forth in the appended claims.

[Brief description of the drawings]

FIG. 1 shows a phylogenetic tree for primates within the primate hominid group. The order of branching is based on well-supported mitochondrial DNA lines (Mess
ier and Stewart, Nature 385: 151-154, 1997).

FIG. 2 (SEQ ID NOS: 1-3) shows human and chimpanzee ICAM-1 sequences (GenBank accession numbers X06990 and V8, respectively).
6848) (corresponding to 6848). The amino acid translation of the chimpanzee sequence is shown below the column.

FIG. 3 shows the nucleotide sequence of Gorilla ICAM-1 (SEQ.
ID NO: 4).

FIG. 4 shows the nucleotide sequence of orangutan ICAM-1 (SEQ ID NO: 5).

FIGS. 5 (A)-(E) show a comparison of the polypeptide sequences of several primate ICAM-1s.

FIGS. 6 (A)-(B) show a comparison of the polypeptide sequences of several primate ICAM-2s.

FIGS. 7 (A)-(P) show a comparison of the polypeptide sequences of several primate ICAM-3s.

FIG. 8 shows an illustration of a procedure for comparing human / primate brain polynucleotides, selecting sequences with evolutionarily significant changes, and further characterizing the selected sequences. The diagram of FIG. 8 illustrates the priority scheme of the present invention, which description serves to explain the principles of the present invention, and includes details and optional additional steps. Any human / primate polynucleotide sequence can be compared in the same procedure,
It turns out that the procedure is not limited to brain polynucleotides.

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──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF terms (reference) 4B063 QA06 QA08 QA13 QA18 QA19 QA20 QQ02 QQ03 QQ08 QQ43 QQ53 QR62 QS25

Claims (63)

[Claims] 1. A method for identifying a polynucleotide sequence of a non-human primate that encodes a polypeptide, said polypeptide being associated with a physiological condition in a non-human primate, comprising: (A) comparing a polynucleotide sequence encoding a non-human primate protein with a polynucleotide sequence encoding a human protein, wherein the human has no physiological condition; and b) selecting a non-human primate polynucleotide sequence that includes the nucleotide change as compared to the corresponding human sequence, and comprising the change being evolutionarily significant. 2. The method of claim 1, wherein said physiological condition is resistant to a disease. 3. The method of claim 2, wherein said disease is cancer. 4. The method of claim 2, wherein said disease is an infectious disease. 5. The method of claim 4, wherein said infectious disease is a viral disease. 6. The method of claim 5, wherein said viral disease is AIDS. 7. The method of claim 1, wherein the sequence encoding the non-human protein is associated with the occurrence of a physiological condition. 8. The method of claim 1, wherein said non-human primate is a member of a primate human superfamily. 9. The method of claim 8, wherein the non-human primate is selected from the group consisting of a chimpanzee, a chimpanzee, a gorilla, and an orangutan. 10. The method of claim 9, wherein said non-human primate is a chimpanzee. 11. The method of claim 1, wherein said nucleotide change is a non-synonymous substitution. 12. The evolutionary significance of the nucleotide change is determined by the non-synonymous substitution rate (K
The method of claim 1, determined according to A). 13. The method of claim 12, wherein the evolutionary significance of said nucleotide change is determined by the ratio of the non-synonymous substitution rate (KS) to the synonymous rate (KS) of the nucleotide sequence. 14. The method of claim 13, wherein said KA / KS ratio is at least about 0.75. 15. The method of claim 13, wherein said KA / KS ratio is at least about 1.00. 16. The method of claim 13, wherein said KA / KS ratio is at least about 1.25. 17. The method of claim 13, wherein said KA / KS ratio is at least about 1.50. 18. The method of claim 13, wherein said KA / KS ratio is at least about 2.00. 19. A method for identifying a human polynucleotide sequence that encodes a polypeptide, said polypeptide comprising being associated with a physiological condition in a human, comprising the steps of: Comparing the polynucleotide sequence encoding the protein with the polynucleotide sequence encoding the non-human primate protein, wherein the non-human primate has no physiological condition; and (b) non-human Selecting a human polynucleotide sequence that includes a nucleotide change when compared to the corresponding sequence of a primate of said primate, wherein the change is evolutionarily significant. 20. The method of claim 19, wherein said physiological condition is lifespan. 21. The method of claim 19, wherein said physiological condition is brain function. 22. The method of claim 21, wherein said brain function is cognitive ability. 23. The sequence of claim 1, wherein the sequence encoding the human protein is associated with the development of a physiological condition.
9. The method according to 9. 24. The method of claim 19, wherein said non-human primate is a member of a primate human superfamily. 25. The method of claim 24, wherein said non-human primate is selected from the group consisting of a chimpanzee, a cockpit chimpanzee, a gorilla, and an orangutan. 26. The method of claim 25, wherein said non-human primate is a chimpanzee. 27. The method of claim 19, wherein said nucleotide change is a non-synonymous substitution. 28. The evolutionary significance of the nucleotide change is determined by the non-synonymous substitution rate (K
20. The method according to claim 19, determined according to A). 29. The method of claim 28, wherein the evolutionary significance of said nucleotide change is determined by the ratio of the non-synonymous substitution rate (KS) to the synonymous rate (KS) of the nucleotide sequence. 30. The method of claim 29, wherein said KA / KS ratio is at least about 0.75. 31. The method of claim 29, wherein said KA / KS ratio is at least about 1.00. 32. The method of claim 29, wherein said KA / KS ratio is at least about 1.25. 33. The method of claim 29, wherein said KA / KS ratio is at least about 1.50. 34. The method of claim 29, wherein said KA / KS ratio is at least about 2.00. 35. A method for identifying an evolutionarily significant change in a polynucleotide sequence encoding a human brain protein, comprising the following steps: (a) combining a polynucleotide sequence encoding a human brain protein with a non-human Comparing the corresponding sequence of a primate of the invention; and (b) selecting a human polynucleotide sequence comprising a nucleotide change when compared to the corresponding sequence of a non-human primate, wherein the change evolves Including significant significance; 36. The method of claim 35, wherein said non-human primate is a member of a primate human superfamily. 37. The method of claim 36, wherein said non-human primate is selected from the group consisting of a chimpanzee, a cockpit chimpanzee, a gorilla, and an orangutan. 38. The method of claim 37, wherein said non-human primate is a chimpanzee. 39. The method of claim 35, wherein the nucleotide sequence encoding a human brain protein corresponds to a human brain cDNA. 40. The method of claim 35, wherein said nucleotide change is a non-synonymous substitution. 41. The evolutionary significance of the nucleotide change is determined by the non-synonymous substitution rate (K
The method according to claim 35, determined according to A). 42. The method of claim 41, wherein the evolutionary significance of said nucleotide change is determined by the ratio of the non-synonymous substitution rate (KS) to the synonymous rate (KS) of the nucleotide sequence. 43. The method of claim 42, wherein said KA / KS ratio is at least about 0.75. 44. The method of claim 42, wherein said KA / KS ratio is at least about 1.00. 45. The method of claim 42, wherein said KA / KS ratio is at least about 1.25. 46. The method of claim 42, wherein said KA / KS ratio is at least about 1.50. 47. The method of claim 42, wherein said KA / KS ratio is at least about 2.00. 48. A method for large-scale sequence comparison between a polynucleotide sequence encoding a human protein and a polynucleotide sequence encoding a protein obtained from a non-human primate, comprising the following steps: ) Aligning the human polynucleotide sequence with the corresponding polynucleotide sequence from a non-human primate according to sequence homology; and (b) comparing the human sequence when compared to a homologous sequence from a non-human primate Identifying any nucleotide change in which the change is evolutionarily significant. 49. The method of claim 48, wherein said protein-encoding sequence is obtained from human brain. 50. A method for identifying a reagent that modulates a physiological condition, comprising contacting a cell transduced with the polynucleotide sequence identified in claim 1 with at least one reagent to be tested. Wherein said reagent is identified by its ability to modulate the function of a polynucleotide sequence. 51. A reagent identified by the method of claim 50. 52. A method for identifying a reagent that modulates a physiological condition, comprising using at least one of the cells transduced with the polynucleotide sequence identified in claim 19 to be tested.
The method of contacting two reagents, comprising: identifying a reagent by its ability to modulate the function of a polynucleotide sequence. 53. A reagent identified by the method of claim 52. 54. A method of identifying a reagent that modulates a physiological condition, comprising testing a polypeptide encoded within the polynucleotide sequence identified in claim 1 or a composition comprising the peptide. The method comprising contacting at least one reagent to be performed, wherein the method comprises identifying a reagent by its ability to modulate the function of a polynucleotide sequence. 55. A reagent identified by the method of claim 54. 56. A method for identifying a reagent that modulates a physiological condition, comprising testing a polypeptide encoded within the polynucleotide sequence identified in claim 19, or a composition comprising said peptide. The method, comprising contacting at least one reagent to be performed, wherein the reagent is identified by its ability to modulate the function of a polynucleotide sequence. 57. A reagent identified by the method of claim 56. 58. A method of correlating an evolutionarily significant human nucleotide change with a physiological condition in a human, comprising the steps of: comprising, in a suitable model system, the polynucleotide sequence identified in claim 1; Analyzing the possible functional effects and indicating that the presence of the functional effects indicates a correlation between an evolutionarily significant nucleotide change and a physiological state. 59. A method of correlating an evolutionarily significant human nucleotide change to a physiological condition in a human, comprising: in a suitable model system, the polynucleotide sequence identified in claim 19 having Analyzing the possible functional effects and indicating that the presence of the functional effects indicates a correlation between an evolutionarily significant nucleotide change and a physiological state. 60. A method of correlating an evolutionarily significant human nucleotide change to a physiological condition in a human, comprising the steps of: encoding the polynucleotide sequence identified in claim 1 in a suitable model system. Analyzing the functional effects that the polypeptide may have, and indicating that the presence of the functional effects indicates an evolutionarily significant correlation between the nucleotide change and the physiological state. 61. A method for correlating an evolutionarily significant human nucleotide change to a physiological condition in a human, comprising the steps of: encoding the polynucleotide sequence identified in claim 19 in a suitable model system. Analyzing the functional effects that the polypeptide may have and indicating that the presence of the functional effects indicates an evolutionarily significant correlation between the nucleotide change and the physiological state. 62. A method of identifying a target site suitable for therapeutic intervention, which corresponds to a non-human primate polypeptide encoded by the polynucleotide sequence identified by the method of claim 19. Such a method, comprising indicating a target site at which the location of the molecular difference, as compared to the human polypeptide, may be present. 63. A method for identifying a target site suitable for therapeutic intervention, comprising a non-human polypeptide corresponding to the human polypeptide encoded by the polynucleotide sequence identified by the method of claim 1. Such a method, comprising indicating a target site at which the location of the molecular difference, as compared to the peptide, may be present.